Accurate genetic maps are the cornerstones of genetic discovery, but their construction can be hampered by missing parental genotype information. Inference of parental haplotypes and correction of phase errors can be done manually on a one by one basis with the aide of current software tools, but this is tedious and time consuming for the high marker density datasets currently being generated for many crop species. Tools that help automate the process of inferring parental genotypes can greatly speed the process of map building. We developed a software tool that infers and outputs missing parental genotype information based on observed patterns of segregation in mapping populations. When phases are correctly inferred, they can be fed back to the mapping software to quickly improve marker order and placement on genetic maps.ParentChecker is a user-friendly tool that uses the segregation patterns of progeny to infer missing genotype information of parental lines that have been used to construct a mapping population. It can also be used to automate correction of linkage phase errors in genotypic data that are in ABH format.ParentChecker efficiently improves genetic mapping datasets for cases where parental information is incomplete by automating the process of inferring missing genotypes of inbred mapping populations and can also be used to correct linkage phase errors in ABH formatted datasets. Lack of knowledge of the parental phase of all alleles segregating in mapping populations can impinge on the accuracy of genetic maps. Recombinant inbred line (RIL) populations developed from two inbred lines are a powerful resource for construction of genetic linkage maps. However, it is not uncommon to observe segregation of markers in RILs that are observed to be fixed in the putative inbred parents of the RIL, and conversely, to observe markers that are polymorphic in the two RIL parents, but fixed in the RIL population. This indicates that the real parents used in the cross to develop the RIL population are different than the available "off parents". This situation probably has two primary causes: 1) where one or both parents were not completely inbred at the time the population was initiated, or 2) from the existence of residual genetic variation within one or both parental lines. This observation is not surprising given that ten or more years can pass between the time when a RIL population is initiated with a cross between two parent plants and the time when it is genotyped along with the presumed parental lines. In both scenarios, one plant of an inbred line was used for the initial hybrid, while another closely related plant of the same inbred line was used for genotyping years later. Thus for case 1) where the original parent plant was heterozygous (Aa) at some fraction of its genome at the time of crossing and then subsequently maintained by inbreeding, the current (more inbred) version of the 'parent' line will have become fixed randomly for either AA or aa, causing the 'unexpected' segregation in the RIL half of the time. For case 2, it is not hard to envisage the existence of limited genotypic differences among individuals within an inbred crop line or variety because it has been standard practice to produce foundation seed-stocks of new cultivars from 'headrow' bulks of 'on-type' highly inbred sublines [1 plant. However, as long as the genotyped parental stocks are genetically very similar to the actual parents, enough information resides in the mapping population to correctly infer the haplotype composition of the actual parents.In other cases, the original parental seed source used to make the RIL population may have been lost as a result of error or project discontinuity, such as personnel changes, which may further complicate the identity of the real parent(s). The problem for the production of a genetic map is that it is advantageous to know the parental phase of all alleles, but the "off-parent" genotypes cannot be used to infer the allele phase of every marker. This "off-parent" problem is most severe when the alleles of both parent stocks are opposite from the alleles in the actual parents of the initial FPrior to the advent of high density genotyping, lack of marker coverage limited the prospects to detect cases of the off-parent problem and to correctly infer the actual parent. High-density genotyping greatly increases the opportunity to observe the off-parent problem and enables the inference of actual parental genotypes ,3. The iHere we present a new software package, ParentChecker, that addresses two common needs in the preparation of genotyping data for mapping with inbred populations in plant species: 1) inference of the actual parental haplotype, which is relevant to biallelic or ACGT format datasets, and 2) automatic correction of the phase of markers in individuals in the mapping population if the markers are expressed in biallelic format and the parental genotypes are unknown.The current version of ParentChecker was developed to handle single-nucleotide polymorphism (SNP) data (in ACGT format). However, it also works for other co-dominant markers that are coded in A, B, H or AA, AB, BB format. ParentChecker is very efficient in terms of memory storage and computational speed. On a desktop computer with CPU 2.0 GHz/2 GB RAM, ParentChecker only needs a few seconds to process genetic data from a relatively large segregating population . The algorithms implemented by ParentChecker to infer the unknown parental genotypes and linkage phase are as follows:2 population. The ratio becomes 3/8: 2/8: 3/8 for an F3. For each additional generation of selfing, the proportion of heterozygotes is reduced by half and the reduced part is equally divided and added to the two homozygotes. Therefore, the theoretical ratio between the two homozygotes is always 1:1. However, there is no theoretical genotype proportion for the two homozygotes when the cross is made by crossing a homozygote and a heterozygote during the construction of the population. Therefore, a χ2 test can be used to determine whether the expected proportions of homozygous individuals are statistically different than 1:1 and thus infer the cross type (e.g. whether it was AA × aa or Aa × aa) for the parents by comparing the observed genotype proportions and the theoretical values listed in Table Parents used to derive inbred mapping populations are usually assumed to be pure lines. In practice, the parents are often heterozygous for some limited number of loci. Table where the major homozygote is defined as the homozygote with frequency higher than that of the other homozygote. The test statistics is calculated asN is the population size, Ohomozgous1 and Ohomozgous2+ heterozygotes are observed frequencies for major homozygote and that of the other two genotypes, respectively. ParentChecker assigns the cross type with the smallest statistics value. If (a) is accepted, the segregating population is assumed to be derived from homozygous parental genotypes; otherwise, the initial cross is assumed to have been made between a homozygote and a heterozygote. Although a cross between two heterozygotes can also produce the same ratio as (a), ParentChecker only suggests the cross type of two homozygotes because the probability of a mating between two heterozygotes can be assumed to be very low for known inbred lines of self-pollinated species and safely ignored. In addition, the initial step for cross type Aa × Aa can also be regarded as the cross between two F1 individuals. Therefore, there is no fundamental difference between Aa × Aa and AA × aa, especially for advanced generations.where Consider three adjacent markers that are dispersed along a linkage group as follows:During meiosis, the frequency of crossovers for each interval is assumed to be independent of other intervals, which means that the recombination frequency between two adjacent markers depends only on the interval size bracketed by the two markers and is not affected by other intervals. Therefore, only the genotypic information of the two markers is relevant for the inference of the linkage phase. This feature allows the use of a hidden Markov model.Assume that the two alleles for marker M1 are A and a and the two alleles for marker M2 are B and b. The parental haplotype for generating the segregating population is either in coupling phase (AABB and aabb) or in repulsion phase (AAbb and aaBB). Since the linkage phase is a dichotomous event, we consider the coupling phase as status 1 and the repulsion phase as 0. If the hypothesis of coupling phase is rejected, the repulsion phase is accepted.Frequencies of the two-locus genotypes are listed in Table Pp = Pr = 0.5 while the alternative hypothesis is Pp >Pr,Table whereandr1 and is calculated from dl using Haldane's [Pp and Pr and are 0.9218 and 0.0782, respectively. Suppose that there are only 50 individuals in total for the four genotypes in the diagonal of Table p-value is 0.0006. Therefore, if the observed counts for AABB + aabb are larger than that of AAbb + aaBB, it is statistically safe to suggest that the linkage between M1 and M2 is coupling if the observed Pp is larger than Pr without calculating the test statistics.The recombination frequency between M1 and M2 is denoted by aldane's  or Kosamaldane's  map funcParentChecker uses the segregating patterns of markers and a linkage map to infer the parental genotypes that produced the segregating population. The formulas that are implemented in the current release of ParentChecker rest on two assumptions: the molecular markers are codominant and markers exhibiting distorted segregation have been removed from the dataset. Users are strongly suggested to use the built-in functions of ParentChecker to remove incompetent markers from the genotypic data before exporting the final outputs. Although the fundamentals of phase inference in linkage analysis has been discussed in detail -9, the sFor SNP data, a recommended workflow for ParentChecker would be to load data in ACGT format and use the output information (inferred parent) from ParentChecker for subsequent analysis such as building improved maps and QTL detection. For SNP data inputted in ACGT format, ParentChecker can generate an output in ABH format suitable for mapping and QTL detection. Furthermore, ParentChecker can directly export input files for popular genetic software packages including FlapJack , GGT 1111, MapQTThe input data format for ParentChecker is flexible. ParentChecker can take data directly from tab-delimited text files or import data from an Excel clipboard. The order of the markers in the genotype file does not have to match the order of the markers in the map as long as the marker names are consistent between the two files.4 mapping populations that had been genotyped with the Illumina 1536-SNP GoldenGate Assay [4 populations had at least one case of missing parental genotype information, with the number of missing parent data totalling 310 instances and ranging from 1 to 107 per mapping population : Windows XP/7Programming language: DelphiLicense: FreewareAny restrictions to use by non-academics: NoneAdditional materials: Two sample datasets from our cowpea project are provided in the ParentChecker package for testing and demonstration purposes.The authors declare that they have no competing interests.ZH designed and implemented the software used in this project. JDE conceived of the study, participated in the design of the application and helped draft the manuscript. PAR, TJC, SW, ML and SX participated in the design of the application and helped draft the manuscript. All authors read and approved the final manuscript.ZH is working under the direction of SX conducting research in the analysis of quantitative genetic variation. JDE is a cowpea breeder developing modern breeding methods. PAR, TJC, SW, and ML are conducting genetic research and genomic resource development on cowpea in support of breeding and trait discovery.


Sub-population structure and intricate kinship dynamics might introduce biases in molecular anthropology studies and could invalidate the efforts to understand diseases in highly admixed populations. In order to clarify the previously observed distribution pattern and morbidity of Chagas disease in Gran Chaco, Argentina, we studied two populations (Wichí and Criollos) recruited following an innovative bio-cultural model considering their complex cultural interactions. By reconstructing the genetic background and the structure of these two culturally different populations, the pattern of admixture, the correspondence between genealogical and genetic relationships, this integrated perspective had the power to validate data and to link the gap usually relying on a singular discipline. Although Wichí and Criollos share the same area, these sympatric populations are differentiated from the genetic point of view as revealed by Non Recombinant Y Chromosome genotyping resulting in significantly high Fst values and in a lower genetic variability in the Wichí population. Surprisingly, the Amerindian and the European components emerged with comparable amounts (20%) among Criollos and Wichí respectively. The detailed analysis of mitochondrial DNA showed that the two populations have as much as 87% of private haplotypes. Moreover, from the maternal perspective, despite a common Amerindian origin, an Andean and an Amazonian component emerged in Criollos and in Wichí respectively. Our approach allowed us to highlight that quite frequently there is a discrepancy between self-reported and genetic kinship. Indeed, if self-reported identity and kinship are usually utilized in population genetics as a reliable proxy for genetic identity and parental relationship, in our model populations appear to be the result not only and not simply of the genetic background but also of complex cultural determinants. This integrated approach paves the way to a rigorous reconstruction of demographic and cultural history as well as of bioancestry and propensity to diseases of Wichí and Criollos. The genetic structure of a population is shaped by its evolutionary history that encompasses all the past demographic events and the complex gene-culture coevolution Genes and culture evolve at different rates and under different rules, but at the same time they are in a close interaction. The dynamic equilibrium between cultural and molecular anthropology has been scarcely investigated so far, mainly due to the fact that the two subjects traditionally had a very low level of interplay. This aspect becomes even more striking when considering host-pathogens interaction, in particular with health emergencies in developing countries. A paradigmatic example is constituted by Chagas disease, which is endemic in almost all the South American continent Trypanosoma cruzi, and a study on 283 human mummies demonstrates that the sylvatic cycle  of Chagas was likely established at the very first time of human colonization (Chinchorro culture) of the Andean coast T. cruzi and humans coevolved for more than 9000 years consistently shaping the genetic and immunological background of native populations. In such a framework it is likely that south Amerindians have developed a peculiar way to cope with T. cruzi infection and this has to be taken into account in Chagas disease epidemiology and treatment strategies. Therefore when dealing with South American populations, which have complex evolutionary histories The Chagas disease is caused by the infection of Sub-population structure, the intricate kinship dynamics and the incompleteness and unreliability of the birth, marriage and death registers might introduce biases in such studies and could invalidate the efforts to understand and challenge Chagas and other diseases in these geographical areas.In Misión Nueva Pompeya, a community in the inner part of the Gran Chaco region in Argentina, the Native American Wichí cohabit with the admixed population generally named Criollos, composed by historical intermarriages between various Chaquean populations and first European male colonizers. The two populations dwell the same geographical environment and the same social space, but they are deeply different in terms of cultural and ethnographical structure. As they belong to different cultural traditions they do not share cultural rules, but in the real practice they are mixed creating a complex society stratification that only the genetic analysis can clarify. Identity, in these populations, is a matter of decision and social affiliation, so defining oneself as Wichí and Criollo has strong fallouts, from the economic, political and social point of view. To be aborigines in Misión Nueva Pompeya has indeed strong consequences both on a social level and in respect to the national government, the most important of which is to have rights upon the land. On the other hand the Criollos are the ones who have access to political decision making, because they have higher degree of education and a considerable knowledge of the law and the rules Usually human population genetic studies analyze a sample of putative unrelated individuals living in a small geographic area, but the risk of using such sampling strategy in Misión Nueva Pompeya is to bundle both native and admixed individuals into a unique population. To prevent this bias our sampling strategy started on a field work based on a detailed knowledge of family relationships, demographic patterns, social and cultural behavior and was based on a large sample, likely the entire population of the area of Misión Nueva Pompeya.T. cruzi, we decided to follow an innovative bio-cultural model in population genetic studies, taking into account the complex cultural interactions between them In order to understand and clarify the pattern of distribution and morbidity of Chagas disease in Wichí and Criollos parajes  parajes . Wichí ath and the 17th centuries, moving later into the dry forest to better feed their cattle. This event took place between the 19th and 20th centuries, thus affecting aboriginal lands. Criollos are the descendants of the first “mestizo” inhabitants of the Hispano Quechua Andean area of the north east, who later expanded and colonized the region in the early 20th century. Also called “chaqueños”, they are the descendants of those who first populated the North West area of the Andes – an area of Hispanic-Quechua roots – and who then spread and colonized the region at the beginning of the 20th century The Criollos of Chaco descend from families settled at the borders of the region, in the land that nowadays is defined as province of Salta and Santiago del Estero, since the 16Current Criollos are really mixed with Wichí due to hidden interaction along decades; phenotypic traits are fused with Wichí and morphological differences are in many cases undetectable. A traditional sampling approach could involve both native and admixed individuals, considering them as a meta-population.parajes population in the panorama of Misión Nueva Pompeya. The strategy to collect all the individuals and not just a representative subgroup of the two populations will enable the identification of the actual population structure that normally remains underestimated in genetic studies.In order to understand which is the influence of the genetic structure on the prevalence of Chagas infection in these two sympatric populations, we sampled, after a long-term fieldwork carried out by cultural anthropologists, not only a sample of putative unrelated individuals from the two groups, but likely the entire population of the area. 552 subjects (342 Native Wichí and 210 admixed Criollos) were then recruited on the basis of their self-reported ethnicity. The great number of samples collected represents almost the whole of Whole blood samples were taken from venous blood in adults (the serum was separated and stored at −20°C until use) and capillary blood in children .The sampling was in accordance with the ethical standards for human experimentation and with the Helsinki Declaration of 1975, as revised in 1983. The study was approved from the ethical committees of the University of Bologna  and of the University Hospital of Maternity and Neonatology, Universidad Nacional de Córdoba, Argentina. The Ministry of Health of the province of Chaco provided further authorization to perform the survey. People who voluntarily participated to the study, were previous informed of its objectives through autochthonous translators and then they signed the consent form written in Spanish and Wichí languages.parajes inhabitants were reconstructed by the cultural researchers during six years of fieldwork (2004–2010). Beside the usual genealogical diagrams taken for a population, specific questionnaires were developed and employed in both languages aiming at obtaining a wide report upon the genealogical memory of the two populations . Furthermore data on cultural, economic and social-health conditions were collected in order to reconstruct the social panorama of the population of Misión Nueva Pompeya. Data collected on serological prevalence of Chagas disease were reported in previous study A deep study on demographic data and their revision was preliminarily performed on field. Genealogies of all the http://www.genopro.com) and was integrated with male and female lineage data obtained from the genetic analysis.All the participants were identified by anonymized codes. The genealogies of Wichí and Criollos individuals were reconstructed using detailed self reported information obtained during the fieldwork and by the deep analysis of pedigrees, combining with data coming from official sources of demographic registry (CENSUS). A pedigree for each paraje was drawn using GenoPro® 2007 v. 2.0.1.6 (DNA was extracted from 2 mL whole blood using the QIAamp DNA Blood Midi Kit® (QIAGEN). All DNAs were then quantified by fluorometric dsDNA assay using PicoGreen® , and normalized to a concentration of 100 ng/µl.The Non Recombining Y chromosome (NRY) genetic variability was tested by 16 SNPs and 17 STRs in the 243 males of the total sample. Details on NRY analysis methods are provided in For the mitochondrial haplogroup classification, all the available 552 samples were analysed using the methods described in Basic parameters of molecular diversity and population genetic structure, including analyses of molecular variance (AMOVA), were calculated using the Arlequin package version 3.5 For the admixture estimation we applied two different methodologies implemented in Admix 2.0 http://www.fluxus-technology.com).Median joining (MJ) networks http://www.r-project.org/), using Fst pairwise matrices both intra and inter populations.For NRY we assigned weights, inversely proportional to the STR variance, ranged from 1 to 10. For mtDNA we adopted the mutation weights as described in Within the 243 males of Misión Nueva Pompeya we identified by NRY biallelic markers genotyping and by STRs haplotypes, 184 unrelated individuals (100 Wichí and 84 Criollos). As expected NRY haplogroup diversity is extremely low (0.3331+/−0.0579) in Wichí compared to Criollos (0.7461+/−0.0366), due to the presence in the latter of 80% of European ancestry haplogroups, subdivided in R1 (44%), E1b1b1(12%), G and I (both 6%) and J1 (7% ) and J2 (3%) lineages . HoweverThe genetic analysis of the Amerindian NRY highlighted the absence of the Q1a3* paragroup (M346) and only the presence of Q1a3a* (xM19xM194xM199) with frequencies of 80% and 20% in Wichí and Criollos respectively.STRs genotyping in Wichí population identified 58 different haplotypes (H = 0.9622+/−0.0118) with a strong founder effect in the Native Q1a3a* lineage, where 17 individuals share the same haplotype. A complete list of the NRY haplotypes is reported in Relationships between haplotypes in the two investigated populations were analyzed by Median Joining Network (MJN) analysis , which eA cluster analysis  based onIn order to measure the extent of the different genetic pattern observed in Wichí and Criollos, AMOVA was carried out considering STRs and SNPs data. Both the results  highlighted a significant differentiation between the two populations, in agreement with the self-identity information collected on the field.To assess the degree of admixture in the two Gran Chaco populations, we collected from the literature a list of SNPs and STRs allele frequencies of the putative ancestral populations from South America and Europe . Wichí gIn order to dissect the European component in this admixed group we used two STRs haplotype datasets from Italian and Iberian populations. The results underlined the major Iberian contribution (>50%) and a comparable contribution by Italian and Amerindian ancestral populations  and the unrelated sample subsets  using 12Furthermore MJN analysis allowed us to better investigate the relationships between native Q haplotypes in Wichí and Criollos and other South American populations . We founThe observed variability of the mitochondrial control region revealed that 99,3% of our samples belong to 4 out of the 5 Native American haplogroups . Furthermore of the 106 different haplotypes we found  and S7, To test selective neutrality and potential departures from a null hypothesis of mutation-drift equilibrium and constant population size we performed Tajima's D considering total and unrelated Wichí and Criollos samples . A negatFst genetic distances for sequences data and haplogroup frequencies confirmed a significant differentiation between Wichí and Criollos , although more weakly than the male perspective. In fact the haplogroup frequencies distributions between Wichí and Criollos are statistically significant (p<0.01), considering both total and unrelated samples . It is nInter-populations comparisons with five other samples from South American available in literature , exhibitSlatkin’s genetic distance matrix was used to perform nmMDS. Two main clusters are highlighted, the first gathering the majority of Amazonian populations and the second the bulk of the Andean populations. The analysis carried out on the total Wichí and Criollos individuals  showed tA MJN based on d-loop sequence from nucleotide 16047 to nucleotide 300 was performed, considering all the populations included in In order to test the effectiveness of our sampling strategy and of the proposed integrated anthropological model, we compared kinship trees obtained from data retrieved by the cultural fieldwork with those from genetic data. To perform this comparison and to test the reliability of the model, we considered one of the most studied and well characterized families in our sample set.The integration of male and female lineages data in the genealogical trees reconstructed by fieldwork marked some inconsistencies see .From the maternal perspective, in particular, the mismatch between the genetic results and the reconstructed relationships based on “self-reported identity” has revealed that ID398 (Wichí) despite having twelve children  followed by DYS458 (three changes) and DYS393 with two changes, whereas GATA H4.1 and DYS391 change just once during generations. Furthermore, in three cases, haplotypes differ by two loci from the related haplotype and in one pair a mutation involves two changes (gain of repetition) in the same locus. These facts highlight the possibility of divergence among lineages in a very short time span, jumping from ancestral to derived haplotype. These very rare occurrences are previously reported by Moreover, if, considering the maternal perspective, the reported 20% of Criollos individuals with Amerindian NRY have a Wichí ancestry (data not shown), and a comparable amount of Wichí has an European NRY, we can confirm the presence of bidirectional flows.In genetic studies associations between genotypes and phenotypes may be confounded by unrecognized population structure and/or admixture. Genetic ancestry in recently admixed populations varies largely among individuals particularly when this variation is the result of the combination of genetic and/or cultural factors although self-reported population ancestry likely provides a suitable proxy for many applications in epidemiology, as well as for assessing disease risks The individuals, whose Wichí or Criollos ethnicity was preliminarily self-reported, were analyzed for mitochondrial genetic variability and 243 males for NRY diversity. On the basis of the Y-STRs haplotype relatedness and the genealogical and demographic reconstruction, performed according to the social relationships and the mating traditions, we identified 184 unrelated males in order to carry out a reliable survey, comparable with literature data. These individuals constituted the bulk of unrelated samples even for mitochondrial analyses. Nevertheless, analyses for both NRY and mtDNA carried out on the total samples, including closely related individuals on the basis of self-reported identity, gave us a unique perspective to dissect the complex kinship patterns of the families of the two sympatric groups.The deep knowledge of the family structures, the mating dynamics and the genealogical trees, carried out during a long time fieldwork, revealed among Wichí some traditional complex kinship patterns that override the real genealogical definition. Some practices, as sororal polygyny , post-maIn order to disentangle this complex scenario and to reconstruct the genetic structure and the pattern of admixture of Misión Nueva Pompeya population, we elaborated an integrated cultural and molecular anthropological model that reconsidered the sampling strategy in the fieldwork of human population genetic studies. This approach allowed us to unravel in a genetic survey all the mismatches between cultural and biological relationships. In this context it was critical the comparison with available literature data, carried out by common sampling methods, both to detect intra population genetic diversity and phylogenetic/phylogeographic relationships. Even though we are aware that regarding to uniparental markers genetic estimates they reflect only a little fraction of any person's total genetic ancestry, but, escaping recombination, they are able to detect the ancestral and admixed components of the two communities, our multidisciplinary study allowed us to understand the correlation between cultural rules and actual mating patterns that shapes the gene pool of the population.Therefore, according to the aims of the present study, we were able to reconstruct the genetic structure and the pattern of admixture, also leading to verify the correspondence between genealogical and genetic relationships. This integrated perspective had the power to validate each other the data, to check the meaning of the results and to link the gap that usually relies on a singular source of information.(parajes), are differentiated also from the genetic point of view. In fact, AMOVA computations support the structure of two distinct populations, with significant values of Fst, for both female and male perspective. Moreover, the admixture analysis revealed more than 90% Amerindian ancestry for Wichí, while in Criollos this value is about 25%.Wichí and Criollos, that are culturally two different populations even sharing the dwelling space NRY genetic variability was very low in the Wichí and higher in the Criollos population. In general, among Wichí a higher homogeneity within the Amerindian lineages with a strong founder effect is observed, in contrast to the higher heterogeneity showed among Criollos  and 4. TThe higher frequency of the Amerindian M3-Q1a3a lineage among Criollos in comparison with previously studied admixed populations suggests a significant gene flow from the Natives to the immigrants among Criollos, and an opposite smaller flow towards the Wichí group. Cultural traditions usually state that the flow is unidirectional, namely a mixed mating always generates a Criollo descendent, never a Wichí. Actually, other mechanisms preserve bidirectional flows, although preferably the Native/Criollo offspring adopt the status of Criollo, rather than Native Wichí.th and 20th centuries from all over Europe but especially from Italy and Spain  showed higher values for Criollos, increasing from the total to the unrelated sample, while in Wichí an opposite trend was shown. An explanation for this result is that the usual practice of reiterated marriages of a Wichí male with different females leads to an underestimation of the mtDNA variability when unrelated samples were considered.Previous nucleotide diversity estimates in Gran Chaco region showed midrange values in comparison to higher values in Andean areas and lower in Tropical Forest The Multidimensional scaling representation of the genetic distances among a set of Amerindian populations from mtDNA haplotype estimates is remarkable in many aspects. The first image  reveals Finally, the MJN  on the pparajes and even households. Nearly 20% is the quote of exchange for NRY between the two demes, in both directions, although the cultural practices suggest uni-directionality from Wichí to Criollos, and not the opposite. Phenotypic traits do not help in the distinction between the two populations. This fact generates a present day appearance of meta-population. Even the self identification often creates some mismatches with actual kinship, due to the local mating practices and social rules, deeply investigated in this work by means of Cultural Anthropology tools. This new insight in approaching anthropological cases leads to new research opportunities: first of all, we can discern actual components in human populations’ genetic structure. Starting from this basic step, some consequences come up. The population relationships and the reconstruction of past migrations as well as the investigation of different ancestries which originated current variability, are now much more reliable and close to the reality, avoiding bias caused by the relatedness among the samples. In such a way, clusterization and phylogenetic relationships in networks, MDS or other statistical methods suitable to represent the spatial distribution of the genetic distances or affinities, fit better with the reality.Summarizing, Wichí and Criollos still preserve their genetic identity and peculiar characterization as a population, despite their sharing of the environment, of some In conclusion, we can affirm that anthropological genetics and cultural anthropology do not overlap but are complementary. The temporal diachronicity and synchronicity do not share the same pattern but are integrated; the populations are structured and differentiated, both from genetic and cultural points of view. Actual knowledge of mating dynamics sheds new insights in the population genetics results. These results state a very strong founder effect in Wichí lineages and a differentiated origin for Wichí and Criollos Amerindian component.The pattern of admixture and the dissection of stratified populations will enable to apply a population-disease approach to test the hypothesis of a different genetic susceptibility to Chagas disease and its clinical outcomes, starting from the comparison between the distribution of the Chagas seroprevalence and the genetic lineages Figure S1H values for Wichí and Criollos at each NRY locus.Θ(TIF)Click here for additional data file.Figure S2Median Joining Network performed on 12 NRY loci haplotypes from Wichí and Criollos (in yellow and red respectively) and other Amerindian populations available in literature.(TIF)Click here for additional data file.Table S1Target sequences for NRY variability assessment.(DOC)Click here for additional data file.Table S2Amplification primers for NRY variability assessment.(DOC)Click here for additional data file.Table S3Populations used in NRY STRs haplotypes comparison.(DOC)Click here for additional data file.Table S4NRY haplotypes in the two populations. .(DOC)Click here for additional data file.Table S5Populations used for NRY haplogroups frequencies comparison and the relative references.(DOC)Click here for additional data file.Table S6mtDNA haplotypes, from 1 to 53, in the two populations.(DOC)Click here for additional data file.Table S7mtDNA haplotypes, from 54 to 106, in the two populations.(DOC)Click here for additional data file.Table S8Main genetic diversity parameters in Wichi and Criollos for mitochondrial D-loop sequences comparing unrelated and total samples.(DOC)Click here for additional data file.Table S9mtDNA sequences publicly available (only one per sub-haplogroup) included in MJ Network analysis .(DOC)Click here for additional data file.Text S1Methods for NRY variability assessment.(DOC)Click here for additional data file.Text S2Methods for mtDNA Haplogroup assignation.(DOC)Click here for additional data file.Text S3Sororate definition.(DOC)Click here for additional data file.


P. falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (∼3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average Nd/Sd ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the  Plasmodium falciparum, which still claims almost one million deaths per year in vitro susceptibility to ARTs The malignant malaria parasite 2+ ATPase (PfSERCA or PfATP6), since the heterologously expressed PfATP6 in Xenopus laevis oocytes can be inhibited by ARTs as well as thapsigargin, a sesquiterpene lactone that specifically inhibits mammalian SERCAs P. falciparum through allelic exchange did not result in similarly dramatic changes in parasite’s susceptibility to ARTs et al. found a S769N substitution in a limited number of parasite field isolates from French Guiana, which was associated with significantly reduced in vitro susceptibility to artemether 50s to artesunate (ATS) in three isolates from Senegal in vitro analysis P. falciparum populations, the S769N mutation was found at high frequency in isolates in travelers returning from a range of African countries pfatp6 appeared inadequate in vitro sensitivity to DHA, ATS or ATM P. falciparum needs further examination.The exact mode of antimalarial action of ART drugs is still under debate pfatp6 gene from global P. falciparum populations prior to the deployment of ACTs has provided baseline information about the spontaneous mutations in pfatp6pfatp6 were the molecular target of ARTs, we would expect to find potential signatures of selection resulted from extensive deployment of this family of drugs in the GMS. To address this question, we undertook a comprehensive analysis of the diversity of pfatp6 from isolates obtained from the GMS. We examined polymorphisms in this gene in different geographic locations to infer whether the observed level of diversity is greater between populations than within populations. Based on the genetic diversity of pfatp6, we tested the underlying selective forces that acted on this gene. Our study did not detect apparent selection of this gene in worldwide parasite populations, suggesting that pfatp6 is not a suitable marker for monitoring ART resistance.A recent large-scale analysis of P. falciparum were collected after 2005 from patients presenting with uncomplicated falciparum malaria at local clinics in these GMS countries. P. falciparum infections were confirmed by light microscopy on Giemsa-stained thin and thick blood smears. The geographic locations of the parasite populations are depicted in In recent years, the malaria situation in the GMS has been improved significantly with most of countries becoming malaria-hypoendemic except Myanmar pfatp6 are listed elsewhere, which do not cover the introns DNA was extracted from filter papers using QIAamp DNA Mini Kit . Parasites were genotyped at three polymorphic loci to exclude multiclonal infections www.phrap.org), and CONSED http://www.mbio.ncsu.edu/bioedit/bioedit.html) using the ClustalW algorithm pfatp6 sequence (PF3D7_0106300) as the reference (www.plasmodb.org). The nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers . In addition, we have retrieved 649 additional pfatp6 sequences from the GenBank for comparison.PHRED S), haplotype diversity (h), and nucleotide diversity (π) were estimated for each sampling site. To study the molecular evolution of pfatp6, we performed three tests of natural selection: 1) The rates of nonsynonymous (dN) and synonymous (dS) substitutions in pfatp6 were estimated (http://www.hiv.lanl.gov). If positive selection has operated since the divergence of the alleles, the ratio of dN/dS will exceed one; conversely, when purifying selection is acting on the gene, the dN/dS ratios will be less than one. For dN/dS ratios, significance was determined by the two tailed Z-test (P<0.05) of selection using the Nei and Gojobori method atp6 gene from P. reichenowi was used as outgroup. The neutrality index (NI) was calculated from the equation NI = (Pn/Ps)/(Fn/Fs), where Pn and Ps are the numbers of nonsynonymous and synonymous polymorphisms, respectively, whereas Fn and Fs are the nonsynonymous and synonymous fixed differences, respectively. Fisher’s exact test was used for the significance of the MK test. 3) Tajima’s DF* tests D test was based on the difference between θ (Watterson’s population nucleotide diversity parameter) and π (average pairwise nucleotide diversity). Under the neutral model, the expectation of θ should be the same as that of π, but under balancing selection, a positive D value is expected F* test takes into account the difference between the observed number of singleton nucleotide polymorphisms and the number expected under neutrality from S  and θSequences were imported into DNASP v5.10 Aligned sequences were imported into Bionumerics software version 6.1  for estimating gene genealogies, and minimum spanning trees (MSTs) were constructed using the method of Templeton et al. FST (based on haplotype frequencies) and ΦST (based on genetic distances between haplotypes) contained within Arlequin version 3.1. Whereas an FST and ΦST score value of 0 between two populations would indicate that they are completely undifferentiated, a score of 1 would indicate that every observable genetic difference between individuals of the two populations can be used to distinguish between the populations. The significance of the two fixation indices was tested through 10,000 permutations for each pairwise comparison. Because the TVM+I+G model given by MODELTEST Genetic population structure was investigated using hierarchical analysis of molecular variance (AMOVA), using both To detect clustering of the haplotypes within the GMS populations, a metric multidimensional scaling (MDS) analysis was conducted. A distance matrix (identity by state) was constructed used to calculate three eigenvectors or principal components (PCs). The three PCs were then mapped onto two dimensional graphs for easy visualization. The data were analyzed using the “bios2mds” package for R pfatp6 gene  in 213 P. falciparum clinical samples from six countries of the GMS  (indels not considered), of which 13 were nonsynonymous substitutions. All polymorphic sites were dimorphic with two alternative nucleotides. Eight SNPs at positions 73, 231, 250, 676, 924, 1945, 2128, and 2340 were novel in the GMS dataset, among which five (in bold) were nonsynonymous. SERCA has three conserved domains: cytoplasmic domain, transmembrane gate and luminal loops pfatp6 molecule, 19 SNPs were located in the cytoplasmic domain, whereas the predicted transmembrane and luminal domains had only one and four SNPs, respectively  We sequenced the entire coding region of  the GMS . Togetheectively . A slidiectively . InteresMost SNPs found in the GMS were relatively rare; the highest prevalence of synonymous and nonsynonymous mutations was 9.8% and 8.5% at nucleotides 2694 and 266 (I89T), respectively Table 1.h) was relatively high with an average of 0.654 (π) of pfatp6 was also low, ranging from 0.00020 to 0.00043, with the population from China showing the highest π value , whereas eight are located within the luminal domain (two nonsynonymous). When all samples were grouped by continent, the average haplotype diversity of the populations from Africa (0.922) was the highest, followed by South America, and Asia (pfatp6 haplotypes were circulating in the sampling areas. The rank order of nucleotide diversity (π) was similar to that of haplotype diversity>Africa (0.00061)>Asia (0.00034) >Pacific Islands (0.00018) with the exception of South America (0.00083)  .pfatp6 gene, we first compared the ratio of nonsynonymous to synonymous substitutions (dN/dS). The results showed that dN/dS ratio for the GMS samples or the entire sample set was below one, suggesting purifying selection. Z-test revealed that either within continent or the entire sample collection there was no significant difference between dN and dS, suggesting that dN/dS ratio did not significantly deviate from the neutral expectation  were well above one pfatp6 gene in most parasite populations did not deviate significantly from neutrality and there was significant heterogeneity between individual populations, suggesting population differentiation.To detect signatures of natural selection on the ectation . To corrhe tests . Tajima’ulations . In agrepfatp6 sequences from the world identified 106 haplotypes based on 71 polymorphic sites. The relationships among these haplotypes were inferred by the MST method, which revealed that three haplotypes were in high frequencies and common for most parasite populations studied (P. reichenowi atp6 gene (data not shown). It was also the most common haplotype occurring in approximately half of all individuals sampled  and had the widest geographic distribution across 16 populations . H1 is connected to several low-frequency haplotypes, generating a star-like genealogy, which is indicative of a possible past range expansion. The reference sequence from 3D7, referred hitherto as haplotype H2, differed from H1 by a T to C transition at position 3236, and was the second most common haplotype in the entire population . Although H2 was restricted to nine populations, it was present in all continents analyzed. The third most represented haplotype (H3)  was also found in nine populations in Asia, Africa and South America. All remaining haplotypes, including 65 singleton (unshared) haplotypes, were found in the remaining 318 samples. Derivatives with lower frequencies were connected to the dominant haplotype by up to eight mutational steps  , suggesting little population differentiation, whereas 54 comparisons had FST values of 0.05–0.148, suggesting of low – intermediate levels of population differentiation. The highest level of subdivision was observed when comparing the parasite population from Venezuela with any other populations  .We used AMOVA to evaluate the degree of population subdivision. We calculated the d (ΦST) . Compari >0.469) . Of the to 0.228 . PopulatFST values among Asia, Pacific Islands and Africa ranged from 0.017 to 0.137, suggesting of low to modest levels of population subdivision. However, the South American population differed substantially from populations from other continents. The high ΦST values obtained from comparing the South American population with populations from other continents (0.540–0.663) were highly significant even after Bonferroni corrections. At the continent level, parasite population from the GMS had a high degree of differentiation from the South American population . A moderate degree of differentiation was noticed for the GMS and Africa populations . In contrast, there was little genetic differentiation between the GMS and Pacific Island populations . Pairwise comparisons revealed that all continental groups were significantly different from each other, and the values remained significant even after Bonferroni correction.We next grouped the populations by continent and analyzed population structure between continents . The FSTo further support this finding, we performed MDS analysis of the GMS parasite populations. From the distance matrix, three PCs were identified with much of the variance  explained by PC1 . PCA ploSERCA is an evolutionarily conserved protein and plays an important role in cellular homeostasis and calcium signaling functions P. falciparum has only one SERCA gene, pfatp6, and it has been proposed as a specific target of ARTs pfatp6 using a large collection of parasite isolates (n = 213) from the six GMS countries, where ART drugs have been used for the longest time. Our results confirmed the high level of genetic diversity (π = 0.00030) in pfatp6 in the GMS, which is slightly lower than that observed for the entire sample set of 862 samples. The nucleotide diversity in pfatp6 gene from the GMS was also somewhat lower than the recent estimate based on genome-wide SNPs pfatp6, including five novel mutations. Among them, three were located in the luminal regions and 10 in the cytoplasmic region, but none was found in the putative ART-binding domain. Similar to previous reports, the majority of the SNPs were uncommon with frequencies below 5% and a substantial number of them were continent- and population-specific pfatp6 in a large global collection of parasite isolates from regions of both prior to and after ART usage, but failed to identify potential mutations that might be indicative of directional ART-driven selection.pfatp6 polymorphisms . China had a drug use history that is drastically different from other countries within the GMS, and ARTs have been applied to treating falciparum malaria for nearly 30 years Parasite population structures are well mirrored from the estimates of pfatp6 as a prime target of ART family drugs based on heterologous expression and biochemical studies has spurred significant interests in validating mutations in this gene as molecular markers of ART resistance in P. falciparum. Sequencing of global P. falciparum field isolates has identified numerous mutations, most of which were population-specific and at low prevalence. The presence of abundant resistance-unrelated SNPs would make it difficult to identify resistance-associated mutations until they gain considerable prevalence. Some studies showed that deployment of ACTs were associated with changes in frequency of certain pfatp6 mutations pfatp6 gene in parasites from this region in recent years would be expected to contain signature mutations designating ART resistance if pfatp6 were indeed the primary target of ART drugs. However, mutations in pfatp6 from the GMS parasite populations, albeit relatively abundant, were either infrequent or present also in other malaria endemic regions with more recent ART use histories. Besides, the clinical ART mutation detected in western Cambodia is not linked to mutations in pfatp6pfatp6 locus pfatp6 as a molecular marker of ART resistance in P. falciparum.The initial postulation of Figure S1A sliding window plot  of nucleotide diversity (π) of pfatp6 gene in P. falciparum populations from the GMS.(PDF)Click here for additional data file.Figure S2Unrooted minimum spanning tree network showing genetic relationship among parasites from four continents. A) Asia; B) Pacific Islands; C) South America; D) Africa.(PDF)Click here for additional data file.Figure S3Unrooted minimum spanning tree network showing genetic relationship among pfatp6 amino acid sequence for the worldwide P. falciparum populations.(PDF)Click here for additional data file.Figure S4Principal component analysis of the GMS parasite populations. A) Screen plot indicating the proportion of variance accounted for in the first 3 eigenvectors or principal components . B–D) Multidimensional scaling plots for each combination of PCs. Figures indicate the relative distances between each of the isolates, color coded by nation.(PDF)Click here for additional data file.Table S1Statistics for pfatp6 gene from the entire falciparum populations.(DOCX)Click here for additional data file.Table S2Genetic differentiation between continent-groups measured by FST  and ΦST  values.(DOCX)Click here for additional data file.


Pan troglodytes, is still poorly understood. An issue of particular controversy is whether the proposed fourth subspecies of chimpanzee, Pan troglodytes ellioti, from parts of Nigeria and Cameroon, is genetically distinct. Although modern high-throughput SNP genotyping has had a major impact on our understanding of human population structure and demographic history, its application to ecological, demographic, or conservation questions in non-human species has been extremely limited. Here we apply these tools to chimpanzee population structure, using ∼700 autosomal SNPs derived from chimpanzee genomic data and a further ∼100 SNPs from targeted re-sequencing. We demonstrate conclusively the existence of P. t. ellioti as a genetically distinct subgroup. We show that there is clear differentiation between the verus, troglodytes, and ellioti populations at the SNP and haplotype level, on a scale that is greater than that separating continental human populations. Further, we show that only a small set of SNPs (10–20) is needed to successfully assign individuals to these populations. Tellingly, use of only mitochondrial DNA variation to classify individuals is erroneous in 4 of 54 cases, reinforcing the dangers of basing demographic inference on a single locus and implying that the demographic history of the species is more complicated than that suggested analyses based solely on mtDNA. In this study we demonstrate the feasibility of developing economical and robust tests of individual chimpanzee origin as well as in-depth studies of population structure. These findings have important implications for conservation strategies and our understanding of the evolution of chimpanzees. They also act as a proof-of-principle for the use of cheap high-throughput genomic methods for ecological questions.In spite of its evolutionary significance and conservation importance, the population structure of the common chimpanzee,  Chimpanzees are viewed with fondness as our closest animal relatives and are valued by scientists for the biological and evolutionary insights they provide. In spite of this, the relationships between different populations of common chimpanzees are still relatively poorly understood, a situation that potentially threatens conservation efforts. Here we have used information gathered in the Chimpanzee Genome Project to design comprehensive tests of genetic variability that show unambiguously the existence of four genetically distinct groups (or populations) of common chimpanzee. We demonstrate that previous methods based on mitochondrial DNA sequences alone are not always accurate and show the feasibility of cheap new genetic tests of individuals' origins that could play an important role in conservation. Pan troglodytes, are incompletely understood. Traditionally, three subspecies have been described: the western chimpanzee (P. t. verus), central chimpanzee (P. t. troglodytes) and eastern chimpanzee (P. t. schweinfurthii). Analysis of mitochondrial DNA (mtDNA) variation led to the proposal of a fourth, “Nigerian” chimpanzee subspecies  from the chimpanzee genome project The development of modern high-throughput SNP genotyping technologies has revolutionized many aspects of human genetics, including our understanding of the history and demography of human populations Pan t. ellioti is genetically distinct from other populations, we obtained DNA samples from Cameroonian chimpanzees which we analysed along with samples from captive Western (verus) and troglodytes chimpanzees. Eastern chimpanzees (P. t. schweinfurthii), with their distinct geographical distribution, were not sampled in the current study. We sequenced 12 autosomal fragments of ∼1 kb and genotyped 691 SNPs from 22 autosomal regions of 40–80 kb HV-I locus.To address the question of whether tructure) were based on the marginal data at each genotyped SNP. We then calculated FST from the DNA sequence data, and finally applied recently developed methods which exploited information on the joint distribution of SNP alleles within haplotypes.We applied a number of different methods to the analysis of these data to assess the relationships and genetic clustering amongst the sampled individuals. The first set of methods  chimpanzees form one cluster while Cameroonian chimpanzees are divided into two genetically distinct clusters, one of which we infer to correspond to P. t. ellioti, whose existence had been the subject of uncertainty. We note that two individuals in the P. t. ellioti cluster had previously been designated P. t. troglodytes based on mtDNA sequence, a point to which we return below. Two individuals  with P. t. troglodytes-like mtDNA lie between the presumptive P. t. verus and P. t. troglodytes clusters, and records have subsequently revealed that these are indeed first-generation hybrids produced in captivity.Using the first two principal components of the data from all 818 SNPs, 52 of the 54 chimpanzees studied clustered into three distinct, non-overlapping groups . These ctructureellioti and troglodytes groups, rather than involving verus chimpanzees. This suggests more recent interaction between P. t. ellioti and P. t. troglodytes than either has had with P. t. verus, although an effect of SNP ascertainment could not be ruled out. We note that the model underlying Structure assumes no linkage disequilibrium between loci, whereas our data do exhibit such correlations because of the clustering of SNPs. The expected effect of this in the Structure model is an over-estimation of precision, rather than bias tructure analysis should be interpreted with some caution.A similar conclusion comes from a different perspective when the software SST, a commonly-used measure of the proportion of total genetic variation occurring between populations. Potential confounding effects from SNP ascertainment complicate interpretation of FST values calculated from the genotype data, so we restricted these analyses to our re-sequencing data alone  tructure's view of relative amounts of co-ancestry, FST between P. t. ellioti and P. t. troglodytes  is slightly lower than, but cannot be formally distinguished from, that between P. t. troglodytes and P. t. verus  or between P. t. ellioti and P. t. verus . The troglodytes – verus figure in our data is lower than the 0.29 for Central vs. Western chimpanzees previously estimated from re-sequencing data Next, we calculated pairwise FWhen genetic data is collected from tightly linked variable sites, exploiting patterns of non-random association (i.e. linkage disequilibrium) can increase power to identify population structure over single-SNP analyses P. t. verus individuals, for whom the most closely related haplotype is virtually always in the same population. Haplotypes of P. t. ellioti and P. t. troglodytes chimpanzees respectively are typically most similar to those of other individuals within the same population, but occasionally to those of individuals from the other (P. t. troglodytes and P. t. ellioti respectively) population. The two previously noted hybrid individuals are clearly identified, and in addition it emerges that two of the P. t. ellioti chimpanzees had a higher level of shared ancestry than the other chimpanzees. The qualitative conclusions from the haplotype-based analysis thus mimic those from principal components and Structure, although reassuringly they explicitly model the correlations between nearby SNPs, in contrast to Structure.We analysed similarities in patterns of haplotype variation among individuals for the 691 clustered autosomal SNPs using a so-called copying model applied to estimated haplotypes from each individual P. t. troglodytes for 3 of 100 re-samples). In By applying the haplotype-based copying model to human data, we can compare quantitatively the extent of differentiation between the three chimpanzee groups with that between various human populations. Importantly, such analyses can allow for ascertainment effects. We show the copy model results for human data from the Phase II HapMap  in An equivalent analysis of the HapMap III African populations We note that while it is theoretically possible to use the lengths of copied fragments in the copying model to estimate the timescale over which differentiation has occurred, our data is not well-suited to this because the shortness of the assayed regions means that relatively few breakpoints are observed, providing little information about the times of events in the history of chimpanzee populations.P. t. verus, P. t. troglodytes, and P. t. ellioti, with the latter two groups sharing somewhat more similarity with each other than either does with P. t. verus. P. t. troglodytes and P. t. verus are two securely defined populations estimated to have diverged 0.4–0.6 million years ago P. t. ellioti to be clearly distinct from P. t. troglodytes with both groups equally distinct from P. t. verus, so that whatever terminology (“population” or “subspecies”) is applied to verus and troglodytes should equally be applied to ellioti.We have applied a number of different analytical methods to an extensive set of SNP data from 54 chimpanzees. All of the methods point clearly to the existence of three distinct population groups, corresponding to three of the previously-described “subspecies” of chimpanzee troglodytes and ellioti, the chimpanzee populations are considerably more distinct than the African populations sampled in HapMap III, suggesting rather differing demographic histories for the two sister species.By way of comparison, we have shown that these three chimpanzee populations are more differentiated than even continental human populations, and also that in spite of the relatively close geographic proximity of the groups, particularly ST approaches, we also calculated pairwise FST values for each of the 100 resamples of individuals and SNPs in our analyses of the three continental population samples. The results are summarized in ST across the 100 samples show the same pattern as copying proportions in the copying model, the sample-to-sample variation is larger. For example, the FST intervals for the central 95% of resamples for Europe-East Asia overlap those of Africa-Europe and Africa-East Asia, and for example for five of the 100 resamples the pairwise FST between Africa and Europe was actually smaller than that between Europe and East-Asia. In contrast, for the copying model analysis the 95% intervals for the proportion that Europe and East Asia copy from each other do not overlap with the 95% intervals for either copying from Africa, and the proportion that Europe copied from Africa was lower than the proportion Europe copied from East Asia in each of the 100 re-samples. This accurately reflects the fact that on average East Asia and Europe share more recent ancestry with each other than with Africa.In order to compare population comparisons based on the copying model with those based on more traditional Ftroglodytes and one as ellioti seems reasonable. Whilst our data alone could not rule out two distinct populations, one or both of which extends across the Sanaga River, this seems a priori unlikely – the river provides a natural barrier between the distinct populations, whereas if both were to exist on the same side of the river there seems no reason for their reproductive isolation—and at variance to other available evidence. Notwithstanding our lack of complete geographical information on sampled chimpanzees, the clear separation between all three populations, relative to the similarities within the populations, seems hard to reconcile with the suggestion that chimpanzee genetic variation is distributed more or less continuously across the species range (cf One weakness of our study (and some others) is that we do not have definitive information on the geographic origin of all of the chimpanzees we have studied. All our analyses point to two very distinct population groups for the chimpanzees originating from eastern Nigeria and Cameroon. In the light of other genetic evidence for distinctiveness of individuals sampled from either side of the Sanaga River range cf .P. t. ellioti was based on mtDNA sequence analysis P. t. verus, to the exclusion of P. t. troglodytes, a description made more robust by a recent analysis of complete mitochondrial genomes troglodytes-like mtDNA but ellioti autosomal SNP genotypes. . Thus the two systems generally agree, but, not surprisingly, single-locus mtDNA data is less reliable for classification than genome-wide data.The initial genetic description of P. t. verus and P. t. ellioti are sister taxa P. t. ellioti, on the basis of extensive autosomal data, have mtDNA which clusters with P. t. troglodytes. Thus, mtDNA from ellioti individuals does not fall into a single clade on a mtDNA tree. If mtDNA is used both to classify individuals and to estimate trees for the resulting groups, there is always a danger, as seems to have occurred in this instance, that misclassification of individuals will lead to a simpler-looking tree than is actually the case. Secondly, the suggestion from the mtDNA data that  ellioti individuals have mtDNA types which are closer to verus than to troglodytes individuals is strikingly different from the results of our analyses based on many independent autosomal loci, which places P. t. ellioti clearly closer to P. t. troglodytes than to P. t. verus. It is interesting to note that a study of morphological variation agreed with the picture obtained from autosomal loci The mtDNA-based picture of demographic relationships suggests that For many conservation applications, it would be desirable to be able to assign or classify individuals to populations based on a small number of loci. We developed and applied a method for choosing subsets of SNPs for classification based on their contribution to assignment probabilities see . To avoiP. t. verus individual (‘Clint’), a further 4% from four other verus, and less than 5% from three P. t. troglodytesellioti and pooled troglodytes and verus chimpanzees. Our study thus confirms the utility of genomic resources even when ascertainment is sub-optimal.For our data, we could essentially reproduce the discrimination obtained with the complete dataset of 818 SNPs with as few as 8 carefully selected SNPs in distinct regions of the genome . While tP. t. ellioti as a genetically distinct population of chimpanzee strongly supports efforts to treat this population as a separate management unit for conservation P. t. ellioti, with an estimated 6,500 individuals remaining, is the least numerous population.The confirmation of Pan troglodytes ellioti, as a genetically distinct group. More generally, our results confirm the utility of high throughput SNP typing for evolutionary genetic and conservation analysis. However, we recognize that a full appraisal of chimpanzee population structure would require denser sampling from all four populations in addition potentially to comparative studies across primates that go beyond great apes and humans.In conclusion, using genomic resources we have assembled the largest SNP-based dataset for investigating chimpanzee population structure. It resolves an outstanding controversy in clearly establishing the fourth putative subspecies, P. t. verus (from Sierra Leone) and 4 putative P. t. troglodytes (unknown geographic origin) chimpanzees held at the Biomedical Primate Research Centre in the Netherlands  before genotyping. DNA samples were also obtained from 15 herlands . For chiCCR5, SDF, CXCR4, CX3CR1, RANTES, CCR2, SEC22L3, ZNF445, PTPN23, CCRL2, MC1R and HBB  were removed from the analysis.Mitochondrial HV-I fragments of 534 bp and fragments of ∼1 kbp from the genes  and HBB  were amphttp://www.ncbi.nlm.nih.gov/projects/SNP/]. The SNPs, arranged in 22 clusters of size 40–80 kbp on several autosomes, were screened using BLAST to ensure unique context. The panel has also been used to assess recombination rates in the 22 regions, orthologous to recombination hotspots in humans A panel of 768 SNPs was designed for the GoldenGate Genotyping Assay , using polymorphism information from the Chimpanzee Genome Project ST in Arlequin (with 95% CIs estimated by jackknifing) tructurePopulation structure was assessed by pairwise FFor the haplotype-based analysis, we inferred haplotypes and population-scaled recombination rates between adjacent SNPs using PHASEv2.1.1 ST for each re-sample was calculated using the approach described in For comparisons with human data, we matched features of the chimpanzee dataset by randomly selecting 18 individuals per population using HapMap Phase 2 Release 21 or HapMap Phase 3 Release 2 consensus haplotypes. For each analysis, we then randomly selected 22 autosomal genomic regions, randomly selecting SNPs to match the SNP density and minor allele frequency distribution  for the respective 22 chimp regions. We ran the copying model using fixed genetic map estimates (build 35 estimates for HapMap2 populations and build 36 estimates for HapMap3 populations) scaled by an effective population size value of 30000, the value that maximized the expected log-likelihood over a fixed grid of , though we note that results were similar for all scaling factors we considered. Ascertaining SNPs on a single randomly selected HapMap Phase2 CEPH individual or HapMap Phase3 Luhya (Kenya) individual not included in the sample gave similar results to those presented. Pairwise FFigure S1P. t. troglodytes, red - P. t. ellioti, blue - P. t. verus) and intermediate colours show intermediate probabilities. Chimpanzees have individual- and fragment-based copying probabilities that are more extreme (closer to 0 or 1) than human Continental populations, indicating greater population differentiation.Assignment of population of origin by genomic fragment: Chimpanzee Data. Each line in the figure shows an individual with its inferred population of origin and 22 autosomal fragments for which SNP genotype data was collected. Each line is divided into two coloured strips showing the two haplotypes for each fragment. Colours show the copying model-estimated probabilities of origin of each fragment for each chromosome (yellow - (PDF)Click here for additional data file.Figure S2Assignment of population of origin by chromosomal fragment: Human Data. Figure as in (PDF)Click here for additional data file.Figure S3Assignment of population of origin by chromosomal fragment: African Populations Data. Figure as in (PDF)Click here for additional data file.Figure S4Haplotype-based analyses of population relationships. Figures as in Main (PDF)Click here for additional data file.Table S1P. t. troglodytes; E, P. t. ellioti; W, ‘Western’ i.e. P. t. verus.Chimpanzees Studied. BPRC = Biomedical Primate Research Centre, The Netherlands. mtDNA classification: T, (DOC)Click here for additional data file.Table S2Amplification Targets.(DOC)Click here for additional data file.Table S3PCR and Sequencing Primers. PCR primers are in bold. All primers were used for sequencing.(DOC)Click here for additional data file.Table S4Highly Differentiated SNPs. For each population, SNPs with highest frequency difference between chimpanzees in that population cluster and the other two clusters. Bold SNPs are in the minimal panel of 10 markers used in (DOC)Click here for additional data file.


Dermatemys mawii) is the only remaining member of the Dermatemydidae family, yet little is known about its population structuring. In a previous study of mitochondrial (mt) DNA in the species, three main lineages were described. One lineage  was dominant across most of the range, while two other lineages were restricted to Papaloapan  or the south-eastern part of the range (1D). Here we provide data from seven polymorphic microsatellite loci and the R35 intron to re-evaluate these findings using DNA from the nuclear genome. Based on a slightly expanded data set of a total of 253 samples from the same localities, we find that mtDNA and nuclear DNA markers yield a highly congruent picture of the evolutionary history and population structuring of D. mawii. While resolution provided by the R35 intron (sequenced for a subset of the samples) was very limited, the microsatellite data revealed pronounced population structuring. Within the Grijalva-Usumacinta drainage basin, however, many populations separated by more than 300 kilometers showed signals of high gene flow. Across the entire range, neither mitochondrial nor nuclear DNA show a significant isolation-by-distance pattern, but both genomes highlight that the D. mawii population in the Papaloapan basin is genetically distinctive. Further, both marker systems detect unique genomic signals in four individuals with mtDNA clade 1D sampled on the southeast edge of the Grijalva-Usumacinta basin. These individuals may represent a separate cryptic taxon that is likely impacted by recent admixture.The critically endangered Central American River Turtle ( Dermatemys mawii, is the last surviving species of giant river turtles of the family Dermatemydidae. This turtle was an important economic and cultural resource for local people throughout its historical distributional range. Anthropogenic overexploitation by consumption of meat has caused drastic population declines The Central American River Turtle, D. mawii includes parts of Belize, the Atlantic Coast of Guatemala and large parts of the Mexican States of Veracruz, Tabasco, Campeche, Chiapas and the southern part of Quintana Roo  of lower Mesoamerica shows pronounced genetic structure not only across the Cordilleran Mountains, but also within each of the contiguous Caribbean and Pacific coastal forest habitats Dermatemys mawii might therefore show previously unknown genetic structuring across its range, with potential conservation implications.The distribution range of tana Roo . DermateD. mawii. Recently, a study that characterized mtDNA variation in the species found signatures of phylogeographic structuring and phylogenetic splits among lineages up to a divergence of 2% D. mawii individuals  carrying a rare, divergent mtDNA haplotype (1D), co-occurring with individuals that carry common haplotypes of the “Central” clade. Based on single-locus evidence from maternally inherited mtDNA, however, it was not possible to assess whether members of the different mitochondrial genetic lineages are capable of interbreeding with each other. Microsatellite markers are commonly used to assess population genetic structuring, impacts of demographic bottlenecks This study is part of a long-term conservation project to develop management plans by identifying management units MUs  for ensuEmys orbicularis shows low divergence and lack of population genetic structure throughout the Iberian Peninsula when analyzed using mtDNA, while results of nuclear microsatellite loci revealed relatively high levels of genetic variability within populations and population structuring Podocnemis expansa), the overall differentiation among populations estimated by FST values was fourfold greater for mtDNA than for nuclear microsatellite markers Nuclear DNA markers have been used to complement the results of mtDNA studies with a bi-parental perspective in other population genetic studies of turtles. For instance, D. mawii populations from across the species' distribution range. We test to what extent the results from the nuclear genome show concordance with previously published mtDNA structuring D. mawii, (3) to determine if D. mawii populations have genetic signatures of a recent bottleneck(s), and (4) to compare the patterns observed at microsatellite loci with those recovered previously from mtDNA In this study we investigate genetic variation at 7 polymorphic microsatellite loci and one nuclear intron in 2). Before cutting, the area was disinfected with 62% ethanol gel and, after sampling, we covered the wound with antiseptic “new skin” (8-hydroxyquinoline at 1%) in order to avoid infections. We avoided cross-individual contamination by using scissors thoroughly cleaned with a 10% bleach solution. We followed the guidelines for use of live amphibians and reptiles in field research from the American Society of Ichthyologists and Herpetologists (ASIH), the Herpetologists' League (HL) and the Society for the Study of Amphibians and Reptiles (SSAR) We collected 253 tissue samples from the hind-foot webbing by cutting small skin snips  GP96, that was designed for Gopherus polyphemus but cross-amplifies in other turtle species Dm3A-32, Dm3A-37, Dm3A-58, Dm3A-13, Dm3A-17, and Dm3A-42, an initial step at 94°C for 7 min was followed by 45 cycles of 92°C for 1 min, annealing at 50–65°C for 1 min depending on the specific primer pair , extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The conditions for the locus Dm3A-72 were: one initial step at 94°C for 7 min, followed by 38 cycles of 94°C for 40 seconds, annealing at 64°C for 40 seconds, 72°C for 45 seconds, and a final extension at 72°C for 7 min. The conditions for locus GP-96 were: 95°C for 7 min, followed by 38 cycles of 95°C for 40 s, 50°C for 30 sec, 72°C for 1 min, and a final extension at 72°C for 15 min. The resulting PCR products were analyzed on a 3130XL Genetic Analyzer using GeneScan ROX 500™ size standard and genotyped with GeneMapper 4 software (Applied Biosystems).We amplified seven dinucleotide-repeat polymorphic microsatellite loci that we previously designed for bp of the first nuclear intron of the RNA fingerprint protein R35 in 11 samples from the three main basins, covering the three main mtDNA lineages , comparing our data to a sequence previously used in a phylogenetic study of turtles We also sequenced 779E) and observed (HO) heterozygosities Dm3A-17 was excluded from the study because of a significantly lower than expected observed heterozygosity in most populations, indicating the widespread presence of null alleles at this locus. Our analyses are therefore based on the 7 loci that did not deviate from neutral HW expectations.We calculated the mean number of alleles (MNA) per locus, allele frequencies, expected (HR) within localities. HP-RARE uses rarefaction approach to account for differences in sample size among populations. As a baseline, we standardized sample size to the smallest number of samples per locality included in this analysis  and then determined, using rarefaction, the expected number of alleles at each locality. We also used HP-RARE to determine the private allelic richness (PA We used the program HP-RARE hness PA . Due to ST/(1-FST) Levels of nuclear DNA differentiation among populations were calculated based on estimates of the fixation index Θ We used the program STRUCTURE v2.3.1 Haplotypic phase of the sequences from the nuclear R35 intron was obtained from the program PHASE, as implemented in DNASP 5.01 Evidence of recent population reductions was assessed using BOTTLENECK v 1.2.02 ST values estimated by Gonzalez-Porter et al. b and ND4 markers), against those estimated using microsatellites, we conducted a correlation analysis using Mantel tests. A Factorial Correspondence Analysis (FCA) was conducted in GENETIX 4.05.2 In order to analyze the possible concordance between the results of FDm3A-17, was excluded due to strong deviations from HW expectations; see above). Among the 7 remaining microsatellites, no locus alone deviated significantly from HW equilibrium frequencies  at any locality. Across loci, and following sequential Bonferroni correction, only the 25 Salinas samples deviated significantly from HW equilibrium (P = 0.003) (Dm3A32 and Dm3A13) showed significant heterozygote deficit . This population harbors genetic material from an apparently differentiated lineage , which may explain these deviations from HWE. We did not find any evidence of significant linkage disequilibrium between any pair of loci at any locality . No locus exhibited large-allelic dropout as assessed in MICROCHECKER. We thus kept all remaining 7 loci for further analyses. These 7 microsatellite loci exhibited a total of 72 alleles, with an allelic richness (AR) ranging from 2.1 at Salpeten to 4.5 for Jonuta. Private allelic richness (PA) ranged from zero in Peru and Yala, to 0.64 in Papaloapan. Observed heterozygosity ranged from 0.329 for Sacnab to 0.609 for Jonuta  . For thir Jonuta . The latSTOverall genetic differentiation at microsatellite loci was moderate, with a global ΘStandard assignment test results revealed that 155 of 251 (61.8%) of individuals were assigned to their known  collection localities, and most cases of mis-assignment were to neighboring populations from within the same river basin . The sitΔK, the statistic of Evanno et al. No signal of a recent bottleneck was detected in any of the reasonably extensively sampled localities (n≥15) using BOTTLENECK: no comparison was significant for the Wilcoxon test or the Mode-shift test for the TPM or SMM models.ST values were significantly correlated with pairwise mtDNA ΦST,mt values from the same localities .Across all localities, patterns of nuclear genetic differentiation were concordant with results obtained using mtDNA from the same 15 populations D. mawiiThe results from mtDNA in bp fragment in PHASE to determine haplotypes. Sequences obtained for D. mawii have been deposited in Genbank (accession numbers JN655665-JN655668). Only three sites were variable in the alignment, limiting the power to detect any population differentiation. We found four haplotypes  has caused drastic population declines Genetic variability did not vary drastically among populations, although some populations in the eastern parts of the distribution appeared to have somewhat reduced levels of variation  have likely buffered against rapid loss of genetic variability during phases of population size reduction Podocnemis unifilis populations in the Orinoco and Amazon basins However, the population reduction tests indicated that none of the analyzed ST tests confirmed that this population is divergent from the other populations. The STRUCTURE analysis revealed that all individuals from Papaloapan were homogeneously grouped into one cluster for K = 3  of southern California exhibit population structure between the unconnected wetlands but not inside each wetland A. callidryas) shows pronounced genetic structure across the Cordilleran Mountains D. mawii, the genus Bufo has different genetic lineages to the West and East of the Isthmus of Tehuantepec and Sierra de Santa Marta D. mawiiThe uniqueness of the Papaloapan population could be explained by the fact that this is the only population located northwest of the Isthmus of Tehuantepec and the Sierra de Santa Marta . SeveralDermatemys mawii populations on the Yucatan Peninsula are only weakly differentiated from each other for mtDNA, and two haplotypes that differ by only a single mutation dominate this entire region Dermatemys mawii is highly aquatic and the rivers in this basin have been interconnected for thousands of years. The relatively high levels of gene flow between localities that we observed are, therefore, not unexpected. However, it is notable to find such high levels of gene flow between localities that are separated by long geographic distances (more than 300 km), as suggested by the mis-assignment of individuals to populations that are located at considerable distance from their collection site. Our results also support human-mediated gene flow due to the translocation of turtles to distant areas as an important factor that may have influenced the low level of contemporary genetic structuring.Most of the populations surveyed in this study are located to the southeast of the Isthmus of Tehuantepec in the Grijalva-Usumacinta river basin. D. mawii has been of great economic importance as food, and it has probably been used for trade or as part of peace offerings to the dominant ruling culture since the time of the Olmecs circa 3000 years ago D. mawii were found in the Teotihuacan archeological site more than 200 kilometers from the natural range of distribution of this species Macrochelys temminckii. This turtle also has a high commercial value, and is distributed in different drainages of the Gulf of Mexico in the USA, but shows very low levels of gene flow for both microsatellites and mtDNA markers D. mawii individuals were frequently assigned to localities far away from their collection sites. Although assignment quality might improve with a larger number of loci, far-reaching gene flow in D. mawii could be explained by the long distance connection of rivers within the Grijalva-Usumacinta basin. Alternatively, far-reaching gene flow could be the result of human translocation of individuals, as further supported by mtDNA data Malaclemys terrapin), where mixing of lineages was found to be due to animal translocation during the early twentieth century Geochelone radiata) Gopherus polyphemus), which have been moved by humans, show high levels of incorrect assignment to their collection localities One example of a species of turtle that was apparently not strongly affected by recent human translocations is the alligator snapping turtle For individuals with the divergent mtDNA haplotype 1D  to investigate if some level of assortative mating among individuals belonging to different mitochondrial lineages may exist. In addition, implementation of morphometric analyses of individuals from these populations in order to determine if there are patterns of morphological differentiation correlated with genetic differentiation Figure S1Plot of isolation by distance for D. mawii populations, with genetic distance plotted against geographic distance .(TIF)Click here for additional data file.Figure S2Evaluation of STRUCTURE  results: plot of Evanno et al.'s ΔK  for different numbers of clusters .(TIF)Click here for additional data file.Figure S3Evaluation of STRUCTURE  results: plot of lnP(D) for different numbers of clusters .(TIF)Click here for additional data file.


HincII-5‘ ε, HindIII-G γ, HindIII-A γ, HincII- ψβ1 and HincII-3‘ ψβ1) were analyzed in three populations (n = 114) from Reconcavo Baiano, State of Bahia, Brazil. The groups included two urban populations from the towns of Cachoeira and Maragojipe and one rural Afro-descendant population, known as the “quilombo community”, from Cachoeira municipality. The number of haplotypes found in the populations ranged from 10 to 13, which indicated higher diversity than in the parental populations. The haplotypes 2 (+ - - - -), 3 (- - - - +), 4 (- + - - +) and 6 (- + + - +) on the βA chromosomes were the most common, and two haplotypes, 9 (- + + + +) and 14 (+ + - - +), were found exclusively in the Maragojipe population. The other haplotypes  had lower frequencies. Restriction site analysis and the derived haplotypes indicated homogeneity among the populations. Thirty-two individuals with hemoglobinopathies  were also analyzed. The haplotype frequencies of these patients differed significantly from those of the general population. In the sickle cell disease subgroup, the predominant haplotypes were BEN (Benin) and CAR , with frequencies of 52.9% and 32.4%, respectively. The high frequency of the BEN haplotype agreed with the historical origin of the afro-descendant population in the state of Bahia. However, this frequency differed from that of Salvador, the state capital, where the CAR and BEN haplotypes have similar frequencies, probably as a consequence of domestic slave trade and subsequent internal migrations to other regions of Brazil. Five restriction site polymorphisms in the β-globin gene cluster ( For more than 20 years haplotypes from the 5' region of the β-globin gene cluster have been used to study the origin and distribution of human populations , 5 (- + - + +) and 6 (- + + - +) are the most prevalent in Eurasians whereas in Africans haplotype 3 (- - - - +) reaches a frequency ≥ 50%  had the Central African Republic (CAR) haplotype, 17 (23.0%) had the Benin (BEN) haplotype and one (1.3%) had the Senegal (SEN) haplotype  in the southeast of Brazil, Salvador (State of Bahia) in the northeast and Belém (State of Pará) in the north  and Maragojipe (S: 12°47' W: 38°56'). Of the 48 individuals living in central Maragojipe, 44 had HbAA, 1 had HbAC and 3 had HbAS electrophoretic profiles. The subjects from Cachoeira consisted of two groups: one of 34 individuals living in the town center  and the other of 32 individuals from the Afro-derived population of Santiago do Iguape (26 HbAA 1 HbAC and 5 HbAS), a village located 44 km from central Cachoeira. These individuals were selected in community health programs run by the Faculdade Adventista da Bahia in partnership with the Fourth Regional Health Division of the State of Bahia and local health boards. The programs offer various health services such as vaccination, prenatal supervision, sex education, dental treatment and blood pressure and glycemia measurements.In addition to these subjects, a further 22 individuals  bearing the sickle-cell trait (HbAS) and 32 unrelated patients with hemoglobinopathies  diagnosed in the Genetics Laboratory of the Department of Physiotherapy at the Faculdade Adventista were selected from other towns in the region.Blood samples (5 mL) for electrophoretic and molecular analyses were collected in tubes containing EDTA (0.03%) as anticoagulant. DNA from leukocytes in 100 μL of venous blood was isolated using GFX Genomic Blood DNA purification kits . The presence of the variant hemoglobins HbS and HbC detected by the electrophoresis of hemoglobin in alkaline medium was confirmed by the polymerase chain reaction (PCR) followed by digestion with an appropriate restriction enzyme. The reaction protocol was a modified version of the Amplification Refractory Mutation System (ARMS) described by HincII, in the 5' region of the ε gene, 2) HindIII in IVS-2 of the Gγ gene, 3) HindIII in IVS-2 of the Aγ gene, 4) HincII in the ψβ gene and 5) HincII in the 3' region of the ψβ gene, were amplified by PCR. The amplifications were done in a final reaction volume of 25 μL containing 25 pM of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphate  (Amersham Lifesciences), 1.0 U of Taq polymerase  and 100 ng of genomic DNA. The reactions consisted of an initial step of DNA denaturation at 95 °C for 2 min, followed by 35 cycles of 1 min at 94 °C for denaturation, 1 min at 54-57 °C for primer pairing, 2 min at 72 °C for polymerization and a final step of 7 min at 72 °C. The amplified products were digested with appropriate restriction enzymes and visualized in 1.5% agarose gels stained with ethidium bromide (0.5 μg/mL); φX174 DNA digested with HaeIII was used as the size ladder.DNA polymorphism was assessed by using a slightly modified version of the technique proposed by ST calculation as provided by Fstat software based on equation 8.27 described by ST in their analyses of inter-populational diversity. Ethnic admixture was estimated by the gene identification method  and the gene differentiation coefficients (Gst and Gst') for the populations from Reconcavo Baiano and other ethnic groups are shown in 2 = 0.99).The extent of ethnic racial admixture was assessed by considering the three populations as a single sample. A three-hybrid population model was used to estimate the percentages for each parental population and yielded the following results: 80.4% African, 10.8% European and 8.8% native Amerindian, with a significant coefficient for multiple correlation  in βC chromosomes agreed with historical data ascribing the origin of this haplotype to Central and West Africa, a region from which large numbers of slaves came to Brazil, especially to northeastern Brazil. However, the frequency of the type II haplotype in the Reconcavo Baiano region was higher than in African populations (HincII 5'ε site (0.217 to 0.325) were intermediate between those of the African population (0.108) and those of the European (0.618) and Amerindian (0.81) populations  and Iguape (0.453) were closer to those of the African population (0.463), whereas the Cachoeira sample had a lower frequency (0.323). Maragojipe (0.260) and Iguape (0.196) showed the highest frequencies for the HindIII IVS2 Aγ restriction site, and the frequency for Cachoeira (0.088) was similar to that of the African population (0.079). The frequency of the HincII ψβ1 site in the Iguape sample (0.018) was closer to the Amerindian population (0.04). The latter data indicated only a distant relationship (based on frequency) with the Maragojipe (0.156) and Cachoeira (0.100) populations, which were closer to the African population (0.158). The HincII 3'ψβ1 site had the lowest variability among restriction sites in the three samples (0.633 and 0.672), with values between those of the European (0.359) and African (0.931) populations.Analysis of the distribution frequencies of the restriction sites showed that the frequencies for the ulations . For theFourteen haplotypes identified in the three populations accounted for 43.7% of the 32 possible haplotypes. This linkage-disequilibrium resulted from the proximity among restriction sites in a region of ~32 kb where the overall recombination rate was estimated at 0.0017%  and in Colombian Amerindians (Haplotypes 18 and 19 were first described in Japanese subjects by The low coefficient of gene differentiation (Gst) for the three populations from Reconcavo Baiano 0.004;  indicateS haplotypes among HbSS individuals in northern, northeastern and southern Brazil have indicated that the average frequency for the predominant CAR haplotype in the three regions (65.9%) agrees with historical data showing that about 70% of the African slaves transported to Brazil were from Angola, the Congo and Mozambique  . Other studies have also identified this haplotype in northeastern Brazil (The Cameroon (CAM) haplotype that we identified comes from the West African coast, especially Nigeria  differ from those reported by The frequencies of the type I and type II βC chromosomes was higher than in Africa, where it ranges from 8% to 23%  in β