This study aims to establish the likely origin of EEJ (Eastern European Jews) by genetic distance analysis of autosomal markers and haplogroups on the X and Y chromosomes and mtDNA.According to the autosomal polymorphisms the investigated Jewish populations do not share a common origin, and EEJ are closer to Italians in particular and to Europeans in general than to the other Jewish populations. The similarity of EEJ to Italians and Europeans is also supported by the X chromosomal haplogroups. In contrast according to the Y-chromosomal haplogroups EEJ are closest to the non-Jewish populations of the Eastern Mediterranean. MtDNA shows a mixed pattern, but overall EEJ are more distant from most populations and hold a marginal rather than a central position. The autosomal genetic distance matrix has a very high correlation (0.789) with geography, whereas the X-chromosomal, Y-chromosomal and mtDNA matrices have a lower correlation .The close genetic resemblance to Italians accords with the historical presumption that Ashkenazi Jews started their migrations across Europe in Italy and with historical evidence that conversion to Judaism was common in ancient Rome. The reasons for the discrepancy between the biparental markers and the uniparental markers are discussed.This article was reviewed by Damian Labuda (nominated by Jerzy Jurka), Kateryna Makova and Qasim Ayub (nominated by Dan Graur). The genetic affinities of the Jewish populations have been studied since the early days of genetics, yet the origin of these populations is still obscure. Some of the studies, trying to establish the origins of the Jewish populations with autosomal markers, claimed that the Jewish populations have a common origin, but others concluded that the Jews are a very diverse group. This corpus of studies has already been critically reviewed .The origin of Eastern European Jews, (EEJ) by far the largest and most important Ashkenazi population, and their affinities to other Jewish and European populations are still not resolved. Studies that compared them by genetic distance analysis of autosomal markers to European Mediterranean populations revealed that they are closer to Europeans than to other Jewish populations -3.EEJ are the largest and most investigated Jewish community, yet their history as Franco-German Jewry is known to us only since their appearance in the 9th century, and their subsequent migration a few hundred years later to Eastern Europe ,5. WhereThe aim of this study is to establish the likely origin of this major Jewish population by using a larger dataset of autosomal markers, and compare the results to analyses based on the available data for the X and Y chromosomes and for mtDNA.Six Jewish populations: EEJ, Moroccan Jews, Iraqi Jews. Iranian Jews, Yemenite Jews and Ethiopian Jews, which have been studied for all the autosomal markers used in this study, are included in the analysis. EEJ are defined on the basis of history as those Jews originating from the areas of the Polish-Lithuanian Kingdom and their descendants in bordering regions, encompassing the territories of Russia, Poland, the Baltic States, Belarus, Moldavia, Moldova (the north-eastern part of Romania) and the Ukraine. The Data on the non-autosomal markers were also available for other Jewish populations: Bulgarian Jews , Turkish Jews , Tunisian Jews (mtDNA), Libyan Jews  and Djerban Jews (Y).The seventeen autosomal markers are: AK, ADA, PGM1, PGD, ACP, ESD, GPT, HP, GC, J311 MspI & MetH TaqI (both on chromosome 7 near the CF locus), FV G1691A, FII G20210A, MTHFR C677T, CBS 844ins68, ACE ID and PAH XmnI. All the markers are unique-event-polymorphisms, and apart from two insertions  are all SNPs. The first nine markers are polymorphisms of red cell enzymes and serum proteins, and were typed mostly by protein electrophoresis, but the variation at the protein level is directly related in a 1:1 manner to the SNP variation at the DNA level. Indeed, some of the results for the Jewish populations were obtained by PCR methods . The polGower (cited in ) recommeThe autosomal genetic distances table  do not sX-chromosomal haplogroups demonstrate the same relatedness of EEJ to Italians and other Europeans table . In contCorrelations between genetic distance and geography and between genetic distance matrices based on different markers (excluding the non-Caucasoid populations Ethiopians and Ethiopian Jews) are shown in table The autosomal genetic distance analysis presented here clearly demonstrates that the investigated Jewish populations do not share a common origin. The resemblance of EEJ to Italians and other European populations portrays them as an autochthonous European population. A study conducted in a New York college in the 1920s point to the same Ashkenazi - Italian similarity on basis of physical characteristics. Freshmen were asked before they knew one another to indicate the origin of their fellow students. Forty percent of the Italians were taken to be Ashkenazi Jews, and the same percentage of Ashkenazi Jews was adjudged Italians . EEJ seeThe high correlation between the autosomal genetic distances and geography and the reduced correlation when EEJ are taken to originate from the Land of Israel reinforce the European origin of EEJ. In fact the correlation of the autosomal markers with geography is higher than previously described for 49 classical markers (0.503) or ~300,000 autosomal SNPs (0.661) in Europe . If for At first sight it seems that there is more than one explanation for the differing results produced by the analysis of the NRY haplogroups. It thus seems possible that EEJ founder population in Rome was composed of exiled Israelite males and local Roman females. In its simple form this clearly contradicts the facts, because both the autosomal and X-chromosomal polymorphisms demonstrate that EEJ do not occupy an intermediate position between European and Middle Eastern populations, but rather a strict European one. From table nd-3rd centuries AD [th century. In the 14th century the estimates for Baghdad alone range from 70,000 to hundreds thousands [th century because of the partitions of the Polish-Lithuanian Kingdom and the immigrations of Jews to Central and Western Europe and America, the estimation of the number of EEJ becomes more difficult, but there is no doubt that the increase in numbers was impressive, as the number of EEJ under Russian rule alone was 5,200,000 in 1897 [The inference that the NRY points to a Middle Eastern origin of EEJ is erroneous not only because the Y chromosomal analysis contradicts the analyses based on the other chromosomes, and because the NRY is a single uniparental marker that does not represent the whole history of the population, but also because its smaller effective population size makes it much more vulnerable to severe genetic drift caused by demographic bottlenecks. The demographic histories of three Jewish populations exemplify how different demographic patterns make the uniparental markers more reliable for Iraqi (Babylonian) Jews and Yemenite Jews and less reliable for EEJ. Both Yemenite Jews and Iraqi Jews resemble populations from their regions of origin according to autosomal markers ,3,30-32.uries AD . Gilberturies AD  estimateuries AD  it was ihousands . By 1939housands . In conthousands . In 1500housands . In the  in 1897 .The existence of severe demographic bottlenecks in the history of EEJ has also been suggested by genetic studies of disease-causing-mutations and mtDNA -46. The The data on the Y chromosome itself also support the unreliability of the uniparental markers for discovering the origin of EEJ. Nebel et al.  studied Some previous studies based on classical autosomal markers concluded that EEJ are a Middle Eastern population with genetic affinities to other Jewish populations. The problems with these studies have been previously discussed in detail . These sRecently, Cochran et al.  used 251th century) component in their gene pool. It is not possible at this stage to say what is the source of this resemblance, since we don't know what is the origin of Sephardic Jews, but considering all the genetic affinities of both groups it likely stems from Sephardic Jews being the descendants of converts in the Mediterranean basin rather than from a common Jewish origin in the Land of Israel. When one compares the autosomal distances of EEJ (current study) or Ashkenazi Jews  from thThe studies of Atzmon et al.  and BehaEEJ are Europeans probably of Roman descent who converted to Judaism at times, when Judaism was the first monotheistic religion that spread in the ancient world. Any other theory about their origin is not supported by the genetic data. Future studies will have to address their genetic affinities to various Italian populations and examine the possibility of other components both European and Non-European in their gene pool.The author declares that he has no competing interests.Damian Labuda, Pediatrics Department, Montreal University Sainte-Justine Hospital Research Center, Montreal, PQ Canada .The author compiled and reanalyzed the data on autosomal and sex chromosomes polymorphisms collected by different laboratories on different Jewish and West-Eurasiatic populations. His analysis indicates much greater European component of Eastern European Jews, EEJ  than of other Jewish groups. Moreover the analysis points to Italians as the closest population to EEJ.The question is how to interpret this evidence. Imperial Rome was a very cosmopolitan city culturally and genetically diverse. To what extent a sample of contemporary Italians preserves the genetic link to its population? It can simply reflect a mixture of historical influences from different centers around the Mediterranean Sea. We should thus keep in mind that the Italian connection may simply indicate Southern European and Mediterranean links with the latter including Middle Eastern roots.Interestingly, this analysis that is based on a limited number of markers provided results that are very similar to a paper of Atzmon and colleagues, published five days ago in the American Journal of Human Genetics, and based on the microarray-based genotyping genome of wide distributed markers. I would like the author to comment on this paper in the context of his findings and his thoughts and reflections on the origin of Jewish Diasporas. Should we go back to the single locus analyses, as in the case of uniparentally transmitted markers, but targeting one by one different individual segments of the nuclear genome? Perhaps, in this way we could partition and identify genetic ancestries of different populations, which due to their history of relative isolation, are considered as genetically homogenous.The author refers to Sangvi's G2 as the most appropriate distance metrics. Could you make it more clear when this metric was used and when that of Reynolds (only to produce a tree?).The historical sources listed above show that conversion to Judaism was common in ancient Rome among all ranks of the Roman society including the imperial families. It is thus unlikely that the original Roman population did not constitute a significant portion of the proselytes. What else can explain the resemblance of EEJ to a general sample of Italians in this study and to more local samples in the two array studies ,54? In aMy comments on the papers by Atzmon et al.  and BehaKateryna Makova, Department of Biology, Penn State University, Pennsylvania USA.This is an interesting manuscript that presents intriguing results. I have only a few comments:1. The introduction is very short, while the discussion is lengthy. I suggest moving parts of the Discussion to the Introduction.2. Some of the statements in the Discussion are too strong. I disagree with statements about "erroneous Y chromosomal genetic distances", "both uniparental markers should not be used to trace their origin", "uniparental markers being unreliable". The author should modify them.I moved the paragraph on the history of EEJ to the Introduction. The current revised version of the paper includes a new comparison based on mtDNA. I maintain that it adds more weight to my assertion that the uniparental markers should not be used to trace the origin of EEJ. In no way did I mean that the uniparental markers are always unreliable; to clarify it I modified the relevant sentence in the discussion. Indeed from the demographic examples that I give in the Discussion, it seems that the uniparental markers can be used to study the origins of Iraqi Jews and Yemenite Jews.Qasim Ayub, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK .The paper by Zoossmann-Diskin entitled 'The origin of Eastern European Jews revealed by autosomal and sex chromosomal polymorphisms' explores autosomal and sex chromosomal polymorphisms in six Jewish populations using previously published and additional unpublished data. The author concludes that the Jewish populations examined do not share a common origin and that Eastern European Jews are closer to the Italian population.My major concern is the choice of markers and populations used in this study. The author has analyzed 17 autosomal loci, including 9 polymorphic protein electrophoretic variants in which the genotype was assumed. Although phenotypes often do correlate with genotypes assuming that they do can lead to erroneous results. Of the remaining 8 it is unclear whether the same samples were genotyped as the sample numbers for each locus vary widely (Supplementary Tables 2-4).The author also uses Y hapologroup frequencies and shows a multidimensional scaling plot of Y chromosomal genetic distance matrix. However, the supplementary data  using a much larger dataset clearly demonstrate a common genetic thread linking the diverse Mizrahi, Sephardic and Ashkenazi Jewish populations with the populations from the Levant and Middle East. The Ashkenazi show a European component but this is shared with many Eastern and Southern Europeans populations. These studies contradict the author's conclusion and demonstrate the power of using unbiased markers and host populations in corresponding geographic regions to address issues such as genetic relatedness among Jewish and non-Jewish populationsI am not sure what Dr Ayub means by "assumed", but I suspect that he means something like the relationships between phenotype and genotype in certain blood groups, in which one (or more) allele is dominant over the other and the gene frequencies of the alleles have to be inferred from the phenotypes assuming Hardy-Weinberg equilibrium. In such cases there may indeed be errors in the gene frequencies. Protein electrophoretic markers are completely different. Nothing is inferred! As mentioned in Methods all the protein electrophoretic markers in this study represent a SNP at the DNA level. This SNP causes an amino acid change that can be detected at the protein level. Both alleles are directly viewed on the gel in the same way as both alleles of an RFLP are directly viewed on the gel. Gene frequencies are determined in both cases by simple gene counting and the error rate in protein electrophoresis is no greater than in DNA studies. There is no need to type the same samples for all the polymorphisms, because the unit of study is the population, not the individual. One can use polymorphisms typed by different researchers using different samples and combine them to create a genetic profile of each population. Typing all the polymorphisms on the same sample does not add more credibility to the study. Indeed the renowned works that employed classical autosomal markers to portray the genetic affinities of human populations were based on many different samples typed by many different researchers ,57.The nomenclature in the Y chromosome supplementary table has been updated. Following the publication of the study by Behar et al.  it was pAs written in the Discussion the genetic distance matrices of Atzmon et al.  and BehaAllele frequencies tables, Tables S1-S7. The file contains seven tables that give the allele frequencies of the employed polymorphisms.Click here for file


Podarcis gaigeae, to demonstrate that this species is polymorphic with respect to throat color, and the morphs form discrete phenotypic clusters with limited overlap between categories. We use divergence in throat color morph frequencies and compare that to neutral genetic variation to infer the evolutionary processes acting on islet- and mainland populations.Patterns of spatial variation in discrete phenotypic traits can be used to draw inferences about the adaptive significance of traits and evolutionary processes, especially when compared to patterns of neutral genetic variation. Population divergence in adaptive traits such as color morphs can be influenced by both local ecology and stochastic factors such as genetic drift or founder events. Here, we use quantitative color measurements of males and females of Skyros wall lizard, e:s < 100). Population divergence (FST) for throat color morph frequencies fell within the neutral FST-distribution estimated from microsatellite markers, and genetic drift could thus not be rejected as an explanation for the pattern. Moreover, for both comparisons among mainland-mainland population pairs and between mainland-islet population pairs, morph frequency divergence was significantly correlated with neutral divergence, further pointing to some role for genetic drift in divergence also at the phenotypic level of throat color morphs.Geographically close islet- and mainland populations of the Skyros wall lizard exhibit strong divergence in throat color morph frequencies. Population variation in throat color morph frequencies between islets was higher than that between mainland populations, and the effective population sizes on the islets were small (NGenetic drift could not be rejected as an explanation for the pattern of population divergence in morph frequencies. In spite of an expected stabilising selection, throat color frequencies diverged in the islet populations. These results suggest that there is an interaction between selection and genetic drift causing divergence even at a phenotypic level in these small, subdivided populations. The role of genetic drift in population divergence and speciation has been a much debated topic in evolutionary biology -5. Earlye), and from such data, the expected probability of fixation of neutral and selected alleles can be calculated. The strength of genetic drift is inversely proportional to Ne and hence drift is a more important force in small populations [e is small, the potential for genetic drift would increase, both at the phenotypic and at the molecular levels. It is important to emphasize that stochastic factors such as drift, historical contingencies and the initial genetic makeup of populations, could interact with deterministic factors like selection [solely influence population divergence of phenotypic traits, but rather if, and to what extent, stochastic factors might interact with deterministic factors such as selection during evolutionary diversification [It is an empirical challenge to argue for a role for genetic drift at the level of phenotypes, since most biologists, for good reasons, believe that phenotypic evolution is mainly influenced by the deterministic forces of natural or sexual selection ,14. One ulations . If it celection -20. Thusfication .Uta stansburiana) where throat color segregates as a single locus (Mendelian) character [Ctenophorus pictus) where a similar simple inheritance pattern might operate [Anolis sagrei), a two-locus inheritance system with epistatic interactions seems to be involved in the determination of back pattern morphs [Discrete and visible color polymorphisms are excellent phenotypic model systems for the study of evolutionary processes in natural settings -24. Pastharacter -32 and t operate . In the n morphs .Lacerta vivipara [Although a genetic background for the throat color polymorphism in lizards is often assumed -34, no cvivipara  is a convivipara  argues tvivipara , can notvivipara  indicateMechanisms that might contribute to maintain color polymorphisms in populations include negative frequency-dependent male-male competition ,38, negaPolymorphic taxa can also shed light on speciation processes; such morphs can serve as starting material for the formation of new species, a process that might be caused by social selection in sympatry ,47. Two Podarcis-clade, color polymorphisms are widespread and multiple throat color morphs coexist within populations [Podarcis gaigeae) where we study the frequency distribution of throat color morphs in different populations to infer the nature of the selective processes that are acting on the throat color morphs in mainland- and islet populations of this species. This lizard occurs on the island of Skyros in Greece and also on many of the small islets located along the coast of Skyros [In many lizard species, including the European ulations ,48,49 . Predation rates on islands are often reduced [The Skyros wall lizard hipelago  with thef Piperi . For comn period ,53. Toda reduced  which is reduced . The isl reduced .P. g. gaigeae was caught at four different "mainland" localities on the island of Skyros and on three islets close to the coast of Skyros. Lizards of the subspecies P. g. weigandi were caught on the island of Piperi , yellow-orange (YO), orange-white (OW), yellow (Y), yellow-white (YW) or white (W)). All these six phenotypes exhibited either one or a combination of any two of the three colors  present in the system, from inspection of the photographic images. No instances of all three colors on the throat of the same lizard were found. To evaluate the reliability of this discrete visual classification system, another observer (E. I. Svensson) independently re-classified a total of 40 lizards that had previously been classified by A. Runemark. In all but two cases (38 out of 40), the independent classifications were identical. This suggests that these color morphs are easily distinguishable with little ambiguity between different observers, possibly reflecting the discrete nature of these morphs.P. g. gaigeae and 31 P. g. weigandi, were photographed and visually classified to different color morphs from the images. Due to the variance in intensity, which was related to the imaged scenario geometry (flash distances and surface orientation) rather than the intrinsic optical properties of the lizard, the analysis was pursued with only the relative unit less quantities . The intensity of the throat images was removed by a transformation of the colorspace from Cartesian to spherical which decreases dimensionality through removing the intensity dimension. Since the aim of the study was to quantify the red green blue (RGB) composition rather than the intensity of the throat colors the subsequent analysis was facilitated by the discarding of the intensity. To preserve the discrete patchy colors of the throat, the chromaticities of individual pixels were summarized in 2 D histogram planes for each sample [® (MathWorks™) was used for all image analyses.To further quantify the discreteness of these color morphs, we analyzed the throat area from the images of the individual lizards and used the data in a more quantitative analysis. A total of 351 lizard throats, 320 h sample . The hish sample , and the2-tests to investigate if the frequencies of the throat color morphs differed among populations and between the habitat groups ("mainland" vs. "islets"). Our starting assumption was a simple (Mendelian) inheritance system: we thus assume that the throat color morph is governed by a single locus with three color alleles, codominant inheritance and hence six visible phenotypes. Based on this assumption, we also tested if the populations deviated from Hardy-Weinberg equilibrium with respect to the throat color morph frequencies, using χ2-tests.We subsequently used a discriminant function analysis (DFA) to assess if the visually classified color morphs could be distinguished from each other using the principal component data that we extracted from the images. The software STATISTICA  was usedAlthough we have no experimental data (e.g. breeding experiments) on the possible inheritance of throat color in this system, our assumption of a simple genetic basis of color is well-supported by several recent previous studies on color polymorphisms in both lizards and many other animal species (see references in Introduction). We do certainly not claim that there are no environmental effects on color and no role for phenotypic plasticity in these morphs. However, the discrete nature of these color morphs in combination with previous studies on animal coloration makes it highly likely that there is some genetic influence on coloration, perhaps through some major effect gene. Previous studies on color polymorphisms, including studies on birds, fish, lizards and many insect species indicate that variation in color is often heritable, and it is certainly not unusual that color polymorphisms are governed by simple systems of inheritance, e.g. single locus systems with several alternate alleles  between populations was estimated with FSTAT [ST for throat color morph was also calculated in FSTAT; the three colors were coded as alternative alleles at one locus. This analysis thus assumes that throat color is a heritable trait with Mendelian inheritance and codominant expression. We assumed that single color and combinations of two colors correspond to homo- and heterozygous stages, respectively. The program Structure version 2.2 [K ranging from 1 to 12 using a burn-in length of 50,000 and a run length of 100,000 iterations. The likelihood for the data given each of K clusters was recorded, but since the main aim of the analysis was to illustrate the degree of genetic differentiation between populations rather than to find the number of genetic clusters for which the likelihood is highest, the additional method [K was not applied. Effective population size Ne was estimated with LDNE [ST as dependent variable and neutral FST and category  as main effects and used a design including all the two-way combinations as well as the three-way combination, to test both for dependence on the two main effects and interactions between them. Since each population is involved in more than one comparison the data points are not independent, so we used a resampling approach [ST-values calculated from the color morphs ("morph divergence") and the FST-values based on the 18 microsatellite loci .We obtained genetic data from 289 individuals from the eight study populations  and neutral population divergence (FST), should utilize the whole neutral FST-distribution to see if the adaptive traits fall within this distribution, before the null hypothesis of genetic drift can be safely rejected [ST-distribution and its relationship to our adaptive trait (color morph divergence). We used a paired t-test to investigate if FST for throat color morph differed from the FST for the microsatellite loci. This procedure assumes that the pair-wise population comparisons are statistically independent, which is questionable, since each population is involved in several different comparisons. We therefore confirmed the results from the paired t-test using the re-sampling procedure described above [P. g. weigandi was not included in this analysis since the habitat of Piperi is expected to differ from that of Skyros and its' surrounding islets and thus not reflect an islet- or mainland habitat effect.Selection can be inferred from the genetic signature of different molecular markers; markers that have been under directional selection are expected to be more diverged than neutral markers and markers that have been subjected to stabilizing selection should be less diverged ,45. Recerejected ,44. We fed above , but ourF = 31.81, df = 7, 318, P < 0.001) , with the only exception for the yellow-orange morph that did not differ from the orange (P = 0.11). The results from these analyses, showing variation from the first two canonical roots is depicted in Fig. A discriminant function analysis based on the seven first principle components extracted from the throat images revealed that the six visually classified throat color morph groups were highly statistically different from each other (P < 0.001). The frequencies also differed significantly between the mainland group and islet group within the subspecies P. g. gaigeae  , only O and W were found (N = 31). We found a significant positive relationship  between the proportion of females in the populations with partly or fully orange throats  and the proportion of males with partly or fully orange throats  Fig. . All maieae Fig. . On the ats Fig. . This suats Fig. .ST-values between populations) ranged from 0.0057 to 0.52 when P. g. weigandi was included and between 0.0057 and 0.36 within P. g. gaigeae only (Table P < 0.001). FST-values estimated from the color morph frequency data ranged from 0.0025 to 0.45 when all populations and P. g. weigandi were included, and between 0.0025 and 0.24 within P. g. gaigeae only  that we could find support for in our data set . At K = 2, two of the island populations form a separate cluster and at K = 3 the P. g weigandi population form a third cluster. The next two clusters to form are the remaining island populations, and at K = 6 the southern mainland population separates from the three northern mainland populations  based on all allele frequencies > 0.01 varied between 39.4 and 97.7 for the islet populations of P. g. gaigeae and between 315.3 and 375.0 for the mainland (Skyros) populations .Estimates of effective population sizes , P = 0.04). There was no such trend when the islet populations of P. g. gaigeae, or when P. g. weigandi and the islet populations, were included in the analysis, however . When we separated these comparisons into three different geographic categories , we found a significant positive relationship within two of these categories , indicating that the correlation between color morph divergence and neutral divergence differed between the geographic settings . Hence these data do not allow us to firmly exclude that population divergence in color morph frequencies have been affected solely by genetic drift. This conclusion remains the same if the categories are analyzed separately . Islet populations had a significantly higher proportion of orange throat color alleles than mainland populations , although no orange throat color alleles were found on one of the three islets.Mean Foci Fig. . No diffWhat is the role of genetic drift in phenotypic evolution, if any? Even if most evolutionary biologists have strong reasons to believe that genetic drift is a weak force in phenotypic evolution, in comparison to selection ,70, geneP. gaigeae and the natural geographical replicates , as well as the variation in throat color throughout the Balkan clade of Podarcis where the sister species P. milensis does not exhibit throat color morphs whereas for example P. melisellensis has retained it, make this an excellent study system to address how selective processes operate in subdivided islet populations.Comparisons of phenotypes from island and mainland populations played a central role in Ernst Mayr's theory of founder effect speciation ,74 and cP. gaigeae can not be excluded. If throat color morph would be entirely plastic with no heritable basis, the significant correlations between neutral genetic variation and the throat color morph frequencies for mainland-mainland and islet-mainland population pairs . In such small populations, stochastic frequency fluctuations in selected alleles might under some periods temporarily override the effects of selection [ST-values of traits subjected to directional selection are expected to differ significantly from the FST-values for neutral genetic variation [ST for throat color morph frequency fell in the lower range of the distribution of the FST-values for neutral genetic variation  should, however, increase our statistical power to detect stabilizing selection in this system that might have favored a certain stable morph frequency equilibrium in all populations. Third, we found a significant positive correlation between the population differentiation in throat color and neutral genetic divergence  are more depauperate on the islets, and the seabird colonies on the islets might alter the nutrient cycles considerably, which in turn might affect the quantity and quality of the food sources of the lizards . It is pP. g. weigandi from P. g. gaigeae, the orange morph increased in frequency on Piperi , genetic drift could potentially indirectly also change the optimal strategies for the remaining morphs since the selective environment will change if one morph and its strategy is removed. For instance, populations of election . Thus, telection ,88,89 anP. gaigeae differs among populations, with more pronounced frequency differences among islet populations. This might indicate a role for local genetic drift on color morph frequency divergence, although it is likely that these color morphs are maintained by long-term balancing selection, given the trans-species nature of these polymorphisms. Comparisons of neutral and morph frequency divergence did not reveal any evidence for either directional or stabilizing selection, which might partly be explained by the low statistical power and weak selection, rather than color morphs being entirely neutral. We therefore suggest that an interaction between selection and genetic drift explains the patterns of divergence in throat color morphs. In isolated populations with small effective population size, genetic drift is particularly likely to operate and to interact with selection which might cause divergence even in phenotypic characters.The color morph frequency in AR participated in the design of the study, carried out the field work, the color measurements and spectral analysis, the genetic analysis and drafted the manuscript. BH planned the genetic labwork and helped out with the design and conduction of the genetic analysis. PP and EDV helped out with the field work. EIS conceived of the study, and participated in its design and and helped to draft the manuscript. All authors read and approved the final manuscript.Number of photographed males and females per population. Contains breakdown sample sizes for number of individuals per population and sex for which throat color morph was determined.Click here for fileTests for deviation from Hardy-Weinberg equilibrium per population. Contains tests for deviations from Hardy-Weinberg equilibrium for all populations and the breakdown sample sizes for number of genotyped individuals per population.Click here for file


NAT2) gene plays a crucial role in the metabolism of many drugs and xenobiotics. As it represents a likely target of population-specific selection pressures, we fully sequenced the NAT2 coding region in 97 Mandenka individuals from Senegal, and compared these sequences to extant data on other African populations. The Mandenka data were further included in a worldwide dataset composed of 41 published population samples  from four continental regions that were adequately genotyped for all common NAT2 variants so as to provide further insights into the worldwide haplotype diversity and population structure at NAT2.The N-acetyltransferase 2 , defining 16 haplotypes. High diversity and no molecular signal of departure from neutrality were observed in this West African sample. On the basis of the worldwide genotyping survey dataset, we found a strong genetic structure differentiating East Asians from both Europeans and sub-Saharan Africans. This pattern could result from region- or population-specific selective pressures acting at this locus, as further suggested in the HapMap data by extremely high values of FST for a few SNPs positions in the NAT2 coding exon  in comparison to the empirical distribution of FST values accross the whole 400-kb region of the NAT gene family.The sequencing analysis of the NAT2 are consistent with selective neutrality in all sub-Saharan African populations investigated, whereas the high level of population differentiation between Europeans and East Asians inferred from SNPs could suggest population-specific selective pressures acting at this locus, probably caused by differences in diet or exposure to other environmental signals.Patterns of sequence variation at  NAT2) gene plays a crucial role in the metabolism of xenobiotics, including many clinically useful drugs and exogenous chemicals present in the diet, cigarette smoke and the environment [NAT2 gives rise to a wide interindividual variation in N-acetylation capacity. In particular, a clear bimodal distribution is observed that segregates the rapid acetylator phenotype, associated with a normal acetylation capacity, from the slow acetylator one, characterized by a reduced enzyme activity. These two main metabolic phenotypes occur with varying prevalence in populations of different ethnic origin [The N-acetyltransferase 2 (ironment . ExtensiNAT2 have led to conflicting results among (and even within) human populations and most association findings have been difficult to replicate. One reason for these inconsistencies may relate to the fact that almost all studies focused on a limited number of candidate polymorphisms, which were not necessarily the same from one study to another [NAT2, this approach should resolve much of the controversial issues of candidate-polymorphism studies.The clinical consequences of the acetylation polymorphism can be severe if standard drug doses are applied, exposing patients to an increased risk of adverse drug reactions or a lack of therapeutic efficacy . In addi another ,6. A shi another . By captNAT2 genetic variants all over the world [NAT2 allele detection, omitting analysis of several polymorphic positions . They focused on a limited number of "indicator" mutations, thought to be tightly linked with other mutations and predictive of acetylator status, and have based allele designation on this. Such incomplete genotyping methods wrongly type different alleles as the same and may lead to misclassification of genotypes and deduced phenotypes [NAT2 allele distribution in worldwide populations.There is now a large body of information on the distribution of he world . HoweverNAT2, we performed an extensive survey of the literature to identify those samples that were adequately genotyped for all common variants in NAT2. In total, 41 population samples  from four continental regions  were selected and jointly analyzed. In addition, we performed full sequencing analysis of the NAT2 coding region in a large and ethnically well defined Mandenka sample from Eastern Senegal so as to further characterize African diversity at this locus and to detect novel variants not yet reported. Beyond a simple description of NAT2 gene diversity, the goal of the present study was to provide further insights into the evolutionary forces that most likely shaped NAT2 genetic variation in humans. In particular, three levels of diversity  were used to investigate to what extent present NAT2 variation patterns solely reflect stochastic events of human evolution, or are distorted by natural selection.In an attempt to better characterize the worldwide haplotype diversity and LD structure of NAT2 in the Mandenka sample are reported in Table NAT2 coding exon. Two of them were singletons confirmed by resequencing. Apart from the seven SNPs that are commonly found in human populations, we found three additional variants that have been recently reported by Patin et al. [NAT2*12g and NAT2*12H) contain a nonsynonymous mutation (G609T or C403G) whose impact on phenotype is unknown. From diplotype configurations at NAT2 in the Mandenka, we inferred 48.5%, 39.2%, and 7.2% of slow, intermediate, and rapid acetylators, respectively. The remaining 5.1% individuals had an unknown acetylator status as they were carriers of either a NAT2*12g or a NAT2*12H haplotype.Results of the sequencing analysis of n et al.  in severn et al.  and fourNAT2 coding region (870 bp) in the Mandenka and the 12 African samples of Patin et al. [π and θw, for the 13 samples are 0.268% and 0.221%, respectively. None of the tests of selective neutrality, performed on each sample both at the intrapopulation and at the interspecies levels, yielded significant results (not shown). This suggests that patterns of diversity at NAT2 are consistent with the hypothesis of selective neutrality and constant population size. The average sequence divergence between human and chimpanzee for NAT2 coding exon was of 1.6%, and the average substitution rate of 1.6 × 10-9 per nucleotide and year. All samples provided similar estimates both for Ne, the current effective population size, and TMRCA, the coalescence time back to the most recent common ancestor: average values were 14,218 individuals and 1.077 My, respectively, for the NAT2 coding exon. The age of mutations in the gene genealogy ranged from 96,200 (G857A) to 496,300 (A803G) years for the seven SNPs that commonly occur in human populations. The other polymorphisms, only reported to date in sub-Saharan Africans, had all estimated ages < 50,000 years.Summary statistics of genetic variation at the n et al.  are repoNAT2 gene. All SNPs and populations were in Hardy-Weinberg equilibrium after Bonferroni correction for multiple testing. The seven SNPs defined 21 distinct haplotypes, whose composition in terms of SNP variants is given in Table A total of 6,727 individuals from 41 worldwide samples were analyzed for their genotype at the seven common SNPs of the NAT2 in the 41 sampled populations is shown in Figure NAT2 haplotypes is displayed in Figure NAT2*5B haplotype. This variant occurs at a high frequency in Europe but is rare in populations of Asian origin. In sub-Saharan Africa, a poorer correlation between 803G and the two other variants is observed. This can be explained by the higher frequency of NAT2*12A in Africa, in which the 803G variant occurs in isolation. The 191A variant (which defines the NAT2*14 cluster) is exclusively present in sub-Saharan Africa, and the 857A variant (which defines the NAT2*7 cluster) is mainly found in Asia and Central America. By contrast, the 590A variant occurs at roughly similar frequencies (~15–40%) in all worldwide samples, except in Amerindians where it is rare . The frequency of the 282T variant only slightly varies among human populations.Allele frequency variation of the seven common SNPs of NAT2 haplotypes are observed. A larger number of haplotypes of similar frequencies indeed occurs in these populations, generating a huge number of distinct genotypes. By contrast, in populations of Asian origin, only a few major haplotypes are found, namely NAT2*4, NAT2*6A and NAT2*7B. The mean haplotype diversity was estimated to be 0.80 ± 0.05 in Africans and 0.56 ± 0.09 in East Asians. Europeans displayed an intermediate value of 0.70 ± 0.04 [see Additional file It is clearly apparent from Figures NAT2*4 haplotype associated with the rapid acetylator-phenotype accounts for more than 50% of the global variation (Thai excepted), while in Africa and Europe, the derived haplotypes associated with the slow-acetylator phenotype are predominant over NAT2*4. This observation explains the lower proportion of slow acetylators in East Asians compared to other human populations [see Additional file NAT2*12A, NAT2*13), are essentially found in Africa and are particularly frequent in Baka and Bakola Pygmies, which display a similar proportion of rapid acetylators as East Asians .In East Asia, the ancestral FST's at the 5% level within each continental area was 35% and 44% in Europe and East Asia, respectively, while it was only of 11% in sub-Saharan Africa. Among Africans, Somali were genetically the most similar to Europeans. Bakola Pygmies were found highly differentiated from all other populations (except from Baka Pygmies) which could easily be explained by strong genetic drift due to isolation and small effective size. Embera and Ngawbe Amerindians did not differentiate statistically from each other; while in close proximity to East Asians, they displayed significant FST values with all other samples. In contrast, Nicaraguans were found very close to Europeans, in agreement with the Amerindian-European mixed origin of the sampled individuals. Turkmen and Kyrgyz displayed NAT2 allele frequencies intermediary to those observed in Europeans and East Asians, which points to the continuous nature of human genetic variation worldwide [NAT2 genetic differentiation patterns are related to geography, which is confirmed by a high and significant correlation coefficient  observed between genetic and geographic distances.Several differentiated clusters of populations clearly appeared in the graphical representation of the MDS Figure . The subFST gives a measure of the proportion of the genetic variance explained by differences among populations. The global FST value estimated for the 41 worldwide samples was of 0.123 (P < 10-5). When grouping these 41 populations into five major geographic areas , the vast majority of genetic variation was shown to occur within populations (83.5%), a high proportion  among geographic groups, and a mere 1.5% (P < 10-5) among populations within groups. When only three geographic groups were considered , the among groups component increased to 19%. The highest continental FST value was found in sub-Saharan Africa , whereas greater homogeneity was observed within East Asia , and even more so within Europe/North Africa . In agreement with the MDS analysis, differentiation was very low between Europe (including Moroccans) and sub-Saharan Africa , in contrast to the high FST values observed between Asia and both Europe and Africa . Likewise, differences in the level of population differentiation were observed among the individual SNPs (Table NAT2*6A haplotype) displayed strikingly low FST values. By contrast, high FST values were observed for the three SNPs T341C, C481T, and A803G which define the NAT2*5B haplotype. The highest FST values at these three SNPs were observed between European and East Asian populations (around 40%).r2 coefficient between SNP pairs) differed between human populations. All population samples displayed similar levels of LD within each geographic area, except Somali who showed higher LD at NAT2  than other sub-Saharan African samples and were, in that respect, more similar to Europeans. The mean pairwise r2 value between the seven SNPs in the European samples  was significantly higher  than in both East Asians (0.276 ± 0.023) and Africans . No statistical differences in the level of LD were found between East Asian and African populations. However, the proportion of SNP pairs with r2 ≥ 0.5 was far smaller in sub-Saharan Africans (6.7%) than in both Europeans (40%) and East Asians (33.3%). Ashkenazi Jews exhibited the highest level of LD ; such an excess of LD is often observed in founder populations that recently grew from relatively small sizes [r2 matrices of LD. Mantel's tests gave highly significant correlation values both between population pairs within geographic areas and for pairs of continental regions . Thus, although Europeans exhibited higher levels of LD at NAT2, the pattern of LD in this gene was found to be similar across human populations.We first tested whether the amount of LD . A similar observation has been made for several other African populations by Patin et al. [NAT2 sequence variation in not yet investigated human populations.This study provides a thorough description of  to 100% -17. SuchNAT2 are largely consistent with those reported in many other studies of different gene regions in the human genome. The inferred levels of sequence diversity , resulting in a larger fraction of slow acetylators in this sample (50% versus 5–20%). Interestingly, among the East Asian populations investigated, the Thai sample is the only representative of the variation of NAT2 in Southern East Asia. Because the full NAT2 gene diversity has not yet been investigated in other Southeast Asian populations, it is not possible to conclude whether this population harbors a specific profile with respect to this genetic system, or if it resembles other Southeast Asians. Further data from these latter populations are needed to speculate on a possible genetic differentiation pattern between Northern and Southern East Asian populations at the NAT2 locus.We observed a particular pattern of genetic diversity at NAT2 gene has long been considered as a likely target of population-specific selective pressures. But many questions remain about the roles that population history and natural selection have played in shaping the diversity of NAT2. An intriguing point about this gene concerns the high frequency of poor-metabolizers and slow acetylator alleles in most human populations. This might represent the evolution of balanced polymorphisms, maintained by natural selection through heterozygote advantage or spatial-temporal selection of alternative alleles, as it has been shown for G6PD deficiencies or phenylketonuria [NAT2 may evolve under no evolutionary constraint, this enzyme being not essential from an evolutionary perspective, maybe because it is dispensable or redundant with other enzymes. In terms of the detoxification of potentially harmful environmental aromatic amines, NAT1 seems indeed to be more active than NAT2, which preempts the latter's role as a key adaptation to increase the fitness of our species [Because of its role in the detoxification of exogenous substances, the etonuria ,28. An a species .NAT2 were consistent with a standard, neutral equilibrium model in 13 populations of sub-Saharan Africa. All neutrality tests found no evidence of a departure from selective neutrality. But making robust inferences on the action of natural selection at a particular locus requires a thorough characterization of population history, since the neutral null hypothesis is a composite hypothesis that also makes assumptions regarding the demography of the populations. It is typically assumed that the population is in equilibrium at constant size and with no population subdivision. But suppose that African populations did pass through a relatively narrow bottleneck in the late Pleistocene and then expand. In this case, the observed data might reflect the antagonistic effects of a bottleneck  and balancing selection (which decreases that frequency), and the apparent evidence in favour of a neutral model of evolution would be an artefact, produced by the confounded effects of these two opposing forces. However, the results of several studies have suggested that sub-Saharan African sequence diversity was compatible with an equilibrium model of long-term constant population size and random mating [NAT2 can be considered as a neutrally evolving gene, at least in the sub-Saharan African populations investigated. Further studies are needed to determine whether this finding can be generalized to all African populations. It would be also useful to increase both the number of individuals studied and the size of the genomic region surveyed (by investigating for instance the entire NAT2 gene sequence which spans around 10 kb) to increase the power of neutrality tests which remains weak when the sample sizes and the numbers of segregating sites are small.In this study, we investigated whether the patterns of sequence variation at m mating -32. TheyFST = 0.123, P < 10-5) was found to be remarkably consistent with the average FST value for the human genome [r = 0.47, P < 10-5) implies that patterns of human diversity at NAT2 can largely be accounted for by the simple interaction of drift and geographically-structured gene flow. However, while the overall degree of population structure at NAT2 was similar to previously reported values for neutral markers [FST values was observed for individual SNPs . These values were compared to the empirical distribution of FST across a 400-kb region encompassing the NAT gene family  [see Additional file FST were computed for 550 individual markers spanning the 400-kb NATs region by using publicly available HapMap SNP data  is very rare in East Asians and could represent a different selective advantage within the gene pools of these separate populations. 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. A thorough survey of NAT2 sequence variation in East Asians will be necessary to determine whether the predominance of the rapid-acetylator NAT2*4 allele over the slow ones is the result of local positive selection or whether it can be explained by stochastic processes such as genetic drift.A possible explanation for the unusually large difference in allele frequencies observed between European and East Asian populations for the n et al.  found evNAT2 gene. We found that patterns of NAT2 sequence variation are consistent with selective neutrality in all sub-Saharan African populations investigated, whereas the high level of population differentiation between Europeans and East Asians inferred from SNPs may suggest population-specific selective pressures acting at this locus, probably caused by differences in diet or exposure to other environmental signals.This study provides a thorough description of the worldwide haplotype diversity and LD structure of the NAT2 exon 2, which contains the entire protein-coding region, was determined in 97 healthy unrelated individuals  from the Niokholo Mandenka. This agriculturalist population from Eastern Senegal speaks a language belonging to Mande, a major primary branch of the Niger-Congo language family. We used DNA extracted from the lymphoblastoid cell lines (LCL) described in Excoffier et al. [NAT2 variants present at a frequency ≥ 3%, with a probability of at least 99%.Full sequence diversity of r et al. . The samNAT2 was amplified by PCR and subsequently sequenced on both DNA strands on an ABI 3730xl automated sequencer (Applied Biosystems) using three internal primer pairs, as described in Cascorbi et al. [NAT2 coding exon and 318 bp of non-coding flanking parts. All singletons were verified by PCR reamplification and resequencing the PCR products in both directions. The observed genotype frequencies at all polymorphic sites were in conformity with the assumptions of Hardy-Weinberg equilibrium when tested using Fisher's exact tests. Haplotype reconstruction was performed using the Bayesian method implemented in PHASE v.2.1 [A 1,211-bp fragment covering the entire coding region of i et al.  (sequencSE v.2.1 . All 194NAT2 sequence data set with a collection of previously published sequences of the NAT2 coding exon (870 bp) [For comparison purposes, we merged the (870 bp) . This adNAT2 and for which genotype data was available. Among these seven SNPs, all located in the coding exon, four result in an amino acid substitution that leads to a significant decrease in acetylation capacity . The other three are either silent mutations  or a non-synonymous substitution that does not alter the phenotype (A803G).We selected from published reports up to 2006 all the population samples that were genotyped for the seven most common SNPs at A complete list of the selected samples is provided in Additional file Pan troglodytes; Ensembl Chinpanzee genome) and one rhesus monkey  were used to infer SNPs' ancestral state. Each of these also served as an outgroup for evolutionary and population genetic tests.All sequence analyses were performed on both the Mandenka sample and on each of the 12 African samples of Patin et al. . Homologπ) and haplotype (H) diversity, Watterson's θw [D [F* and D* [Fs [H [DnaSP v.4.10  was usedson's θw , as well's θw [D , Fu and * and D* , Fu's Fsd D* [Fs , and Fay* [Fs [H . The sta* [Fs [H  was applNAT2 gene. The time to the most recent common ancestor (TMRCA) and mutation ages were estimated from the NAT2 gene tree, conditional on a maximum-likelihood estimate of θ(6 replications), under a standard coalescence model assuming neutrality, the infinite-sites mutation model , random mating, and constant population size. All estimates were inferred on individual population samples, to avoid biases due to population structure. Time, scaled in 2Ne units, was converted into years by use of a 25-year generation time and the value of the effective population size (Ne) obtained as μ. The neutral mutation rate per gene per generation (μ) was estimated based on human-chimpanzee sequence divergence, assuming a divergence time of 5 million years (My) [A coalescence model for the ancestral history of a sample of genes was used to estimate the time scale of polymorphic variation in the ee v.9.0  (runningars (My) .NAT2 haplotypes from the unphased multi-locus genotypes using PHASE v.2.1 software [NAT2, in accordance with the acknowledged classification of NAT2* alleles based on their functional impact [see Table For the worldwide genotyping survey, we inferred software . The indNAT2 haplotypes were generated using Network 4.1.1 software [Median-joining networks  describisoftware .NAT2 haplotypes. Population differentiation was tested by permutation tests  based on the FST statistic. Coancestry coefficients, or linearized FST values [Population structure in the worldwide genotyping survey set was investigated by an analysis of molecular variance (AMOVA)  that incT values , were coT values  performeT values . A ManteT values .r2 statistic [r2 matrices were performed using the program CADM [r2 values were recalculated for populations pooled into geographical groups and Mantel tests were applied.Pairwise LD between the seven genotyped SNPs was estimated by computing the tatistic  with Dnatatistic , after tram CADM . CompariNAT2 gene sequencing in the Mandenka sample. EP provided the conceptual framework for the study, supervised the statistical analyses and finalized the manuscript. All authors read and approved the final manuscript.AS conceived of the study, collected data, performed the majority of the statistical analyses and wrote the initial draft of the manuscript. AL and PD participated to study design, supervised analyses, and were involved in drafting the manuscript. AL and EP provided the Mandenka sample. NG and RK participated in the Summary description of the 41 samples included in the worldwide genotyping survey.Click here for fileNAT2 predicted acetylation phenotypes in the 41 samples of the worldwide genotyping survey.Distribution of Click here for fileST Fvalues between Europeans and East Asians across a 400-kb segment encompassing the human NAT gene family on chromosome 8.Distribution of Click here for fileNAT gene family on chromosome 8.LD/block structure of the European HapMap sample across a 400-kb segment encompassing the human Click here for fileNAT gene family on chromosome 8.LD/block structure of the East Asian HapMap sample across a 400-kb segment encompassing the human Click here for file


Drosophila subobscura populations recently sampled from different wild sources.Natural selection and genetic drift are major forces responsible for temporal genetic changes in populations. Furthermore, these evolutionary forces may interact with each other. Here we study the impact of an ongoing adaptive process at the molecular genetic level by analyzing the temporal genetic changes throughout 40 generations of adaptation to a common laboratory environment. Specifically, genetic variability, population differentiation and demographic structure were compared in two replicated groups of We found evidence for a decline in genetic variability through time, along with an increase in genetic differentiation between all populations studied. The observed decline in genetic variability was higher during the first 14 generations of laboratory adaptation. The two groups of replicated populations showed overall similarity in variability patterns. Our results also revealed changing demographic structure of the populations during laboratory evolution, with lower effective population sizes in the early phase of the adaptive process. One of the ten microsatellites analyzed showed a clearly distinct temporal pattern of allele frequency change, suggesting the occurrence of positive selection affecting the region around that particular locus.Genetic drift was responsible for most of the divergence and loss of variability between and within replicates, with most changes occurring during the first generations of laboratory adaptation. We also found evidence suggesting a selective sweep, despite the low number of molecular markers analyzed. Overall, there was a similarity of evolutionary dynamics at the molecular level in our laboratory populations, despite distinct genetic backgrounds and some differences in phenotypic evolution. Evolution in a novel environment involves a complex array of processes that produces both genetic and phenotypic changes. The extent of these changes varies as a function of several forces, such as the selective pressures imposed and the magnitude of genetic drift, as well as the genetic background and prior evolutionary history of the populations concerned. Natural selection is an important evolutionary process affecting differentiation between populations. Different selective regimes foster evolutionary divergence, while common novel selective forces are expected to lead to convergence . NeverthAn important evolutionary factor leading to differences among populations is genetic drift, particularly in populations with low effective size . MoreoveExperimental evolution can help address these issues through the use of controlled selection regimes, controlled population sizes, and replication, both simultaneous and sequential . In partThe joint study of evolutionary changes in selectively-important quantitative traits, such as those that define life histories, and highly polymorphic molecular markers, such as microsatellites, allows us to analyse in further detail the effects of natural selection and genetic drift in the genome of evolving populations.Multilocus screens have been used as a tool to identify regions of the genome that have undergone positive selection e.g., ,12). The. The12])Drosophila subobscura derived from different foundations in the wild  as they undergo adaptation to a common laboratory environment, based on molecular markers.Here we present a detailed study of the temporal genetic changes in two sets of replicated populations of The phenotypic evolution of these populations in the laboratory environment has already been analyzed through evolutionary trajectories for several life history traits, revealing a clear adaptive response, particularly for fecundity-related traits. Nevertheless, these populations also showed differences in their phenotypic evolutionary rates, particularly during an early phase of the laboratory adaptation process . Bearingdsub14 presenting the lowest mean allele number and the lowest expected heterozygosity in all generations analyzed .AR and TW groups of populations already differed significantly at generation 3  or between TW populations . On the other hand, all populations within each group were significantly differentiated by generation 14  and 40 . In each generation, genetic differentiation between populations within each group was not significantly different between TW and AR [see also Additional file Fst comparisons within and across laboratory generations]. In both groups of populations genetic differentiation increased significantly between generation 3 and 14 (AR and TW)  but not between generations 14 and 40 .Genetic differentiation within each group of populations (AR and TW) was also analyzed, in each generation and across generations. No genetic differentiation was obtained at generation 3 either between AR populations (e estimates for both AR and TW populations during the two periods of laboratory adaptation: the first period (generations 3 to 14), the second period (generations 14 to 40) and also during the overall study (generations 3 to 40) using both a pseudo-likelihood approach and the loss of heterozygosity method. Ne values were estimated excluding microsatellite locus dsub14 from the data, given its extremely low diversity and its increase in heterozygosity between generations 3 and 14. Furthermore, the disparity between dsub14 and all other microsatellite loci may be due to non-neutrality at this locus, an assumption of all models estimating Ne. The case of this particular locus will be addressed further below.Table t-tests using as data points the √Ne estimates of the three replicate populations; p < 0.05 for all estimates; see Table Using either method, effective population size estimates for the first period of laboratory adaptation were significantly lower than those obtained for the second period for both TW and AR groups of populations . Nevertheless, the AR and TW Ne estimates obtained using the pseudo-likelihood method for this first period did not differ significantly . Ln RH values for locus dsub14 were significantly different from those obtained for all other loci in all six populations  as a result of the increase in heterozygosity at this locus. Ln RH values between all other pairs of loci were not significantly different . Also, standardized Ln RH values for microsatellite locus dsub14 fell outside the 95% confidence interval of the standard normal distribution for all replicates  in all TW populations and the AR3 population, while a different allele (with 116 bp) increased in frequency in both AR1 and AR2 populations. In TW populations, the allele that increased in frequency (120 bp) rose from an average initial frequency of 11.5% at generation 3 to 31.6% at generation 14. In the AR3 population, the 120 bp allele increased from 5% to 19.2% while the 116 bp allele increased in AR1 and AR2 populations from an average frequency of 5.2% to 15.5%.Heterozygosity ratios (Ln RH ratios) were calculated for both groups of populations by comparing data between generations 3 and 14 as well as between generations 14 and 40. When comparing generations 3 and 14, Ln RH values were significantly different between loci, both in TW and AR populations . This was also observed for each replicate population by analyzing the standardized Ln RH values . Nevertheless, in the AR3 population the allele that had previously increased in frequency (120 bp) slightly decreased (from 19.2% to 13.5%), being the high Ln RH ratio due to the increase in frequency of other alleles. For the TW populations, no significant differences between loci were detected with the general ANOVA or the Scheffé test. However, the analysis of the standardized Ln RH values for each TW replicate population showed some significant results, though they were not consistent among replicates. Specifically, locus dsub14 showed a significant decrease in heterozygosity in the TW2 population, due to a decrease in frequency of the 120 bp allele. Heterozygosities for this particular locus remained almost constant in the TW1 and TW3 populations during this period  and expected heterozygosity (Hexp = 0.877–0.898) in these populations were similar to the values observed for the same seven common loci studied in other European natural populations .Fst value of 0.015 at generation 3 was slightly higher than the values obtained from other comparisons among European populations of Drosophila subobscura . This sDrosophila. This depletion in genetic variability was generally observed throughout laboratory culture for both AR and TW populations, as shown by the parallel declines among variability indexes. However, it is important to note that this loss of genetic variability was relatively mild, since after 40 generations of laboratory adaptation AR and TW populations retained, respectively, 95% and 90% of their initial genetic diversity. The careful maintenance and overall high census sizes  in our populations may explain these results. This is in accordance with the high levels of genetic variability that we had already found for our NW Drosophila subobscura populations after 49 generations in the laboratory, with 87 to 89% of the genetic diversity of the third generation of TW populations .During the course of 40 generations of laboratory culture, the initially high genetic variability was progressively eroded: both allele number and heterozygosity showed signs of decline during this period. This was predictable, because two forces that are both expected to erode genetic variability – random genetic drift and sustained directional selection – are likely to be pronounced in laboratory cultures, particularly given that effective population sizes are likely to be much lower than those occurring in natural populations of Drosophila melanogaster )Fst values) increased among all six populations through time. This is expected to be particularly important in smaller populations, due to genetic drift . The aim of this test is to search for loci with a pattern of variability which is significantly different from that expected with neutrality.Effects of positive selection were tested for each microsatellite locus by applying the Ln RH test statistic . This teTo apply this test, ratios of expected heterozygosities were calculated for each locus using data from generations 3 and 14 (G14/G3 ratios) and also generations 14 and 40 (G40/G14 ratios) for each AR and TW populations. To account for the different effective population sizes of X chromosomes, a correction was introduced for the X chromosomal loci heterozygosities (see ):k used was 1.33, assuming a balanced sex ratio [the correction factor ex ratio . Since Lex ratio , signifiex ratio . This telocus as factor and the Ln RH values (of the three AR or TW populations) as the dependent variable. To search for differences between loci a post hoc Scheffé test was also performed. Normality in Ln RH data was previously tested. All these analyses were done in Statistica 5.0.To detect potentially selected loci, we also performed a one-way ANOVA, defining PS, JS and MM performed the life history trait assays and maintained laboratory populations. PS and JS performed the microsatellite analyses. PS and MM carried out the statistical analyses. PS, MP, MRR and MM designed the experiment. PS and MM wrote the first draft of the manuscript. MP, MRR, and JS contributed to the final draft of the manuscript. All authors read and approved the final manuscript.Genetic Variability of AR and TW populations. Mean allele number and expected heterozygosity per locus for the AR and TW groups of populations at generations 3, 14 and 40 of laboratory evolution.Click here for fileFst comparisons between AR and TW populationsPairwise . Genetic differentiation between AR and TW populations at each of the three generations analyzed: generation 3, 14 and 40.Click here for fileFst comparisons within and across laboratory generationsPairwise . Genetic differentiation in each set of replicate populations (AR or TW) within and across generations analyzed.Click here for file


Brazil holds the largest commercial cattle populations worldwide. Local cattle breeds can be classified according to their origin, as exotic or Creole. Exotic breeds imported in the last 100 years, both zebuine and taurine, currently make up the bulk of the intensively managed populations. Locally adapted Creole breeds, originated from cattle introduced by the European conquerors derive from natural selection and events of breed admixture. While historical knowledge exists on the Brazilian Creole breeds very little is known on their genetic composition. The objective of this study was to assess the levels of genetic diversity, phylogenetic relationships and patterns of taurine/zebuine admixture among ten cattle breeds raised in Brazil.Significant reduction of heterozygosity exists due both to within-population inbreeding and to breed differentiation in both subspecies (taurine and zebuine). For taurine breeds the number of markers that contribute to breed differentiation is larger than for zebuine. A consistently similar number of alleles was seen in both subspecies for all microsatellites. Four Creole breeds were the most genetically diverse followed by the zebuine breeds, the two specialized taurine breeds and the Creole Caracu. Pairwise genetic differentiation were all significant indicating that all breeds can be considered as genetically independent entities. A STRUCTURE based diagram indicated introgression of indicine genes in the local Creole breeds and suggested that occasional Creole introgression can be detected in some Zebuine animals.This study reports on a comprehensive study of the genetic structure and diversity of cattle breeds in Brazil. A significant amount of genetic variation is maintained in the local cattle populations. The genetic data show that Brazilian Creole breeds constitute an important and diverse reservoir of genetic diversity for bovine breeding and conservation. The genetic data was able to shed light on a number of issues related to the local breeds origin and structure. The Brazilian Creole breeds are all important and viable targets for conservation for they display peculiar traits both phenotypic and of cultural and historical nature that deserve conservation efforts. Brazil holds the largest commercial cattle populations worldwide, with over 190 million animals raised both for dairy products and meat  estimateLikewise most European countries and the US, the rapid growth of these commercially proeminent breeds has happened to the expense of the second group of locally adapted, genetically heterogeneous breeds. This group of Creole breeds, also referred as native, local or naturalized breeds includes those derived from the first cattle populations introduced by the European conquerors around 1500. While all other South American countries received only Spanish breeds, due to its peculiar colonial origin Brazil was the only one that received Portuguese breeds . NaturalWhile historical knowledge has been accumulated on the Brazilian Creole breeds -8, very In the context of the Guidelines for Development of National Farm Animal Genetic Resources Management Plans , the FAOFollowing the project proposed for the Animal Genetic Resources by FAO  the objeBos subspecies (taurine and zebuine) and for both subspecies together. Expected locus heterozygosities in both subspecies and all the breeds combined were nominally larger that the observed heterozygosity for all loci. The only exception was observed at locus ETH3 in the group of zebuine breeds although it did not result in a statistically significant excess of heterozygotes. In the group of taurine breeds only loci INRA63 and HEL1 and in the zebuine group only loci INRA35, INRA37, CSSM66, SPS115, TGLA227, INRA23, ETH3 and BM1824 were found to be in HWE. All other loci displayed deviations from HWE. When all breeds combined were analyzed all loci deviated from HWE (Table IS) and to breed differentiation estimated both under the infinitesimal model (FST) and the step-wise mutation model (RST). A higher estimate of within subspecies inbreeding was seen for zebuine (0.113) when compared to taurine (0.074), although the larger sample size assayed for taurine could be partly accountable for this difference. However when an analysis was carried out on equalized samples sizes of 292 animals per subspecies, results were the same.A total of 915 animals representing ten Brazilian breeds was analyzed  were in general more pronounced than by the FST statistics in absolute values. The overall loci estimates of FST and RST were similar in both subspecies groups however RST was much higher that FST when all breeds together were analyzed. The global deficit of heterozygosity when all breeds of both subspecies were combined amounted to 0.176 and the global differentiation among all breeds was estimated by FST at 0.098 and at 0.1861 by RST. Most of this differentiation is likely due to the well known and marked subspecies genetic difference although genetic variation between breed of the same subspecies is also significant (see below).The contribution of the microsatellite markers for breed differentiation was estimated by the significance of the FIS were seen for JER followed closely by the three zebuine breeds GYR, GUZ and NEL and the taurine CUR. The average proportion of shared alleles among animals within breeds were similar for all breeds although PAN, MON and CRL had lower values consistent with their highest observed heterozigosities.Diversity measures for each breed showed a remarkably similar mean number of alleles per locus fluctuating around 8.5. Table . The CreThe partitioning of the genetic variation at different levels resulted in small but significant (p < 0.001) between breed proportions of the variation in all structures tested Table . Among tST) were all significant after Bonferroni corrections (p < 0.01), indicating that all breeds can be considered as genetically independent entities. Different genetic distance measures were estimated but all showed a very high correlation so that only Nei distance, DA, is reported  in the sample of 915 animals studied was relatively high, higher than estimates in other studies that involved local breeds both of taurine and zebuine origin [ST. The observed overall reduction of heterozygosity is therefore due in almost equivalent proportions to within-population inbreeding (FIS = 0.086) and genetic drift among all ten breeds (FST = 0.098). All breeds displayed a significant reduction in heterozygosity due to non-random matings within populations (Table IS). This result most likely reflects the more intense reproductive management that the zebuine breeds and JER have been subjected to, with the use of a relatively small number of high value bulls as semen donors in assisted reproduction practices.The global deficit of heterozygotes  [ST = 0.088 and RST = 0.144 [ST when compared to FST suggests that differences among breeds involve not only allele frequencies but also allele size differences due to the mutational behavior of microsatellites.Significant genetic differentiation was observed among all ten breeds estimated both by F61 Table . Similar = 0.06) ; 0.112 a = 0.06) ; 0.035 a = 0.06) ; 0.107 i = 0.06) ; around  = 0.06) ,32 and 0 = 0.06) . In a st = 0.144 . The mucST among all ten breeds for the 22 microsatellites are useful indicators of markers that could be powerful tools for breed differentiation. Differentiation of breeds that belong to different subspecies, taurine or indicine, is a relatively trivial task as several markers with a significant FST could easily diagnose the most likely breed as well as the proportions of zebuine and taurine genomes. Within each subspecies however it would be more difficult. In taurine, for example, out of the twenty markers significantly contributing to interbreed differences, markers INRA63, INRA5, CSSM33, ETH10 and TGLA227, the top five ranked by FST values, could be tested for this purpose. In zebuine, only eight markers showed a significant FST, and all of very low value, so that breed differentiation in this subspecies could demand other kinds of markers such as carefully selected and validated ancestrally informative single nucleotide polymorphisms. Both FST and RST estimates within the taurine and zebuine groups taken separately showed a lower differentiation among the three zebuine breeds when compared to the taurine group. The possible explanation resides in the way that these two groups were introduced and are currently managed in Brazil. No specific breed segregation was practiced at the time and all animals coming from the Indies were generically classified as Zebu [The significance and values of the overall estimates of F as Zebu . Finally as Zebu .et al. [Four of the five Creole breeds CRL, CUR, MON and PAN displayed a higher allelic richness than all other breeds. The same comparative pattern of genetic variation was observed by Liron et al. . The intet al. . Howeveret al. . The diset al. [The partitioning of the genetic variation from an AMOVA also revealed that the largest amount of variation was always found among individuals within breeds, irrespective of the different structures tested Table . Maximumet al.  however,et al.  that havet al. . Second,et al. [A comparison of autossomal microsatellite, mtDNA haplogroups and Y-chromosome microsatellite haplotypes has shown that for Bolivian and Argentinean Creole breeds significant male mediated zebuine introgression has taken place ,36. The et al.  when anaMuch controversy has been going and several approaches have been proposed to assess conservation priorities on the basis of molecular markers ,37. No aThis study reports on a comprehensive study of the genetic structure and diversity of bovine cattle breeds in Brazil. The genetic analysis showed that a significant amount of genetic variation is maintained in the local cattle populations and all breeds studied could be considered as distinct genetic entities. Four of the five Creole Brazilian breeds displayed a markedly higher allelic richness than all other breeds most likely as a result of a combination of natural selection in diverse environmental conditions, mild artificial selective pressure and recurrent breed hybridizations including introgression from zebuine breeds. The genetic data corroborate historical records in that they indicate that variable patterns of breed admixture have occurred since colonial times shaping the current genetic status of the local breeds. Brazilian Creole breeds constitute an important and diverse reservoir of genetic diversity for bovine breeding and viable targets for conservation for they display peculiar traits both phenotypic and of cultural nature. As pointed out by several authors, many other aspects besides the amount and distribution of genetic diversity have to be taken into account when dealing with conservation strategies of livestock species. Historical, cultural and traditional aspects regarding the use of particular breeds are relevant issues. Furthermore one should not forget the fact that directional selection practiced by man has shaped animal genomes in unexpected ways favoring alleles or genes complexes for which the surrogate neutral markers used in diversity surveys are not necessarily fully representative.Ten Brazilian bovine breeds were analyzed, involving a total of 915 animals. The breeds studied can be classified into three groups: (a) Taurine Creole breeds ; (b) European taurine breeds (Holstein – HOL and Jersey – JER) and (c) Brazilian zebuine breeds  (Table a = 56°C); a 2-plex of markers CSSM9, CSSM33 ; a 2-plex of markers HEL1, INRA05 (Ta = 56°C); a 5-plex of markers BM2113, ETH10, SPS115, TGLA122, ETH225 (Ta = 61°C) and a 5-plex of markers TGLA227, TGLA53, INRA23, ETH3, BM1824 (Ta = 61°C). Microsatellite CSSM66 was amplified alone (Ta = 61°C) and the PCR product injected together with markers HEL1 and INRA5 before electrophoresis. Only markers CSSM9 [Twenty-two microsatellites were amplified by polymerase chain reaction (PCR) in five different multiplex systems where the forward primer of each microsatellite was labeled either with 6-FAM, HEX or NED fluorochromes according to the expected allele size range. Several of these microsatellites have been commonly used by other groups worldwide thus making possible future comparative analysis or consolidation of data sets. The multiplex systems used were: a 7-plex composed by markers INRA35, INRA37, HEL9, HEL5, INRA63, ILSTS5, ETH152  and data collected under virtual filter D using GeneScan 2.0 and Genotyper 2.1 (Applied Biosystems) to declare alleles. An internal size standard labeled with ROX  was usedF-statistics for each locus were calculated using Weir and Cockerman's method [F-statistics  for each microsatellite locus were obtained by constructing 95% and 99% confidence intervals based on the standard deviations estimated by jackknifing across populations using FSTAT.Allele frequencies were estimated by direct counting. Parameters of locus diversity were estimated for all microsatellite markers in all breeds using the Cervus software , includis method  using FSs method . A signiF-statistics  and the indicative P-value was adjusted by a Bonferroni procedure using the same software package [An exact test was used to determine deviations from Hardy-Weinberg proportions and heterozygosity deficiency using the GENEPOP software package . The Mar package . Using b package .A [ST) was calculated using FSTAT. The log-likelihood G-statistics [ST [A distances using the Dispan package. Bootstrap values were computed over 1,000 replicates. Additionally a Neighbor-Net graph [A distances was constructed with SplitsTree4 program [Genetic distances between breeds was estimated by DA  using DIA . The traatistics  was usedatistics . RST [57et graph  based on program .The pairwise genetic distances between all individual animals were estimated by the logarithm of the proportions of shared alleles (Dps) , using MBased on genotypes at the 22 marker loci, individual animals were clustered into a given number of populations and assigned probabilistically to clusters inferred with a Bayesian approach implemented by the STRUCTURE software . The tesAs part of her PhD thesis, AAE participated in the project conception, carried out most of the experimental work, including microsatellite genotyping and data analysis, and drafted the first version of the manuscript. SRP contributed to the statistical analysis. MSMA, STC and LDA helped with DNA extraction and PCR assays. ARS participated in the project conception and design, provided critical information about Creole breeds and helped reviewing the manuscript. As thesis advisor, DG contributed to the design and execution of the experiments and data analysis and to the writing of the final version of the manuscript.Distribution of allele frequencies for the 22 microsatellites in the 10 cattle breeds raised in Brazil. Estimates of allele frequencies for the 22 microsatellites markers in the ten Creole and commercial cattle breeds raised in Brazil.Click here for file


Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of disease genes. In these populations the disease allele reveals Linkage Disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. In a previous study we examined the LD extension on the Xq13 region in three Corsican sub-populations from the inner mountainous region of the island. On the basis of those previous results we have proposed a multistep procedure to carry out studies aimed at the identification of genes involved in complex diseases in Corsica. A prerequisite to carry out the proposed multi-step procedure was the presence of different degrees of LD on the island and a common genetic derivation of the different Corsican sub-populations. In order to evaluate the existence of these conditions in the present paper we extended the analysis to the Corsican coastal populations.Samples were analyzed using seven dinucleotide microsatellite markers on chromosome Xq13-21: DXS983, DXS986, DXS8092, DXS8082, DXS1225, DXS8037 and DXS995 spanning approximately 4.0 cM (13.3 Mb). We have also investigated the distribution of the DXS1225-DXS8082 haplotype which has been recently proposed as a good marker of population genetic history due to its low recombination rate.the results obtained indicate a decrease of LD on the island from the central mountainous toward the coastal sub-populations. In addition the analysis of the DXS1225-DXS8082 haplotype revealed: 1) the presence of a particular haplotype with high frequency; 2) the derivation from a common genetic pool of the sub-populations examined in the present study.These results indicate the Corsican sub-populations useful for the fine mapping of genes contributing to complex diseases. Isolates have been of considerable use in genetic studies aimed at identifying mutations underlying rare diseases . MoreoveAssociation studies are critically dependent on the extent of LD. Several reports have underlined how Linkage Disequilibrium (LD) is more extended in founder populations . LD exteGenetic homogeneity found in isolated populations is a great advantage in the identification of large genomic regions containing the disease-associated locus, while fine mapping could require recently expanded population.The aim of this paper is an evaluation of some Corsican sub-populations as candidate to identify genes implicated in complex disease. Genetic structure of Corsican population has been studied by several authors -9. CorsiCorsica has been invaded several times . In the great majority of cases, these invasions were limited to the coastal areas and left slight marks on the gene pool of the native populations . Strong The close by isolated founder Sardinian population has been extensively investigated and has provided a marked contribution towards the mapping of rare monogenic and complex diseases ,11,12. WIn the present study we report the LD pattern on Xq13-21 microsatellite markers in a Corsican coastal sub-population in order to assess the presence of a decrease of LD extension in island. We have also examined the distribution of the DXS1225-DXS8082 haplotypes ,18 in thIn a previous paper, we have studied the LD extent in 3 sub-populations located in the central mountainous area  . Here weThe use of microsatellite markers in LD mapping is being substituted by SNPs. Nevertheless microsatellites markers remain a valuable tool for the first screening of background LD in a given population.DNA samples were collected from unrelated male individuals. These samples were analyzed using seven dinucleotide microsatellite markers on chromosome Xq13-21: DXS983, DXS986, DXS8092, DXS8082, DXS1225, DXS8037 and DXS995 -21.Microsatellites were analysed using an ABI prism 377 DNA analyser. Genotypes were processed by Genescan v3.1 and Genotyper v2.5 software. A DNA standard, consisting of the CEPH control individual number 1347.02 (Applied Biosystems) was incorporated in all the runs to verify accuracy of typing.corrected = 1 - (1 - p)n, where n is the number of p values smaller or equal to that being corrected [The non-random allelic association between pairs of microsatellite loci was tested by an extension of Fisher exact test on contingency tables. p values were corrected by the step-down Holm-Sidak procedure . The method, based on allelic distribution of alleles in the various populations, are described by Raymond and Rousset (1995) . The unbAn unbiased estimate of gene diversity g to Nei  as folloVariance of gene diversity is given by:where r is the numbers of loci andGene diversity is often referred to as expected heterozygosity and is defined as the probability that two randomly chosen alleles from the population are different.The study complies with the Declaration of Helsinki and received ethical approval by the University of Corte. Each subject, of Corsican ancestry up to grandparents, gave informed consent.The extent of linkage disequilibrium was assessed in each Corsican sub-populations, using seven markers on Xq13-21 and two different approaches: Fisher's exact test was used to evaluate statistical significance of the allelic association between all pairs of loci and Lewontin's coefficient D' was applied to assess the strength of LD.Table The coastal population showed a low level of LD, rather similar to values obtained for Corte. In both sub-populations only one marker pair (DXS8082- DXS1225) displayed a significant p value following correction for multiple testing with a high D' value . This marker pair has shown a strong association in all the populations analyzed so far .The lowest levels of D' was shown by the pooled Corsican sample. Only the DXS8082- DXS1225 pair showed a corrected D' exceeding 0.5. Nevertheless, significant values were revealed in four marker pairs by means of Fisher's exact test.Bozio sub-population exhibited the highest level of LD in Corsica followed by Niolo . AccordiTable The distribution of DXS1225-DXS8082 haplotypes was also analyzed Table . The mosIn addition we analysed the number of alleles and the gene diversity estimates in each sub-population and in the pooled sample. None of the samples showed a different mean gene diversity with respect to other sub-populations Table . MoreoveThe findings of the present study illustrate a decline in the LD extent in Coastal Corsican population compared to the inner mountainous regions of Niolo and Bozio. We have also shown the presence of an high frequency of a particular DXS1225-DXS8082 haplotype in most of the Corsican sub-populations examined, implying a common genetic origin (with the possible exception of Corte). The decrease observed in the level of LD in the coastal population  either in the Coastal sub-population sample as in sub-populations from Niolo and Bozio regions, suggest their origin from a common genetic pool. Being recombination events between these two markers extremely rare, new variants arise prevalently from a mutation in one of the two loci. The footprint of a demographic event persists for a longer period in haplotype distribution within a region characterized by a low crossing-over rate than in a single marker or between actively recombining markers . NeverthAll the sub-populations examined show a strong degree of differentiation, according to the differentiation test, with the exception of Bozio and Niolo in agreement with previous results . These dAverage gene diversity estimate for each Corsican sub-population was quite similar to values reported for other populations and indicates that they possess similar homogenous genetic architectures. The pattern of gene diversity obtained for all single markers showed no clear correlation with the extent of LD, thus suggesting that the increase in linkage disequilibrium is probably not due to selection, but rather due to demographic history of the studied populations .The island of Corsica is geographically very close to the other Mediterranean Island of Sardinia. The isolated founder Sardinian population has been extensively investigated and has provided a marked contribution towards the mapping of rare monogenic and complex diseases ,3. The CThe most frequent haplotypes of microsatellite loci DXS1225-DXS8082 in the Corsican sub-populations under investigation Table  have not and therefore could represent a good candidate for association studies.Taken together, this data suggests the suitability of the Corsican sub-populations as a candidate for use in identifying genes underlying complex diseases by means of LD association studies performed according to a multistep procedure as described previously . As prevLastly, it should be underlined how particular caution should be taken when using "isolated populations", such as those of the Sardinian and Corsican Islands as a whole, for the purpose of performing long range LD mapping, due to the possibility that micro differentiation may limit LD extension as reported here for the Corsican population.The authors declare that they have no competing interests.VL: genotyping, contribution to statistical analysis, interpretation of the data and drafting the manuscript. GS: main statistical analysis, contribution to interpretation of the data and drafting the manuscript. LV: revising critically the manuscript. GV: revising critically the manuscript MSR: Concept and design of the study, interpretation of the data, drafting the manuscript and final approval of the version to be published.The pre-publication history for this paper can be accessed here: