BACKGROUND
for over a decade researchers interested in the genetic basis of brain evolution have sought clues in the molecular evolution of genes associated with the neurodevelopmental disorder, primary microcephaly  <cit> .
microcephaly is a congenital disorder characterized by an early cessation of brain growth, specifically affecting cortical development.
mendelian inheritance of microcephaly has now been linked to deleterious mutations at seven unlinked loci  <cit> .
these loci encode proteins with central roles in neurogenesis, largely in the formation and function of the centrioles, which in turn control the way in which neural progenitor cells divide  <cit> .
disruption of these crucial functions causes microcephaly, and it is hypothesized that modification of their function through evolutionary time could tip the cell fate switch towards greater neurogenic output, underpinning the evolution of larger brains.
much of the early focus on the molecular evolution of microcephaly genes centered on the role of two loci  in human evolution  <cit> .
early studies suggested an increase in the rate at which these genes evolved along the lineage leading to humans  <cit> .
intriguingly, as more microcephaly loci were identified each has been shown to evolve adaptively  <cit>  suggesting they may be a persistent target of positive selection.
in addition, as more species were incorporated into analyses the signature of positive selection extended beyond humans first to great apes  <cit> , then to all anthropoid primates  <cit> .
similarly, the potential phenotypic relevance of this selection was extended from a role in the rapid expansion of human brain size  <cit>  to a more widespread, conserved role in primate brain evolution  <cit> .
this shift has been accompanied by more rigorous hypothesis testing on larger datasets  <cit> .
for example, it has previously been found that the molecular evolution of aspm and cdk5rap <dig> co-evolves with brain mass, particularly neonatal brain mass  <cit> .
the association between aspm evolution and brain mass is particularly interesting as it is found in primate clades which experienced both increases and decreases in brain mass  <cit> .
elsewhere, tentative evidence has been found linking the evolution of mcph <dig> to sexual dimorphism in brain mass in primates  <cit> , a surprising finding supported, in part, by human population studies of sex-specific associations between snps in microcephaly genes and brain size  <cit>  and functional analyses of base pair substitutions that may interact with sex-specific developmental pathways  <cit> .
other studies have extended the taxonomic scope beyond primates to test the hypothesis that microcephaly genes may contribute to the evolution of brain size in other mammals.
notably, so far these studies have been limited to species with relatively large brains.
in cetaceans, both mcph <dig> and aspm have been shown to have evolved under positive selection  <cit> .
however, evidence linking selection on either locus to brain size in cetaceans is lacking  <cit> .
in addition, exclusively studying large brained clades inevitably leads to an ascertainment bias.
indeed, there is evidence that both of these genes evolved under positive selection across placental mammals  <cit> .
if this is the case, there are clear implications for our understanding of brain evolution.
first, if there is an evolutionary link between microcephaly loci and changes in neurogenesis, such pervasive selection may suggest a conserved genetic basis to some aspects of mammalian brain size evolution.
second, the diversity of mammalian brain sizes could provide a good comparative framework in which to test for gene-phenotype co-evolution.
finally, if microcephaly genes do have a conserved evolutionary role in brain size, or any other phenotype, the results of functional assays within and between more practically tractable species than primates, such as rodents, may generalize to other mammals.
in this study, we examine patterns of molecular evolution in seven microcephaly genes across placental mammals, including the first comprehensive interspecific analysis of three of these loci, stil, cep <dig> and wdr <dig>  and the first mammal-wide analysis of two further loci, cdk5rap <dig> and cenpj.
we perform tests for adaptive evolution within primates, across non-primate mammals and across the combined data to test how pervasive the signature of positive selection is.
we further test whether the pattern of evolution of these genes differ in primates compared to non-primates, and finally provide a preliminary assessment of the link between microcephaly genes and brain size in non-primate clades.
we find a signature of positive selection that is strong and widespread for all but one of the loci and identify intriguing evidence of an association between two loci and neonatal brain mass in two non-primate clades.
greater sampling both at the molecular and phenotypic level are necessary to perform robust tests of gene-phenotype associations to confirm this hypothesis, but this preliminary extension of the role of microcephaly genes in brain evolution beyond primates may have a number of implications.
RESULTS
tests for positive selection
full coding sequences for seven loci  were obtained for  <dig> primates and  <dig> non-primate eutherian mammals .
two site model tests were applied to these datasets, the m1a/m2a and m8a/m <dig> pairs implemented in paml  <cit> .
these allow the ω  to vary among sites but not across lineages  <cit> .
across primates
across the  <dig> primate species for which full coding sequences are available for all  <dig> loci, four loci - aspm, cdk5rap <dig>  cenpj and cep <dig> - show a consistent signature of positive selection .
of the three that do not, one of these, mcph <dig>  has previously been shown to have evolved under positive selection across a larger dataset of partial coding sequence in anthropoid primates  <cit>  suggesting that the lack of significance may be due to the small sample size.
we therefore expanded the anthropoid dataset to include  <dig> species by amplifying selected exons of the remaining two loci , targeting regions with high dn/ds based on a sliding window analysis of the full coding sequence .
consistent with an effect of sample size these additional data yield evidence for positive selection acting on wdr <dig>  =  <dig> , p =  <dig> ; m8a/m <dig> lr =  <dig> , p =  <dig> ).
the results for stil are inconsistent between tests .
across mammals
consistent evidence for positive selection is found across non-primate mammals under both site model tests for  <dig> loci - aspm, cdk5rap <dig>  mcph <dig>  cenpj and cep <dig> .
when the site model tests are repeated after incorporating the  <dig> primate sequences extensive positive selection is again found, with the m8a/m <dig> test for stil also becoming narrowly significant .
among the loci which experienced positive selection across mammals the proportion of sites targeted by selection varies from ~2% to ~10% with dn/ds estimates for these sites typically between  <dig>  and  <dig> .
diversifying selection on microcephaly loci across mammals
we next examined patterns of divergent selection in different mammalian clades using branch and clade models.
branch models allow dn/ds to vary across branches in the phylogeny but not across sites  <cit> , whereas clade models allow a proportion of sites to undergo divergent selection pressures in two or more clades defined a priori <cit> .
comparisons of the gene-wide average dn/ds between primates and non-primate mammals are significant for all microcephaly loci except wdr <dig> .
whilst this may suggest a greater influence of positive selection in primates, the clade models do not support this general conclusion.
here, only two loci, cdk5rap <dig> and wdr <dig>  are supported as having a proportion of sites under significantly different selective regimes between primates and other mammals .
it therefore seems likely that the significant branch model results reflect technical issues, such as branch length, or neutral effects such as differences in population size or life history  <cit> .
the possibility of divergent selective pressure was next explored on a wider taxonomic scale comparing three major eutherian clades: the euarchontoglires , the laurasiatheria , and the afrotheria + xenarthra  .
under the branch models there is little evidence for divergent selective regimes between these three clades and clade model tests are only significant for two loci, aspm and mcph <dig> .
the overarching pattern of selection on microcephaly loci is therefore a consistent signature of positive selection across all eutherian mammals, perhaps with the exception of stil, but with relatively little evidence of clade-specific differences in overall rates of evolution.
linking molecular and phenotypic evolution
understanding the phenotypic relevance of this widespread positive selection is clearly of major interest.
given the long held hypothesis that selection on microcephaly genes in primates is linked to brain expansion  <cit>  and the comparative evidence linking rates of evolution of aspm and cdk5rap <dig> to variation in brain mass across anthropoid primates  <cit> , a particular interest is the possibility that microcephaly genes may have a conserved role in mammalian brain evolution  <cit> .
one approach to test a link between molecular evolution of candidate genes and brain size has been to compare pairs of species that differ in brain size .
in our phylogeny three episodes of brain expansion are relatively well documented in the fossil record and form taxon-pairs with smaller brain species; the expansion of human  <cit> , dolphin  <cit>  and elephant brain sizes  <cit> .
branch models were used to test for significant differences in dn/ds between the bottlenose dolphin and the cow , between the elephant and the hyrax , and between humans and the bushbaby .
only two loci showed significant differences between elephant and hyrax , of which only one, mcph <dig>  had a higher dn/ds in the elephant lineage.
comparing the dolphin and cow branches no loci showed significant differences, though cdk5rap <dig> approached significance and had a higher dn/ds on the dolphin branch.
finally, only cep <dig> showed a significantly higher dn/ds along the human lineage compared to the bushbaby lineage.
hence, there is no consistent pattern of acceleration of microcephaly evolution along lineages leading to selected large brain species, compared to their smaller brained sister-lineage as represented in this dataset.
again, as these branches are relatively long dn/ds estimates are likely influenced by variation in life history and population size.
an alternative test of a gene-phenotype link is to take a more quantitative approach and test for co-evolution between molecular rates and the trait of interest.
we tested for associations between root-to-tip dn/ds and two traits: neonatal and adult brain mass.
comparisons between any associations with these traits are informative as we expect genes involved in the evolution of neurogenesis to show a stronger relationship with neonatal brain mass as mammalian neurogenesis is predominantly prenatal  <cit> .
we applied this test to three sub-clades within the mammalian phylogeny; anthropoid primates , the focus of previous studies, and two additional grand-orders  <cit> , the glires  and euungulata  chosen to reflect a trade-off between a relative consistency in life history parameters, the number of species and sample density.
within primates we found no evidence for an association with any of the seven loci .
using larger datasets of partial coding sequence the evolution of aspm and cdk5rap <dig> have been linked to brain size  <cit> .
we repeated the tests using only the exons sequenced for previous studies  <cit>  and again found no association suggesting sample size, which was much lower in the current study, and the phenotypic diversity within the dataset contribute to the difference in results between the present analysis and previous studies.
within the glires and euungulata datasets there is an intriguing pattern suggesting a link between brain size and selection on some microcephaly loci.
although based on small samples sizes, these results provide the first evidence of a microcephaly gene-phenotype association outside primates.
in glires only aspm is significantly associated with neonatal brain mass , whilst cdk5rap <dig>  and stil  show non-significant trends.
in all three cases the strength of the association is reduced, or lost, with adult brain mass .
within euungulata aspm , cdk5rap <dig>  and wdr <dig>  show significant associations with neonatal brain size and again the significance falls when adult brain size is considered .
in contrast stil shows a significant association with adult brain size , which is reduced to a non-significant trend when neonatal brain size is considered .
discussion
our results indicate that the majority of loci linked to microcephaly, a severe neurodevelopmental disorder, have been targeted by positive selection throughout the evolution of eutherian mammals, in both primates and non-primates.
given the large evolutionary time under consideration it is remarkable that such a consistent pattern should be found on a functionally related set of genes that share a key role in neural development.
only stil shows a weak or inconsistent signature of adaptive evolution.
given the paucity of brain expressed coding-genes with high rates of evolution identified in the majority of genome scans  this raises the intriguing possibility that microcephaly genes are hotspots for positive selection among brain expressed coding genes.
whether this is true or not will require a further examination of genome wide patterns of selection across a greater number of species, as no study has included comparable numbers of species.
a recent exome-wide analysis across  <dig> species of primates, to our knowledge the largest to date, did not report any enrichment for brain-expressed genes or neurodevelopmental processes among positively selected genes  <cit> , nor did a study of six mammalian genomes  <cit> .
however, these sample sizes are towards the lower limit at which site-based models have power to detect positive selection  <cit> .
to fully assess whether microcephaly genes are targeted by positive selection more frequently than other neurodevelopmental genes it will be necessary to perform genome-wide analyses of the selective regimes acting on mammalian protein coding genes with much larger sample sizes than previous studies.
the frequent targeting of microcephaly genes by selection also raises important questions about the functional effects of substitutions in these loci.
the majority of the microcephaly genes contribute to the development and function of the spindle poles, or astral microtubule network  <cit> , and disruption of this function is linked to changes in spindle or microtubule behavior  <cit> .
the spindle poles play a key role in the cell fate switch of neural progenitor cells.
in the developing brain a pool of neural progenitor cells undergo successive symmetric and proliferative divisions, their number increasingly exponentially  <cit> .
after a certain number of divisions these cells begin to divide asymmetrically, with each division contributing a neuron to a radial column of cortical neurons before terminally dividing into two neurons  <cit> .
this switch between proliferative, symmetric divisions to asymmetric neurogenic divisions is controlled by the angle of cell division, which is in turn controlled by the spindle poles  <cit> .
hence, functional changes in microcephaly genes could conceivably alter the duration of symmetric divisions to ultimately change the number of neurons produced during brain development.
a key role for modification of this cell fate switch is consistent with evo-devo models of brain expansion  <cit> .
the radial unit hypothesis suggests a general mechanism for rapidly increasing brain size in mammals is to prolong the period of symmetric divisions, resulting in more radial units of neurons and an expanded volume  <cit> .
microcephaly genes have precisely the functions the radial unit hypothesis would predict as being targeted during episodes of brain expansion.
notably, another candidate gene, nin, which functions in the maintenance of asymmetric divisions of neural progenitor cells  <cit> , and has not been linked to microcephaly, also shows a signature of adaptive molecular evolution across anthropoid primates  <cit> .
maintaining this asymmetric cell division would result in larger numbers of neurons/radial unit and comparative data suggest an association between selection at this locus and interspecific variation in the number of neurons/unit area in the cortex, a suggested proxy for the number of neurons per radial unit  <cit> .
both cell fate switches highlighted by the radial unit hypothesis therefore involve proteins that were targeted by positive selection across long periods of evolutionary time.
although there is some variation in cell developmental pathways leading to neuron production among mammals, such as the emergence of additional progenitor cells associated with increased gyrification  <cit> , much of the developmental program is conserved  <cit> .
comparative data across mammals also suggest the timing of brain development is conserved  <cit>  indicating strong constraints act on brain development, limiting the potential ways in which selection can modify brain size and structure.
these constraints may be due to pleiotropic effects of shared developmental pathways or to adaptive, functional co-evolution  <cit>  but regardless, provide little reason to suspect genes targeted by selection in relation to primate brain evolution should differ from those targeted in non-primate mammals.
such convergence, or parallelism, in the genetic basis of mammalian phenotypes may be more widespread than perhaps expected, with examples including sensory perception  <cit> , energy metabolism  <cit> , digestive enzymes  <cit> , immunity genes  <cit>  and coloration  <cit> .
the results of our phylogenetic comparative analyses provide direct evidence that evolution of brain size is indeed linked to four microcephaly genes  in two mammalian clades, glires and euungulata.
furthermore, as predicted by the neurodevelopmental models above, the relationship is generally stronger for neonatal brain size, a time point by which most neurons have already arisen  <cit> , than adult brain size.
these results raise the possibility that aspm and cdk5rap <dig> play a consistent role in mammalian brain evolution, as these two loci have previously been implicated in brain evolution in primates  <cit>  .
however, we stress that the sample sizes are small, the significance of our gene-phenotype tests are all > <dig> , and it will be necessary to confirm and further explore these results with larger datasets.
regardless, the key implication of our analysis is that diversifying evolutionary studies of brain size beyond enigmatic, large brained clades may offer new avenues for testing evolutionary or functional hypotheses.
it is likely however, that testing such gene-phenotype hypotheses over large evolutionary distances will be a challenging endeavor for a number of reasons  <cit> .
first, effects of non-adaptive processes such as variation in life history or population size may affect dn/ds <cit>  introducing noise to any genuine gene-phenotype association making larger, more densely sampled datasets desirable.
similarly, variation in phenotypic structure, such as neuron density, which may affect the linearity of the relationship between brain mass and neuron number  <cit> , may impact upon gene-phenotype comparisons across large evolutionary distances.
careful consideration is therefore needed as to what phenotype is most relevant, and molecular studies should target species where phenotypic data is available, when these data are a limiting factor.
second, evolutionary reversals may be common in some mammalian clades and this may obscure gene-phenotype associations.
for example, if a gene is targeted by selection during both increases and decreases in brain size there could be a mismatch between high rates of evolution at the molecular level and a small perceived difference in brain size.
understanding the evolutionary history of a phenotype then becomes a key component of the search for that phenotype’s molecular basis.
an example of this comes from callitrichids, a subfamily of new world monkeys that experienced a decrease in brain mass  <cit>  and gyrencephaly  <cit>  in association with major episodes of phyletic dwarfism  <cit> .
callitrichids appear as outliers to the positive association between brain mass and dn/ds for aspm across anthropoids  <cit> , but when considered alone show a negative association between brain mass and dn/ds for aspm suggesting functional changes in aspm may have contributed to decreases in brain mass in this clade  <cit> .
this is also a potential explanation for the lack of an evolutionary association between aspm and brain mass in cetaceans, where reversals are more common  <cit> .
a third problem could be encountered if a gene is associated with a phenotype but has an intermittent role in its evolution.
a potential example here is cdk5rap <dig> that coevolves with brain mass in primate taxa where it has increased  <cit> , but not when it has decreased  <cit> .
in the present case the positive trends found in glires and euungulata would be expected if phenotypic differences within these clades are mostly due to increases in brain size on lineages leading to larger brained species, rather than decreases leading to smaller brained species.
finally, a major challenge may occur when the gene-phenotype association is limited to a small subset of domains or sites within a gene.
site-based methods for detecting positive selection were developed because it is thought that selection is unlikely to act across a whole gene equally  <cit> .
this positive selection is presumably associated with some phenotypic, functional or fitness-related change, and it is therefore likely that gene-phenotype associations may be limited to a subset of domains or codons.
developing methods, which are capable of testing gene-phenotype associations on a site-by-site basis, or which incorporate a form of sliding window analysis, may be a worthwhile endeavor .
again, to gain sufficient statistical power such analyses will require large, densely sampled datasets.
the pervasive signature of positive selection across mammals, combined with the limited evidence of divergence in selection pressures, suggests results from experimentally tractable clades may be applicable to wider taxonomic groups.
the link between selection on microcephaly genes and the evolution of brain size has yet to be confirmed  by functional data.
mice transgenic for aspm, and in vitro assays for mcph <dig> confirm changes in the coding sequence have functional affects  <cit> .
however, although the human sequence of aspm rescued the phenotype of transgenic mice with a disrupted copy of aspm it did not lead to an increase brain size  <cit> .
this has been interpreted as being indicative of functional conservation  <cit> , but without the reciprocal experiments this conclusion may be premature.
it could be, for example, that the effects of microcephaly genes are background dependent, or are combinatorial such that changing individual genes has only a minor or no affect in isolation.
given the importance placed in functional confirmation of gene-phenotype associations a shift towards examining phenotypic diversity within experimentally tractable clades may be worthwhile.
for example, greater sampling density in rodents could permit comparative analyses to test for positive selection, and subsequently test for gene-phenotype associations.
a range of data exists for a number of rodents that vary widely in brain size, neuron number and gyrencephaly  <cit> , and the results presented above for glires provide some encouragement for pursuing studies in this clade.
both micro and macroevolutionary gene-phenotype associations could be performed and, if an association were found, supporting evidence could be sought through evolutionary developmental studies.
rather than doing one-way human/mouse transgenic experiments  <cit> , it may be just as informative to perform, for example, reciprocal rat/mouse transgenic experiments.
although we note that recent developments in in vitro human organoids may render two-way human/mouse transgenics technically feasible  <cit> , ethical considerations may still limit this approach.
regardless of the results of such a study, greater sampling within clades with smaller brains will provide a useful comparison with larger brained mammalian orders, such as primates and cetaceans, and are clearly necessary to avoid ascertainment biases.
CONCLUSIONS
we have shown that microcephaly genes have experienced pervasive positive selection not just across primates but across placental mammals.
we find little evidence to suggest that these loci experienced divergent selective pressures in different clades, which may suggest conservation in function and imply a common phenotypic relevance for this wide spread positive selection.
developmental models of cortical expansion and evidence for conservation in brain developmental pathways provide a clear basis with which to hypothesize that the phenotype of relevance is brain mass, or more specifically the number of neurons produced during cortical neurogenesis.
we provide evidence to support this hypothesis in glires and euungulata that, combined with previous work in primates, suggests that this phenotypic association may be common across mammals.
whilst several challenges face attempts to test gene-phenotype hypotheses, the persistent signal of positive selection should permit useful studies in more experimentally tractable species.

 for over a decade researchers interested in the genetic basis of brain evolution have sought clues in the molecular evolution of genes associated with the neurodevelopmental disorder, primary microcephaly. microcephaly is a congenital disorder characterized by an early cessation of brain growth, specifically affecting cortical development. mendelian inheritance of microcephaly has now been linked to deleterious mutations at seven unlinked loci. these loci encode proteins with central roles in neurogenesis, largely in the formation and function of the centrioles, which in turn control the way in which neural progenitor cells divide. disruption of these crucial functions causes microcephaly, and it is hypothesized that modification of their function through evolutionary time could tip the cell fate switch towards greater neurogenic output, underpinning the evolution of larger brains. much of the early focus on the molecular evolution of microcephaly genes centered on the role of two loci in human evolution. early studies suggested an increase in the rate at which these genes evolved along the lineage leading to humans. intriguingly, as more microcephaly loci were identified each has been shown to evolve adaptively suggesting they may be a persistent target of positive selection. in addition, as more species were incorporated into analyses the signature of positive selection extended beyond humans first to great apes, then to all anthropoid primates. similarly, the potential phenotypic relevance of this selection was extended from a role in the rapid expansion of human brain size to a more widespread, conserved role in primate brain evolution. this shift has been accompanied by more rigorous hypothesis testing on larger datasets. for example, it has previously been found that the molecular evolution of aspm and cdk5rap co-evolves with brain mass, particularly neonatal brain mass. the association between aspm evolution and brain mass is particularly interesting as it is found in primate clades which experienced both increases and decreases in brain mass. elsewhere, tentative evidence has been found linking the evolution of mcph to sexual dimorphism in brain mass in primates, a surprising finding supported, in part, by human population studies of sex-specific associations between snps in microcephaly genes and brain size and functional analyses of base pair substitutions that may interact with sex-specific developmental pathways. other studies have extended the taxonomic scope beyond primates to test the hypothesis that microcephaly genes may contribute to the evolution of brain size in other mammals. notably, so far these studies have been limited to species with relatively large brains. in cetaceans, both mcph and aspm have been shown to have evolved under positive selection. however, evidence linking selection on either locus to brain size in cetaceans is lacking. in addition, exclusively studying large brained clades inevitably leads to an ascertainment bias. indeed, there is evidence that both of these genes evolved under positive selection across placental mammals. if this is the case, there are clear implications for our understanding of brain evolution. first, if there is an evolutionary link between microcephaly loci and changes in neurogenesis, such pervasive selection may suggest a conserved genetic basis to some aspects of mammalian brain size evolution. second, the diversity of mammalian brain sizes could provide a good comparative framework in which to test for gene-phenotype co-evolution. finally, if microcephaly genes do have a conserved evolutionary role in brain size, or any other phenotype, the results of functional assays within and between more practically tractable species than primates, such as rodents, may generalize to other mammals. in this study, we examine patterns of molecular evolution in seven microcephaly genes across placental mammals, including the first comprehensive interspecific analysis of three of these loci, stil, cep and wdr and the first mammal-wide analysis of two further loci, cdk5rap and cenpj. we perform tests for adaptive evolution within primates, across non-primate mammals and across the combined data to test how pervasive the signature of positive selection is. we further test whether the pattern of evolution of these genes differ in primates compared to non-primates, and finally provide a preliminary assessment of the link between microcephaly genes and brain size in non-primate clades. we find a signature of positive selection that is strong and widespread for all but one of the loci and identify intriguing evidence of an association between two loci and neonatal brain mass in two non-primate clades. greater sampling both at the molecular and phenotypic level are necessary to perform robust tests of gene-phenotype associations to confirm this hypothesis, but this preliminary extension of the role of microcephaly genes in brain evolution beyond primates may have a number of implications.  tests for positive selection full coding sequences for seven loci were obtained for primates and non-primate eutherian mammals. two site model tests were applied to these datasets, the m1a/m2a and m8a/m pairs implemented in paml. these allow the ω to vary among sites but not across lineages. across primates across the primate species for which full coding sequences are available for all loci, four loci - aspm, cdk5rap cenpj and cep - show a consistent signature of positive selection. of the three that do not, one of these, mcph has previously been shown to have evolved under positive selection across a larger dataset of partial coding sequence in anthropoid primates suggesting that the lack of significance may be due to the small sample size. we therefore expanded the anthropoid dataset to include species by amplifying selected exons of the remaining two loci, targeting regions with high dn/ds based on a sliding window analysis of the full coding sequence. consistent with an effect of sample size these additional data yield evidence for positive selection acting on wdr  = , p = ; m8a/m lr = , p =  ). the results for stil are inconsistent between tests. across mammals consistent evidence for positive selection is found across non-primate mammals under both site model tests for loci - aspm, cdk5rap mcph cenpj and cep. when the site model tests are repeated after incorporating the primate sequences extensive positive selection is again found, with the m8a/m test for stil also becoming narrowly significant. among the loci which experienced positive selection across mammals the proportion of sites targeted by selection varies from ~2% to ~10% with dn/ds estimates for these sites typically between and. diversifying selection on microcephaly loci across mammals we next examined patterns of divergent selection in different mammalian clades using branch and clade models. branch models allow dn/ds to vary across branches in the phylogeny but not across sites, whereas clade models allow a proportion of sites to undergo divergent selection pressures in two or more clades defined a priori. comparisons of the gene-wide average dn/ds between primates and non-primate mammals are significant for all microcephaly loci except wdr. whilst this may suggest a greater influence of positive selection in primates, the clade models do not support this general conclusion. here, only two loci, cdk5rap and wdr are supported as having a proportion of sites under significantly different selective regimes between primates and other mammals. it therefore seems likely that the significant branch model results reflect technical issues, such as branch length, or neutral effects such as differences in population size or life history. the possibility of divergent selective pressure was next explored on a wider taxonomic scale comparing three major eutherian clades: the euarchontoglires, the laurasiatheria, and the afrotheria + xenarthra. under the branch models there is little evidence for divergent selective regimes between these three clades and clade model tests are only significant for two loci, aspm and mcph. the overarching pattern of selection on microcephaly loci is therefore a consistent signature of positive selection across all eutherian mammals, perhaps with the exception of stil, but with relatively little evidence of clade-specific differences in overall rates of evolution. linking molecular and phenotypic evolution understanding the phenotypic relevance of this widespread positive selection is clearly of major interest. given the long held hypothesis that selection on microcephaly genes in primates is linked to brain expansion and the comparative evidence linking rates of evolution of aspm and cdk5rap to variation in brain mass across anthropoid primates, a particular interest is the possibility that microcephaly genes may have a conserved role in mammalian brain evolution. one approach to test a link between molecular evolution of candidate genes and brain size has been to compare pairs of species that differ in brain size. in our phylogeny three episodes of brain expansion are relatively well documented in the fossil record and form taxon-pairs with smaller brain species; the expansion of human, dolphin and elephant brain sizes. branch models were used to test for significant differences in dn/ds between the bottlenose dolphin and the cow, between the elephant and the hyrax, and between humans and the bushbaby. only two loci showed significant differences between elephant and hyrax, of which only one, mcph had a higher dn/ds in the elephant lineage. comparing the dolphin and cow branches no loci showed significant differences, though cdk5rap approached significance and had a higher dn/ds on the dolphin branch. finally, only cep showed a significantly higher dn/ds along the human lineage compared to the bushbaby lineage. hence, there is no consistent pattern of acceleration of microcephaly evolution along lineages leading to selected large brain species, compared to their smaller brained sister-lineage as represented in this dataset. again, as these branches are relatively long dn/ds estimates are likely influenced by variation in life history and population size. an alternative test of a gene-phenotype link is to take a more quantitative approach and test for co-evolution between molecular rates and the trait of interest. we tested for associations between root-to-tip dn/ds and two traits: neonatal and adult brain mass. comparisons between any associations with these traits are informative as we expect genes involved in the evolution of neurogenesis to show a stronger relationship with neonatal brain mass as mammalian neurogenesis is predominantly prenatal. we applied this test to three sub-clades within the mammalian phylogeny; anthropoid primates, the focus of previous studies, and two additional grand-orders, the glires and euungulata chosen to reflect a trade-off between a relative consistency in life history parameters, the number of species and sample density. within primates we found no evidence for an association with any of the seven loci. using larger datasets of partial coding sequence the evolution of aspm and cdk5rap have been linked to brain size. we repeated the tests using only the exons sequenced for previous studies and again found no association suggesting sample size, which was much lower in the current study, and the phenotypic diversity within the dataset contribute to the difference in results between the present analysis and previous studies. within the glires and euungulata datasets there is an intriguing pattern suggesting a link between brain size and selection on some microcephaly loci. although based on small samples sizes, these results provide the first evidence of a microcephaly gene-phenotype association outside primates. in glires only aspm is significantly associated with neonatal brain mass, whilst cdk5rap and stil show non-significant trends. in all three cases the strength of the association is reduced, or lost, with adult brain mass. within euungulata aspm, cdk5rap and wdr show significant associations with neonatal brain size and again the significance falls when adult brain size is considered. in contrast stil shows a significant association with adult brain size, which is reduced to a non-significant trend when neonatal brain size is considered. discussion our results indicate that the majority of loci linked to microcephaly, a severe neurodevelopmental disorder, have been targeted by positive selection throughout the evolution of eutherian mammals, in both primates and non-primates. given the large evolutionary time under consideration it is remarkable that such a consistent pattern should be found on a functionally related set of genes that share a key role in neural development. only stil shows a weak or inconsistent signature of adaptive evolution. given the paucity of brain expressed coding-genes with high rates of evolution identified in the majority of genome scans this raises the intriguing possibility that microcephaly genes are hotspots for positive selection among brain expressed coding genes. whether this is true or not will require a further examination of genome wide patterns of selection across a greater number of species, as no study has included comparable numbers of species. a recent exome-wide analysis across species of primates, to our knowledge the largest to date, did not report any enrichment for brain-expressed genes or neurodevelopmental processes among positively selected genes, nor did a study of six mammalian genomes. however, these sample sizes are towards the lower limit at which site-based models have power to detect positive selection. to fully assess whether microcephaly genes are targeted by positive selection more frequently than other neurodevelopmental genes it will be necessary to perform genome-wide analyses of the selective regimes acting on mammalian protein coding genes with much larger sample sizes than previous studies. the frequent targeting of microcephaly genes by selection also raises important questions about the functional effects of substitutions in these loci. the majority of the microcephaly genes contribute to the development and function of the spindle poles, or astral microtubule network, and disruption of this function is linked to changes in spindle or microtubule behavior. the spindle poles play a key role in the cell fate switch of neural progenitor cells. in the developing brain a pool of neural progenitor cells undergo successive symmetric and proliferative divisions, their number increasingly exponentially. after a certain number of divisions these cells begin to divide asymmetrically, with each division contributing a neuron to a radial column of cortical neurons before terminally dividing into two neurons. this switch between proliferative, symmetric divisions to asymmetric neurogenic divisions is controlled by the angle of cell division, which is in turn controlled by the spindle poles. hence, functional changes in microcephaly genes could conceivably alter the duration of symmetric divisions to ultimately change the number of neurons produced during brain development. a key role for modification of this cell fate switch is consistent with evo-devo models of brain expansion. the radial unit hypothesis suggests a general mechanism for rapidly increasing brain size in mammals is to prolong the period of symmetric divisions, resulting in more radial units of neurons and an expanded volume. microcephaly genes have precisely the functions the radial unit hypothesis would predict as being targeted during episodes of brain expansion. notably, another candidate gene, nin, which functions in the maintenance of asymmetric divisions of neural progenitor cells, and has not been linked to microcephaly, also shows a signature of adaptive molecular evolution across anthropoid primates. maintaining this asymmetric cell division would result in larger numbers of neurons/radial unit and comparative data suggest an association between selection at this locus and interspecific variation in the number of neurons/unit area in the cortex, a suggested proxy for the number of neurons per radial unit. both cell fate switches highlighted by the radial unit hypothesis therefore involve proteins that were targeted by positive selection across long periods of evolutionary time. although there is some variation in cell developmental pathways leading to neuron production among mammals, such as the emergence of additional progenitor cells associated with increased gyrification, much of the developmental program is conserved. comparative data across mammals also suggest the timing of brain development is conserved indicating strong constraints act on brain development, limiting the potential ways in which selection can modify brain size and structure. these constraints may be due to pleiotropic effects of shared developmental pathways or to adaptive, functional co-evolution but regardless, provide little reason to suspect genes targeted by selection in relation to primate brain evolution should differ from those targeted in non-primate mammals. such convergence, or parallelism, in the genetic basis of mammalian phenotypes may be more widespread than perhaps expected, with examples including sensory perception, energy metabolism, digestive enzymes, immunity genes and coloration. the results of our phylogenetic comparative analyses provide direct evidence that evolution of brain size is indeed linked to four microcephaly genes in two mammalian clades, glires and euungulata. furthermore, as predicted by the neurodevelopmental models above, the relationship is generally stronger for neonatal brain size, a time point by which most neurons have already arisen, than adult brain size. these results raise the possibility that aspm and cdk5rap play a consistent role in mammalian brain evolution, as these two loci have previously been implicated in brain evolution in primates. however, we stress that the sample sizes are small, the significance of our gene-phenotype tests are all >, and it will be necessary to confirm and further explore these results with larger datasets. regardless, the key implication of our analysis is that diversifying evolutionary studies of brain size beyond enigmatic, large brained clades may offer new avenues for testing evolutionary or functional hypotheses. it is likely however, that testing such gene-phenotype hypotheses over large evolutionary distances will be a challenging endeavor for a number of reasons. first, effects of non-adaptive processes such as variation in life history or population size may affect dn/ds introducing noise to any genuine gene-phenotype association making larger, more densely sampled datasets desirable. similarly, variation in phenotypic structure, such as neuron density, which may affect the linearity of the relationship between brain mass and neuron number, may impact upon gene-phenotype comparisons across large evolutionary distances. careful consideration is therefore needed as to what phenotype is most relevant, and molecular studies should target species where phenotypic data is available, when these data are a limiting factor. second, evolutionary reversals may be common in some mammalian clades and this may obscure gene-phenotype associations. for example, if a gene is targeted by selection during both increases and decreases in brain size there could be a mismatch between high rates of evolution at the molecular level and a small perceived difference in brain size. understanding the evolutionary history of a phenotype then becomes a key component of the search for that phenotype’s molecular basis. an example of this comes from callitrichids, a subfamily of new world monkeys that experienced a decrease in brain mass and gyrencephaly in association with major episodes of phyletic dwarfism. callitrichids appear as outliers to the positive association between brain mass and dn/ds for aspm across anthropoids, but when considered alone show a negative association between brain mass and dn/ds for aspm suggesting functional changes in aspm may have contributed to decreases in brain mass in this clade. this is also a potential explanation for the lack of an evolutionary association between aspm and brain mass in cetaceans, where reversals are more common. a third problem could be encountered if a gene is associated with a phenotype but has an intermittent role in its evolution. a potential example here is cdk5rap that coevolves with brain mass in primate taxa where it has increased, but not when it has decreased. in the present case the positive trends found in glires and euungulata would be expected if phenotypic differences within these clades are mostly due to increases in brain size on lineages leading to larger brained species, rather than decreases leading to smaller brained species. finally, a major challenge may occur when the gene-phenotype association is limited to a small subset of domains or sites within a gene. site-based methods for detecting positive selection were developed because it is thought that selection is unlikely to act across a whole gene equally. this positive selection is presumably associated with some phenotypic, functional or fitness-related change, and it is therefore likely that gene-phenotype associations may be limited to a subset of domains or codons. developing methods, which are capable of testing gene-phenotype associations on a site-by-site basis, or which incorporate a form of sliding window analysis, may be a worthwhile endeavor. again, to gain sufficient statistical power such analyses will require large, densely sampled datasets. the pervasive signature of positive selection across mammals, combined with the limited evidence of divergence in selection pressures, suggests results from experimentally tractable clades may be applicable to wider taxonomic groups. the link between selection on microcephaly genes and the evolution of brain size has yet to be confirmed by functional data. mice transgenic for aspm, and in vitro assays for mcph confirm changes in the coding sequence have functional affects. however, although the human sequence of aspm rescued the phenotype of transgenic mice with a disrupted copy of aspm it did not lead to an increase brain size. this has been interpreted as being indicative of functional conservation, but without the reciprocal experiments this conclusion may be premature. it could be, for example, that the effects of microcephaly genes are background dependent, or are combinatorial such that changing individual genes has only a minor or no affect in isolation. given the importance placed in functional confirmation of gene-phenotype associations a shift towards examining phenotypic diversity within experimentally tractable clades may be worthwhile. for example, greater sampling density in rodents could permit comparative analyses to test for positive selection, and subsequently test for gene-phenotype associations. a range of data exists for a number of rodents that vary widely in brain size, neuron number and gyrencephaly, and the results presented above for glires provide some encouragement for pursuing studies in this clade. both micro and macroevolutionary gene-phenotype associations could be performed and, if an association were found, supporting evidence could be sought through evolutionary developmental studies. rather than doing one-way human/mouse transgenic experiments, it may be just as informative to perform, for example, reciprocal rat/mouse transgenic experiments. although we note that recent developments in in vitro human organoids may render two-way human/mouse transgenics technically feasible, ethical considerations may still limit this approach. regardless of the results of such a study, greater sampling within clades with smaller brains will provide a useful comparison with larger brained mammalian orders, such as primates and cetaceans, and are clearly necessary to avoid ascertainment biases.  we have shown that microcephaly genes have experienced pervasive positive selection not just across primates but across placental mammals. we find little evidence to suggest that these loci experienced divergent selective pressures in different clades, which may suggest conservation in function and imply a common phenotypic relevance for this wide spread positive selection. developmental models of cortical expansion and evidence for conservation in brain developmental pathways provide a clear basis with which to hypothesize that the phenotype of relevance is brain mass, or more specifically the number of neurons produced during cortical neurogenesis. we provide evidence to support this hypothesis in glires and euungulata that, combined with previous work in primates, suggests that this phenotypic association may be common across mammals. whilst several challenges face attempts to test gene-phenotype hypotheses, the persistent signal of positive selection should permit useful studies in more experimentally tractable species. 
we discuss the implications of our results for studies of the genetic basis of brain evolution, and explicit tests of gene-phenotype hypotheses. using phylogenetically corrected comparative analyses, we find that the evolution of two microcephaly loci, aspm and cdk5rap are correlated with neonatal brain size in glires and euungulata, the two most densely sampled non-primate clades. we find extensive evidence for positive selection having acted on the majority of these loci not just in primates but also across non-primate mammals. nevertheless, several limitations of currently available data and gene-phenotype tests are discussed, including sparse sampling across large evolutionary distances, averaging gene-wide rates of evolution, potential phenotypic variation and evolutionary reversals. together with previous results, this suggests that aspm and cdk5rap may have had a consistent role in the evolution of brain size in mammals. comparative data suggest a link between selection on some of these loci and the evolution of primate brain size. whether or not either positive selection or this phenotypic association are unique to primates is unclear, but recent studies in cetaceans suggest at least two microcephaly genes evolved adaptively in other large brained mammalian clades. furthermore, the patterns of selection in major mammalian clades are not significantly different. genes associated with the neurodevelopmental disorder microcephaly display a strong signature of adaptive evolution in primates.
 genes associated with the neurodevelopmental disorder microcephaly display a strong signature of adaptive evolution in primates. comparative data suggest a link between selection on some of these loci and the evolution of primate brain size. whether or not either positive selection or this phenotypic association are unique to primates is unclear, but recent studies in cetaceans suggest at least two microcephaly genes evolved adaptively in other large brained mammalian clades.  here we analyse the evolution of seven microcephaly loci, including three recently identified loci, across eutherian mammals. we find extensive evidence for positive selection having acted on the majority of these loci not just in primates but also across non-primate mammals. furthermore, the patterns of selection in major mammalian clades are not significantly different. using phylogenetically corrected comparative analyses, we find that the evolution of two microcephaly loci, aspm and cdk5rap are correlated with neonatal brain size in glires and euungulata, the two most densely sampled non-primate clades.  together with previous results, this suggests that aspm and cdk5rap may have had a consistent role in the evolution of brain size in mammals. nevertheless, several limitations of currently available data and gene-phenotype tests are discussed, including sparse sampling across large evolutionary distances, averaging gene-wide rates of evolution, potential phenotypic variation and evolutionary reversals. we discuss the implications of our results for studies of the genetic basis of brain evolution, and explicit tests of gene-phenotype hypotheses. aspmadaptive evolutionbrain sizecdk5rap2mammalsmicrocephaly genesneurogenesis 