BACKGROUND
all along human evolution and continuing in present times, populations have developed a wide variety of cultural innovations.
many of these may have affected the interactions between humans and their environment in such a way as to leave gene-culture coevolution imprints in the genome, due to both demographic and selective processes .
among these innovations, new subsistence strategies represent major shifts with notable consequences on the pathogenic and dietary environments in which populations have been living, thus possibly also inducing new selective pressures on phenotypes .
subsistence strategies are broadly classified in two opposite categories, i.e., foragers , relying on the collection of nutriments naturally occurring in the wild, and food-producers whose appearance in the prehistoric record is generally associated with the emergence of the neolithic .
among the latter, two major modes of production are distinguished, namely agriculture based on the cultivation of domesticated plants, and pastoralism based on the herding of domesticated animals.
these two modes of subsistence also imply differential usage of space, in that most agricultural societies developed sedentarism whereas communities relying on animal husbandry developed nomadism or transhumance to exploit seasonal variations in the location of pasture areas.
it is currently held that these differential developments in subsistence strategies also influenced the selective regimes to which past populations were subjected, including through the action of new population-specific  selective pressures induced by dietary changes .
lactase persistence, a heritable condition in which the physiological ability to digest lactose  is maintained throughout adulthood, probably represents the best-known example of an adaptation related to diet  <cit> .
indeed, convergent evolution of the trait has been demonstrated  <cit> , and is explained by the emergence of similar selective pressures resulting from adopting a diet heavily relying on milk.
however, demographic processes  playing a significant role in the spread of the trait constitutes a valid, mutually non-exclusive alternative explanation .
hence, disentangling the respective contributions of selective and demographic forces to extant levels and patterns of genetic differentiation between populations represents a challenging task  <cit> .
reduced arylamine n-acetyltransferase  <dig>  activity is another trait whose evolution was probably also shaped by differential, population-specific selective pressures .
due to its early discovery linked to its major role in the treatment of tuberculosis with isoniazid, inherited variation in n-acetylation activity is currently one of the best known pharmacogenetic traits  <cit> .
the phenotype is driven by the existence of a polymorphic n-acetyltransferase  <dig>  enzyme.
this cytosolic enzyme, encoded by the gene nat <dig> of chromosome  <dig>  is mainly expressed in the liver, small intestine and colon  <cit>  where it catalyzes a phase ii acetylation reaction, i.e., the transfer of an acetyl functional group to the terminal nitrogen of aromatic amines, heterocyclic amines, and hydrazines  <cit> .
hydrazines are used in the synthesis of numerous organic molecules, like the anti-tubercular agent isoniazid, while aromatic and heterocyclic amines are known to be produced, for instance, in meat and fish cooked at high temperatures, as well as in combustion smokes such as those from tobacco, grasses and wood chips  <cit> .
thus, the nat <dig> enzyme plays a crucial role in the detoxification of numerous xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment  <cit> .
acetylation capacity  can be measured by an individual’s response to drugs metabolized by the enzyme, and this phenotype is now known to depend on the individual’s genotype at the single coding exon of the nat <dig> gene.
mutations in nat <dig> result indeed in diverse acetylation phenotypes that explain inter-individual variation in response to standard drug dose administration   <cit> .
moreover, mutations in nat <dig> may also act as risk factors for different types of cancers  <cit> .
the gene displays a high degree of polymorphism in humans, with  <dig> alleles  listed to date by the official consensus gene nomenclature of human nat <dig> alleles .
inter-population variation in nat <dig> allele frequencies has been intensively documented, particularly so for those alleles defined by the combination of nucleotides at four major functional single nucleotide polymorphisms  of the coding exon  <cit>  .
although response to drug intake is a quantitative variable, the distribution of the phenotype in tested human groups was shown to be at least bi-  modal  <cit> .
a simplified model of genotype-phenotype relationships was therefore adopted from the early studies onwards, in which those alleles considered as fully functional are responsible for the fast acetylator status, whereas decreased function alleles are responsible for the slow acetylator status .
hence, acetylation status of an individual that is carrier of two fully functional alleles is classified as fast, that of a carrier of one fully functional allele and one allele with decreased function is classified as intermediate, and that of a carrier of two decreased function alleles as slow.
also, many studies do not distinguish between fast and intermediate acetylators, categorizing both types of subjects as fast  acetylators.
similarly to lactase persistence, nat <dig> phenotypes prevalence varies substantially between populations , and hypotheses of gene-culture coevolution of nat <dig> polymorphisms have been put forwards, notably the idea that the slow acetylator phenotype became selectively advantageous in those populations adopting a food-producing mode of subsistence in the neolithic.
indeed, given its role in the detoxification of numerous exogenic compounds, the nat <dig> enzyme acts at the interface between the organism and its chemical environment, and hence is a likely target for natural selection.
however, in contrast to lactose for lactase persistence, a dietary or environmental causal factor driving nat <dig> evolution is not identified, but hypotheses involving meat consumption and availability in food supplied folates related to nutritional shifts during the neolithic have been proposed  <cit> .
these hypotheses have been fuelled by the finding of significantly different frequency distributions of acetylation phenotypes between agriculturalists and hunter-gatherers, both in sub-saharan africa  <cit>  and globally at the worldwide scale  <cit> .
in addition, significant differential acetylation prevalence between sedentary agriculturalists and nomadic pastoralists has been reported in central asia  <cit> .
here, a higher proportion of slow acetylators was found among tajik agriculturalists than among kirghiz nomadic pastoralists.
these two distinct food-producing strategies also prevail among sympatric populations of the african sahel, but knowledge on nat <dig> genetic variation among african pastoral nomads was up to now very scarce, thus hindering a meaningful statistical analysis of the likely differences between food-producing subsistence modes in sub-saharan africa .
current research holds that the earliest food-producing communities in sub-saharan africa relied on nomadic cattle herding, while sedentary farming would be a more recent phenomenon .
as the southern part of the african continent was occupied rather recently by bantu herders, the most diversified pastoral societies live nowadays in the sahelian belt, and these are, from west to east , the moors, fulani, tuareg, tubu, zaghawa, nomadic arabs  and beja.
this region forms a unique ecosystem bordered by the saharan desert and the tropical rainforests, extending from east to west all across the continent.
its climate is characterized by annual cycles of wet and dry seasons allowing the co-existence of sedentary farmers and nomadic pastoralists who move with their livestock between wet- and dry-season pastures  <cit> .
in this study, we carried out an investigation of nat <dig> sequence variation among six sahelian populations relying either on nomadic pastoralism or on sedentary agriculture sampled in an area extending from western burkina faso to northeastern chad, in order to explicitly address the hypothesis that different selective pressures associated to these lifestyles acted on the evolution of nat <dig>  moreover, by combining our dataset with published african samples of nat <dig> sequences , we also address the hypothesis that selective pressures, stemming from an environmental factor linked to the ecoregion in which populations have been living , might also have shaped the evolution of this gene.table  <dig> african population samples studied for nat <dig> sequence variation
african americans2
1classification according to climatic zone and biome , based on  <cit>
2african americans: americans of african ancestry in southwestern us
3nd: not defined
RESULTS
nat <dig> diversity in the sahel
we analyzed a total number of  <dig> samples from six well-defined sahelian populations relying on two different modes of subsistence, namely pastoralism  and agriculture .
we completed this new dataset of nat <dig> sequences with those from other african population published samples .
for each of the  <dig> sahelian samples,  <dig>  base pairs  encompassing the 870 bp nat <dig> coding exon were successfully sequenced .
in total,  <dig> polymorphic positions were observed,  <dig> of which are located in the coding exon, and  <dig> being non-synonymous .fig.
<dig> schematic diagram of the nat <dig> locus on 8p <dig>  including the non-coding  <dig> bp-long exon  <dig>  the  <dig>  kb-long intronic region, and the  <dig> bp-long single protein-coding exon  <dig>  adapted from  <cit>  and  <cit> .
the first  and last positions  of the open reading frame  of exon  <dig> are indicated at the bottom of it, as well the relative positions of exon  <dig> and of the polyadenylation signals.
the positions of the  <dig> polymorphic sites  observed among the  <dig> individuals from the six sahelian samples sequenced in this study are shown as heavy-black  or light-gray  vertical bars below the diagram.
segments link the snps positions to the list of  <dig> haplotypes inferred from the combination of the  <dig> snps.
haplotypes’ associated acetylation activity  and average frequency among the six sahelian samples are shown on the left and right sides of the list, respectively.
a diagram displaying the position of the  <dig> snps observed in protein-coding exon  <dig> among the  <dig> african population samples  analyzed in this study is shown in additional file 3: figure s2
from the  <dig> single nucleotide polymorphisms  observed, a total of  <dig> haplotypes were inferred with the expectation-maximisation  algorithm of arlequin for the six sahelian populations .
among these, four haplotypes differed from known haplotypes at positions outside the coding exon, and were named with alphabetical suffixes , while their predicted effect on the enzyme’s activity was considered unchanged from the defining haplotype .
the em algorithm also led to the inference of three new haplotypes, and the following names were attributed by the official nat <dig> nomenclature committee : nat2*12n, nat2*13d and nat2*14k.
all three are defined by a new combination of recognized signature snps from the official nat <dig> gene nomenclature with other snps, so that enzymatic activity could not be predicted for any of these three haplotypes.table  <dig> haplotype frequencies and molecular diversity of the six sahelian samples in a  <dig>  bp sequence encompassing the nat <dig> coding exon
nat2*4
nat2*4a
nat2*12a
nat2*13a
nat2*5a
nat2*5b
nat2*5ba
3
nat2*5c
nat2*6a
nat2*6aa
3
nat2*6c
nat2*7b
nat2*14a
nat2*14b
nat2*14ba
3
nat2*6f
nat2*6o
nat2*12h
nat2*12n
4
nat2*13d
4
nat2*14k
4
nucleotide diversity  x 10−3
tajima’s d 5
<dig>
1population codes as in table 1
2reported activity in the official nat <dig> gene nomenclature
3small caps alphabetical suffixes were added to the names of haplotypes that differ from known haplotypes in the flanking region of the nat <dig> coding exon
4new haplotypes submitted to the official nat <dig> gene nomenclature and included in it
5
p-value associated with tajima’s d test for departure from selective neutrality: it is given as the proportion of random d values generated under the neutral equilibrium model that are smaller than, or equal to the observed value.
the sole significant result is shown in bold; it corresponds to a type i error rate of  <dig> , and it remains significant after bonferroni correction for multiple testing
frequency distributions of the  <dig> nat <dig> haplotypes are reported in table  <dig>  low-activity haplotype nat2*5b is the most common haplotype in all six populations .
it is followed by nat2*6a , also a low-activity haplotype.
thus, these two slow haplotypes together account for  <dig>  % of the gene copies in the total sample of our study.
further low-activity haplotypes detected in most of our samples include nat2*5c, *14a, and *14b, as well as *5a, *7b and 6c.
in turn, only three different fast haplotypes were detected in the sahelian samples , and these represent on average less than 18 % of the gene copies.
finally, six haplotypes could not be classified according to their effect on acetylation status.
their total frequency is of 3 % or less in the fulani samples, whereas it is of  <dig>  % and  <dig>  % in the kanembou and daza, respectively.
thus, when considering the whole  <dig>  bp sequenced segment,  <dig> to  <dig> segregating sites, and  <dig> to  <dig> haplotypes were observed in each of the six sahelian samples, and similar levels of diversity were estimated, as indicated by gene diversity  values ranging from  <dig>  to  <dig> , and nucleotide diversity  values ranging from  <dig>  to  <dig>  .
no departure from hardy-weinberg equilibrium, nor from selective neutrality and demographic equilibrium  was found .
on another hand, a significant departure from the neutral equilibrium model in favor of overdominant selection was found with tajima’s d test for the fulani nomads from the banfora area .
this value remained significant after bonferroni correction for multiple testing.
we note that in the five other tested populations, estimates of d were all positive although not significant.
nat <dig> diversity in african populations
the complete list of sixty-one nat <dig> haplotypes detected in the  <dig> african samples of coding-exon sequences is provided in additional file 3: figure s <dig> and additional file 4: table s <dig>  among these, seventeen haplotypes are newly described , and were assigned new names by the official nat <dig> nomenclature committee .
analysis of estimated diversity levels for the 870 bp nat <dig> coding exon evidenced substantial variation between populations, ranging from  <dig>  to  <dig>  for expected heterozygosity , and from  <dig>  to  <dig>  for nucleotide diversity  .
heterozygosity levels estimated for the six sahelian populations are located in the lower half of the distribution .
for nucleotide diversity instead, four of our sahelian samples display intermediate values , while the fulani from bongor display a slightly lower value , and the kanembou a slightly higher one .
however, these estimates are associated with large standard deviations thus precluding finding significant differences in diversity levels between populations.
this is especially the case of many of the published datasets, which include samples of less than  <dig> individuals.
in support of this, we found a high and significant correlation between sample size and number of haplotypes detected , and it remained significant even after removal of the four samples with sizes larger than  <dig> individuals .
substantial variation in frequency distributions of nat <dig> haplotypes was observed among african populations, particularly so for slow haplotypes nat2*5b and *6a, and the fast one nat2*12a .
the variance in haplotype frequency among african populations is highest for nat2*5b .
this low-activity haplotype is indeed generally more frequent in populations living in the sahel or in the dry savannas of east africa  than in populations living to the south of it , such as the yoruba or the pygmy populations .
nat2*6a, also a slow haplotype, displays a similar trend.
conversely, the fast haplotype nat2*12a displays a somewhat opposite trend, in that it is frequent in the pygmy populations and in the san as well , whereas its frequency is lower than 14 % in most sahelian and east african populations.
it is also rather frequent in those two yoruba samples of small size , but not in the larger sized ones .
when considering only the  <dig> bp of the nat <dig> coding exon, a positive and significant tajima’s d value was found again for the fulani from banfora, as well as for the fulani from ader, the fulani tested by mortensen et al.
<cit> , the mbuti pygmies, and the egyptians .
these five rejection cases result from an excess of intermediate frequency variants .
for the mbuti pygmies, a significant excess of observed heterozygosity was detected also with the ewens-watterson test , thus leading to the rejection of selective neutrality and demographic equilibrium.
however, none of these results remained significant after benjamini and hochberg false discovery rate  adjustment for multiple testing.
nat <dig> population structure in africa
to investigate for the presence of a genetic structure differentiating populations with distinct food-producing subsistence modes , analyses of population structure were performed on three subsets of the population data , namely:  on the african populations dataset ,  on the african food-producing populations dataset , and  on an african food-producing populations dataset comprising only those samples with size ≥  <dig> individuals .
taking into account the molecular diversity of nat <dig> sequences , the global level of population structure estimated for the african continent is of  <dig> % , which means that  <dig> % of the total genetic variation is attributed to differentiation among populations, while  <dig> % is due to differences among individuals within populations.
in the afr dataset,  <dig>  % of the pairwise genetic distances between populations were found to be significant at the  <dig> % level.
this proportion drops to  <dig>  % in the fp dataset, thus suggesting substantial nat <dig> genetic differentiation both among hunter-gatherer populations and between hunter-gatherer and food-producing societies.
consistently, the proportion of genetic variance explained by differences among populations drops to  <dig> % in the fp dataset, but it is still significant .
thus, while differentiation of hunter-gatherer populations contributes to nat <dig> population structure in the african continent, population structure among food-producing communities is also significant.
among the non-significant pairwise genetic distances, a rather large proportion could actually be due to lack of power in significance testing because of small sample sizes.
indeed, the proportion of significant distances increases again to  <dig> % in the fpls dataset, i.e., in the dataset that excludes food-producing population samples of less than  <dig> individuals.
in line with this, the proportion of genetic variance explained by differences among populations also raises to  <dig> % , thereby suggesting that the inclusion of samples of small size in the afr and fp analyses leads to a lowering of the power to differentiate populations.
most of the significant pairwise genetic distances in the fpls dataset differentiate the yoruba and akele populations from the others .
conversely, none of the six sahelian populations was found significantly differentiated from the others, irrespective of language affiliation or lifestyle, and most of the genetic distances between our samples and the east african samples were also found statistically not significant.
accordingly, no correlation of genetic distances with geographic distances was found for any of the afr and fp datasets , whereas the mantel test with the fpls dataset led to a significant negative correlation .
this somewhat surprising result stems from both a set of large genetic distances between populations located in close geographic proximity and a set of small genetic distances between populations geographically far apart , and does not fit the expectation of an isolation-by-distance model of evolution.
in line with this, no significant correlogram of moran’s i spatial autocorrelation indices was found with any of the nat <dig> haplotypes observed in africa, except for haplotype nat2*5c, a rather infrequent haplotype.
association of nat <dig> genetic structure with geography, culture, or climatic zone and biome
hierarchical analyses of molecular variance  were carried out to gain further insight into a possible association of the genetic structure of populations with factors related to their demographic and cultural history.
four categorization criteria were tested : geography, language, subsistence mode and ecoregion .
the results highlight a marked genetic structure associated with the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it .
indeed, with a classification of populations in ecoregions, Φct indices were found high and significant for all three data subsets  and greater than Φsc indices in all cases .
significant genetic structure was also detected with a classification according to subsistence strategy, but under this categorization criterion and for each of the three datasets, the Φct index is lower than the Φsc index, thereby meaning that more differentiation is found among populations in groups with matching subsistence mode than between those groups, even if only slightly more so.
finally, the results indicate that neither differentiation among geographic groups nor among linguistic groups does associate with the genetic structure of populations displayed by nat <dig> sequences, since Φct indices were never found significant for any of the afr, fp, or fpls datasets .table  <dig> analysis of molecular variance  under four criteria of classification of populations, based on the  <dig> bp long nat <dig> coding exon
dataset1
p-value3
p-value4
<dig> as described in methods, the three population data subsets are afr:  <dig> samples, excluding the americans of african ancestry ; fp:  <dig> samples of african food-producing populations; fpls:  <dig> samples of african food-producing populations with sample size ≥  <dig> individuals.
thus, the asw sample was not considered in any of the amova analyses
<dig> categories as in table  <dig> : classification according to geographical region , subsistence mode , linguistic affiliation , and ecoregion , namely climatic zone and biome, which defines the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it
<dig> significance of the Φct index and of the corresponding percentage of variation due to differences between groups.
significant p-values  are shown in bold, and adjusted p-values after bonferroni correction for multiple testing  are provided in brackets
<dig> significance of the Φsc index and of the corresponding percentage of variation due to differences between populations within groups.
significant p-values  are shown in bold, and adjusted p-values after bonferroni correction for multiple testing  are provided in brackets
<dig> only three geographical regions are considered here  because the fourth region  is represented by one population sample only
<dig> because variance components in amova are actually defined as covariances, negative values can occur  <cit> .
a negative Φct value would be expected if gene copies were more correlated between groups than between populations within groups.
however, none of the negative Φct values in the table are statistically significant, thus indicating that they are equal to zero
<dig> only two linguistic families are considered here  because the third family  is represented by one population sample only
the two-dimensional plots resulting from the nonmetric multidimensional scaling  analyses of genetic distances among populations are shown in fig.
<dig> for the afr dataset, and in additional file 11: figure s <dig> and additional file 12: figure s <dig> for the fp and fpls datasets, respectively.
in each of these figures, the mds plot is displayed four times, with populations being highlighted according to our four categorization criteria, namely geography, language, subsistence mode and biome.
consistent with the amova results, categorization according to the environment seems to better fit with the location of populations in the plots than any of the three other criteria .fig.
<dig> mds plot of pairwise reynolds genetic distances between the  <dig> populations of the afr dataset.
the stress value is  <dig> .
the same plot is reproduced  <dig> times, with populations color-coded according to:  geographical region,  linguistic affiliation,  subsistence mode, and  biome
nat <dig> phenotypes
the frequency of slow acetylators in each population sample was predicted from individuals’ nat <dig> genotypes .
predicted proportions of slow acetylators in african populations  are displayed on a map in fig.
<dig>  boxplots of predicted prevalence of slow acetylators  indicate higher median proportion of slow acetylators for pastoralists than for the other subsistence modes , although the variances of these proportions among populations are very large.
on another hand, slow acetylation is more frequent in populations living/dwelling in the sahelian belt or in the dry savanna surrounding it  than in populations living to the south of it , apparently irrespective of their subsistence strategy .
actually, crossing the information on subsistence mode with that on biome suggests higher slow acetylation in agricultural and hunter-gatherer populations living in the seasonally dry zones  than in those living in the humid tropical and equatorial zones.
this trend seems even more marked for agriculturalists when only population samples of at least  <dig> individuals are considered .fig.
<dig> map showing the frequency distributions of predicted nat <dig> phenotypes in african populations screened for sequence variation in the coding-exon .
map created with the qgis open source software  <cit> , with climatic zones defined according to  <cit>
fig.
<dig> boxplots of predicted prevalence of slow acetylators in populations classified according to subsistence mode and further to ecoregion.
each box extends from the first to the third quartile and displays the median , and the dispersion of populations’ individual values  is shown by dashed lines.
thirty-eight african populations ;  same dataset crossed with biome information, i.e., separating populations living in the seasonally dry zones  from those living in the humid tropical and equatorial zones;  same cross-analysis as in  but excluding population samples of less than  <dig> individuals
kruskal-wallis tests for homogeneity of slow acetylation frequency among groups corroborated these observations .
neither groups defined by geographic locations  nor groups defined by linguistic families  associate with differences in slow acetylation prevalence among populations.
in turn, a significant difference in the frequency of slow acetylators was found among populations relying on different subsistence strategies .
this difference remains both when hunter-gatherers and when small samples are excluded from the analysis .
when hunter-gatherers are excluded , only three subsistence modes are represented in the data, namely agriculturalists, agro-pastoralists, and pastoralists .
when small samples are excluded , only agriculturalists and pastoralists are represented, thus implying that slow acetylation prevalence is significantly higher among pastoralist than among agriculturalist populations.
these results were confirmed by pairwise wilcoxon tests of equality in average slow acetylation frequencies among subsistence strategies .
a significant difference in slow acetylation prevalence was consistently found between pastoralists and agriculturalists .
nevertheless, high prevalence of slow acetylation was also predicted for several agriculturalist populations from dry savanna regions, such as the kanembou  and luhya , thus suggesting a possible association of acetylation status with ecoregion, namely with the climatic zone and biome populations live in .
in line with this observation, a highly significant difference in average slow acetylation frequency was consistently found between populations living within the dry savanna biome or outside of it , irrespective of subsistence strategy.table  <dig> kruskal-wallis test for equality of frequency of the slow acetylation phenotype across geographical regions, linguistic families, subsistence modes, and climatic zones
dataset1
p-value3
fp5
<dig> as described in methods, the three population data subsets are afr:  <dig> samples, excluding the 1kg americans of african ancestry ; fp:  <dig> samples of african food-producing populations; fpls:  <dig> samples of african food-producing populations with sample size ≥  <dig> individuals.
thus, the asw sample was not considered in any of the kruskal-wallis tests
<dig> categories as in table  <dig> : classification according to geographical region , subsistence mode , linguistic affiliation , and ecoregion , namely climatic zone and biome, which defines the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it
<dig> significant p-values  are shown in bold, and adjusted p-values after bonferroni correction for multiple testing  are provided in brackets
<dig> only three geographical regions are considered here  because the fourth region  is represented by one population sample only
<dig> only two linguistic families are considered here  because the third family  is represented by one population sample only
single factor analyses of variance  corroborated the results of the kruskal-wallis rank sum tests in that predicted slow acetylation frequency is observed to differ significantly between populations classified both by ecoregion  and subsistence mode , but not by geography  or linguistic classification  .
moreover, ecoregion and subsistence mode were shown to be independently associated with differences in mean predicted slow acetylation frequency by multi-factor anovas, as no significant interaction between these two factors  was found.
finally, tukey’s honest significant difference  tests for multiple comparisons of means confirmed the significant difference in predicted frequency of slow acetylators between populations living/dwelling in seasonally dry zones  and those living in humid tropical and equatorial zones , as well as between populations relying on different subsistence modes, notably between those relying on pastoralism and those relying on agriculture .
taken together, our results thus indicate that factors associated both to the ecoregion in which populations have been living/dwelling and the mode of subsistence on which they have been relying independently explain a significant proportion of nat <dig> phenotypic diversity in africa.
discussion
in this study, we have generated  <dig>  bp of sequence encompassing the nat <dig> coding exon in  <dig> chromosomes sampled in six sahelian populations.
the activity of the enzyme encoded by this highly polymorphic gene is of clinical importance as it influences individuals’ physiological response to the absorption of numerous chemical compounds present in the diet and the environment, including several nowadays useful medications, and it is also involved in cancer development risk  <cit> .
five of the populations studied rely on pastoralism as a mode of subsistence, four of which are the fulani nomads from niger, burkina faso and chad, all speaking dialects of a niger-congo language   <cit> , whilst in the fifth one, the daza from chad, a nilo-saharan language  is spoken  <cit> .
the sixth population sample studied represents the kanembou, a sedentary population, also from chad, but relying on agriculture, and speaking a nilo-saharan language  <cit> .
intermediate to low nat <dig> molecular diversity in sahelian populations
twenty-one haplotypes were parsimoniously inferred for the six sahelian samples, of which three haplotypes not yet described to the best of our knowledge .
in order to compare our newly generated dataset with published nat <dig> sequences from other african samples, we restricted the analysis to the  <dig> bp of the nat <dig> single coding exon.
we have thus shown that levels of genetic diversity in the six sahelian populations are amidst the low to intermediate range of values in the scale of nat <dig> polymorphism known for the african continent, especially so when measured by expected heterozygosity .
this result could be due to faster genetic drift in these populations compared to others, which is congruent with the idea, based on analyses of polymorphisms in uniparentally transmitted dna  showing that some sahelian populations experienced a demographic contraction in their history  <cit> .
actually, in line with mitochondrial dna , nat <dig> diversity was found somewhat lower in the fulani pastoral groups than in the kanembou agriculturalists , consistent with recent genomic results that inferred smaller expansion rates for pastoral populations than for those having adopted agriculture  <cit> .
moreover, while nearly none of the ewens-watterson homozygosity or fu’s fs tests on nat <dig> variation rejected the null hypothesis of selective neutrality and demographic equilibrium, tajima’s d tests were found significantly positive for three fulani groups, although not anymore after correction for multiple testing.
nevertheless, for one of those fulani groups , tajima’s d was positive when tested on the entire sequenced segment of  <dig>  bp, and it remained significant after correction .
thus, this trend towards tajima’s d positive values could also point to a reduction in population size  <cit> , particularly so in the pastoral nomads.
however, irrespective of subsistence mode, most african populations analyzed here displayed the same trend towards positive d values, which are also expected if the gene is submitted to balancing selection.
the signature left by balancing selection should be detected by the ewens-watterson homozygosity test, since this selective regime should lead to an excess of heterozygotes, as has been shown for the human leukocyte antigen  loci  <cit> .
as already stated, among the  <dig> ewens-watterson tests performed on the complete population dataset, a single case of significant rejection of neutrality was found for the mbuti pygmies , because of heterozygosity in excess , but not anymore after adjustment for type i error rate.
while all other tests were not significant, the proportion of cases in which the observed heterozygosity was lower than the expected was of nearly 40% .
however, positive d values can also result from the presence, in the populations’ gene pools, of several molecularly rather distant haplotypes at frequencies higher than expected under neutrality.
such frequency distributions can develop if several standing variants become submitted to positive directional selection  <cit> .
this interpretation was favored in a recent study that screened variation of the entire nat <dig> locus sequence  in  <dig> world populations from the  <dig> genomes project  <cit> , but no pastoral population was represented in that collection.
the genetic structure of nat <dig> in africa is neither associated to geography nor to linguistics
consistent with previous studies  <cit> , we found a significant genetic structure of nat <dig> in africa .
here we further showed that this structure is not only attributable to the genetic differentiation of hunter-gatherer populations but is also found among african food-producing communities, as attested by the significant Φst value of  <dig>  % for the fpls dataset of  <dig> populations.
this significant genetic structure is mainly due to the variation in frequencies of three major haplotypes, two of which are categorized as decreased function alleles , which are particularly frequent in populations living in the dry savanna biome, and one fast allele , more frequently detected outside this region  than within it.
accordingly, both the amova  and mds analyses  indicate that neither differentiation among geographic groups nor among linguistic groups does associate with the genetic structure of populations displayed by nat <dig> sequences, contrarily to what is known for polymorphisms at other loci of the genome, such as mtdna  <cit> , y chromosome and classical polymorphisms  <cit> , or even for genome-wide variation  <cit> .
furthermore, the genetic structure of nat <dig> in africa does not strongly associate with subsistence strategy either, mainly because most sahelian and east african populations display little to no genetic differentiation between them , although both regions are populated by nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities.
actually, this low genetic differentiation between most sahelian and east african populations suggests a possible climate and biome link that is supported by the significant association of nat <dig> genetic structure with a classification of populations according to ecoregions.
however, in contrast to biological functions for which the link with environmental pressures is obvious, such as immunity and pathogens , a climate and biome related factor that would affect nat <dig> evolution, if any, remains to be determined  <cit> .
differences in slow acetylation prevalence across the southern sahelian limit
the frequency of slow acetylators in each population sample was predicted from individuals’ nat <dig> genotypes .
kruskal-wallis tests of homogeneity in proportions of slow acetylators among groups of populations did not evidence significant variation between geographic groups, nor between linguistic groups.
in turn, significant difference of slow acetylators were found between subsistence strategies, and specifically between pastoralist and agriculturalist populations .
considering only those populations for which sample sizes included at least  <dig> individuals, average frequency of slow acetylators among pastoralists is of  <dig>  % , while it is of  <dig>  %  among agriculturalists .
the higher standard deviation estimated for the latter group reflects a high variance in slow acetylator frequencies among agriculturalist populations such as the yoruba  and the kanembou .
by contrast, among pastoralists, these frequencies vary from more than  <dig> % in the daza to around  <dig> % in the somali and the fulani from ader.
for comparison, slow acetylators average to  <dig>  %  among hunter-gatherers.
similarly to the finding of a significant difference in proportions of acetylation phenotypes between sedentary farmers and nomadic pastoralists in central asia  <cit> , these results are compatible with the hypothesis of a differential evolution of nat <dig> acetylation capacity according to lifestyle among food-producing societies in africa.
it is thus tempting to suppose that the driving selective force behind this differential evolution is to be found in the development of dietary differences due to lifestyle.
polymorphisms in the nat <dig> gene or downstream from it and tagging its phenotypic expression have been recently identified by genome-wide association studies for their role in the metabolism of sugars and carbohydrates  <cit> , or in that of lipids  <cit> , thus making a strong case for dietary influences.
however, we have also found a significant difference in frequencies of the slow acetylator phenotype across the southern sahelian limit, thus showing that such difference is actually enclosed in the zones defined by climate and biome in which the populations are living.
irrespective of lifestyle , we found significantly higher proportions of slow acetylators in populations living in the sahelian and tropical with dry seasons zones than in those living in the humid tropical and equatorial zones .
multi-factor anovas further showed that no significant interaction between subsistence mode and ecoregion explained the observed differences in predicted mean slow acetylation frequency .
altogether, these results point to a possible independent influence of dietary habits on one hand , and of the chemical environment on the other hand , in the evolution of nat <dig> diversity.
thus, this hypothesis does not necessarily call for the existence of specific dietary components associated with subsistence modes, and hence has the advantage of being compatible with the opposite pattern of variation observed in sub-saharan africa with respect to central asia, where higher slow acetylation prevalence is observed in sedentary agriculturalists than in nomadic pastoralists  <cit> .
further population sampling will thus be needed to substantiate it.
at present, we can only acknowledge that the difference in slow acetylation frequency between african sedentary agriculturalist populations living within the dry savanna biome or outside of it was not found significant .
causative factors driving nat <dig> evolution
consistent with previous investigations on nat <dig> molecular variation  <cit>  , no statistically significant signal of selection was found in our datasets after correction type i error rate.
this suggests that if positive directional selection for slow-causing nat <dig> haplotypes in specific dietary and environmental conditions has been acting on the genetic evolution of populations, such selective pressures might have acted on multiple standing genetic variation rather than on specific new nat <dig> mutations  <cit> .
we acknowledge indeed that classical selective neutrality tests on population molecular data  are known to have little power to detect a signal of selection on standing genetic variation  <cit> .
thus, the possibility exists that the genetic structure of african food-producing populations inferred through the analysis of nat <dig> molecular diversity, which differentiates populations from distinct climatic zones, could result from differentiated selective pressures on standing variation related to the xenobiotic environment of distinct climate and biome zones, and explain the close genetic proximity of sahelian populations with east african populations independently of their lifestyle.
alternatively, archaeological evidence suggests that the sahel could have been first inhabited only by pastoralists, and adoption of farming by some populations would represent a recent, secondary change  <cit> .
thus the lack  of significant genetic structure of nat <dig> molecular variation among sahelian populations would be compatible with the hypothesis of differentiated selective pressures on standing variation related to xenobiotic intake of distinct dietary habits.
this hypothesis has the advantage of explaining the significant difference in frequencies of the slow acetylator phenotype observed between african pastoralists and agriculturalists.
consequently, either secondary adoption of farming by formerly pastoralist populations should also be postulated for some east african populations, such as the luhya from kenya , or a rather common occurrence of gene flow between pastoralists and agriculturalists populations in east africa should be envisioned.
this latter case is compatible with the complex history of east african populations evidenced for mitochondrial dna  <cit> , as well as at the genome-wide level .
finally, as discussed in  <cit> , it is also possible to envision that no selective constraint has acted on the evolution of nat <dig>  notably if the function of the enzyme encoded by this gene is redundant with other enzymes such as nat <dig>  in this case, the low genetic differentiation observed between sahelian and east african populations could be due to a recent common origin.
under this last hypothesis, we would expect a consistent pattern of variation of nat <dig> with the genetic structure inferred from other independent genetic markers in the genome, thus pointing to the demographic history of these populations rather than to specific selective pressures having acted on nat <dig> evolution.
this last alternative is not favored at present, given the patterns of variation known for other genomic segments, as discussed above, but it calls for further investigation because it would allow disentangling the effects of demographic and selective processes in the evolution of nat <dig>
our analysis suffers of course of some drawbacks.
firstly, as already stated, because it makes the simplistic assumption of a discrete categorization model of acetylation phenotypes  on the basis of genotypes, although heterogeneity in these categories has been recently demonstrated  <cit> .
for instance, acetylation status was found to vary significantly between the three genotypes formed by the two slow alleles nat2* <dig> and nat2* <dig>  with nat2* <dig> homozygotes being faster acetylators than nat2*5/nat2* <dig> heterozygotes, and the latter displaying faster acetylation than nat2* <dig> homozygotes.
in addition, the existence of variation in acetylation capacity due to polymorphisms outside the coding-exon of the nat <dig> gene is not considered here, although we know that regulatory polymorphisms could alter levels of protein expression and thereby acetylation activity.
besides, the existence of epistatic relationships between the expression of nat <dig> and its ortholog nat <dig> has been described  <cit> , thus further emphasizing the complexity of the acetylation function in the organism and its evolution.
secondly, the high level of polymorphism displayed by nat <dig> in human populations is probably very often underestimated with the samples sizes currently in use, as shown by the high and significant correlation of the latter with the number of alleles detected, at least for african populations .
interestingly, we found that this correlation holds both for slow-causing variants  and variants with unknown effect on phenotype as well , but not for fast acetylation alleles .
this last result suggests that many more slow-causing variants could exist, probably at low frequencies, which is compatible with the hypothesis of a relaxation of functional constraints on nat <dig> in the course of human evolution, notably in those populations that adopted a food-producing mode of subsistence, where these variants are more frequent.
thirdly, we should also consider that the discrete lifestyle categories in which we have classified our population samples  are rather gross approximations of much more complex subsistence strategies.
extant cultures are not mono-subsistent as their diets also rely on various complementary modes of food supply, such as fishing and gathering, and complex networks of food exchange are concurrently used  <cit> .
for instance, some groups among the kanembou agriculturalists from chad are specialized in hunting and gathering  <cit> .
notably, the collection of a wild grass species known as kreb and of spirulina algae known as dihe’ makes a substantial contribution to the diets of these peoples  <cit> .
at present unfortunately, to the best of our knowledge no accurate estimation of the relative dependence on various forms of subsistence is available for our samples.
finally, the ecological zones considered in our analyses have been shifting in the past, so that populations living in one zone today might have coped with a different environment several generations ago.
extreme environmental changes are documented specifically in the lake chad basin; the current extension of the lake is ~ <dig>  km <dig> but it is estimated that it was ~ <dig>  km <dig> some ten thousand years ago  <cit> .
in fact, with the onset of holocene, the southern sahara changed into a landscape rich in water resources, as evidenced by archaeological findings of items related to an aquatic life in places where we can barely imagine them today: in addition to direct evidence, such as hippopotamus and crocodile bones in dry river systems, there are findings of harpoons, which people used to hunt these animals  <cit> .
CONCLUSIONS
the pattern of variation of nat <dig> in sub-saharan african food-producing populations differs from that expected under a model of isolation-by-distance or from those observed with other genetic systems, but it is compatible with the hypothesis that it was shaped by selective pressures linked to the chemical environment in which populations evolved.
we have shown that it is possible that differences in xenobiotic environments associate with climates and biomes that oppose arid, seasonally dry regions, such as the sahel to tropical humid regions, such as around the gulf of guinea, hence explaining nat <dig> genetic and phenotypic differentiation across the southern sahelian limit.
however, the possibility exists that differences in xenobiotic environments interacting with nat <dig> could also result from differential dietary habits linked to subsistence modes.
under this hypothesis, the genetic similarity between eastern and central-western african populations from the sahel would then be explained by two non-mutually exclusive processes, namely significant gene flow across the sahel, or secondary shift from pastoralism to agriculturalism in those sahelian and east african populations that practice agriculture nowadays.
future studies including measurements of phenotypes and more sampling of populations  will certainly shed a new light on these conjectures.

 all along human evolution and continuing in present times, populations have developed a wide variety of cultural innovations. many of these may have affected the interactions between humans and their environment in such a way as to leave gene-culture coevolution imprints in the genome, due to both demographic and selective processes. among these innovations, new subsistence strategies represent major shifts with notable consequences on the pathogenic and dietary environments in which populations have been living, thus possibly also inducing new selective pressures on phenotypes. subsistence strategies are broadly classified in two opposite categories, i.e., foragers, relying on the collection of nutriments naturally occurring in the wild, and food-producers whose appearance in the prehistoric record is generally associated with the emergence of the neolithic. among the latter, two major modes of production are distinguished, namely agriculture based on the cultivation of domesticated plants, and pastoralism based on the herding of domesticated animals. these two modes of subsistence also imply differential usage of space, in that most agricultural societies developed sedentarism whereas communities relying on animal husbandry developed nomadism or transhumance to exploit seasonal variations in the location of pasture areas. it is currently held that these differential developments in subsistence strategies also influenced the selective regimes to which past populations were subjected, including through the action of new population-specific selective pressures induced by dietary changes. lactase persistence, a heritable condition in which the physiological ability to digest lactose is maintained throughout adulthood, probably represents the best-known example of an adaptation related to diet. indeed, convergent evolution of the trait has been demonstrated, and is explained by the emergence of similar selective pressures resulting from adopting a diet heavily relying on milk. however, demographic processes playing a significant role in the spread of the trait constitutes a valid, mutually non-exclusive alternative explanation. hence, disentangling the respective contributions of selective and demographic forces to extant levels and patterns of genetic differentiation between populations represents a challenging task. reduced arylamine n-acetyltransferase activity is another trait whose evolution was probably also shaped by differential, population-specific selective pressures. due to its early discovery linked to its major role in the treatment of tuberculosis with isoniazid, inherited variation in n-acetylation activity is currently one of the best known pharmacogenetic traits. the phenotype is driven by the existence of a polymorphic n-acetyltransferase enzyme. this cytosolic enzyme, encoded by the gene nat of chromosome is mainly expressed in the liver, small intestine and colon where it catalyzes a phase ii acetylation reaction, i.e., the transfer of an acetyl functional group to the terminal nitrogen of aromatic amines, heterocyclic amines, and hydrazines. hydrazines are used in the synthesis of numerous organic molecules, like the anti-tubercular agent isoniazid, while aromatic and heterocyclic amines are known to be produced, for instance, in meat and fish cooked at high temperatures, as well as in combustion smokes such as those from tobacco, grasses and wood chips. thus, the nat enzyme plays a crucial role in the detoxification of numerous xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. acetylation capacity can be measured by an individual’s response to drugs metabolized by the enzyme, and this phenotype is now known to depend on the individual’s genotype at the single coding exon of the nat gene. mutations in nat result indeed in diverse acetylation phenotypes that explain inter-individual variation in response to standard drug dose administration. moreover, mutations in nat may also act as risk factors for different types of cancers. the gene displays a high degree of polymorphism in humans, with alleles listed to date by the official consensus gene nomenclature of human nat alleles. inter-population variation in nat allele frequencies has been intensively documented, particularly so for those alleles defined by the combination of nucleotides at four major functional single nucleotide polymorphisms of the coding exon. although response to drug intake is a quantitative variable, the distribution of the phenotype in tested human groups was shown to be at least bi- modal. a simplified model of genotype-phenotype relationships was therefore adopted from the early studies onwards, in which those alleles considered as fully functional are responsible for the fast acetylator status, whereas decreased function alleles are responsible for the slow acetylator status. hence, acetylation status of an individual that is carrier of two fully functional alleles is classified as fast, that of a carrier of one fully functional allele and one allele with decreased function is classified as intermediate, and that of a carrier of two decreased function alleles as slow. also, many studies do not distinguish between fast and intermediate acetylators, categorizing both types of subjects as fast acetylators. similarly to lactase persistence, nat phenotypes prevalence varies substantially between populations, and hypotheses of gene-culture coevolution of nat polymorphisms have been put forwards, notably the idea that the slow acetylator phenotype became selectively advantageous in those populations adopting a food-producing mode of subsistence in the neolithic. indeed, given its role in the detoxification of numerous exogenic compounds, the nat enzyme acts at the interface between the organism and its chemical environment, and hence is a likely target for natural selection. however, in contrast to lactose for lactase persistence, a dietary or environmental causal factor driving nat evolution is not identified, but hypotheses involving meat consumption and availability in food supplied folates related to nutritional shifts during the neolithic have been proposed. these hypotheses have been fuelled by the finding of significantly different frequency distributions of acetylation phenotypes between agriculturalists and hunter-gatherers, both in sub-saharan africa and globally at the worldwide scale. in addition, significant differential acetylation prevalence between sedentary agriculturalists and nomadic pastoralists has been reported in central asia. here, a higher proportion of slow acetylators was found among tajik agriculturalists than among kirghiz nomadic pastoralists. these two distinct food-producing strategies also prevail among sympatric populations of the african sahel, but knowledge on nat genetic variation among african pastoral nomads was up to now very scarce, thus hindering a meaningful statistical analysis of the likely differences between food-producing subsistence modes in sub-saharan africa. current research holds that the earliest food-producing communities in sub-saharan africa relied on nomadic cattle herding, while sedentary farming would be a more recent phenomenon. as the southern part of the african continent was occupied rather recently by bantu herders, the most diversified pastoral societies live nowadays in the sahelian belt, and these are, from west to east, the moors, fulani, tuareg, tubu, zaghawa, nomadic arabs and beja. this region forms a unique ecosystem bordered by the saharan desert and the tropical rainforests, extending from east to west all across the continent. its climate is characterized by annual cycles of wet and dry seasons allowing the co-existence of sedentary farmers and nomadic pastoralists who move with their livestock between wet- and dry-season pastures. in this study, we carried out an investigation of nat sequence variation among six sahelian populations relying either on nomadic pastoralism or on sedentary agriculture sampled in an area extending from western burkina faso to northeastern chad, in order to explicitly address the hypothesis that different selective pressures associated to these lifestyles acted on the evolution of nat moreover, by combining our dataset with published african samples of nat sequences, we also address the hypothesis that selective pressures, stemming from an environmental factor linked to the ecoregion in which populations have been living, might also have shaped the evolution of this gene.table african population samples studied for nat sequence variation african americans2 1classification according to climatic zone and biome, based on 2african americans: americans of african ancestry in southwestern us 3nd: not defined  nat diversity in the sahel we analyzed a total number of samples from six well-defined sahelian populations relying on two different modes of subsistence, namely pastoralism and agriculture. we completed this new dataset of nat sequences with those from other african population published samples. for each of the sahelian samples, base pairs encompassing the 870 bp nat coding exon were successfully sequenced. in total, polymorphic positions were observed, of which are located in the coding exon, and being non-synonymous.fig. schematic diagram of the nat locus on 8p including the non-coding bp-long exon the kb-long intronic region, and the bp-long single protein-coding exon adapted from and. the first and last positions of the open reading frame of exon are indicated at the bottom of it, as well the relative positions of exon and of the polyadenylation signals. the positions of the polymorphic sites observed among the individuals from the six sahelian samples sequenced in this study are shown as heavy-black or light-gray vertical bars below the diagram. segments link the snps positions to the list of haplotypes inferred from the combination of the snps. haplotypes’ associated acetylation activity and average frequency among the six sahelian samples are shown on the left and right sides of the list, respectively. a diagram displaying the position of the snps observed in protein-coding exon among the african population samples analyzed in this study is shown in additional file 3: figure s2 from the single nucleotide polymorphisms observed, a total of haplotypes were inferred with the expectation-maximisation algorithm of arlequin for the six sahelian populations. among these, four haplotypes differed from known haplotypes at positions outside the coding exon, and were named with alphabetical suffixes, while their predicted effect on the enzyme’s activity was considered unchanged from the defining haplotype. the em algorithm also led to the inference of three new haplotypes, and the following names were attributed by the official nat nomenclature committee: nat2*12n, nat2*13d and nat2*14k. all three are defined by a new combination of recognized signature snps from the official nat gene nomenclature with other snps, so that enzymatic activity could not be predicted for any of these three haplotypes.table haplotype frequencies and molecular diversity of the six sahelian samples in a  bp sequence encompassing the nat coding exon nat2*4 nat2*4a nat2*12a nat2*13a nat2*5a nat2*5b nat2*5ba 3 nat2*5c nat2*6a nat2*6aa 3 nat2*6c nat2*7b nat2*14a nat2*14b nat2*14ba 3 nat2*6f nat2*6o nat2*12h nat2*12n 4 nat2*13d 4 nat2*14k 4 nucleotide diversity x 10−3 tajima’s d 5 1population codes as in table 1 2reported activity in the official nat gene nomenclature 3small caps alphabetical suffixes were added to the names of haplotypes that differ from known haplotypes in the flanking region of the nat coding exon 4new haplotypes submitted to the official nat gene nomenclature and included in it 5 p-value associated with tajima’s d test for departure from selective neutrality: it is given as the proportion of random d values generated under the neutral equilibrium model that are smaller than, or equal to the observed value. the sole significant result is shown in bold; it corresponds to a type i error rate of, and it remains significant after bonferroni correction for multiple testing frequency distributions of the nat haplotypes are reported in table  low-activity haplotype nat2*5b is the most common haplotype in all six populations. it is followed by nat2*6a, also a low-activity haplotype. thus, these two slow haplotypes together account for  % of the gene copies in the total sample of our study. further low-activity haplotypes detected in most of our samples include nat2*5c, *14a, and *14b, as well as *5a, *7b and 6c. in turn, only three different fast haplotypes were detected in the sahelian samples, and these represent on average less than 18 % of the gene copies. finally, six haplotypes could not be classified according to their effect on acetylation status. their total frequency is of 3 % or less in the fulani samples, whereas it is of  % and  % in the kanembou and daza, respectively. thus, when considering the whole  bp sequenced segment, to segregating sites, and to haplotypes were observed in each of the six sahelian samples, and similar levels of diversity were estimated, as indicated by gene diversity values ranging from to, and nucleotide diversity values ranging from to. no departure from hardy-weinberg equilibrium, nor from selective neutrality and demographic equilibrium was found. on another hand, a significant departure from the neutral equilibrium model in favor of overdominant selection was found with tajima’s d test for the fulani nomads from the banfora area. this value remained significant after bonferroni correction for multiple testing. we note that in the five other tested populations, estimates of d were all positive although not significant. nat diversity in african populations the complete list of sixty-one nat haplotypes detected in the african samples of coding-exon sequences is provided in additional file 3: figure s and additional file 4: table s among these, seventeen haplotypes are newly described, and were assigned new names by the official nat nomenclature committee. analysis of estimated diversity levels for the 870 bp nat coding exon evidenced substantial variation between populations, ranging from to for expected heterozygosity, and from to for nucleotide diversity. heterozygosity levels estimated for the six sahelian populations are located in the lower half of the distribution. for nucleotide diversity instead, four of our sahelian samples display intermediate values, while the fulani from bongor display a slightly lower value, and the kanembou a slightly higher one. however, these estimates are associated with large standard deviations thus precluding finding significant differences in diversity levels between populations. this is especially the case of many of the published datasets, which include samples of less than individuals. in support of this, we found a high and significant correlation between sample size and number of haplotypes detected, and it remained significant even after removal of the four samples with sizes larger than individuals. substantial variation in frequency distributions of nat haplotypes was observed among african populations, particularly so for slow haplotypes nat2*5b and *6a, and the fast one nat2*12a. the variance in haplotype frequency among african populations is highest for nat2*5b. this low-activity haplotype is indeed generally more frequent in populations living in the sahel or in the dry savannas of east africa than in populations living to the south of it, such as the yoruba or the pygmy populations. nat2*6a, also a slow haplotype, displays a similar trend. conversely, the fast haplotype nat2*12a displays a somewhat opposite trend, in that it is frequent in the pygmy populations and in the san as well, whereas its frequency is lower than 14 % in most sahelian and east african populations. it is also rather frequent in those two yoruba samples of small size, but not in the larger sized ones. when considering only the bp of the nat coding exon, a positive and significant tajima’s d value was found again for the fulani from banfora, as well as for the fulani from ader, the fulani tested by mortensen et al., the mbuti pygmies, and the egyptians. these five rejection cases result from an excess of intermediate frequency variants. for the mbuti pygmies, a significant excess of observed heterozygosity was detected also with the ewens-watterson test, thus leading to the rejection of selective neutrality and demographic equilibrium. however, none of these results remained significant after benjamini and hochberg false discovery rate adjustment for multiple testing. nat population structure in africa to investigate for the presence of a genetic structure differentiating populations with distinct food-producing subsistence modes, analyses of population structure were performed on three subsets of the population data, namely: on the african populations dataset, on the african food-producing populations dataset, and on an african food-producing populations dataset comprising only those samples with size ≥ individuals. taking into account the molecular diversity of nat sequences, the global level of population structure estimated for the african continent is of %, which means that % of the total genetic variation is attributed to differentiation among populations, while % is due to differences among individuals within populations. in the afr dataset, % of the pairwise genetic distances between populations were found to be significant at the % level. this proportion drops to % in the fp dataset, thus suggesting substantial nat genetic differentiation both among hunter-gatherer populations and between hunter-gatherer and food-producing societies. consistently, the proportion of genetic variance explained by differences among populations drops to % in the fp dataset, but it is still significant. thus, while differentiation of hunter-gatherer populations contributes to nat population structure in the african continent, population structure among food-producing communities is also significant. among the non-significant pairwise genetic distances, a rather large proportion could actually be due to lack of power in significance testing because of small sample sizes. indeed, the proportion of significant distances increases again to % in the fpls dataset, i.e., in the dataset that excludes food-producing population samples of less than individuals. in line with this, the proportion of genetic variance explained by differences among populations also raises to %, thereby suggesting that the inclusion of samples of small size in the afr and fp analyses leads to a lowering of the power to differentiate populations. most of the significant pairwise genetic distances in the fpls dataset differentiate the yoruba and akele populations from the others. conversely, none of the six sahelian populations was found significantly differentiated from the others, irrespective of language affiliation or lifestyle, and most of the genetic distances between our samples and the east african samples were also found statistically not significant. accordingly, no correlation of genetic distances with geographic distances was found for any of the afr and fp datasets, whereas the mantel test with the fpls dataset led to a significant negative correlation. this somewhat surprising result stems from both a set of large genetic distances between populations located in close geographic proximity and a set of small genetic distances between populations geographically far apart, and does not fit the expectation of an isolation-by-distance model of evolution. in line with this, no significant correlogram of moran’s i spatial autocorrelation indices was found with any of the nat haplotypes observed in africa, except for haplotype nat2*5c, a rather infrequent haplotype. association of nat genetic structure with geography, culture, or climatic zone and biome hierarchical analyses of molecular variance were carried out to gain further insight into a possible association of the genetic structure of populations with factors related to their demographic and cultural history. four categorization criteria were tested: geography, language, subsistence mode and ecoregion. the results highlight a marked genetic structure associated with the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it. indeed, with a classification of populations in ecoregions, Φct indices were found high and significant for all three data subsets and greater than Φsc indices in all cases. significant genetic structure was also detected with a classification according to subsistence strategy, but under this categorization criterion and for each of the three datasets, the Φct index is lower than the Φsc index, thereby meaning that more differentiation is found among populations in groups with matching subsistence mode than between those groups, even if only slightly more so. finally, the results indicate that neither differentiation among geographic groups nor among linguistic groups does associate with the genetic structure of populations displayed by nat sequences, since Φct indices were never found significant for any of the afr, fp, or fpls datasets.table analysis of molecular variance under four criteria of classification of populations, based on the bp long nat coding exon dataset1 p-value3 p-value4 as described in methods, the three population data subsets are afr: samples, excluding the americans of african ancestry; fp: samples of african food-producing populations; fpls: samples of african food-producing populations with sample size ≥ individuals. thus, the asw sample was not considered in any of the amova analyses categories as in table : classification according to geographical region, subsistence mode, linguistic affiliation, and ecoregion, namely climatic zone and biome, which defines the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it significance of the Φct index and of the corresponding percentage of variation due to differences between groups. significant p-values are shown in bold, and adjusted p-values after bonferroni correction for multiple testing are provided in brackets significance of the Φsc index and of the corresponding percentage of variation due to differences between populations within groups. significant p-values are shown in bold, and adjusted p-values after bonferroni correction for multiple testing are provided in brackets only three geographical regions are considered here because the fourth region is represented by one population sample only because variance components in amova are actually defined as covariances, negative values can occur. a negative Φct value would be expected if gene copies were more correlated between groups than between populations within groups. however, none of the negative Φct values in the table are statistically significant, thus indicating that they are equal to zero only two linguistic families are considered here because the third family is represented by one population sample only the two-dimensional plots resulting from the nonmetric multidimensional scaling analyses of genetic distances among populations are shown in fig.  for the afr dataset, and in additional file 11: figure s and additional file 12: figure s for the fp and fpls datasets, respectively. in each of these figures, the mds plot is displayed four times, with populations being highlighted according to our four categorization criteria, namely geography, language, subsistence mode and biome. consistent with the amova results, categorization according to the environment seems to better fit with the location of populations in the plots than any of the three other criteria.fig. mds plot of pairwise reynolds genetic distances between the populations of the afr dataset. the stress value is. the same plot is reproduced times, with populations color-coded according to: geographical region, linguistic affiliation, subsistence mode, and biome nat phenotypes the frequency of slow acetylators in each population sample was predicted from individuals’ nat genotypes. predicted proportions of slow acetylators in african populations are displayed on a map in fig.  boxplots of predicted prevalence of slow acetylators indicate higher median proportion of slow acetylators for pastoralists than for the other subsistence modes, although the variances of these proportions among populations are very large. on another hand, slow acetylation is more frequent in populations living/dwelling in the sahelian belt or in the dry savanna surrounding it than in populations living to the south of it, apparently irrespective of their subsistence strategy. actually, crossing the information on subsistence mode with that on biome suggests higher slow acetylation in agricultural and hunter-gatherer populations living in the seasonally dry zones than in those living in the humid tropical and equatorial zones. this trend seems even more marked for agriculturalists when only population samples of at least individuals are considered.fig. map showing the frequency distributions of predicted nat phenotypes in african populations screened for sequence variation in the coding-exon. map created with the qgis open source software, with climatic zones defined according to fig. boxplots of predicted prevalence of slow acetylators in populations classified according to subsistence mode and further to ecoregion. each box extends from the first to the third quartile and displays the median, and the dispersion of populations’ individual values is shown by dashed lines. thirty-eight african populations; same dataset crossed with biome information, i.e., separating populations living in the seasonally dry zones from those living in the humid tropical and equatorial zones; same cross-analysis as in but excluding population samples of less than individuals kruskal-wallis tests for homogeneity of slow acetylation frequency among groups corroborated these observations. neither groups defined by geographic locations nor groups defined by linguistic families associate with differences in slow acetylation prevalence among populations. in turn, a significant difference in the frequency of slow acetylators was found among populations relying on different subsistence strategies. this difference remains both when hunter-gatherers and when small samples are excluded from the analysis. when hunter-gatherers are excluded, only three subsistence modes are represented in the data, namely agriculturalists, agro-pastoralists, and pastoralists. when small samples are excluded, only agriculturalists and pastoralists are represented, thus implying that slow acetylation prevalence is significantly higher among pastoralist than among agriculturalist populations. these results were confirmed by pairwise wilcoxon tests of equality in average slow acetylation frequencies among subsistence strategies. a significant difference in slow acetylation prevalence was consistently found between pastoralists and agriculturalists. nevertheless, high prevalence of slow acetylation was also predicted for several agriculturalist populations from dry savanna regions, such as the kanembou and luhya, thus suggesting a possible association of acetylation status with ecoregion, namely with the climatic zone and biome populations live in. in line with this observation, a highly significant difference in average slow acetylation frequency was consistently found between populations living within the dry savanna biome or outside of it, irrespective of subsistence strategy.table kruskal-wallis test for equality of frequency of the slow acetylation phenotype across geographical regions, linguistic families, subsistence modes, and climatic zones dataset1 p-value3 fp5 as described in methods, the three population data subsets are afr: samples, excluding the 1kg americans of african ancestry; fp: samples of african food-producing populations; fpls: samples of african food-producing populations with sample size ≥ individuals. thus, the asw sample was not considered in any of the kruskal-wallis tests categories as in table : classification according to geographical region, subsistence mode, linguistic affiliation, and ecoregion, namely climatic zone and biome, which defines the fourth categorization criterion that considers whether populations live within the dry savanna biome or outside of it significant p-values are shown in bold, and adjusted p-values after bonferroni correction for multiple testing are provided in brackets only three geographical regions are considered here because the fourth region is represented by one population sample only only two linguistic families are considered here because the third family is represented by one population sample only single factor analyses of variance corroborated the results of the kruskal-wallis rank sum tests in that predicted slow acetylation frequency is observed to differ significantly between populations classified both by ecoregion and subsistence mode, but not by geography or linguistic classification. moreover, ecoregion and subsistence mode were shown to be independently associated with differences in mean predicted slow acetylation frequency by multi-factor anovas, as no significant interaction between these two factors was found. finally, tukey’s honest significant difference tests for multiple comparisons of means confirmed the significant difference in predicted frequency of slow acetylators between populations living/dwelling in seasonally dry zones and those living in humid tropical and equatorial zones, as well as between populations relying on different subsistence modes, notably between those relying on pastoralism and those relying on agriculture. taken together, our results thus indicate that factors associated both to the ecoregion in which populations have been living/dwelling and the mode of subsistence on which they have been relying independently explain a significant proportion of nat phenotypic diversity in africa. discussion in this study, we have generated  bp of sequence encompassing the nat coding exon in chromosomes sampled in six sahelian populations. the activity of the enzyme encoded by this highly polymorphic gene is of clinical importance as it influences individuals’ physiological response to the absorption of numerous chemical compounds present in the diet and the environment, including several nowadays useful medications, and it is also involved in cancer development risk. five of the populations studied rely on pastoralism as a mode of subsistence, four of which are the fulani nomads from niger, burkina faso and chad, all speaking dialects of a niger-congo language, whilst in the fifth one, the daza from chad, a nilo-saharan language is spoken. the sixth population sample studied represents the kanembou, a sedentary population, also from chad, but relying on agriculture, and speaking a nilo-saharan language. intermediate to low nat molecular diversity in sahelian populations twenty-one haplotypes were parsimoniously inferred for the six sahelian samples, of which three haplotypes not yet described to the best of our knowledge. in order to compare our newly generated dataset with published nat sequences from other african samples, we restricted the analysis to the bp of the nat single coding exon. we have thus shown that levels of genetic diversity in the six sahelian populations are amidst the low to intermediate range of values in the scale of nat polymorphism known for the african continent, especially so when measured by expected heterozygosity. this result could be due to faster genetic drift in these populations compared to others, which is congruent with the idea, based on analyses of polymorphisms in uniparentally transmitted dna showing that some sahelian populations experienced a demographic contraction in their history. actually, in line with mitochondrial dna, nat diversity was found somewhat lower in the fulani pastoral groups than in the kanembou agriculturalists, consistent with recent genomic results that inferred smaller expansion rates for pastoral populations than for those having adopted agriculture. moreover, while nearly none of the ewens-watterson homozygosity or fu’s fs tests on nat variation rejected the null hypothesis of selective neutrality and demographic equilibrium, tajima’s d tests were found significantly positive for three fulani groups, although not anymore after correction for multiple testing. nevertheless, for one of those fulani groups, tajima’s d was positive when tested on the entire sequenced segment of  bp, and it remained significant after correction. thus, this trend towards tajima’s d positive values could also point to a reduction in population size, particularly so in the pastoral nomads. however, irrespective of subsistence mode, most african populations analyzed here displayed the same trend towards positive d values, which are also expected if the gene is submitted to balancing selection. the signature left by balancing selection should be detected by the ewens-watterson homozygosity test, since this selective regime should lead to an excess of heterozygotes, as has been shown for the human leukocyte antigen loci. as already stated, among the ewens-watterson tests performed on the complete population dataset, a single case of significant rejection of neutrality was found for the mbuti pygmies, because of heterozygosity in excess, but not anymore after adjustment for type i error rate. while all other tests were not significant, the proportion of cases in which the observed heterozygosity was lower than the expected was of nearly 40%. however, positive d values can also result from the presence, in the populations’ gene pools, of several molecularly rather distant haplotypes at frequencies higher than expected under neutrality. such frequency distributions can develop if several standing variants become submitted to positive directional selection. this interpretation was favored in a recent study that screened variation of the entire nat locus sequence in world populations from the genomes project, but no pastoral population was represented in that collection. the genetic structure of nat in africa is neither associated to geography nor to linguistics consistent with previous studies, we found a significant genetic structure of nat in africa. here we further showed that this structure is not only attributable to the genetic differentiation of hunter-gatherer populations but is also found among african food-producing communities, as attested by the significant Φst value of % for the fpls dataset of populations. this significant genetic structure is mainly due to the variation in frequencies of three major haplotypes, two of which are categorized as decreased function alleles, which are particularly frequent in populations living in the dry savanna biome, and one fast allele, more frequently detected outside this region than within it. accordingly, both the amova and mds analyses indicate that neither differentiation among geographic groups nor among linguistic groups does associate with the genetic structure of populations displayed by nat sequences, contrarily to what is known for polymorphisms at other loci of the genome, such as mtdna, y chromosome and classical polymorphisms, or even for genome-wide variation. furthermore, the genetic structure of nat in africa does not strongly associate with subsistence strategy either, mainly because most sahelian and east african populations display little to no genetic differentiation between them, although both regions are populated by nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities. actually, this low genetic differentiation between most sahelian and east african populations suggests a possible climate and biome link that is supported by the significant association of nat genetic structure with a classification of populations according to ecoregions. however, in contrast to biological functions for which the link with environmental pressures is obvious, such as immunity and pathogens, a climate and biome related factor that would affect nat evolution, if any, remains to be determined. differences in slow acetylation prevalence across the southern sahelian limit the frequency of slow acetylators in each population sample was predicted from individuals’ nat genotypes. kruskal-wallis tests of homogeneity in proportions of slow acetylators among groups of populations did not evidence significant variation between geographic groups, nor between linguistic groups. in turn, significant difference of slow acetylators were found between subsistence strategies, and specifically between pastoralist and agriculturalist populations. considering only those populations for which sample sizes included at least individuals, average frequency of slow acetylators among pastoralists is of %, while it is of % among agriculturalists. the higher standard deviation estimated for the latter group reflects a high variance in slow acetylator frequencies among agriculturalist populations such as the yoruba and the kanembou. by contrast, among pastoralists, these frequencies vary from more than % in the daza to around % in the somali and the fulani from ader. for comparison, slow acetylators average to % among hunter-gatherers. similarly to the finding of a significant difference in proportions of acetylation phenotypes between sedentary farmers and nomadic pastoralists in central asia, these results are compatible with the hypothesis of a differential evolution of nat acetylation capacity according to lifestyle among food-producing societies in africa. it is thus tempting to suppose that the driving selective force behind this differential evolution is to be found in the development of dietary differences due to lifestyle. polymorphisms in the nat gene or downstream from it and tagging its phenotypic expression have been recently identified by genome-wide association studies for their role in the metabolism of sugars and carbohydrates, or in that of lipids, thus making a strong case for dietary influences. however, we have also found a significant difference in frequencies of the slow acetylator phenotype across the southern sahelian limit, thus showing that such difference is actually enclosed in the zones defined by climate and biome in which the populations are living. irrespective of lifestyle, we found significantly higher proportions of slow acetylators in populations living in the sahelian and tropical with dry seasons zones than in those living in the humid tropical and equatorial zones. multi-factor anovas further showed that no significant interaction between subsistence mode and ecoregion explained the observed differences in predicted mean slow acetylation frequency. altogether, these results point to a possible independent influence of dietary habits on one hand, and of the chemical environment on the other hand, in the evolution of nat diversity. thus, this hypothesis does not necessarily call for the existence of specific dietary components associated with subsistence modes, and hence has the advantage of being compatible with the opposite pattern of variation observed in sub-saharan africa with respect to central asia, where higher slow acetylation prevalence is observed in sedentary agriculturalists than in nomadic pastoralists. further population sampling will thus be needed to substantiate it. at present, we can only acknowledge that the difference in slow acetylation frequency between african sedentary agriculturalist populations living within the dry savanna biome or outside of it was not found significant. causative factors driving nat evolution consistent with previous investigations on nat molecular variation, no statistically significant signal of selection was found in our datasets after correction type i error rate. this suggests that if positive directional selection for slow-causing nat haplotypes in specific dietary and environmental conditions has been acting on the genetic evolution of populations, such selective pressures might have acted on multiple standing genetic variation rather than on specific new nat mutations. we acknowledge indeed that classical selective neutrality tests on population molecular data are known to have little power to detect a signal of selection on standing genetic variation. thus, the possibility exists that the genetic structure of african food-producing populations inferred through the analysis of nat molecular diversity, which differentiates populations from distinct climatic zones, could result from differentiated selective pressures on standing variation related to the xenobiotic environment of distinct climate and biome zones, and explain the close genetic proximity of sahelian populations with east african populations independently of their lifestyle. alternatively, archaeological evidence suggests that the sahel could have been first inhabited only by pastoralists, and adoption of farming by some populations would represent a recent, secondary change. thus the lack of significant genetic structure of nat molecular variation among sahelian populations would be compatible with the hypothesis of differentiated selective pressures on standing variation related to xenobiotic intake of distinct dietary habits. this hypothesis has the advantage of explaining the significant difference in frequencies of the slow acetylator phenotype observed between african pastoralists and agriculturalists. consequently, either secondary adoption of farming by formerly pastoralist populations should also be postulated for some east african populations, such as the luhya from kenya, or a rather common occurrence of gene flow between pastoralists and agriculturalists populations in east africa should be envisioned. this latter case is compatible with the complex history of east african populations evidenced for mitochondrial dna, as well as at the genome-wide level. finally, as discussed in, it is also possible to envision that no selective constraint has acted on the evolution of nat notably if the function of the enzyme encoded by this gene is redundant with other enzymes such as nat in this case, the low genetic differentiation observed between sahelian and east african populations could be due to a recent common origin. under this last hypothesis, we would expect a consistent pattern of variation of nat with the genetic structure inferred from other independent genetic markers in the genome, thus pointing to the demographic history of these populations rather than to specific selective pressures having acted on nat evolution. this last alternative is not favored at present, given the patterns of variation known for other genomic segments, as discussed above, but it calls for further investigation because it would allow disentangling the effects of demographic and selective processes in the evolution of nat our analysis suffers of course of some drawbacks. firstly, as already stated, because it makes the simplistic assumption of a discrete categorization model of acetylation phenotypes on the basis of genotypes, although heterogeneity in these categories has been recently demonstrated. for instance, acetylation status was found to vary significantly between the three genotypes formed by the two slow alleles nat2* and nat2* with nat2* homozygotes being faster acetylators than nat2*5/nat2* heterozygotes, and the latter displaying faster acetylation than nat2* homozygotes. in addition, the existence of variation in acetylation capacity due to polymorphisms outside the coding-exon of the nat gene is not considered here, although we know that regulatory polymorphisms could alter levels of protein expression and thereby acetylation activity. besides, the existence of epistatic relationships between the expression of nat and its ortholog nat has been described, thus further emphasizing the complexity of the acetylation function in the organism and its evolution. secondly, the high level of polymorphism displayed by nat in human populations is probably very often underestimated with the samples sizes currently in use, as shown by the high and significant correlation of the latter with the number of alleles detected, at least for african populations. interestingly, we found that this correlation holds both for slow-causing variants and variants with unknown effect on phenotype as well, but not for fast acetylation alleles. this last result suggests that many more slow-causing variants could exist, probably at low frequencies, which is compatible with the hypothesis of a relaxation of functional constraints on nat in the course of human evolution, notably in those populations that adopted a food-producing mode of subsistence, where these variants are more frequent. thirdly, we should also consider that the discrete lifestyle categories in which we have classified our population samples are rather gross approximations of much more complex subsistence strategies. extant cultures are not mono-subsistent as their diets also rely on various complementary modes of food supply, such as fishing and gathering, and complex networks of food exchange are concurrently used. for instance, some groups among the kanembou agriculturalists from chad are specialized in hunting and gathering. notably, the collection of a wild grass species known as kreb and of spirulina algae known as dihe’ makes a substantial contribution to the diets of these peoples. at present unfortunately, to the best of our knowledge no accurate estimation of the relative dependence on various forms of subsistence is available for our samples. finally, the ecological zones considered in our analyses have been shifting in the past, so that populations living in one zone today might have coped with a different environment several generations ago. extreme environmental changes are documented specifically in the lake chad basin; the current extension of the lake is ~  km but it is estimated that it was ~  km some ten thousand years ago. in fact, with the onset of holocene, the southern sahara changed into a landscape rich in water resources, as evidenced by archaeological findings of items related to an aquatic life in places where we can barely imagine them today: in addition to direct evidence, such as hippopotamus and crocodile bones in dry river systems, there are findings of harpoons, which people used to hunt these animals.  the pattern of variation of nat in sub-saharan african food-producing populations differs from that expected under a model of isolation-by-distance or from those observed with other genetic systems, but it is compatible with the hypothesis that it was shaped by selective pressures linked to the chemical environment in which populations evolved. we have shown that it is possible that differences in xenobiotic environments associate with climates and biomes that oppose arid, seasonally dry regions, such as the sahel to tropical humid regions, such as around the gulf of guinea, hence explaining nat genetic and phenotypic differentiation across the southern sahelian limit. however, the possibility exists that differences in xenobiotic environments interacting with nat could also result from differential dietary habits linked to subsistence modes. under this hypothesis, the genetic similarity between eastern and central-western african populations from the sahel would then be explained by two non-mutually exclusive processes, namely significant gene flow across the sahel, or secondary shift from pastoralism to agriculturalism in those sahelian and east african populations that practice agriculture nowadays. future studies including measurements of phenotypes and more sampling of populations will certainly shed a new light on these conjectures. 
furthermore, we found significantly higher predicted proportions of slow acetylators in pastoralists than in agriculturalists, but also among food-producing populations living in the sahelian and dry savanna zones than in those living in humid environments, irrespective of their mode of subsistence. here we addressed the hypothesis of different selective pressures associated to the agriculturalist or pastoralist lifestyles having acted on the evolution of nat by sequencing the gene in individuals from five pastoralist and one agriculturalist sahelian populations. dietary changes associated to shifts in subsistence strategies during human evolution may have induced new selective pressures on phenotypes, as currently held for lactase persistence. this genetic structure does associate more strongly with a classification of populations according to ecoregions than to subsistence strategies, mainly because most sahelian and east african populations display little to no genetic differentiation between them, although both regions hold nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities. these two subsistence strategies also prevail among sympatric populations of the african sahel, but knowledge on nat variation among african pastoral nomads was up to now very scarce. we show that the significant nat genetic structure of african populations is mainly due to frequency differences of three major haplotypes, two of which are categorized as decreased function alleles, particularly common in populations living in arid environments, and one fast allele, more frequently detected in populations living in tropical humid environments. the environmental causative factor driving its evolution is as yet unknown, but significant differences in prevalence of acetylation phenotypes are found between hunter-gatherer and food-producing populations, both in sub-saharan africa and worldwide, and between agriculturalists and pastoralists in central asia. our results suggest a possible independent influence of both the dietary habits associated with subsistence modes and the chemical environment associated with climatic zones and biomes on the evolution of nat diversity in sub-saharan african populations. similar hypotheses exist for arylamine n-acetyltransferase mediated acetylation capacity, a well-known pharmacogenetic trait with wide inter-individual variation explained by polymorphisms in the nat gene. nat2acetylation polymorphismafrican sahelpastoral nomadssubsistence modeecoregionnatural selectionissue-copyright-statement© the author 2015
 dietary changes associated to shifts in subsistence strategies during human evolution may have induced new selective pressures on phenotypes, as currently held for lactase persistence. similar hypotheses exist for arylamine n-acetyltransferase mediated acetylation capacity, a well-known pharmacogenetic trait with wide inter-individual variation explained by polymorphisms in the nat gene. the environmental causative factor driving its evolution is as yet unknown, but significant differences in prevalence of acetylation phenotypes are found between hunter-gatherer and food-producing populations, both in sub-saharan africa and worldwide, and between agriculturalists and pastoralists in central asia. these two subsistence strategies also prevail among sympatric populations of the african sahel, but knowledge on nat variation among african pastoral nomads was up to now very scarce. here we addressed the hypothesis of different selective pressures associated to the agriculturalist or pastoralist lifestyles having acted on the evolution of nat by sequencing the gene in individuals from five pastoralist and one agriculturalist sahelian populations.  we show that the significant nat genetic structure of african populations is mainly due to frequency differences of three major haplotypes, two of which are categorized as decreased function alleles, particularly common in populations living in arid environments, and one fast allele, more frequently detected in populations living in tropical humid environments. this genetic structure does associate more strongly with a classification of populations according to ecoregions than to subsistence strategies, mainly because most sahelian and east african populations display little to no genetic differentiation between them, although both regions hold nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities. furthermore, we found significantly higher predicted proportions of slow acetylators in pastoralists than in agriculturalists, but also among food-producing populations living in the sahelian and dry savanna zones than in those living in humid environments, irrespective of their mode of subsistence.  our results suggest a possible independent influence of both the dietary habits associated with subsistence modes and the chemical environment associated with climatic zones and biomes on the evolution of nat diversity in sub-saharan african populations. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nat2acetylation polymorphismafrican sahelpastoral nomadssubsistence modeecoregionnatural selectionissue-copyright-statement© the author 2015 