BACKGROUND
the identification of signatures of positive selection in genes is a central theme in molecular evolution  <cit> .
the relative contribution of neutral and adaptive evolution is the subject of ongoing debate .
a powerful approach to identify positive selection in coding sequences is the comparison of substitutions and polymorphic sites using the mcdonald and kreitman  test  <cit> .
this test builds on previously developed approaches to detect deviations from neutral evolution using divergence and polymorphism data  <cit> .
the mk test compares the number of non-synonymous  and synonymous  substitutions in a pair of species to the number of non-synonymous  and synonymous  polymorphisms in one of the species for the same coding sequence.
positively selected mutations reach fixation very rapidly, and thus increase non-synonymous divergence; however, they are rarely observed as polymorphic sites.
as a result, if positive selection has played a significant role in the evolution of a gene, the ratio between non-synonymous and synonymous substitutions is expected to be larger than the ratio between non-synonymous and synonymous polymorphisms.
the first ratio divided by the second ratio has been named the fixation index   <cit> .
fi values larger than  <dig> indicate positive selection in the lineage of interest, and values lower than  <dig> indicate an excess of non-synonymous polymorphisms in the current population.
related statistics are the neutrality index   <cit> , direction of selection   <cit>  and the proportion of adaptive substitutions   <cit> .
several authors have used the mk test to estimate the importance of positive selection in diverse genomes, with contrasting results depending on the species studied  <cit> .
in most studies of humans, the estimated proportion of adaptive amino acid substitutions is very low .
one likely explanation is the presence of slightly deleterious segregating mutations caused by reduced effective population size, resulting in inflated non-synonymous snps relative to non-synonymous substitutions  <cit> .
most deleterious mutations are expected to be present at low frequencies in the population; therefore, removing low frequency variants should improve the estimation of fi and other similar statistics  <cit> .
other approaches to identify signatures of natural selection are based on the comparison of non-synonymous and synonymous substitutions in different branches of a tree obtained from a set of homologous sequences.
using data from several species has the advantage that we can target genes that show lineage-specific positive selection and discard genes that are also evolving under positive selection in related lineages, which is especially relevant when searching for species-specific traits.
genome-wide studies have shown that the pace of evolution of a protein often varies substantially in different branches of a tree , which is in agreement with the so-called over-dispersion of the molecular clock  <cit> .
under lineage-specific positive selection, we expect an increase in the non-synonymous to synonymous substitution rate ratio in the corresponding branch.
however, this pattern is also compatible with relaxation of the protein’s selective constraints.
another widely used method to detect branch-specific natural selection patterns in the context of a phylogenetic tree is the branch-site-test  <cit> .
this test is based on the comparison of the likelihood of a model in which all codons evolve neutrally or under purifying selection in all branches of the tree, with the likelihood of a model in which a fraction of the codons evolve adaptively but only in the branch of interest.
this test and previous similar tests have been used by several authors to identify putative positively selected genes in humans  <cit> .
computer simulation studies indicated that the test tends to be conservative and that its power is limited when closely related species are compared .
statistically relevant comparisons with experimentally validated data are not yet available; however, nozawa and colleagues have noted that known functional changes in vision genes are not always detected by the test  <cit> .
despite strong interest in understanding which evolutionary events have driven humans apart from their closest living relatives, our current knowledge of the associated molecular mechanisms remains very limited  <cit> .
recently, this question has started to be addressed using genome-wide approaches .
studies published to date have either considered human–chimpanzee divergence measures for the mk test, being unable to identify in which of the two species changes have occurred, or have only employed the branch-site test, missing the valuable information provided by polymorphism data.
large-scale scans of positive selection have reported on various functional groups of genes enriched in positive selection signatures, including those involved in sensory perception  <cit> , olfactory receptors  <cit>  and spermatogenesis  <cit> .
other gene-based studies have reported signatures of positive selection in several genes that affect brain size  <cit> , including aspm , cdk5rap <dig> , cenpj  and mcph <dig> .
here, for the first time, we used divergence and polymorphism data specific to the human branch that integrates the results of several divergence based approaches to identify positively selected genes.
the analysis revealed that genes involved in nervous system functions are enriched in positive selection signatures in the human branch.
we estimated that at least 40% of the non-synonymous substitutions in genes accelerated in the human branch, or in genes detected by the branch site test, have been adaptive.
by combining results from the different tests we were able to identify several genes that show multiple lines of evidence of adaptive evolution in the 5– <dig> million years separating humans from chimpanzees.
RESULTS
effect of rare variants on the detection of positive selection
to identify genes that are under positive selection in the human lineage we compared coding sequence divergence in the human branch after the split from the chimpanzee branch, representing 5– <dig> million years of human evolution, and polymorphism data from the current human population.
we obtained gene families comprising one-to-one orthologs from human, chimpanzee, macaque and mouse from ensembl compara  <cit>  .
subsequently, we estimated the number of dn and ds substitutions for each branch of the tree using the codeml maximum likelihood based program  <cit> .
the application of several data quality controls  resulted in  <dig>  genes with human branch specific substitution data .
the average number of dn and ds per gene in the human lineage was  <dig> and  <dig> , respectively .
we mapped all available pn and ps substitutions from dbsnp  to the human coding sequences from our gene set.
the vast majority  of the snps we employed had been identified in the  <dig> genomes project  <cit> .
most genes  contained at least one polymorphic site, and about 35% of them had four or more polymorphic sites .figure  <dig>
one-to-one orthologous gene dataset and identification of human accelerated genes.
fisher’s test using the estimated number of non-synonymous and synonymous substitutions in different branches of the tree.
data are for the fas  binding factor  <dig>  gene.
distribution of the number of non-synonymous  and synonymous  substitutions in the human branch.
for the sake of clarity, the x-axis has been cut at a value of  <dig>  there are an additional  <dig> genes with more than  <dig> non-synonymous substitutions and  <dig> with more than  <dig> synonymous substitutions.
the area of the bars is proportional to the number of genes.
equivalent bins in the two graphs are shown in the same color to facilitate the comparison.
the fi for the complete gene set was  <dig> .
this value was obtained by summing the dn, ds, pn and ps values of individual genes .
the neutrality index , which is 1/fi, was  <dig> .
the nitg, a related index that takes into account gene heterogeneity  <cit> , had a very similar value,  <dig> .
the fi gradually increased as we considered higher frequency snps, reaching a value of  <dig> for snps with derived allele frequency  ≥60% .
this progression is consistent with the presence of slightly deleterious mutations in the population, because such mutations are expected to be comparatively less abundant at higher daf values  <cit> .
as expected, the proportion of adaptive substitutions  and other related statistics showed the same effect .
we observed a similar tendency when we used the minor allele frequency  instead of daf .
for snps with daf ≥60%, we obtained α =  <dig> , which corresponds to  <dig> % adaptive substitutions.table  <dig>
fixation index  and related statistics for different human gene sets
data are for genes with at least one polymorphic site.
dos: direction of selection; α: proportion of adaptive substitutions.
snp: single nucleotide polymorphism.
daf: derived allele frequency.
genes showing accelerated evolution in the human lineage
we identified genes that showed an excess of dn substitutions when compared to the number of ds substitutions in the human branch with respect to the hominoid ancestral branch .
we only selected cases that passed a second test in which this same effect was not observed in the chimpanzee branch.
following this procedure, we obtained  <dig> genes showing human-specific acceleration .
taking all the genes together, we observed an fi of  <dig>  in the human accelerated gene set, indicating a strong impact of positive selection in this group of genes.
when we restricted the analysis to snps with daf ≥60%, the overall fi of the accelerated genes increased to  <dig>  and the proportion of adaptive substitutions reached  <dig> % .
the analysis of the distribution of fi values in different genes confirmed important differences between human accelerated genes and the complete gene set .
as expected, the dn/ds values in the human branch tended to be much larger in the accelerated gene set than in the complete gene set , reflecting the accumulation of a larger number of fixed non-synonymous substitutions in the former set.
in contrast, the distribution of pn/ps values was indistinguishable in the complete gene set and in the accelerated gene set , indicating similar levels of selective constraints in both gene sets.figure  <dig>
comparison of divergence and polymorphism data in different gene sets.
the data shown are for genes in which dn, ds, pn and ps ≠  <dig> and pn + ps ≥  <dig>  test genes).
the area within the box contains 50% of the data; the horizontal line is the median; small circles represent outliers .
the y-axis  has been cut at fi =  <dig>  we performed mann–whitney-wilcoxon tests against the background  distribution: *p <  <dig> ; **p <  <dig> ; ***p <  <dig> .
to better understand the effect of rapid gene evolution on the fi, when unlinked from human-specific evolutionary rate acceleration, we sampled from the complete set of genes a subset of genes that showed the same evolutionary rate distribution as the human accelerated genes, but which did not include any genes showing human-specific acceleration .
the fi value of the rapidly evolving gene set was  <dig>  for snps with daf ≥60% , indicating that some of these genes are evolving adaptively.
however, this value was much lower than the fi value of the human accelerated gene set , indicating that they are not so influenced by positive selection as the latter set.
we also noted that the pn/ps ratio was higher in the set of rapidly evolving genes than in the human accelerated genes .
this indicates that the former genes tend to evolve under more relaxed constraint than the latter ones, which is consistent with the fact that they are evolving rapidly not only in the human branch, but also in other branches.
genes detected by the maximum likelihood based branch-site test
the branch-site  test is a maximum likelihood based test that compares the likelihood of an evolutionary model  that includes branch-specific positive selection  with the likelihood of a model  that does not consider such a possibility   <cit> .
several recent works have used this test to perform genome-wide scans of positive selection in genes from humans or other mammalian species  <cit> .
using this test, we identified  <dig> gene families that showed a signature of positive selection in the human branch at p <  <dig>  .
a large number of these genes  were also significant after false discovery rate correction .
the overlap with the accelerated gene set was relatively small .
the global fi of the human bs test gene set, considering all snps, was  <dig> ; however, this value increased to  <dig>  for snps with daf ≥60%, which corresponded to about 40% adaptive substitutions, similar to the values obtained for human accelerated genes .
the distribution of fi values in genes detected by the bs test set was skewed to values larger than  <dig> compared with the complete gene set , but to a lesser extent than for the accelerated gene set.
the dn/ds values were lower than for the human-specific accelerated genes, but still significantly higher than for the complete gene set .
unexpectedly, the pn/ps values were lower in the bs test set than in the complete gene set , indicating that genes selected by this test are biased towards highly constrained genes.
mammalian-specific genes
another interesting set of genes to include in the comparison was phylogenetically restricted genes, many of which are likely to be important for lineage-specific adaptations  <cit> .
lineage-specific genes typically show higher than average non-synonymous to synonymous substitution rates , as well as intra-specific signatures of relaxed purifying selection  <cit> .
however, it is still unclear if positive selection has significantly affected their evolution  <cit> .
to answer this question, we crossed-checked a list of mammalian-specific genes extracted from the literature  <cit>  with our set of orthologous genes from human, chimpanzee, macaque and mouse .
both the dn/ds and pn/ps were about three times higher in this set than in the complete gene set ; however, the fi, dos and α were not affected .
this indicates that relaxation of purifying selection is the main driver of the rapid evolution of these genes.
adaptive evolution signatures in neural system genes
the comparison of divergence and polymorphism data for each gene can be used to identify putative positively selected genes  <cit> .
to analyze the distribution of fi values, we focused on genes with at least four polymorphic sites .
we used this threshold to avoid very unreliable fi estimates while maintaining a substantial number of genes for comparative analysis.figure  <dig>
histogram of the distribution of the fixation index in human genes.
for the sake of fi estimation robustness, we selected genes in which dn, ds, pn and ps ≠  <dig> and pn + ps ≥  <dig>  genes with fi >  <dig> were not considered for the plot.
the total number of genes included in the plot was  <dig>  genes in blue are significant by the mk test .
mcph <dig>  fi =  <dig> ; cdk5rap <dig>  fi =  <dig> ; aspm  fi =  <dig> ; ddx <dig>  fi = 7; fbf <dig>  fi =  <dig> ; txlnb  fi =  <dig>
applying the g test of independence described in the original mk test  <cit> , we identified  <dig> genes with a positive selection signature at a p-value <  <dig> .
five additional cases were identified when we also considered genes with a lower number of polymorphic sites .
the genes in the list were involved in a variety of functions, including the immune response , cellular adhesion  and neural functions .
aspm, previously reported to be under positive selection in the human lineage  <cit> , showed a high fi, but did not reach statistical significance in the mk test .
this was not surprising when we considered that the fi of the significant genes was ≥  <dig>  whereas the fi of asmp was  <dig> .
the position of some of these genes on the overall fi distribution is shown in figure  <dig>
none of the genes that were marked as significant by the mk test passed a multiple test correction.
this is probably a consequence of the closeness of the two species compared and the low diversity of the human population, both factors limiting the power of the test at the individual gene level.
nevertheless, seven of the genes were also identified by at least one divergence based method , making them particularly strong candidates for human-specific adaptations .
two of these genes are involved in neural development.
the first is taxilin beta , encoding a protein that plays important roles in motor nerve regeneration  <cit> , which was detected by all three methods.
the second gene was syntabulin , which mediates axonal transport and is required for the formation of new presynaptic boutons in developing neurons  <cit> .
this gene was detected by the mk test and also displayed significant human-specific evolutionary rate acceleration.table  <dig>
genes with multiple evidence of positive selection in the human lineage
tle6
ushbp1
sybu
c20orf96
txlnb
tle4
fbf1
mk: mcdonald-kreitman test; bs test: branch-site test; accelerated: showing human-specific evolutionary rate acceleration.
the other genes with multiple evidence of positive selection were fas  binding factor  <dig> , which binds to keratin and plays a role in epithelial cell polarization  <cit> ; the gene encoding usher syndrome 1c binding protein  <dig> , which binds to the protein responsible for usher syndrome type 1c, a sensory defect involving deafness and blindness  <cit> ; and two genes from the enhancer of split groucho family, tle <dig> and tle <dig>  which are involved in cell fate decision and boundary formation  <cit> .
to further investigate the relationship between adaptive evolution and gene function, we examined the fi values of genes annotated under different gene ontology  terms, focusing on terms with more than  <dig> annotated genes and including the group formed by genes with no associated go terms .
among the  <dig> groups with the highest fi value, three presented a fi value distribution that was statistically different from the complete gene set or background .
remarkably, two of these groups were related to nervous system functions: synapsis  and neuronal cell body go terms .
these two groups only shared eight genes, thus they were largely independent.
the third significant group was genes of unknown function .
this indicates that many relevant genes for human evolution are probably uncharacterized.
we also analyzed the data for two additional functional classes reported to be enriched in positively selected genes in previous genome-wide studies: spermatogenesis and response to stimulus  <cit> .
the latter group included many genes involved in sensory perception, such as olfactory receptors.
both classes showed high dn/ds and pn/ps values, but their fi values were not significantly different from the background .figure  <dig>
comparison of divergence and polymorphism data in different gene functional groups.
the set of genes is the same as in figure  <dig>  gene ontology  annotations defined the functional groups.
synapse: genes annotated under the “synapse” term; neuron: “neuronal cell body”; sperm: “spermatogenesis”; sensory: “response to stimulus”; all: complete gene set or background; unknown: genes with no annotated go terms.
the area within the box contains 50% of the data; the horizontal line is the median; small circles represent outliers .
the y-axis  has been cut at fi =  <dig>  we performed a mann–whitney-wilcoxon test to assess if the differences with the background were statistically significant, for cases in which the median value in the gene set was larger than the background median: *p <  <dig> ; **p <  <dig> ; ***p <  <dig> .
discussion
the comparison of the number of non-synonymous to synonymous changes in coding sequences is a powerful tool to uncover the action of natural selection.
in the absence of selection we expect the number of non-synonymous substitutions per non-synonymous site  and the number of synonymous substitutions per synonymous site  to be similar.
for the vast majority of protein coding sequences dn < ds, indicating that purifying selection prevents the fixation of deleterious mutations in coding sequences.
a dn/ds ratio larger than  <dig> indicates positive selection and has been used in the past to identify putative positively selected genes using human and chimpanzee orthologous sequences  <cit> .
however, many sites in a protein are subject to purifying selection, which undermines the detection of positive selection by this method.
additionally, a ratio larger than  <dig> is very rarely statistically significant for closely related species that have accumulated few substitutions since their separation.
increased sensitivity may be achieved by comparing divergence to polymorphism data, as in the mk test, or by comparing the non-synonymous to synonymous substitution ratio in different branches of the tree, either using complete sequence or codon-based divergence measurements.
here, we applied these different methods to a large set of mammalian gene families to gain a novel insight into the nature of human molecular adaptive processes.
our overall estimate of the fraction of fixed adaptive substitutions  in the human lineage is very low, approximately  <dig> %, which is consistent with previous studies .
in contrast, in species with much larger effective population sizes, such as mouse or fruit fly, the estimated α values are typically 40–60%  <cit> .
one likely cause is the presence of segregating, slightly deleterious mutations in humans; for example, those caused by a recent population bottleneck, which will increase the number of pn polymorphisms relative to the neutral expectation and downwardly bias the estimation of the fi and α value  <cit> .
the fact that the estimated α is higher for polymorphisms of increasing allele frequency in the population, as observed here and in other recent studies  <cit> , is consistent with this hypothesis.
another factor that may contribute to the low α in humans is the small population size itself.
species with small effective population sizes are expected to have a lower proportion of adaptive substitutions than species with a larger number of individuals, simply because the overall number of new mutations per generation is lower.
accordingly, a recent study based on  <dig> independent pairs of eukaryotic species showed that the rate of adaptive protein evolution is positively correlated with effective population size  <cit> .
finally, we used data derived from studies that employed individuals from different populations  <cit> , which may have artificially increased the number of low frequency polymorphisms and decreased fi.
using snps with daf ≥60% we obtained an overall fi of approximately  <dig> in the complete gene set.
this value may be an underestimate of the real value if a portion of the deleterious mutations persists at such high frequencies  <cit> .
however, it is still useful to compare the relative contribution of positive selection in different selected subsets of genes.
in contrast to several earlier studies that used substitutions derived from human and chimpanzee sequence pairwise alignments for the mk test  <cit> , we employed human branch-specific substitutions, which means that we automatically discarded cases in which the signal of positive selection was in the chimpanzee branch and not the human branch.
this was important to avoid mixing signals from both branches.
additionally, as the quality of the chimpanzee genome sequence is lower than that of the human genome, much of the signal when using both branches may be caused by false positives in the chimpanzee  <cit> .
branch-specific evolutionary rate acceleration can be caused by positive selection, decreased purifying selection, or a combination of both factors.
as expected, we found strong evidence of positive selection in genes with accelerated evolution in humans.
surprisingly, we found no evidence of relaxed purifying selection in these genes, as they displayed similar pn/ps values than the rest of genes.
genes in the accelerated gene set showed remarkably high non-synonymous to synonymous substitutions; however, this alone was not sufficient to explain their high fi values, as deduced from the comparison with similarly rapidly evolving genes that were not human accelerated or with the set of rapidly evolving mammalian-specific genes.
as shown here for humans, in bacteria, a correlation between species-specific protein evolutionary rate variation and positive selection signatures by the mk test was found  <cit> .
the impact of positive selection on the bs test selected genes, as determined by the observed fi and α values, was similar or even lower than that in the accelerated genes.
this was unexpected, because the bs test has been specifically designed to identify positive selection signatures  <cit> .
given enough data, the test can be very useful to identify putatively positively selected sites, but it is also very sensitive to the presence of misaligned positions in the alignment, something that is difficult to correct for in large-scale analyses  <cit> .
recently, we showed that, whereas the estimation of the ratio between non-synonymous and synonymous substitutions  is relatively robust to alignment errors, the results of the bs test are not  <cit> .
altogether, the detection of branches showing increased dn/ds appears to be a simple but effective method to detect positive selection.
as polymorphism data become available for a larger number species, it will be possible to routinely combine the results of the tree-based methods  with the results of the mk test , improving the detection of positive selection.
in humans, because of reduced diversity, the estimation of fi is only possible for some genes, and the power of the mk test is low.
long protein coding sequences tend to have more snps, which favors their detection by the mk test, compared with shorter coding sequences.
despite the important reduction in dataset size, focusing on the most informative genes  allowed us to compare the gene fi distribution in  <dig> different functional classes, each containing at least  <dig> different genes.
under these conditions, three terms related to the nervous system were among the top  <dig> groups with the highest fi values; synapse, neuronal cell body and axon guidance, of which the first two showed a statistically significant deviation.
nervous system genes tend to evolve in a very constrained manner and few substitutions accumulate, even in cases in which the fi is larger than  <dig>  which is likely to make their detection by the bs test difficult  <cit> .
this might explain why they have not been detected in previous genome-wide scans using this test.
functional groups found to be enriched in positive selection signatures in previous genome-wide studies using divergence data included sensory perception, immunity, spermatogenesis, apoptosis or regulation of transcription  <cit> .
among these functions, genes involved in regulation of transcription and spermatogenesis showed relatively elevated fi values in our study, although the differences with the background were not significant.
the go term ‘response to stimulus’ , containing many genes involved in sensory perception, showed abnormally high pn/ps ratios.
this supports the hypothesis that these genes evolve under very low evolutionary constraints, as also found by  <cit> .
other possible explanations are that this set includes genes that have recently become pseudogenes, or that balancing selection in present-day populations is maintaining high non-synonymous polymorphism levels.
the size of the accelerated and bs test gene sets in our study  was not sufficiently large to perform a systematic search for significantly overrepresented go terms.
nevertheless, we counted how many genes were annotated under synapsis, neuronal cell body, response to stimulus and spermatogenesis in these gene sets.
none of these classes achieved statistical significance, except synapse in the accelerated gene set, with five observed genes compared to one expected by chance .
taken together, our results strongly indicate that positive selection in neural genes is likely to have played a significant role in the human evolution.
a study using human-macaque and mouse-rat comparisons reported that nervous system genes evolved in a more accelerated manner in primates than in rodents  <cit> , also pointing to a link between increased non-synonymous substitutions in neural genes and primate evolution.
CONCLUSIONS
comparing the number of non-synonymous and synonymous substitutions in sister species can help identify sequences that have been the target of positive selection in one of the species.
here, we have combined divergence and variation measures to identify genes containing adaptive substitutions in the human lineage, but not in chimpanzees or the common ancestor or both species.
the study uncovered an unexpected enrichment in positive selection signatures in neural genes.

 the identification of signatures of positive selection in genes is a central theme in molecular evolution. the relative contribution of neutral and adaptive evolution is the subject of ongoing debate. a powerful approach to identify positive selection in coding sequences is the comparison of substitutions and polymorphic sites using the mcdonald and kreitman test. this test builds on previously developed approaches to detect deviations from neutral evolution using divergence and polymorphism data. the mk test compares the number of non-synonymous and synonymous substitutions in a pair of species to the number of non-synonymous and synonymous polymorphisms in one of the species for the same coding sequence. positively selected mutations reach fixation very rapidly, and thus increase non-synonymous divergence; however, they are rarely observed as polymorphic sites. as a result, if positive selection has played a significant role in the evolution of a gene, the ratio between non-synonymous and synonymous substitutions is expected to be larger than the ratio between non-synonymous and synonymous polymorphisms. the first ratio divided by the second ratio has been named the fixation index. fi values larger than indicate positive selection in the lineage of interest, and values lower than indicate an excess of non-synonymous polymorphisms in the current population. related statistics are the neutrality index, direction of selection and the proportion of adaptive substitutions. several authors have used the mk test to estimate the importance of positive selection in diverse genomes, with contrasting results depending on the species studied. in most studies of humans, the estimated proportion of adaptive amino acid substitutions is very low. one likely explanation is the presence of slightly deleterious segregating mutations caused by reduced effective population size, resulting in inflated non-synonymous snps relative to non-synonymous substitutions. most deleterious mutations are expected to be present at low frequencies in the population; therefore, removing low frequency variants should improve the estimation of fi and other similar statistics. other approaches to identify signatures of natural selection are based on the comparison of non-synonymous and synonymous substitutions in different branches of a tree obtained from a set of homologous sequences. using data from several species has the advantage that we can target genes that show lineage-specific positive selection and discard genes that are also evolving under positive selection in related lineages, which is especially relevant when searching for species-specific traits. genome-wide studies have shown that the pace of evolution of a protein often varies substantially in different branches of a tree, which is in agreement with the so-called over-dispersion of the molecular clock. under lineage-specific positive selection, we expect an increase in the non-synonymous to synonymous substitution rate ratio in the corresponding branch. however, this pattern is also compatible with relaxation of the protein’s selective constraints. another widely used method to detect branch-specific natural selection patterns in the context of a phylogenetic tree is the branch-site-test. this test is based on the comparison of the likelihood of a model in which all codons evolve neutrally or under purifying selection in all branches of the tree, with the likelihood of a model in which a fraction of the codons evolve adaptively but only in the branch of interest. this test and previous similar tests have been used by several authors to identify putative positively selected genes in humans. computer simulation studies indicated that the test tends to be conservative and that its power is limited when closely related species are compared. statistically relevant comparisons with experimentally validated data are not yet available; however, nozawa and colleagues have noted that known functional changes in vision genes are not always detected by the test. despite strong interest in understanding which evolutionary events have driven humans apart from their closest living relatives, our current knowledge of the associated molecular mechanisms remains very limited. recently, this question has started to be addressed using genome-wide approaches. studies published to date have either considered human–chimpanzee divergence measures for the mk test, being unable to identify in which of the two species changes have occurred, or have only employed the branch-site test, missing the valuable information provided by polymorphism data. large-scale scans of positive selection have reported on various functional groups of genes enriched in positive selection signatures, including those involved in sensory perception, olfactory receptors and spermatogenesis. other gene-based studies have reported signatures of positive selection in several genes that affect brain size, including aspm, cdk5rap, cenpj and mcph. here, for the first time, we used divergence and polymorphism data specific to the human branch that integrates the results of several divergence based approaches to identify positively selected genes. the analysis revealed that genes involved in nervous system functions are enriched in positive selection signatures in the human branch. we estimated that at least 40% of the non-synonymous substitutions in genes accelerated in the human branch, or in genes detected by the branch site test, have been adaptive. by combining results from the different tests we were able to identify several genes that show multiple lines of evidence of adaptive evolution in the 5– million years separating humans from chimpanzees.  effect of rare variants on the detection of positive selection to identify genes that are under positive selection in the human lineage we compared coding sequence divergence in the human branch after the split from the chimpanzee branch, representing 5– million years of human evolution, and polymorphism data from the current human population. we obtained gene families comprising one-to-one orthologs from human, chimpanzee, macaque and mouse from ensembl compara. subsequently, we estimated the number of dn and ds substitutions for each branch of the tree using the codeml maximum likelihood based program. the application of several data quality controls resulted in genes with human branch specific substitution data. the average number of dn and ds per gene in the human lineage was and, respectively. we mapped all available pn and ps substitutions from dbsnp to the human coding sequences from our gene set. the vast majority of the snps we employed had been identified in the genomes project. most genes contained at least one polymorphic site, and about 35% of them had four or more polymorphic sites.figure one-to-one orthologous gene dataset and identification of human accelerated genes. fisher’s test using the estimated number of non-synonymous and synonymous substitutions in different branches of the tree. data are for the fas binding factor gene. distribution of the number of non-synonymous and synonymous substitutions in the human branch. for the sake of clarity, the x-axis has been cut at a value of there are an additional genes with more than non-synonymous substitutions and with more than synonymous substitutions. the area of the bars is proportional to the number of genes. equivalent bins in the two graphs are shown in the same color to facilitate the comparison. the fi for the complete gene set was. this value was obtained by summing the dn, ds, pn and ps values of individual genes. the neutrality index, which is 1/fi, was. the nitg, a related index that takes into account gene heterogeneity, had a very similar value,. the fi gradually increased as we considered higher frequency snps, reaching a value of for snps with derived allele frequency ≥60%. this progression is consistent with the presence of slightly deleterious mutations in the population, because such mutations are expected to be comparatively less abundant at higher daf values. as expected, the proportion of adaptive substitutions and other related statistics showed the same effect. we observed a similar tendency when we used the minor allele frequency instead of daf. for snps with daf ≥60%, we obtained α = , which corresponds to % adaptive substitutions.table fixation index and related statistics for different human gene sets data are for genes with at least one polymorphic site. dos: direction of selection; α: proportion of adaptive substitutions. snp: single nucleotide polymorphism. daf: derived allele frequency. genes showing accelerated evolution in the human lineage we identified genes that showed an excess of dn substitutions when compared to the number of ds substitutions in the human branch with respect to the hominoid ancestral branch. we only selected cases that passed a second test in which this same effect was not observed in the chimpanzee branch. following this procedure, we obtained genes showing human-specific acceleration. taking all the genes together, we observed an fi of in the human accelerated gene set, indicating a strong impact of positive selection in this group of genes. when we restricted the analysis to snps with daf ≥60%, the overall fi of the accelerated genes increased to and the proportion of adaptive substitutions reached %. the analysis of the distribution of fi values in different genes confirmed important differences between human accelerated genes and the complete gene set. as expected, the dn/ds values in the human branch tended to be much larger in the accelerated gene set than in the complete gene set, reflecting the accumulation of a larger number of fixed non-synonymous substitutions in the former set. in contrast, the distribution of pn/ps values was indistinguishable in the complete gene set and in the accelerated gene set, indicating similar levels of selective constraints in both gene sets.figure comparison of divergence and polymorphism data in different gene sets. the data shown are for genes in which dn, ds, pn and ps ≠  and pn + ps ≥  test genes). the area within the box contains 50% of the data; the horizontal line is the median; small circles represent outliers. the y-axis has been cut at fi =  we performed mann–whitney-wilcoxon tests against the background distribution: *p < ; **p < ; ***p < . to better understand the effect of rapid gene evolution on the fi, when unlinked from human-specific evolutionary rate acceleration, we sampled from the complete set of genes a subset of genes that showed the same evolutionary rate distribution as the human accelerated genes, but which did not include any genes showing human-specific acceleration. the fi value of the rapidly evolving gene set was for snps with daf ≥60%, indicating that some of these genes are evolving adaptively. however, this value was much lower than the fi value of the human accelerated gene set, indicating that they are not so influenced by positive selection as the latter set. we also noted that the pn/ps ratio was higher in the set of rapidly evolving genes than in the human accelerated genes. this indicates that the former genes tend to evolve under more relaxed constraint than the latter ones, which is consistent with the fact that they are evolving rapidly not only in the human branch, but also in other branches. genes detected by the maximum likelihood based branch-site test the branch-site test is a maximum likelihood based test that compares the likelihood of an evolutionary model that includes branch-specific positive selection with the likelihood of a model that does not consider such a possibility. several recent works have used this test to perform genome-wide scans of positive selection in genes from humans or other mammalian species. using this test, we identified gene families that showed a signature of positive selection in the human branch at p < . a large number of these genes were also significant after false discovery rate correction. the overlap with the accelerated gene set was relatively small. the global fi of the human bs test gene set, considering all snps, was; however, this value increased to for snps with daf ≥60%, which corresponded to about 40% adaptive substitutions, similar to the values obtained for human accelerated genes. the distribution of fi values in genes detected by the bs test set was skewed to values larger than compared with the complete gene set, but to a lesser extent than for the accelerated gene set. the dn/ds values were lower than for the human-specific accelerated genes, but still significantly higher than for the complete gene set. unexpectedly, the pn/ps values were lower in the bs test set than in the complete gene set, indicating that genes selected by this test are biased towards highly constrained genes. mammalian-specific genes another interesting set of genes to include in the comparison was phylogenetically restricted genes, many of which are likely to be important for lineage-specific adaptations. lineage-specific genes typically show higher than average non-synonymous to synonymous substitution rates, as well as intra-specific signatures of relaxed purifying selection. however, it is still unclear if positive selection has significantly affected their evolution. to answer this question, we crossed-checked a list of mammalian-specific genes extracted from the literature with our set of orthologous genes from human, chimpanzee, macaque and mouse. both the dn/ds and pn/ps were about three times higher in this set than in the complete gene set; however, the fi, dos and α were not affected. this indicates that relaxation of purifying selection is the main driver of the rapid evolution of these genes. adaptive evolution signatures in neural system genes the comparison of divergence and polymorphism data for each gene can be used to identify putative positively selected genes. to analyze the distribution of fi values, we focused on genes with at least four polymorphic sites. we used this threshold to avoid very unreliable fi estimates while maintaining a substantial number of genes for comparative analysis.figure histogram of the distribution of the fixation index in human genes. for the sake of fi estimation robustness, we selected genes in which dn, ds, pn and ps ≠  and pn + ps ≥  genes with fi >  were not considered for the plot. the total number of genes included in the plot was genes in blue are significant by the mk test. mcph fi = ; cdk5rap fi = ; aspm fi = ; ddx fi = 7; fbf fi = ; txlnb fi =  applying the g test of independence described in the original mk test, we identified genes with a positive selection signature at a p-value < . five additional cases were identified when we also considered genes with a lower number of polymorphic sites. the genes in the list were involved in a variety of functions, including the immune response, cellular adhesion and neural functions. aspm, previously reported to be under positive selection in the human lineage, showed a high fi, but did not reach statistical significance in the mk test. this was not surprising when we considered that the fi of the significant genes was ≥  whereas the fi of asmp was. the position of some of these genes on the overall fi distribution is shown in figure  none of the genes that were marked as significant by the mk test passed a multiple test correction. this is probably a consequence of the closeness of the two species compared and the low diversity of the human population, both factors limiting the power of the test at the individual gene level. nevertheless, seven of the genes were also identified by at least one divergence based method, making them particularly strong candidates for human-specific adaptations. two of these genes are involved in neural development. the first is taxilin beta, encoding a protein that plays important roles in motor nerve regeneration, which was detected by all three methods. the second gene was syntabulin, which mediates axonal transport and is required for the formation of new presynaptic boutons in developing neurons. this gene was detected by the mk test and also displayed significant human-specific evolutionary rate acceleration.table genes with multiple evidence of positive selection in the human lineage tle6 ushbp1 sybu c20orf96 txlnb tle4 fbf1 mk: mcdonald-kreitman test; bs test: branch-site test; accelerated: showing human-specific evolutionary rate acceleration. the other genes with multiple evidence of positive selection were fas binding factor, which binds to keratin and plays a role in epithelial cell polarization; the gene encoding usher syndrome 1c binding protein, which binds to the protein responsible for usher syndrome type 1c, a sensory defect involving deafness and blindness; and two genes from the enhancer of split groucho family, tle and tle which are involved in cell fate decision and boundary formation. to further investigate the relationship between adaptive evolution and gene function, we examined the fi values of genes annotated under different gene ontology terms, focusing on terms with more than annotated genes and including the group formed by genes with no associated go terms. among the groups with the highest fi value, three presented a fi value distribution that was statistically different from the complete gene set or background. remarkably, two of these groups were related to nervous system functions: synapsis and neuronal cell body go terms. these two groups only shared eight genes, thus they were largely independent. the third significant group was genes of unknown function. this indicates that many relevant genes for human evolution are probably uncharacterized. we also analyzed the data for two additional functional classes reported to be enriched in positively selected genes in previous genome-wide studies: spermatogenesis and response to stimulus. the latter group included many genes involved in sensory perception, such as olfactory receptors. both classes showed high dn/ds and pn/ps values, but their fi values were not significantly different from the background.figure comparison of divergence and polymorphism data in different gene functional groups. the set of genes is the same as in figure  gene ontology annotations defined the functional groups. synapse: genes annotated under the “synapse” term; neuron: “neuronal cell body”; sperm: “spermatogenesis”; sensory: “response to stimulus”; all: complete gene set or background; unknown: genes with no annotated go terms. the area within the box contains 50% of the data; the horizontal line is the median; small circles represent outliers. the y-axis has been cut at fi =  we performed a mann–whitney-wilcoxon test to assess if the differences with the background were statistically significant, for cases in which the median value in the gene set was larger than the background median: *p < ; **p < ; ***p < . discussion the comparison of the number of non-synonymous to synonymous changes in coding sequences is a powerful tool to uncover the action of natural selection. in the absence of selection we expect the number of non-synonymous substitutions per non-synonymous site and the number of synonymous substitutions per synonymous site to be similar. for the vast majority of protein coding sequences dn < ds, indicating that purifying selection prevents the fixation of deleterious mutations in coding sequences. a dn/ds ratio larger than indicates positive selection and has been used in the past to identify putative positively selected genes using human and chimpanzee orthologous sequences. however, many sites in a protein are subject to purifying selection, which undermines the detection of positive selection by this method. additionally, a ratio larger than is very rarely statistically significant for closely related species that have accumulated few substitutions since their separation. increased sensitivity may be achieved by comparing divergence to polymorphism data, as in the mk test, or by comparing the non-synonymous to synonymous substitution ratio in different branches of the tree, either using complete sequence or codon-based divergence measurements. here, we applied these different methods to a large set of mammalian gene families to gain a novel insight into the nature of human molecular adaptive processes. our overall estimate of the fraction of fixed adaptive substitutions in the human lineage is very low, approximately %, which is consistent with previous studies. in contrast, in species with much larger effective population sizes, such as mouse or fruit fly, the estimated α values are typically 40–60%. one likely cause is the presence of segregating, slightly deleterious mutations in humans; for example, those caused by a recent population bottleneck, which will increase the number of pn polymorphisms relative to the neutral expectation and downwardly bias the estimation of the fi and α value. the fact that the estimated α is higher for polymorphisms of increasing allele frequency in the population, as observed here and in other recent studies, is consistent with this hypothesis. another factor that may contribute to the low α in humans is the small population size itself. species with small effective population sizes are expected to have a lower proportion of adaptive substitutions than species with a larger number of individuals, simply because the overall number of new mutations per generation is lower. accordingly, a recent study based on independent pairs of eukaryotic species showed that the rate of adaptive protein evolution is positively correlated with effective population size. finally, we used data derived from studies that employed individuals from different populations, which may have artificially increased the number of low frequency polymorphisms and decreased fi. using snps with daf ≥60% we obtained an overall fi of approximately in the complete gene set. this value may be an underestimate of the real value if a portion of the deleterious mutations persists at such high frequencies. however, it is still useful to compare the relative contribution of positive selection in different selected subsets of genes. in contrast to several earlier studies that used substitutions derived from human and chimpanzee sequence pairwise alignments for the mk test, we employed human branch-specific substitutions, which means that we automatically discarded cases in which the signal of positive selection was in the chimpanzee branch and not the human branch. this was important to avoid mixing signals from both branches. additionally, as the quality of the chimpanzee genome sequence is lower than that of the human genome, much of the signal when using both branches may be caused by false positives in the chimpanzee. branch-specific evolutionary rate acceleration can be caused by positive selection, decreased purifying selection, or a combination of both factors. as expected, we found strong evidence of positive selection in genes with accelerated evolution in humans. surprisingly, we found no evidence of relaxed purifying selection in these genes, as they displayed similar pn/ps values than the rest of genes. genes in the accelerated gene set showed remarkably high non-synonymous to synonymous substitutions; however, this alone was not sufficient to explain their high fi values, as deduced from the comparison with similarly rapidly evolving genes that were not human accelerated or with the set of rapidly evolving mammalian-specific genes. as shown here for humans, in bacteria, a correlation between species-specific protein evolutionary rate variation and positive selection signatures by the mk test was found. the impact of positive selection on the bs test selected genes, as determined by the observed fi and α values, was similar or even lower than that in the accelerated genes. this was unexpected, because the bs test has been specifically designed to identify positive selection signatures. given enough data, the test can be very useful to identify putatively positively selected sites, but it is also very sensitive to the presence of misaligned positions in the alignment, something that is difficult to correct for in large-scale analyses. recently, we showed that, whereas the estimation of the ratio between non-synonymous and synonymous substitutions is relatively robust to alignment errors, the results of the bs test are not. altogether, the detection of branches showing increased dn/ds appears to be a simple but effective method to detect positive selection. as polymorphism data become available for a larger number species, it will be possible to routinely combine the results of the tree-based methods with the results of the mk test, improving the detection of positive selection. in humans, because of reduced diversity, the estimation of fi is only possible for some genes, and the power of the mk test is low. long protein coding sequences tend to have more snps, which favors their detection by the mk test, compared with shorter coding sequences. despite the important reduction in dataset size, focusing on the most informative genes allowed us to compare the gene fi distribution in different functional classes, each containing at least different genes. under these conditions, three terms related to the nervous system were among the top groups with the highest fi values; synapse, neuronal cell body and axon guidance, of which the first two showed a statistically significant deviation. nervous system genes tend to evolve in a very constrained manner and few substitutions accumulate, even in cases in which the fi is larger than which is likely to make their detection by the bs test difficult. this might explain why they have not been detected in previous genome-wide scans using this test. functional groups found to be enriched in positive selection signatures in previous genome-wide studies using divergence data included sensory perception, immunity, spermatogenesis, apoptosis or regulation of transcription. among these functions, genes involved in regulation of transcription and spermatogenesis showed relatively elevated fi values in our study, although the differences with the background were not significant. the go term ‘response to stimulus’, containing many genes involved in sensory perception, showed abnormally high pn/ps ratios. this supports the hypothesis that these genes evolve under very low evolutionary constraints, as also found by. other possible explanations are that this set includes genes that have recently become pseudogenes, or that balancing selection in present-day populations is maintaining high non-synonymous polymorphism levels. the size of the accelerated and bs test gene sets in our study was not sufficiently large to perform a systematic search for significantly overrepresented go terms. nevertheless, we counted how many genes were annotated under synapsis, neuronal cell body, response to stimulus and spermatogenesis in these gene sets. none of these classes achieved statistical significance, except synapse in the accelerated gene set, with five observed genes compared to one expected by chance. taken together, our results strongly indicate that positive selection in neural genes is likely to have played a significant role in the human evolution. a study using human-macaque and mouse-rat comparisons reported that nervous system genes evolved in a more accelerated manner in primates than in rodents, also pointing to a link between increased non-synonymous substitutions in neural genes and primate evolution.  comparing the number of non-synonymous and synonymous substitutions in sister species can help identify sequences that have been the target of positive selection in one of the species. here, we have combined divergence and variation measures to identify genes containing adaptive substitutions in the human lineage, but not in chimpanzees or the common ancestor or both species. the study uncovered an unexpected enrichment in positive selection signatures in neural genes. 
genes with multiple lines of evidence of positive selection include taxilin beta, which is involved in motor nerve regeneration and syntabulin, and is required for the formation of new presynaptic boutons. the use of single nucleotide polymorphisms with high derived allele frequencies minimized the influence of segregating slightly deleterious mutations and improved the estimation of the number of adaptive sites. in general, neural genes are enriched in positive selection signatures. the use of variation data, in addition to sequence divergence information, uncovered previously undetected positive selection signatures in neural genes. positive selectionvariation datadivergencenervous systemhuman evolutionissue-copyright-statement© the author 2014 using daf ≥ 60% we showed that the proportion of adaptive substitutions is % in the complete gene set. we compared human branch-specific substitutions with variation data in the current human population to measure the impact of adaptive evolution on human protein coding genes. however, the percentage rose to 40% when we focused on genes that are specifically accelerated in the human branch with respect to the chimpanzee branch, or on genes that show signatures of adaptive selection at the codon level by the maximum likelihood based branch-site test. the recent increase in human polymorphism data, together with the availability of genome sequences from several primate species, provides an unprecedented opportunity to investigate how natural selection has shaped human evolution. we combined several methods to detect adaptive evolution in human coding sequences at a genome-wide level.
 the recent increase in human polymorphism data, together with the availability of genome sequences from several primate species, provides an unprecedented opportunity to investigate how natural selection has shaped human evolution.  we compared human branch-specific substitutions with variation data in the current human population to measure the impact of adaptive evolution on human protein coding genes. the use of single nucleotide polymorphisms with high derived allele frequencies minimized the influence of segregating slightly deleterious mutations and improved the estimation of the number of adaptive sites. using daf ≥ 60% we showed that the proportion of adaptive substitutions is % in the complete gene set. however, the percentage rose to 40% when we focused on genes that are specifically accelerated in the human branch with respect to the chimpanzee branch, or on genes that show signatures of adaptive selection at the codon level by the maximum likelihood based branch-site test. in general, neural genes are enriched in positive selection signatures. genes with multiple lines of evidence of positive selection include taxilin beta, which is involved in motor nerve regeneration and syntabulin, and is required for the formation of new presynaptic boutons.  we combined several methods to detect adaptive evolution in human coding sequences at a genome-wide level. the use of variation data, in addition to sequence divergence information, uncovered previously undetected positive selection signatures in neural genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords positive selectionvariation datadivergencenervous systemhuman evolutionissue-copyright-statement© the author 2014 