Baylisascaris schroederi is one of the most common nematodes of the giant panda, and can cause severe baylisascarosis in both wild and captive giant pandas. Previous studies of the giant pandas indicated that this population is genetically distinct, implying the presence of a new subspecies. Based on the co-evolution between the parasite and the host, the aim of this study was to investigate the genetic differentiation in the B. schroederi population collected from giant pandas inhabiting different mountain ranges, and further to identify whether the evolution of this parasite correlates with the evolution of giant pandas.B. schroederi were collected from 28 wild giant pandas inhabiting the Qinling, Minshan and Qionglai mountain ranges in China. The complete sequence of the mitochondrial cytochrome b (mtCytb) gene was amplified by PCR, and the corresponding population genetic diversity of the three mountain populations was determined. In addition, we discussed the evolutionary relationship between B. schroederi and its host giant panda.In this study, 48 B. schroederi, and high genetic variation within populations and a low genetic distance were observed. Both phylogenetic analyses and network mapping of the 16 haplotypes revealed a dispersed pattern and an absence of branches strictly corresponding to the three mountain range sampling sites. Neutrality tests and mismatch analysis indicated that B. schroederi experienced a population expansion in the past.For the DNA dataset, insignificant Fst values and a significant, high level of gene flow were detected among the three mountain populations of B. schroederi, low genetic structure and rapid evolutionary rate suggest that the B. schroederi populations did not follow a pattern of isolation by distance, indicating the existence of physical connections before these populations became geographically separated.Taken together, the dispersed haplotype map, extremely high gene flow among the three populations of  Baylisascaris schroederi is one of the most common nematodes of giant panda, and can cause severe baylisascarosis in both wild and captive giant pandas, Baylisascaris transfuga (accession number: NC_015924.1 for the mtCytb gene), a closely related species[All sequences were initially aligned using ClustalX 1.81 with theFs and Tajima’s D value), average number of nucleotide differences (K), nucleotide divergence (Dxy) and the net genetic distance (Da) between populations; the Fst statistic and value were computed from the haplotype frequencies using the program Arlequin Version 3.5[The program Dna SP 5.0 was usedsion 3.5. To evalsion 3.5 was usedsion 3.5.B. schroederi parasitizing giant pandas in three different mountain ranges.The pairwise genetic distances between individual haplotypes were calculated following the Kimura 2-parameter model, using MEGA 5.0; phylogeP value for each population, to assess whether population expansion occurred in B. schroederi. To calculate the distribution of the number of pairwise differences between the 48 sample sequences, a mismatch analysis was conducted using Dna SP 5.0[For population expansion analysis, the Arlequin Version 3.5 coupled a SP 5.0 over 100B. schroederi, calibration of the absolute rate of evolution of these parasites is generally problematic. Therefore, the evolutionary rate of the mtCytb gene from the parasite nematode Heligmosomoides polygyrus, [2P) distance between the 48 parasite mtCytb sequences in order to date the isolation time of B. schroederi.Due to the lack of fossil records for n years) in this Cytb in 48 B. schroederi isolates was determined and deposited in GenBank under the accession numbers KC796955-KC797002. The sequence analysis showed 31 variable sites on the sequences (2.80%), including 24 singleton variable sites, defining 16 haplotypes  of the mt107 bp ofB. schroederi, the sequence divergence values to construct hypothetical phylogenetic trees were calculated by the MP method and BI procedure. The two methods of phylogenetic analysis using the mtCytb marker led to very similar trees with shallow branches . Seven of the haplotypes were found in the Minshan population (H1-H7), three in the Qinling population  and twelve in the Qionglai population . While H1 and H2 were present in the populations from all three mountain ranges, haplotype H1, which had a cumulative frequency of 33.33%, was more frequent than H2 (18.8%). The Qionglai and Minshan populations shared haplotypes H3 and H6 , while the lowest value was recorded for the Minshan and Qionglai populations . The pairwise genetic distances between haplotypes are presented in Table 2P. Nucleotide divergence (Dxy) among the three populations was low, ranging from 0.182% to 0.221%. Taken together, the three mountain range populations were not significantly differentiated.To analyze the population genetic structure, several AMOVA analyses at different hierarchical levels were performed. Based on these AMOVA analyses, 98.03% of the variation occurred within the individual populations, and only 1.97% occurred across the three populations. As shown in Table B. schroederi sequences had a significant, large negative Fu’s Fs value . To further explore the demographic history of the B. schroederi population, the Fs statistic values for Cytb for the three populations were also estimated. Overall, the Fs values were −2.69374 (P < 0.05), 1.87372 (P > 0.05) and −4.91076 (P < 0.005) for the Minshan, Qinling and Qionglai populations, respectively. Mismatch distribution analysis of the complete datasets revealed the presence of a major peak, which suggests that at least one expansion event occurred in the population demographic history of the B. schroederi population  have been widely applied for analysis of the phylogenetic relationships between nematodes at the subspecies, species, genus and order levels[Cytb gene was sequenced in 48 B. schroederi isolates sampled from 28 giant pandas inhabiting three different mountain ranges, and the corresponding population genetic diversity of the nematode parasites was subsequently determined.In recent years, successive studies employing molecular tools have attempted to understand the genetic diversity, population differentiation, and evolutionary or taxonomic relationships between closely related species. There is no doubt that there has been relatively wide interest in the genetic diversity of parasites. MtDNA markers have higher Fst values than nuclear sequences, and the mtDNA of nematodes evolves more quickly than the mtDNA of other parasites,20,21. Rr levels,22,23. HB. schroederi from the different mountain ranges had a high haplotype diversity and low nucleotide diversity. This phenomenon is frequently observed in a number of other invertebrate animals with large standing population sizes and an extremely high fecundity[B. schroederi, or indicate the occurrence of expansion after a period of low effective population size, as rapid population growth enhances the retention of new mutations.In general, standard indices of genetic diversity are represented by the number of distinct haplotypes, haplotype diversity (h) and nucleotide diversity (π). Our resecundity,26, and B. schroederi populations (including Qinling mountain), consistent with previous reports based on the ribosome DNA [Nm value indicated the occurrence of strong gene flow among the B. schroederi populations over time. The most likely major contributor to gene flow is the movement of the giant panda. Generally, network approaches are better methods for representing genealogical relationships at a population level than traditional phylogenetic methods, and these approaches take account of several features associated with intraspecific gene evolution, including the persistence of ancestral haplotypes, the existence of multiple descendant haplotypes and often low levels of sequence variation[B. schroederi populations from different mountain ranges, in contrast with that reported in giant pandas[B. schroederi and its host. Additionally, efforts were also made to determine whether a correlation existed between genetic differentiation and geographical distance. The calculated “within population” similarity indices showed that the populations did not follow a pattern of differentiation by distance. Collectively, these results clearly indicate the absence of significant geographical structuring among B. schroederi. Genetic diversity within populations is affected by their effective population sizes, immigration from other populations and mutation rates, whereas gene flow among populations and the time of divergence of a population from a common ancestral population affect the extent of genetic diversity[Fst is appropriate for assessing the levels of differentiation within populations, as genetic drift is assumed to be the major factor that leads to genetic differentiation among closely related populations or over short-term evolution. Our assnd 5.8S),28. Furtariation. Based ot pandas, suggestiversity.B. schroederi samples from different mountain ranges shared the same haplotypes, indicating that some of the mountain systems have a physical connection, perhaps due to human activity or host movement. Additionally, the haplotype network analysis revealed that the H1 haplotype is the most ancient haplotype  samples, especially from Qinling Mountains. Although the limit of three populations used for analysis might not be sufficient, strong correlations emerged when the phylogenetic relationship among these haplotypes, even for individual haplotypes (H16) contradicted the general trend Figure . Some B. Figure , as prevB. schroederi populations is likely to be influenced by a variety of factors. On the one hand, drainage systems and isolation of the mountain ranges, in particular geographical isolation, have been identified as factors leading to evolutionarily distinct clusters. The Min River valley and the existence of six relatively isolated mountain ranges that giant panda inhabit, three of which were sampled in this study, might result in a distinct population structure among the populations. Second, unlike most free-living organisms, not only the parasites own reproductive and transmission patterns but also the genetics and behavior of the host could influence their genomic variation[et al. (2012) carried out whole-genome re-sequencing of 34 wild giant pandas to continuously outline the history of the giant panda population, and revealed that the Minshan and Qionglai-Daxiangling-Liangshan populations were also genetically distinct[B. schroederi genetic exchange due to giant panda and, especially, human movements may have independently led to the presence of a large number of haplotypes in the B. schroederi populations in each area. Indeed, the network map clearly revealed each B. schroederi population has its own unique haplotypes  and network, the H8, H14 and H16 haplotypes clearly have a wide distribution, especially the H16 haplotype which could be derivatized by two paths . This phenomenon of genetic diversity in ariation. Host moariation. The giaariation. Previouariation. Subsequdistinct. Prior tCytb gene evolution rate, particularly in parasitic nematodes including ascaridoid species. In the present study, the nematode H. polygyrus was chosen as a comparison for exploring the evolutionary rate of Cytb gene in B. schroederi due to its known Cytb evolution, taxonomic and phylogenetic relationships with B. schroederi. Based on the molecular evolution rate of H. polygyrus Cytb gene (3.5-3.7% K2P distance per million years)[B. schroederi divergence time is estimated to be 0.108-0.114 million years (Myr). In addition, it is well-known that sequence diversity can result from an accelerated rate of nucleotide substitution[Cytb gene is approximately 7-fold higher in B. schroederi than in the giant panda[Cytb genes were sequenced and clearly demonstrated the genetic relationships and gene diversity of B. schroederi populations from different mountain ranges.Placing a time scale on molecular data remains a difficult problem, since very little information is available on the n years), the B. titution. In our nt panda. This rent panda. A largent panda,35. In tCytb gene in 48 B. schroederi isolates sampled from the three main giant panda habitats revealed a lack of population structure (Fst) and a relatively high gene flow for B. schroederi, and indicates that the low genetic diversity might be a result of either a low DNA mutation rate, frequent movement of the hosts, or a combination of both of these factors. We also presume that B. schroederi has experienced lineage re-arrangement, as indicated by the high rate of gene flow and lack of a sufficient differentiation period. In addition, expansion phenomena have occurred among the B. schroederi populations, as most of the genetic variation identified in this study was found within populations, suggesting that a more intensive sampling strategy could possibly uncover the geographic structure of B. schroederi in more detail.This first investigation of the variability of the complete mtCytb: Mitochondrial cytochrome b; ITS1: Internal transcribed spacer region 1; ITS-2: The second internal transcribed spacer; cox1: Cytochrome c oxidase subunit 1; NAD1: NADH dehydrogenase subunit 1; MP: Maximum-parsimony; BI: Bayesian inference; K2P: Kimura 2-parameter; mtDNA: Mitochondrial DNA; Myr: Million years.The authors declare that they have no competing interests.YGY conceived the concept of the project. ZZH, WCD, GXB, WSX, PXR and YGY collected samples. ZX, XY and SY performed the lab work. ZX and XY carried out the statistical analyses. ZX wrote the initial draft of the manuscript. YGY and XY revised the manuscript. All authors read and approved the final manuscript.Origins and numbers of B. schroederi isolates and their hosts. a, GenBank accession numbers of mitochondrial Cytb sequences of these B. schroederi samples.Click here for filePolymorphic sites in complete mtCytb gene. The nucleotides present at each variable site among the 48 B. schroederi isolates.Click here for file


To examine population genetic structure and hypotheses of the origin of the modern Basque population in Spain using autosomal short tandem repeat (STR) data from individuals living in 27 mountain villages in the provinces of Alava, Vizcaya, Guipuzcoa, and Navarre, by comparing Basque autosomal STR variation with that of neighboring populations in Europe, as well as proposed ancestral populations in North Africa and the Caucasus.SW distance matrix. Allele frequencies for 9 autosomal STR loci  and several population genetic parameters were determined for the 4 provinces in the Basque region of Spain (n = 377). Heterozygosity within the Basque population was measured using a locus-by-locus analysis of molecular variance. Relationships between the Basques and other populations were examined using a multidimensional scaling (MDS) plot of Shriver’s DHeterozygosity levels in the Basque provinces were on the low end of the European distribution (0.805-0.812). The MDS plot of genetic distances revealed that the Basques differed from both the Caucasian and North African populations with respect to autosomal STR variation.Autosomal STR analysis does not support the hypotheses of a recent common ancestor between the Basques and populations either from the Caucasus or North Africa. Euskara, is most widely accepted as an isolate, unrelated to any other extant language in Europe  and a high frequency of RH*cde between 30.5%-35.6%) -15. Sinc.5%-35.6%The present study characterizes autosomal STR genotypes from the Basque region of Spain to examine population substructure and genetic relationships with other groups, including testing of the proposed genetic affinities between the Basques and populations in the Caucasus and North Africa. We predict that if the Basques share a common ancestor (or have experienced more recent migration and resultant gene flow) with either populations in the Caucasus or North Africa, allele frequencies of autosomal STR loci will be similar among the Basques and these proposed related populations and genetic distances between these populations will be low. Alternatively, if the Basques are an autochthonous European population (with no recent gene flow from groups in North Africa or the Caucasus), autosomal STR frequencies will be within the range of other populations on the European continent, and the Basques will be more genetically similar to other European groups. Previous studies presenting STR data from the Basque population either used small samples that were often collected in urban areas of a single province (when collection location was reported) to study relationships between the Basques and other populations ,54,58-60To test the hypotheses of genetic similarity between Basques and other populations, buccal DNA samples were collected from 652 autochthonous (those who claimed 4 Basque grandparents) participants of both sexes, in mountain villages throughout the Basque region of northern Spain. Six villages were sampled in Alava (n = 143), 17 villages in Vizcaya (n = 237), 10 villages in Guipuzcoa (n= 220), and 2 villages in Navarre (n = 52). A sub-sample of individuals from 27 villages (n = 478) was screened for autosomal STR genotypes  Figure . The samDNA extraction was performed using a standard phenol:chloroform protocol. A portion of each sample was reserved for autosomal STR analysis using the Applied Biosystems Profiler Plus Kit . The samples were characterized for 9 STR loci, including D3S1358, D5S818, D7S820, D8S1179, D13S317, D18S51, D21S11, FGA, and vWA, plus the sex-determining amelogenin locus, using a multiplex PCR procedure according to manufacturer’s instructions . AmplifiTo test hypotheses of population origins, autosomal STR data from our sample were compared to geographically and/or proposed related populations, including others from the Iberian Peninsula  ,62-67; EST). GST and gene diversity values for the Basques and comparative populations were calculated using DISPAN software (SW), an adaptation of Nei’s standard distance (D) weighted by the difference in number of repeats between alleles to account for the stepwise mutation pattern of tandem repeat loci. Genetic relationships between groups were examined using a multidimensional scaling (MDS) plot of the genetic distance matrix in NTSYS 2.1 software , so that not all loci were amplified for every individual. Samples that did not amplify for all loci were removed from the analysis. Allele frequencies for the autosomal STR loci in each of the Basque provinces after correction for allelic dropout are available in the web extra material.P values indicate those loci with lower than expected heterzogosity after correction of the data for allelic dropout  to 0.92105 (vWA), with both extremes found in Navarre, likely as a result of small sample size (N = 38) . In Alav dropout . This su dropout .a = -0.095). The lack of structure among provinces was confirmed by the global estimate of the fixation index among groups . A small amount of subdivision was found between villages within provinces . Examination of the locus-by-locus results revealed that 3 loci made significant contributions to the differences between villages: D7S820 , vWA , and D18S51 . The majority of variation, however, was found between individuals within villages .The results of the AMOVA  suggesteT) was high, ranging from 0.724 for D5S818 to 0.878 for D18S51. However, most of this diversity is explained by variation between individuals within subpopulations (HS). The percentage of gene differentiation between subpopulations relative to the total gene differentiation (Gst) ranged from a high of 0.009 for D13S317 and D21S11 to a low of 0.006 for D3S1358, FGA, D7S820, vWA, and D18S51.Average heterozygosity values by population ranged from 0.803 in Morocco to a high of 0.820 in Scotland . Among tr = 0.93498, t test: t = 5.7717, P = 1.0).Visual representation of genetic distances between populations using multidimensional scaling  showed tOur study of the autosomal STR variation did not support the hypotheses of a recent common ancestor between the Basques and populations either from the Caucasus or North Africa. Allelic dropout was noted for several samples in the present analysis, raising the possibility of a technical or genomic error in the typing of samples. At low sample DNA concentrations, the Profiler Kit is known to preferentially amplify short alleles and homozygotes . BecauseThe present analysis of autosomal STR variation does not support either the Caucasian or Vasco-Iberian hypothesis of Basque origins. Caucasian languages themselves are not a cohesive group, and while some linguists see similarities between Basque and some aspects of the northern or southern Caucasian languages, these similarities have been attributed either to poor interpretation, a shared Euro-African substratum, or similarities in the evolution of language itself . ExaminaST values between Basques and Caucasus-dwelling groups were much greater than between Basques and surrounding Indo-European populations (Recent studies of molecular markers also found little similarity between Basques and populations living in the Caucasus region. Analysis of Y-SNP haplogroups found that Fulations . While culations , the addulations . As withThe Vasco-Iberian hypothesis is based partly on craniometrics, the anthropometry of head shape . Broca sThe majority of genetic studies supporting a relationship between Basques and North African populations have been based on HLA data ,107-111.


N-acetyltransferase 2 (NAT2) is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples  representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4%) and herding (48.2%) as compared to populations mostly relying on hunting and gathering (22.4%) (P = 0.0007). This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25%) as compared to hunter-gatherers (8%). These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research questions involving NAT2.Arylamine  N-acetyltransferase 2 (NAT2) gene is involved in human physiological responses to a wide range of xenobiotic compounds, including many clinically useful drugs and a variety of exogenous chemicals present in the diet and the environment. Genetic polymorphisms at the NAT2 locus, giving rise to either the ‘slow’ or the ‘fast’ acetylator phenotype, influence individual variation in cancer susceptibility, responses to environmental toxins, and the effectiveness of prescribed medications NAT2 has generated considerable interest in the field of evolutionary genetics and numerous studies have attempted to decipher the relative roles of population history and natural selection in shaping genetic variation at this locus The arylamine NAT2 gene variation is the high prevalence of slow acetylators in humans (well above 50% worldwide) which calls into question the role that slow acetylation has played in the adaptation of our species. Several non-mutually exclusive hypotheses have been proposed. A first possible explanation is that NAT2 may be a neutrally evolving gene, the NAT2 enzyme having become dispensable or redundant with other detoxifying enzymes such as NAT1, and thus being no more essential to human life and health NAT2 allele NAT2 may have simultaneously become targets of directional selection, thereby generating an excess of intermediate-frequency haplotypes. This complex model of ‘multiallelic’ directional selection seems to better fit the patterns of NAT2 diversity observed in present-day populations than the standard ‘hard sweep’ model which assumes the rapid fixation of a single newly arisen advantageous mutation NAT2 locus A particularly intriguing aspect of NAT2 variation conducted in a large number of human populations have provided compelling evidence that at least some of the slow-causing variants of NAT2 have been driven to present-day frequencies through the action of natural selection, although the observed patterns do not allow to discriminate between balancing selection and directional selection on multiple standing variants NAT2 gene in six Central Asian populations has indeed revealed a clear contrast between populations having different lifestyles and dietary habits, with twice as many slow acetylators in long-term sedentary agriculturalists (55%–63%) as compared to nomadic pastoralists (26%–35%) et al.NAT2 haplotype frequencies in 47 worldwide populations (of which 12 newly studied populations), assigned them to one of the major subsistence strategies , and performed tests for the equality of haplotype frequencies across subsistence modes. The pool of fast haplotypes showed a strong decreasing trend in the order hunter-gatherers/pastoralists/agriculturalists, with average frequencies of 0.52, 0.36 and 0.27, respectively, significantly departing from equality (P<0.001). Among slow haplotypes, NAT2*5 and NAT2*6 showed a similar (inverted) trend, with significantly higher frequencies in agriculturalists  as compared to pastoralists (0.27 and 0.23) and hunter-gatherers (0.23 and 0.11). Luca et al.NAT2-altering variants may have gained a selective advantage in populations shifting from hunting-gathering to pastoralism/agriculture and proposed the diminished folate supply resulting from the nutritional shift as a possible cause of the fitness change.The recent surveys of NAT2 gene polymorphisms in an impressive number of populations of distinct ethnic backgrounds since the discovery of the gene in 1990 NAT2 variation. By systematically retrieving data from the literature, we collected frequency data for the most relevant genetic variants of NAT2 in 128 population samples representing different subsistence modes and dietary habits. This allowed us to perform a thorough analysis of the covariation between NAT2 haplotype frequencies and the main subsistence strategies at both a worldwide and continent scale. Furthermore, the vast amount of data collected provided a comprehensive and up-to-date description of worldwide NAT2 genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research questions involving the NAT2 gene.An overwhelming amount of data has been generated on NAT2 gene by systematically retrieving data from the literature , Europe (28), Asia (39), America (25), and Oceania (2). Sample sizes ranged from 11 to 1,312 individuals, with an average of 115 (±178) individuals per sample. The number of samples genotyped for 7, 6, 5, 4 or 3 SNPs was 74, 32, 3, 8 and 11, respectively. The geographical distribution of the population samples is shown in We created a comprehensive resource of frequency data for the seven most important genetic variants of the terature . These sNAT2 haplotype and phenotype variation, we focused on the 99 population samples adequately characterized for the seven (or six for non-African samples) SNPs of NAT2  and five slow haplotypes . African populations showed the highest level of within-population diversity  and had also the largest number of private haplotypes.To describe the global patterns of NAT2 see . HaplotyNAT2 haplotypes  in the 99 worldwide samples revealed striking differences between continental groups , pointing to a remarkable homogeneity for NAT2 variation in this continent. This sharply contrasted with the high level of population differentiation observed in Asia  and America , the African samples displaying an intermediate value . The magnitude of frequency differences among American populations can be easily explained by the presence of both several small isolated populations undergoing rapid evolution through genetic drift (e.g. Karitiana and Surui) and a few large urban populations that probably include a substantial level of recent European and/or African ancestry (e.g. Rio de Janeiro). Regarding Asia, three distinct features can explain the extreme degree of interpopulation differentiation observed here: (1) the high level of diversity of Omani and Indian populations which display the largest number of distinct haplotypes at NAT2 (two-fold higher than the worldwide average), (2) the unusually large frequency of the NAT2*13A haplotype (31%) in the Vietnamese Khin sample, and (3) the specific profile of North-East Asian populations  which form a remarkably homogeneous group in regard to NAT2 haplotype frequencies . This group is notably characterized by frequencies of the fast NAT2*4 haplotype that are among the highest worldwide, particularly low frequencies of the slow NAT2*5B haplotype  and a poor haplotype diversity  with only three distinct haplotypes occurring at a frequency > 0.01. The slow NAT2*6A haplotype occurs at roughly similar frequencies all over the world (notwithstanding significant disparities within America), whereas the slow haplotypes NAT2*7B and NAT2*14 mainly cluster in specific continental regions . The existence of NAT2*14 haplotypes in the samples from Goiás and Rio de Janeiro is consistent with the high level of African admixture present in the Brazilian population.The distribution of the most common l groups . AfricanSTF value estimated for the 99 worldwide samples was of 0.126 (P<10−5), consistent with the average value estimated for the human genome To assess population genetic structure, the 99 populations were grouped into four continental regions . The vast majority of genetic variation was found to occur within populations (87.4%), a high proportion (8.3%) among continental groups, and a mere 4.3% among populations within groups. The global NAT2*4 haplotype in this group of populations. In Central Asia, the prevalence of slow acetylators varies greatly among populations, mainly according to lifestyle, ranging from 0.34 on average in nomad pastoralists to 0.59 on average in sedentary agriculturalists. The prevalence of slow acetylators is highly heterogeneous in Africa and in America, with striking differences among populations, even at a small geographic scale.The overall population prevalence of the fast/slow acetylation phenotypes in the 99 worldwide samples investigated is reported in n = 73), pastoralists (n = 18) and hunter-gatherers (n = 19). We performed tests for the equality of haplotype frequencies across subsistence modes, considering the four main haplotype series of NAT2 known to be associated with an altered enzyme function: NAT2*5, NAT2*6, NAT2*7 and NAT2*14, defined by the 341T>C, 590G>A, 857G>A and 191G>A slow-causing variants, respectively. The frequency of the fast acetylation phenotype, as inferred from genotype data, was also compared across subsistence categories.Among the 128 population samples collected, 110 could be assigned to one of the three major subsistence strategies: agriculturalists . By contrast, a remarkably similar pattern was observed between pastoralists and agriculturalists for both phenotype and haplotype frequencies.Hunter-gatherers showed a significantly higher prevalence of the fast acetylation phenotype (77.6%) as compared to pastoralists (51.8%) and agriculturalists (54.9%) , and a higher prevalence of fast acetylators was still observed in non-Pygmy hunter-gatherers when compared to the two other subsistence groups (P = 0.0150).The analysis was also performed at a smaller geographic scale, by considering only the samples from Africa (the sole continent where the three subsistence strategies co-exist in our population survey). Consistently with our previous results, a significantly higher prevalence of fast acetylators was observed among hunter-gatherers (79.1±12.0%) as compared to pastoralists (35.1±7.7%) and agriculturalists (49.1±12.8%) , but the results were similar to those obtained previously .Note that we required a minimum sample size of 10 individuals per published sample for it to be included in our database, and thus in the analyses. However, many published samples are still of very small size, thus preventing a precise estimation of allele and phenotype frequencies. Among the 110 samples included in the correlation analysis of eviously . The sigNAT2 may represent a further example of a gene exposed to culturally-driven selective pressures arising from new dietary and xenobiotic exposure.Recent genomic studies have provided growing evidence that cultural processes can have a profound impact on the human genome, triggering significant changes in allele frequencies in response to culturally modified environmental conditions NAT2 slow acetylators in human populations is related to the subsistence strategy historically adopted by the past populations. Compared to the study of Luca et al.NAT2 haplotype frequencies, we analysed the prevalence of the fast acetylation phenotype as inferred from genotype data in the samples adequately genotyped for the four slow-causing variants of NAT2, while Luca et al. focused on the individual slow haplotype series and on the pool of fast haplotypes. Third, in addition to a global analysis performed at a worldwide scale, we conducted an analysis at a smaller scale, within the African continent where the three main modes of subsistence are represented in geographically close populations. Our results demonstrated a significantly higher prevalence of slow acetylators in populations practicing farming and herding as compared to populations mostly relying on hunting and gathering, thus confirming the previous findings of Luca et al.NAT2 slow-acetylation alleles. However, this model supposes that NAT2 slow variants shifted from deleterious alleles, eliminated or maintained at low frequencies through the action of purifying selection, to neutral or nearly neutral polymorphisms evolving through random genetic drift. Yet, many previous studies support an adaptive evolution of the NAT2 gene  on NAT2 slow-acetylation alleles, rather suggesting a selective advantage associated with a slower acetylation rate in food-producing communities NAT2 sequence data. Patin et al. NAT2*5B haplotype in Western and Central Eurasian populations in the last ∼6,500 years in response to positive selection, suggesting that this slow allele probably conferred some selective advantage to its carriers in this part of the world. While the other studies could not demonstrate clear signals of strong positive selection as those expected under the ‘hard sweep model’ using conventional approaches for detecting selection, they all nevertheless highlighted patterns of variation compatible with the action of natural selection, either in the form of balancing selection or directional selection acting on multiple standing slow-causing variants.In the present study, we tested the hypothesis that the prevalence of n = 7) and America (n = 12) are located on different continents and yet display very similar frequencies of fast acetylators . Second, the contrasting pattern observed between food collectors and food producers in the prevalence of the fast acetylation phenotype was also found on a smaller spatial scale, within Africa, where the geographic distances between populations sharing the same subsistence mode are very close to those separating populations with different cultural practices. If the similarity in the prevalence of the fast acetylation phenotype between populations was mainly related to the geographic proximity of these populations due to more extensive gene flow, we would expect a positive and significant correlation between the difference in prevalence of fast acetylators and geographic distance. Yet, no such correlation was observed between the 26 African populations included in our analysis  as compared with food producers (∼25%) is in sharp contrast with the homogenous distribution reported for this variant in the 99 populations of the worldwide diversity survey . Such a low level of differentiation between widely dispersed populations may be interpreted as a signature of homogenizing selection, favouring the same allelic variant in otherwise disparate populations . In view of the marked correlation of the 590A variant with subsistence mode, we can speculate that the 590A slow-causing variant probably increased in frequency in populations shifting to agricultural and pastoral activities in response to new selective pressures and that the low level of differentiation observed at this locus results from the convergent selection of the 590A variant in agriculturalist and pastoralist populations which are now present in most parts of the world. Contrary to the results of Luca et al.NAT2*5 and NAT2*14 haplotype series across subsistence modes . The significant result observed for NAT2*7 (P = 0.021) must be interpreted with caution due to the geographic clustering of this haplotype in Asia and America and due to its highly heterogeneous frequency among hunter-gatherers, being very rare in hunter-gatherers from Africa (0.001±0.004) and much more frequent in hunter-gatherers from America (0.19±0.11).Consistently with the results of Luca NAT2 genetic diversity and provided an up-to-date picture of the global patterns of NAT2 variation in a wide range of populations throughout the world. This considerably extended the range of our previous worldwide survey NAT2 genetic variation in Central, South and North Asian populations NAT2 one of the best characterized pharmacogenetic gene for interethnic and geographic variation. With this better coverage of human genetic diversity, NAT2 variation no longer appears to be composed of discrete clusters roughly corresponding to continental regions but rather describes a broad geographic cline of allele frequencies that parallels those observed for presumably neutral genetic markers NAT2 genetic system and a true genetic differentiation between Northern and Southern East Asian populations at this locus. In the present report, the input of 25 additional populations from different parts of Asia enabled to highlight a clearly distinct pattern of diversity characterizing Northeast Asian populations , with a particularly high prevalence of the fast ancestral NAT2*4 haplotype  and a quasi-absence of the slow NAT2*5B haplotype. Besides, Thais displayed a similar profile to that seen in other populations from Southeast and Central Asia. The unexpected pattern of variation of NAT2 in the Khin ethnic group deserves further investigations to confirm the unusually high frequency of the fast NAT2*13A haplotype in other samples from the Vietnamese population.As a second contribution of this study, the vast amount of data collected from the literature allowed a comprehensive analysis of NAT2 haplotype frequencies in Northeast Asian populations can hardly be explained by a distinctive subsistence mode since these populations share, with those of Europe and many other parts of Asia, the same mode based primarily on agriculture. However, population-specific dietary habits and/or environmental exposures may still be valuable hypotheses to explain the specific pattern of NAT2 haplotype frequencies in this geographic area. Unfortunately, these hypotheses cannot be tested within the framework of this study since we mainly focused on the major subsistence categories rather than on specific dietary components or xenobiotics. A dedicated study investigating NAT2 sequence variation in Northeast Asians would be required to determine whether the increased frequency of the ancestral rapid NAT2*4 allele and/or the rarity of the slow NAT2*5B allele in these populations are the result of the action of local selective pressures or whether the observed pattern of frequencies is to be explained only by stochastic processes.The specific pattern of NAT2 genetic diversity is structured in the human species is not only of anthropological importance, but also of medical relevance for both pharmacogenetic and epidemiological applications. For example, if major differences in allele frequencies exist between populations, individuals from different ethnic or geographic origins may respond differently to acetylated drugs. Our study confirmed a wide variation across ethnic groups in NAT2 gene polymorphism and acetylation status at both global and microgeographic scales. The development of ethnically tailored therapies, however, appears irrelevant in the case of the NAT2 gene polymorphism since (1) there were only few region-specific haplotypes and (2) most genetic diversity occurred between individuals rather than between populations. In this context, the ethnicity of an individual does not represent a good proxy for the acetylation status. The present report points to several geographic regions of potential interest for pharmacogenetic applications that remain poorly characterized for NAT2 variation. A better description of NAT2 genetic diversity in sub-Saharan African and Indian populations would be particularly interesting in view of the considerable genetic, cultural, and phenotypic variation found in these world regions. Furthermore, characterizing patterns of NAT2 genetic diversity constitutes an imperative pre-requisite in the context of association studies aiming to better understand the role of NAT2 in drug-induced side-effects, drug response and disease susceptibility. Spurious associations can indeed arise from an unknown population structure and significant differences in allele frequencies and haplotype structure among populations may explain some of the contradictory observations of positive, negative and no associations of NAT2 gene polymorphisms with specific phenotypes. In this respect, the high genetic heterogeneity observed among populations from different parts of Asia, as well as among populations from Africa and America has imperatively to be taken into account when performing association studies in these populations. By contrast, the remarkable homogeneity of European populations in regard to NAT2 allele frequencies and haplotype structure facilitates the replication of association findings across populations of European background. Interestingly, a recent report indicates that a single SNP (rs1495741), located approximately 14 kb 3′ of NAT2, can be substituted for the panel of seven NAT2 SNPs, as an accurate marker of the NAT2 phenotype in molecular epidemiology studies performed in populations of European ancestry An understanding of how NAT2 in a large collection of human populations, allowing the investigation of specific research questions interesting both the biomedical and anthropology genetic communities.In conclusion, we provided clear evidence for a correlation between the prevalence of slow acetylators in humans and the subsistence strategy adopted by the past populations in the last 10,000 years, suggesting that a slower rate of acetylation may have gained a selective advantage in populations shifting from foraging to pastoralism/agriculture in the Neolithic period. In addition, we provided a comprehensive resource of frequency data for the most important genetic variants of NAT2 (191G>A (rs1801279), 282C>T (rs1041983), 341T>C (rs1801280), 481C>T (rs1799929), 590G>A (rs1799930), 803A>G (rs1208), and 857G>A (rs1799931)) and for which allele and/or genotype frequency data were available in the published reports. We also included population samples genotyped for only a subset of the seven SNPs of NAT2 (at least three) only in those cases where the ethnic origin of the sample was not already represented in the set of samples genotyped for all seven SNPs. Significantly heterogenous samples  or samples without a specific geographic or ethnic origin were excluded from the population survey, as well as samples composed of related individuals and with sample sizes below 10. In most cases, the selected samples were composed of apparently healthy, randomly selected volunteers of defined ethnicity. Information on each subject was confirmed not to be doubly included . The final data set included 128 population samples from throughout the world, representing 14,679 individuals. A full description of the selected samples is provided in NAT2 allele frequency data.We performed an extensive survey of the literature (up to March 2010) to identify all the population samples that were genotyped for the seven most common SNPs of NAT2 gene variation, only the 74 samples genotyped for the seven common SNPs of NAT2 were used so as to avoid possible haplotype and phenotype misclassifications due to incompleteness of genotype data. As the SNP 191G>A has been shown to be rare in non-African populations To adequately characterize the worldwide patterns of NAT2 haplotypes were either directly resolved using molecular-haplotyping techniques  or computationally inferred from the unphased multilocus genotypes using statistical algorithms . For some samples, a combination of the two approaches was used. The specific haplotyping method used in each sample is specified in NAT2 haplotypes were named in accordance with the consensus gene nomenclature of human NAT2 alleles (http://www.louisville.edu/medschool/pharmacology/NAT2.html). The fast NAT2*4 haplotype was considered as the ancestral human haplotype, as inferred from primate sequences (unpublished data).In each sample, NAT2, following the acknowledged classification of NAT2* alleles into fast and slow haplotypes. The acetylation phenotype for each individual was inferred by assuming that the homozygous or compound heterozygous genotype for two haplotypes of the series NAT2*4, NAT2*11, NAT2*12 or NAT2*13 results in the rapid acetylator status, the occurrence of one of these haplotypes in combination with a haplotype of the series NAT2*5, NAT2*6, NAT2*7 or NAT2*14 results in the intermediate acetylator status and the occurrence of two haplotypes of the series NAT2*5, NAT2*6, NAT2*7 or NAT2*14 results in the slow acetylator phenotype. The proportions of slow, intermediate and fast acetylators in the 99 samples studied are provided in Thanks to the well-established genotype-phenotype correlation FST statistic NAT2 haplotypes was included in AMOVA and FST computations.Analysis of molecular variance (AMOVA) P-value was less than or equal to 0.05. The NAT2*5, NAT2*6, NAT2*7 and NAT2*14 haplotype series were represented by the 341T>C, 590G>A, 857G>A and 191G>A slow-causing variants, respectively. Data on the prevalence of the fast acetylation phenotype were considered only for the samples genotyped for all four slow-causing variants, to which we added the non-African samples genotyped for all SNPs except 191G>A  or because of divergence between sampling location and region of origin (samples 43 and 87); these samples are displayed in a box beneath the caption.(TIF)Click here for additional data file.Figure S2n = 26 populations). (B) In America (n = 12 populations). The population pairs are colour-coded (shown in the captions) according to the compared subsistence categories . No significant correlation between geographic distance and difference in prevalence of fast acetylators was found in either one of these two continental regions .Plot of the difference in prevalence of fast acetylators between population pairs as a function of geographic distance. (A) In Africa ((DOC)Click here for additional data file.Table S1Description of the 128 population samples collected from the literature, along with SNP, haplotype and phenotype frequencies.(XLS)Click here for additional data file.Table S2Test for equality of frequency of phenotype and haplotype series across subsistence modes when using only those samples with a minimum size of 20 individuals.(XLS)Click here for additional data file.


Alsophila spinulosa as an example species, the main purpose of this study was to assess the relative impact of sexual and asexual reproduction on the level and structure of population genetic variation.Essentially all ferns can perform both sexual and asexual reproduction. Their populations represent suitable study objects to test the population genetic effects of different reproductive systems. Using the diploid homosporous fern Inter-simple sequence repeats analysis was conducted on 140 individuals collected from seven populations  in China. Seventy-four polymorphic bands discriminated a total of 127 multilocus genotypes. Character compatibility analysis revealed that 50.0 to 70.0% of the genotypes had to be deleted in order to obtain a tree-like structure in the data set from populations HSG, LCH, MPG, BPC, GX, and LD; and there was a gradual decrease of conflict in the data set when genotypes with the highest incompatibility counts were successively deleted. In contrast, in population ZHG, only 33.3% of genotypes had to be removed to achieve complete compatibility in the data set, which showed a sharp decline in incompatibility upon the deletion of those genotypes. All populations examined possessed similar levels of genetic variation. Population ZHG was not found to be more differentiated than the other populations.A. spinulosa. However, somatic mutation contributes most to the genetic variation in population ZHG. This change of the primary mode of reproduction does not cause a significant difference in the population genetic composition. Character compatibility analysis represents an effective approach to separate the role of sexual and asexual components in shaping the genetic pattern of fern populations.Sexual recombination is the predominant source of genetic variation in most of the examined populations of  Almost all ferns are homosporous, producing only one type of spore that germinates to form a bisexual gametophyte Hemionitis palmataPleopeltis species Polystichum otomasuiAsplenium trichomanes subsp. quadrivalensDryopteris aemulaBotrychium virginianum show little interpopulation genetic differentiation despite the possession of extremely high levels of within-population inbreeding Equisetum arvense and E. hyemale exhibit considerable among-population divergence even though they reproduce primarily by outcrossing Equisetum, i.e. the inefficiency of spore germination and gametophyte reproduction in noncolonizing situations, which was considered to limit the gene flow between populations Taraxacum agamosperms Botrychium pumicolaLife-history traits, such as reproductive system and frequency of sexual versus asexual reproduction, are considered to be particularly important for shaping the level and structure of genetic variation in plant populations Alsophila spinulosa (Hook.) Tryon (Cyatheaceae) is a diploid homosporous fern (2 n = 138) with a single, erect, tree-like rhizome up to 8 m tall A. spinulosa is an outcrossing species, mainly producing sporophytes by intergametophytic crossing. In mainland China, natural populations of A. spinulosa have been recorded in Fujian, Guangdong, Hainan, Guangxi, Guizhou, Yunnan, Sichuan, and Chongqing A. spinulosa was officially declared a first-grade protected plant by the State Environmental Protection Commission of China A. spinulosa has the potential to maintain multiple reproductive modes and therefore affords a suitable study object to test the population genetic effects of different mating systems. The main purpose of the present study was to assess the relative contribution of sexual and asexual reproduction in shaping the population genetic composition by using inter-simple sequence repeat (ISSR) markers. In this respect, a novel molecular genetic approach, namely character compatibility analysis, has been shown to be very useful for separating the role of different modes of reproduction As a homosporous fern, Twenty ISSR primers  were useMeasures of genetic variation indicated that populations MPG, BPC, and GX might maintain higher levels of genetic variation than other populations . NeverthThe polymorphic bands discriminated a total of 127 multilocus genotypes. The number of genotypes within each population varied from 15 to 20 . In popuBθ values and their 95% confidence intervals (CI) between the Alsophila spinulosa populations were estimated by running the f-free analysis in Hickory. The estimated Bθ values between population pairs (excluding population ZHG) ranged from 0.1710 (CI: 0.0812–0.2935) to 0.3862 (CI: 0.2799–0.4968) (Pairwise –0.4968) .Bθ values between population ZHG and the six other populations ranged from 0.1654 (CI: 0.0808–0.2854) to 0.3686 (CI: 0.2628–0.4725)  . TherefoAll populations except ZHG displayed considerable matrix incompatibility. It took 14 of 20 (70.0%), 12 of 18 (66.7%), 11 of 18 (61.1%), 11 of 19 (57.9%), 10 of 19 (52.6%), and 9 of 18 (50.0%) genotypes to eliminate all the matrix incompatibility in the data of populations HSG, LCH, MPG, BPC, GX, and LD, respectively . The conA. spinulosa.Molecular markers have been increasingly used to infer the influence of reproductive systems on patterns of population genetic variation in plants A. spinulosa populations examined, sexual recombination had been the predominant source of genetic variation. In populations HSG, LCH, MPG, BPC, GX, and LD, between 50.0% and 70.0% of genotypes contributed to incompatibility and in each population the contribution of genotypes to incompatibility varied considerably. In particular, there was only a gradual decrease of conflict in the data set when genotypes with the highest incompatibility counts were successively deleted . Population ZHG is located in Zi Huang Gou, Chishui, Guizhou, growing by streamsides under bamboo forest. Its habitat demonstrates no appreciable difference from that of other populations, especially HSG and LCH, which are also from Guizhou. However, our own survey, as well as Zhou et al.’s In contrast to the above-mentioned six populations, genetic variation in population ZHG appears to be primarily caused by somatic mutations. The incompatibility in its data set was quickly driven to zero upon deletion of only five of 15 (33.3%) genotypes; and the ten remaining genotypes exhibited a strictly tree-like data structure . In addiA. spinulosa have existed in population ZHG before the predominance of asexual reproduction, which account for most of the observed genetic variation Saxifraga cernuaIt has been predicted that asexual reproduction is expected to reduce population genetic variation and enhance genetic structure A. spinulosa, asexual reproduction can indeed occur and dominate on a local scale. Similar patterns have been identified in a number of other fern species Our results indicate that although sexual reproduction is predominant in most of the natural populations of This study was conducted in accordance with all People’s Republic of China laws. No specific permits were required for the described field studies. No specific permissions were required for access to the locations or for activities described in this study. The location is not privately owned or protected in any way.Alsophila spinulosa were sampled across its range in China. The locations are given in Ten natural populations of Total genomic DNA was isolated from ground frond tissue by means of the modified cetyltrimethylammonium bromide (CTAB) protocol Taq polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.0 mM MgCl2, 0.2 mM each dNTP, 0.3 µM primer, 80 ng genomic DNA, and DNA-free water. Amplifications were conducted in an MJ-Research PTC-100TM Peltier thermal cycler. Initial denaturation was at 94°C for 5 min, followed by 40 cycles of 94°C for 40 s, 53°C for 35 s, 72°C for 70 s, and a final elongation at 72°C for 5 min. A total of 80 ISSR primers  were initially screened for amplification. Twenty primers that generated clear and reproducible banding patterns were chosen for the final ISSR analysis  or absence (0) and entered into a data matrix. The binary matrix was used to calculate the percentage of polymorphic loci, observed number of alleles, effective number of alleles, and Nei’s gene diversity f and Bθ are analogues to the inbreeding coefficient (ISF) and the fixation index (STF) of F-statistics f and Bθ were estimated numerically through Markov Chain Monte Carlo (MCMC) simulations by running HICKORY v1.0 f-free” model option was preferred due to its lower deviance information criterion and pD (measure of model complexity) values than for other models. Point estimates of Bθ, as well as their 95% confidence intervals, were achieved with a burn-in of 50 000 iterations and a sampling run of 250 000 iterations from which every 50th sample was retained for posterior calculations. Three replicates were run to ensure that the results were consistent.Holsinger et al. Character compatibility analysis was conducted to reveal the relative contribution of asexual and sexual reproduction in the population


The present sero-genetic study is the first of its kind to present the baseline data of Bharia tribe of Madhya Pradesh. The main aim of this study is to provide phenotype and allele-frequency data to characterize the population genetically and to fill the void on the genetic map of Madhya Pradesh.For this, blood samples from 92 unrelated healthy individuals of Bharia tribe from Chhindwara district (Tamia block) were collected. Hemolysates prepared were analyzed for two serological (A1A2BO and Rh) and six biochemical  parameters, following the standard electrophoretic techniques.The Chi-square test for goodness of fit revealed no significant deviation between the observed and expected numbers in any of the seven genetic markers, suggesting that the tribe is in genetic equilibrium. A high incidence of B allele in A1A2BO blood group and low incidence of the A1 allele, with presence of A2 in only one individual, and a low frequency of Rh(D)  was observed in serological markers. Also, no rare variant was observed for biochemical markers.Principal Component Analysis done in order to detect the genetic affinity of Bharia tribe with other populations from the adjoining states of Madhya Pradesh based on the allele frequencies, showed a close association of Bharia with Gujarat and Rajasthan. Hence, this study has been helpful in revealing the genetic structure and affinity of Bharia tribe. In the past three decades, a great deal of human genetic data has been generated, but not much work has been carried out on the serological and biochemical markers of the tribal population from the central Indian state of Madhya Pradesh. A large amount of data on ethnic, regional and social distribution of genetic markers has already been reported on Indian populations, but our knowledge about genetic variation of different genetic markers is still limited.–6 Althoudhaya (a system of shifting cultivation) cutters, but nowadays they rear cattle and sell milk and milk products. They are also farmers and mostly produce small millet called Kodo-Kutki. Other areas of activities include forest wealth collection and hunting.Tribes are the assets of a country and the central Indian state of Madhya Pradesh is one of the largest states of India inhabiting the bulk of tribal population of the country , constituting 20.3% of the total population  of the state. Amongst a total of 46 scheduled tribes of the state, the main focus of this study was on Bharia tribe, mainly the inhabitants of the district Chhindwara . Bharia i1A2BO and Rh) and biochemical  data of Bharia tribe from Chhindwara district of Madhya Pradesh.The present study is therefore planned to present the serological (AThe present study comprises blood collection (about 1 ml each) from a total of 92 healthy unrelated individuals of either sex from Tamia block of Chhindwara district, after obtaining informed consent, in accordance with the ethical standards of Institutional Ethical Committee, following ICMR guidelines. Hemolysates, prepared by freezing and thawing technique, were electrophoresed for studying variabilities of different red cell enzymes in Bharia tribe. The electrophoretic typing of the six erythrocyte enzymes was performed following the original techniques for ACP locus 1 (ACP1), PGM locuFrom phenotype data obtained after electrophoresis, allele frequencies were calculated using the gene counting method. Chi-squa12The phenotypic distribution of two serological markers (A1A2BO and Rh) and six erythrocyte enzymes  are presented in In the A1A2BO system, blood group B was found to be the most preponderant and A2 incidence was much lower, whereas the occurrence of A1 was observed to be more than that of O. In terms of allele frequency, highest frequency was that of B (0.2491) and lowest was of A2 (0.0108). In case of Rh system, Rh(D) was observed in all the samples of Bharia tribe. In each of the five enzyme systems considered, all the possible common phenotypes were found, but no example of any rare variant was encountered. For ADA, high allele frequency was recorded for ADA*1 (0.8939), which is found frequently in Indian population. Similar result was obtained for AK1 and PGM1 with high allele frequency of AK1*1 (0.9278) and PGM1*1 (0.6667), while for ACP1, allele ACP1*B showed high occurrence. Interestingly, ESD showed equal value for both the alleles, whereas ESD*1 is the most frequently occurring among the Indian population. Goodness of fit Chi-square test revealed that the distribution of phenotypes in each of the blood groups and enzyme systems typed was in genetic equilibrium in the studied Bharia tribe.By and large, in the case of A1A2BO blood group system, it was observed that the average value of allele B was more as compared to allele A. Among the tribes, the value of B was high as compared to A; however, the difference between A and B frequencies was less among the tribes. Similar 125As far as the erythrocyte enzyme markers are concerned, variations at different enzyme loci were observed. The present ADA*1 frequency in Bharia tribe was found similar to that observed in Keer, while Bh10ESD showed remarkable equal frequency for both the alleles, ESD*1 and ESD*2, despite the fact that the frequency of common allele ESD*1 is reported to be 75% in the Indian population. Low sample size can be a possible reason for this, as earlier publications have shown an average allele frequency of 0.7315 for the other tribal populations of Madhya Pradesh.12 ACP frBoth the PGM1*1 and PGM1*2 alleles are present in all populations in appreciable frequencies  in Indians. No rare variant was observed in Bharia, while so far only Korku tribe from Hoshangabad have shown the occurrence of PGM1*7. For GPI,26Locus-by-locus comparison in Bharia suggests considerable intra-tribal variation in phenotype and allele frequencies. Chi-square value showed a good agreement between the observed and expected values in Bharia tribe. Both serological and biochemical markers were in genetic equilibrium as the differences between observed and expected frequencies were statistically not significant. Also, a considerable range of heterozygosity values across the loci was examined. The highest value of heterozygosity was found for the A1A2BO locus (0.6059) and the lowest for GPI (0.0109), with an overall mean of 0.291. On the other hand, PGM1 and ACP1 showed similar heterozygosity values (0.4444). The variation in average heterozygosity may simply be the genetic consequence of the population structure or it may indicate the effect of polymorphic or directional natural selection operating in the present Bharia tribal population of Madhya Pradesh. This wide variation in heterozygosity values from locus to locus also stresses the need for their estimation at a large number of loci to provide an adequate average heterozygosity estimate. Estimates of average heterozygosity ranging from 0.127 to 0.513 have been reported for Indian populations.29 Some oThe dendrogram  construcThe present study has witnessed variation at different loci in the Bharia tribe, suggesting intra-population genetic differentiation. Every population is different from the other population in terms of its genetic constitution, but this diversity is more at the intra-population level than at the inter-population level. The present study has thus presented a possible illustration of the genetic structure, and the affinity of Bharia tribe of Madhya Pradesh has been revealed. Moreover, genetic studies on Bharia tribe featuring the variations at molecular level, which would help to further support the above presented data, are still under consideration.


Larimichthys crocea) is the most important mariculture fish species in China and the wild stocks of this croaker have collapsed in the past decades due to high fishing pressure and habitat degradation. Due to a lack of wild croaker samples, however, studies concerning the genetic changes of the cultured croaker stocks compared to their wild counterparts were never conducted. Here, we collected three wild populations in the northern and central East China Sea during fisheries survey and investigated the differences in terms of genetic diversity and differentiation between and within cultured stocks and wild populations. Our results demonstrated that the cultured croaker had significantly reduced genetic diversity in contrast to the wild populations, and also presented statistically significant differentiation from the wild, indicating that enhancement of the current wild stock should be conducted with caution. These changes may be caused by founder effects, artificial selection and random genetic drift. With a relatively high level of genetic diversity, the wild populations showed important value for improving the ongoing breeding program of this croaker. Further, we detected no differentiation among the wild populations, suggesting that the wild croaker in the northern and central East China Sea should be considered as one unit for management and conservation.The large yellow croaker ( Larimichthys crocea, is an economically important marine fish species endemic to China. The production of this croaker reached about 200,000 tons in the mid 1970s and it was once ranked in the top three commercial fish species in mainland China [The large yellow croaker, nd China . In ordend China . The aqund China .However, aquaculture practices are likely to reduce the genetic diversity and further to cause the loss of disease resistance and environmental adaptability, which greatly limit the potential for selective breeding –9. RecenWe successfully collected wild populations of the large yellow croaker in fisheries survey from 2007 to 2010, which allowed us to conduct genetic studies as described above. In addition, microsatellite markers have been successfully used in genetic monitoring of the changes between hatchery stocks and wild populations –17. HereP = 0.0007; Under exact tests, no consistent deviations from HWE in each sample were detected. After sequential Bonferroni correction, only two locus-population pairs (H37-NB and H54-ZJW) were significant  and in cultured stock DQ (4.307), respectively. In terms of heterozygosities including HO and HE, the highest and the lowest values were also identified in wild population and cultured stock, respectively (R varied from 4.307 (DQ) to 5.037 (NB) with a mean of 4.686 in cultured stocks, which was significantly lower than in wild populations ranging from 5.913 (ZJW) to 7.816 (YCW) with a mean of 6.730 (P < 0.01). Similar to AR, HO and HE values were also significantly lower in the cultured stocks  than in the wild populations . However, FIS values showed no significant differences between the cultured stocks and the wild populations (P > 0.05). The Bottleneck analysis did not detect signals of recent population reduction for each sample whether in test under TPM or in mode-shift test.In terms of genetic variation, one locus, H80, was monomorphic in two cultured stocks, DQ and MY . The othectively . FurtherST was 0.070 with 95% confidence interval ranging from 0.023 to 0.173 and was highly significant (P < 0.001). Pairwise FST analysis showed that the wild populations were significantly divergent from the cultured stocks after Bonferroni correction for multiple comparisons (P < 0.002). Among five cultured stocks, only DQ was significantly different from the others. Interestingly, there was no differentiation detected among the three wild populations , sea bream (Pagrus major) and the orange-spotted grouper (Epinephelus coioides), suggesting these microsatellites are sufficient to detect genetic variation in the large yellow croaker [E per locus were 5.750 and 0.541, respectively, which is much lower than that (20.6 and 0.79 per locus) found in marine fishes [Investigation of genetic variation of cultured and wild populations of domesticated animals can provide valuable information for breeding programs and also for conservation genetics ,18. In t croaker –21. Acroe fishes . This reR, HO and HE. Typically, AR is independent of sample size and is more sensitive to be detected in populations of reduced genetic diversity than heterozygosity, as loss of rare alleles shows little effect on heterozygosity [R but also in terms of heterozygosity measures, which likely demonstrates that the reduction of genetic variation in the cultured stocks is not caused by sampling and genotyping bias.Aquaculture practices have been broadly reported to have the tendency to reduce genetic variability in cultured stocks of fish species ,17,23. Azygosity ,18,25. HThe decline of genetic diversity in cultured stocks is typically considered to be the result of interacting founder effects, random genetic drift, and artificial and natural selection in the cultured environments ,26,27. IST, AMOVA, FCA and Structure analysis consistently supported the significant differentiation between cultured stocks and wild populations , grass carp (Ctenopharyngodon idella) and orange-spotted grouper, which was considered to result from artificial selection, founder effects and genetic drift [With respect to genetic divergence, pairwise Fulations  and 3. Sic drift ,21,29. Iic drift . This reIn total, we detected statistically significantly less genetic diversity in the cultured stocks of the large yellow croaker in contrast to their wild counterparts, which was mainly caused by founder effects, random genetic drift and artificial selection involved in aquaculture practices. In addition, significant genetic divergence between cultured stocks and wild populations was also observed. Possessing a high level of genetic variation, the wild croaker showed great value for ongoing selective breeding programs by providing more genetic variation. At the same time, due to the lack of differentiation, the wild populations can be considered as one unit for conservation. However, ongoing artificial wild stock enhancement by releasing of cultured croaker fingerlings should be conducted with caution, as previous studies have suggested that release of hatchery fingerlings with low genetic variation would likely reduce the genetic diversity of the wild populations and further lead to loss of adaptation to variable environments for the wild populations –33. The Cytb and COI genes. The results revealed that this species was in the process of population expansion after Pleistocene glaciations, which also suggested few signals of genetic bottleneck in the wild croaker populations [Since the 1990s, sample collections of the wild populations of this croaker have rarely been reported. In our study, we collected three samples of the wild croaker in the northern and central East China Sea during fisheries survey for several years, which may suggest that this area is “refugia” of this species. Although the wild stock of this croaker has collapsed, we did not detect the signals of recent bottleneck in the three wild croaker populations using microsatellites. In another study, we analyzed the phylogeography of this croaker using the same wild samples as in this study by sequencing mitochondrial ulations . Howeverulations . CombiniWe analyzed five cultured stocks and three wild populations of the large yellow croaker. Among these five cultured stocks, three  were collected from Ningbo, Zhejiang province, while the other two (XP and SD) were from Ningde, Fujian province. In detail, two (MY and NB) and three stocks  were the third and second generation offspring of the local fish farms, respectively. The broodstocks of these cultured stocks were founded using brooders collected from the central East China Sea. Due to stock collapse in the wild resources of this croaker, however, it is very difficult to capture the wild fish. Fortunately, we collected three wild populations  from the northern and central East China Sea during several fisheries surveys between 2007 and 2010. Judging from the body size, all the wild individuals were adult. Detailed information about sample size and location is shown in Genomic DNA was isolated using the standard phenol-chloroform extraction protocol . All samO), expected heterozygosities (HE) and allele riches (AR) to measure the genetic variation in each sample. These parameters were calculated in FSTAT version 2.9.3.2 [IS) was also estimated using FSTAT version 2.9.3.2 [U test. Recent population size reduction was examined in the form of heterozygote excess using the program Bottleneck version 1.2.02 [et al. [We employed the number of alleles (A), observed heterozygosities . The significance was tested by a permutation with 10,000 replicates using ARLEQUIN 3.5 [6 iterations with a burn-in length of 106. The most likely K value was inferred by investigating mean Ln likelihood values.Genetic differentiation among samples was estimated using pairwise Wright’s F-statistics (FQUIN 3.5  with seqQUIN 3.5 . We alsoQUIN 3.5  to detecQUIN 3.5 . This prIn total, our study demonstrated that the cultured croaker had significantly reduced in genetic diversity in contrast to the wild and also presented statistically significant differentiation from the wild, indicating the current wild stock enhancement should be conducted with caution. With a relatively high level of genetic diversity, the wild populations showed important value for the ongoing breeding programs of this croaker. Simultaneously, we detected no differentiation among the wild populations, suggesting that the wild croaker in the northern and central East China Sea should be considered as one unit for management and conservation.


When studying the genetic structure of human populations, the role of cultural factors may be difficult to ascertain due to a lack of formal models. Linguistic diversity is a typical example of such a situation. Patrilocality, on the other hand, can be integrated into a biological framework, allowing the formulation of explicit working hypotheses. The present study is based on the assumption that patrilocal traditions make the hypervariable region I of the mtDNA a valuable tool for the exploration of migratory dynamics, offering the opportunity to explore the relationships between genetic and linguistic diversity. We studied 85 Niger-Congo-speaking patrilocal populations that cover regions from Senegal to Central African Republic. A total of 4175 individuals were included in the study.By combining a multivariate analysis aimed at investigating the population genetic structure, with a Bayesian approach used to test models and extent of migration, we were able to detect a stepping-stone migration model as the best descriptor of gene flow across the region, with the main discontinuities corresponding to forested areas.Our analyses highlight an aspect of the influence of habitat variation on human genetic diversity that has yet to be understood. Rather than depending simply on geographic linear distances, patterns of female genetic variation vary substantially between savannah and rainforest environments. Our findings may be explained by the effects of recent gene flow constrained by environmental factors, which superimposes on a background shaped by pre-agricultural peopling. Understanding how human populations interact and admix is one of the primary aims of human evolutionary genetics. To date, three main factors have been studied in detail which could be possible determinants of gene flow within and among human groups: geography, language and social structure.Geographical factors have been shown to play an important role in shaping genetic structure, at both inter and intra-continental levels e.g. -7). Alon. Alon7])The relationship between linguistic and genetic diversity has been investigated in numerous studies aimed at understanding how cultural factors may shape gene pools e.g. -10). The. The10])Finally, following the seminal study by Seielstad et al. , there hEven though the vast literature accumulated over twenty years e.g. ,9) has p has p9])In this context, given their high cultural and linguistic diversity and their complex history, African populations probably represent one of the most interesting case studies. Recent studies on large-scale datasets regarding autosomal markers (both STRs and SNPs) support the role of both geography and language in explaining the distribution of genetic variation in Africa ,25. Amonex novo and 80 collected from the literature) of individuals settled in an area spanning from Central to Western Africa. The populations under study inhabit both the savannah and the rainforest regions, and all speak languages belonging to the Niger-Congo phylum [In this work, we investigated the genetic structure and the patterns of gene flow in a broad dataset . Thereao phylum -41. By cIntra-population diversity parameters are shown in Table Pairwise genetic distances were calculated among all populations and the matrix represented in a MDS plot, shown in Figure From a linguistic point of view . Cluster 6 was the less geographically homogeneous, including two populations from Nigeria (Yoruba and Idoma) and the three nomadic groups from north Cameroon  along with Western Africans. On the other hand, the ellipses of dispersion indicated that clusters 3 and 7, even though they account for the highest number of populations, had lower internal variances. This is probably due to the fact that they include the geographical areas with the densest sampling coverage, which results in a higher number of genetically more closely related populations.Most clusters were found to group populations that are geographically close together, with few exceptions  and stronger than in the latter (R2 = 0.73 vs R2 = 0.53) which was non-significant .In addition, the Mantel test showed a low but statistically significant correlation between geographic and genetic distances for the whole sample . When dividing the populations according to their habitat, geographic and genetic distances were highly correlated within the savannah region , while the rainforest area seemed characterized by a weaker but still significant correlation between the parameters . This trend was confirmed when plotting the linear regression for the genetic and geographic distances of the clusters in directions West to East , while the others ranged from a mean value of −14.052 to −21.832 , a linear stepping-stone (B) and an intermediate model  as well as including 5 out of the 7 populations with non-significant Fs values  .As an important evolutionary consideration, we should take into account the possibility that differences in Fu's statistical values between savannah and rainforest could be also explained by the role of selection. However, although the worldwide distribution of mtDNA lineages has been proposed to be driven by selective processes related to temperature changes, the geographic region here analysed appears to be quite homogeneous for this putative temperature effect . In futua priori definition of population units, based on the sampling location and the languages spoken by the individuals. We are aware that such a definition may lead to an approximation in the estimate of the spatial distribution of allele frequencies, since each population is considered as a sampling point. In the present case, we believe that, despite the vast geographical area covered by our dataset, the homogeneous nature of sampling helps overcoming this limitation and is allowing a reliable representation of the distribution of maternal lineages.Another caveat of the present study may be the The complexity of the migratory patterns observed here is further emphasized by a discontinuity detected between clusters 7 and 2, which overlaps with a broad area of the rainforest region  where the sampling coverage is fairly homogeneous. Cultural factors do not seem to offer an explanation for this separation. In fact, the populations composing the two clusters speak languages that are closely related, within the Narrow Bantu family and show no major differences in their subsistence economy. On the other hand, environmental factors could have played a role if one considers that the rainforest habitat may decrease the intensity of gene flow among populations after their initial settlement in deforested areas, making migration more difficult. Another discontinuity in the pattern (between clusters 1 and 6) overlapped with a gap in the sampling coverage of the dataset under study, corresponding to the area of Guinea, the Ivory Cost and Liberia, where tropical rainforest vegetation generally prevails. In the absence of these samples, any further inference on the validity of the observed discontinuity would be very speculative. However, their analysis could contribute to a more exhaustive testing of the influence of different environments on the intensity of migrations among human populations.Considering all the previous observations, we suggest that farming rainforest populations have probably undergone a local, more recent, and less intense demographic expansion than other food producer populations of the Niger-Congo phylum, which has been previously observed in Gabon through the analysis of Y chromosome lineages . EvidencIt is interesting to note the unexpected association observed in cluster 6 where populations of nomadic shepherds from Northern Cameroon  and Guinea Bissau and Senegal (clusters 6 and 5), which present a high linguistic diversity, seem to be characterized by a rather continuous gene flow and show smaller inter-population differences. This contradicts the expectations described above, based on linguistic data, of a recent demic expansion from the area of Nigeria-Cameroon towards Central Southern and Eastern Africa, and an earlier diffusion from Western to Central Africa .As a general conclusion, language does not seem to be the main predictor for the distribution of genetic variation among Niger-Congo-speaking populations. Despite the general belief that language is transmitted by migrating women, genetic analyses have repeatedly shown its preferential correlation with paternal rather than maternal genetic variation ,13,21.Unfortunately, we were unable to find a reliable approach for the definition of linguistic distances. Comprehensive classifications based on a quantitative measure of lexical similarities are only available for the Narrow Bantu languages, and not systematically for other Niger-Congo branches . Since in this dataset only 28/85 populations belong to the Narrow Bantu family, we decided to avoid this approach in order not to introduce interpretation biases due to inaccurate or questionable linguistic classification.Even though the genetic clusters here reported cannot be considered as random mating units, the picture presented in our study suggests that, in particular thanks to female-biased movements, gene flow occurs among human populations speaking very different languages.The analysis of paternal patterns of migration would be useful to shed light on the substructure and the random mating areas among patrilocal populations, while autosomal and X-chromosomal data could be productively investigated to explore whether sex-biased movements are detectable in the distribution of genome variation.In this paper, we present a genetic study on female patterns of migration in populations from Central and Western Africa which share a patrilocal tradition and belong to the same linguistic phylum. Our results show how macro habitats seem to play a major role in determining population genetic structure. Population samples from Guinea, the Ivory Coast and Liberia could allow us to test whether this working hypothesis applies to an even larger area of the continent. However, we highlight here how fundamental the knowledge of cultural factors is when planning a population genetic study. In fact, having reliable information about matrimonial behaviour, even the resolution provided by a relatively small region of mtDNA, proved useful in inferring complex patterns of migration and isolation.http://www.ethnologue.com/), while a tree representing structure within Niger-Congo and relations among languages spoken in the populations analysed is presented in Additional file Our dataset contains 4175 individuals from 85 Niger-Congo speaking populations from Western-Central sub-Saharan Africa  , with mi89 , withIntra-population diversity parameters, Fu's neutrality test, pairwise genetic distances, AMOVA and Mantel test statistics were calculated using Arlequin 3.5 software . The disGenetic structure was inferred through the Discriminant Analysis of Principal Components . To anak-means approach, which relies on classical ANOVA. This method maximizes the variance among groups and minimizes the variance within groups. The Bayesian Information Criterion (BIC) was used to detect the best number of groups comparing the decrease of the residual variance among different numbers of clusters, with the best number corresponding to the minimum BIC value [The first step of the structure analysis consisted in assigning populations or individuals to clusters through the IC value .k-means in order to investigate their separation which is summarized by the discriminant components [discriminant functions, thus maximize the ratio of the variance among groups and the variance within groups. Group positions, defined by the discriminant functions, are presented in a scatterplot. The residual of the probability of population assignment to true clusters versus randomly permuted clusters (a.score) was calculated to test the goodness-of-fit of the discriminant analysis [DAPC was performed on the clusters inferred with the mponents . This ananalysis ,69,70.A simple linear regression analysis was performed to evaluate the correlation between genetic and geographic distances among the clusters using the geographic coordinates of their centroids  and mean(long) of the populations in the cluster). This was then plotted for both East to West and West to East directions .Mega 5.05 software was used to calculate the alpha value of the gamma distribution for the mutation rate of the whole dataset and to obtain trees of Minimum Evolution for the sequences included in each cluster .Once the unbiased structure of the populations under study was determined, the migration pattern among the clusters identified was tested through a Bayesian approach, which is implemented in migrate-n software version 3.2.9 ,41. The In order to reduce the prohibitive computational time, migration estimates were carried out on a proportional sub-sampling of each cluster. A random sub-set accounting for 30% of each cluster, for a total of 1024 individuals, was pooled five times. Considering the high amount of samples included in cluster 3 and the fact that they belong to a very small geographical area, which is overrepresented in comparison to the rest of the region, the cluster 3 sampling was reduced to 15% in order to obtain a comparable sample size for all clusters. Each model was then run 3 times for each different sub-dataset for a total of 45 runs. Log Bayes Factors were calculated as follows for a total of 45 crossed comparisons among pairs of models:Log BayesFactor = 2ln (Prob(D|Model1)-Prob(D|Model2)ST values among each cluster and its subsets  Map of geographical positions of the 85 populations analysed in the present study: central (green), central-west (red), west (blue). b) Phylogenetic relationships among the languages spoken by the 85 populations analysed in the present study graphically reproduced according to ethnologue.com.Click here for filea) Curve of BIC decreasing in relation to number of clusters considered. The minimum BIC value corresponds to number of clusters = 7. b) Assignation of the populations to the clusters. The intensity of the colour is proportional to probability of assignation.Click here for fileSummary statistics for the 45 sub-datasets compared with the original sample belonging to each of the 7 clusters individuated. N is the number of individuals, K the number of haplotypes, k/N is ratio between the two previous values, S is the number of segregating sites and %S the percentual retain of variability in comparison with the original sample. HD is the haplotype diversity, MNPD the mean number of pairwise differences.Click here for fileMinimum Evolution tree topologies for the 7 DAPC clusters . The tree for cluster 3 was divided across 9 pages for a better graphical visualization.Click here for filePlot of the linear regression between genetic vs geographic distances based on the clusters' centroids. a) The linear distances are calculated starting from cluster 5 in direction West to East. b) The linear distances are calculated starting from cluster 2 in direction East to West.Click here for filePosterior distributions of the parameters estimated with migrate-n (θ and M) for one of the 15 runs.Click here for fileHaplotypes found in the populations typed in the present study.Click here for fileFrequencies of the main haplogroups in the populations analysed.Click here for file