BACKGROUND
work on the molecular basis of animal adaptation is providing ample evidence that small genetic changes can underlie striking phenotypic differences, both within and between species  <cit> .
of recent particular interest is the role of short tandemly repeating dna elements  as evolutionary ‘tuning knobs’  <cit> .
microsatellites are mutation-prone and often show substantial length variation in repeat number  <cit> .
these repeats are common in mammalian genomes, often occurring within coding exons, potentially translating into expansions and contractions of poly-amino acid stretches  <cit> .
moreover, proteins containing such repeats are often involved in development  <cit> .
thus, the function of a protein can be easily modified via alterations in repeat tract length  <cit>  and such modifications may have less drastic pleiotropic effects than other types of mutation  <cit> .
an example of this is the correlation between specific tandem repeats and variation in midfacial length  in carnivorans  <cit> .
variation in this striking morphological trait appears to be causatively associated with variation in coding sequence repeats within the gene runx <dig> .
runx <dig>  codes for a transcription factor that plays an important role in mediating osteoblast differentiation and activity  <cit> .
its function is critical for building and repairing bone.
the activity of runx <dig> is mediated by a few functional domains, one of which consists of a stretch of glutamines  followed by a stretch of alanines  .
for runx <dig>  the ratio of glutamines to alanines  appears to be positively correlated with the transcriptional activity of the protein  <cit> .
runx <dig> mutations in humans cause the skeletal disease cleidocranial dysplasia  and have been associated with a shortening or protruding of the face and other skeletodental pathologies  <cit> , some of which correspond to changes in certain clades of nonhuman mammals  <cit> .
in general, work on runx <dig> in humans and mice indicates that when runx <dig> is up-regulated, bone development is accelerated  <cit> .
interestingly, in comparisons of the modern human and neanderthal genomes, runx <dig> is specifically highlighted by green et al.
<cit>  as one of few genes showing a genetic signature of a selective sweep in the modern human lineage.
assuming the neanderthal sequence data are valid, this would suggest a potentially important role for runx <dig> in human-specific cranial/skeletal features.
the qa ratio of runx <dig> is correlated with facial length across breeds of dog .
specifically, dog breeds and other carnivoran species with a high qa ratio tend to have a relatively long rostrum or "face".
furthermore, sears et al.
<cit>  noted that the correlation was stronger among dogs, bears, raccoons and their relatives  than among cats, civets, and mongooses , related to the fact that the rostrum in caniforms shows more positive allometry during growth than in feliforms.
that is, compared to a cat, a dog shows more growth in the face relative to the braincase during its ontogeny.
the possibility that this correlation represents a general mechanism responsible for at least some aspects of skull growth across non-carnivoran mammals has not yet been thoroughly investigated.
facial shape and length are highly variable in many mammalian clades, including afrotherians , xenarthrans  and euarchontoglires .
morphometrically, the skull shows a tremendous amount of continuous variation across mammals, an observation which lends itself to the metaphor of the "tuning knob" as a genetic basis behind this variation  <cit> .
runx <dig>  specifically the qa ratio, is thus a good candidate for examining the genetic basis of variation in skull proportions across mammals.
here we assess the extent to which the correlation between skull shape and runx <dig> qa  ratio holds true across a variety of non-carnivoran placental mammals.
in particular, we focus on xenarthrans, afrotherians, and primates as these orders show marked variations in face length, which are thought to be adaptively associated with diet and ecology  <cit> , and for these orders we have access to morphometric and genetic data.
in analyzing data for  <dig> placental mammal species representing  <dig> orders , we find no correlation between runx <dig> sequence and face length  across our sample as a whole.
*see figure  <dig> for measurement details.
when greater than one, the number of individuals is given in parentheses.
1all variables were natural log transformed before analysis.
see figure  <dig> for measurement details.
1see figure  <dig> for measurement details; * statistically significant; ns not significant.
RESULTS
runx <dig> tandem repeat ratios
the translated amino acid sequences for the  <dig> species are provided in the supplementary material .
the runx <dig> amino acid sequences flanking the tandem repeat region were generally conserved across taxa providing evidence that orthologous rather than paralogous sequences were successfully retrieved.
in support of this, all publicly-available mammalian genomes contain a single annotated runx <dig> locus, suggesting that this gene has 1: <dig> orthology across mammals and has not been subject to duplication.
qa ratios calculated from the translated sequences range from  <dig>   to  <dig>   .
with the exception of equus, these qa ratios were generally lower than those of carnivorans  <cit> .
in their analysis of carnivora, sears et al.
<cit>  report a lowest value of  <dig>  for the qa ratio in potos flavus, which is close to the range of domesticated dog breeds .
glutamine and alanine amino acid counts for non-carnivoran mammals  were not markedly different than those of domestic dog breeds , although carnivorans are on the low end of alanine counts .
across the mammals included in our dataset, the majority of the qa ratios lie between  <dig> and  <dig> .
xenarthrans had the lowest variation in qa ratio across sampled species  and laurasiatherians had the highest .
we make such taxonomic comparisons tentatively, as species within clades are not equally distant to each other and the species sampled here represent only a subset of the extant diversity for each clade.
across the  <dig> mammals tested, both the polyq and polya are variable, but the polyq has a greater range of values than the polya repeat  .
also, intraspecific variation was detected in two  of the four species where multiple individuals were sequenced .
although runx <dig> is generally conserved across mammals, three of the runx <dig> sequences obtained via genome browsers  contain distinctive amino acid changes .
we cannot conclusively exclude the possibility of errors in the online genome sequence annotations, and this is of particular concern for the horse where there is extensive divergence in the 3’ flanking sequence.
the unusually high qa ratio of  <dig>  for equus caballus is mostly due to the highly shortened poly a sequence , and, thus, this result should be viewed with caution.
however, there is no a priori reason to suspect sequence or annotation errors for the three-lined squirrel or bottlenose dolphin; in these cases the sequences flanking the unusual amino acids are highly conserved, as are other mammalian sequences.
we also note that the flanking sequences for some of the novel species  are short, but in these cases we can confirm the sequence from original trace files.
the removal of flanking sequence was done when an ambiguous base pair was present in the sequence trace file.
sequences were only included in both the alignment and further analysis if the qa region of the sequence contained no ambiguous trace file calls; thus the qa ratio is likely to be correct in these cases, even though the flanking sequence was partially truncated.
the polya repeat was often interrupted by a single valine .
in fact, the presence of an interrupting amino acid has an interesting phylogenetic distribution , as most afrotherians and xenarthrans exhibit an interrupted polya sequence but laurasiatherians or euarchontoglires do not.
regarding the polyq repeat, only microcebus murinus, the grey mouse lemur, has an interrupted glutamine sequence .
in further analyses , we considered both the total qa ratio  and the qa ratio for uninterrupted sequences only.
for example, we scored the total qa ratio for the mouse lemur as  <dig>   in the initial analysis, but we also ran the analyses with a mouse lemur qa ratio of  <dig>   .
whether we included total qa ratios or uninterrupted qa ratios, the direction and significance of the correlation results did not change .
since the goal of this study is to examine whether the pattern sears et al.
<cit>  describe for carnivorans holds for mammals in general, we took morphological measurements comparable to those described in sears et al.
<cit>  .
after adjusting measurements relative to body size by dividing by proxy measures of overall size , dasypus  and myrmecophaga  had the longest ‘nose’ .
these taxa, along with sus  and tursiops  had the longest ‘face’ .
at the other extreme, callithrix , bradypus  and mustela  showed the shortest face; callithrixpongo  and nomascus  had the shortest nose.
those species with the longest and shortest rostra described above were not those with the largest and smallest runx <dig> qa ratios, especially in xenarthrans and afrotherians .
also, we did not recover a significant correlation between qa ratio and indices of rostrum size in non-carnivorans.
none of our phylogenetic generalized linear models showed a significant relationship between qa ratios and relative face and nose lengths .
this was true regardless of our method of accounting for body size.
in fact, closely related pairs of species that differ substantially in relative rostrum size  did not show correspondingly different qa ratios .
for example, the extraordinarily long-faced myrmecophaga had a slightly lower qa ratio  than the short-faced chlamyphorus .
similar results were found for primates.
the hamadryas baboon  and the rhesus macaque  have identical qa ratios  but the baboons have markedly longer faces .
as is the case for the armadillo, anteater, and tenrec examples mentioned above , gibbons have a qa ratio similar to that of orangutans  and this also matches the ancestral reconstruction of the ratio at the great ape node.
however, gibbons have much shorter faces than the great apes .
thus, in contrast to the trend apparent among carnivorans in which long-faced species possess high qa ratios  <cit> , this correlation does not seem to hold across placental mammals in general, and particularly not among xenarthrans or afrotherians .
in order to characterize these observations further, we used the newly developed bayesian comparative method of lartillot and poujol  <cit>   to study the correlation between runx <dig> qa ratios and relative face/nose lengths .
as a proof of concept example, the method was first applied to the sears et al.
carnivoran dataset  <cit> .
using  <dig> mt-cytb  sequences and fossil calibrations to control for phylogeny and divergence times, the bayesian method retrieved the positive correlation between runx <dig> qa ratio and relative facial length in carnivora with a posterior probability of  <dig>  .
however, similar analyses of our placental dataset using  <dig> vwf  nuclear sequences and fossil calibrations to control for phylogeny and divergence times showed no significant correlation between runx <dig> qa ratio and the four relative face/nose size-controlled variables tested .
these results confirm the results of the pgls  analyses in demonstrating statistical independence between runx <dig> qa ratio and facial length at the global level of placentals.
moreover, the bayesian method also allowed the joint ancestral reconstruction of both qa and facial length ratios under the brownian assumption .
figure 3a illustrates the relative homogeneity of the runx <dig> qa ratio in non-carnivoran placentals.
the 95% credibility interval for the ancestral placental qa ratio is  <dig> - <dig> , whereas the ancestral carnivoran qa ratio is estimated at a larger value .
xenarthrans , afrotherians , and primates  showed remarkably comparable ancestral values.
also the relatively modest value  reconstructed for the laurasiatheria ancestral node suggests that the runx <dig> qa ratio has increased in carnivora possibly as a response to selection for increasing facial length in this clade.
the bayesian joint reconstruction of relative facial length shows a much greater variability among placentals than the qa ratio .
a larger heterogeneity is observed in both xenarthrans and afrotherians with relatively large ancestral facial ratios being inferred for each group.
this contrasts with the homogeneity of their runx <dig> qa ratios .
also, as previously noted, primates are characterized by relatively reduced facial length ratios compared to the other placental groups.
this suggests that the relative facial length has been reduced in the ancestral branch leading to living primates.
overall, the comparison between the two panels of figure  <dig> highlights the general lack of correlation between the runx <dig> qa ratio and relative facial length in placental mammals.
discussion
our study provides the first example of using a fully integrative bayesian method for testing a correlation between genetic and morphometric data while controlling for phylogeny, evolutionary rates, and divergence times.
our results show that the runx <dig> qa ratio is not correlated with facial length across non-carnivoran placental mammals.
the runx <dig> qa tandem repeat may yet play an important role in influencing variation in face length in these mammals.
however, striking changes in prognathism have occurred along several mammalian lineages without associated changes in qa ratio.
variation in qa ratio has similarly occurred without corresponding changes to facial length phenotype .
another interesting result is the unusually low qa ratio for both choloepus species , the only placental mammals in our sample with a qa ratio below  <dig> .
however, this cannot be the sole factor determining the short face length of sloths as bradypus tridactylus  had a qa ratio above  <dig> .
it should be noted, however, that the two-toed and three-toed sloths are not particularly closely related.
genetic evidence suggests that bradypus and choloepus evolved an arboreal lifestyle independently  <cit>  and separated more than  <dig> million years ago  <cit> .
if face length also shortened independently, then it may be that selection has acted differently upon runx <dig> in the two-toed than in the three-toed sloth, explaining the higher qa ratio in bradypus.
some work suggests that the relationship between tandem repeats and anatomical form might not be linear  <cit> .
however, with our preliminary data set of 1- <dig> data points per order, fitting a quadratic or exponential line seems arbitrary  <cit>  and does not markedly improve fit.
although the nature of this study is a broad comparison across  <dig> orders of mammals, future work focusing on deep sampling within individual orders would be worthwhile.
in addition, sears et al.
<cit>  noted a weaker qa ratio in carnivorans without positively allometric growth of the face .
a larger sample within orders, particularly for primates  <cit> , that accounts for variation in the degree of positive allometry across species  may similarly reveal a stronger correlation within clades characterized by positively allometric growth of the face .
another potential problem is the presence of intraspecific variation  <cit> .
although only likely to produce slight variation of the qa ratio for each species, across a phylogeny it will add to the noise within the independent contrasts analysis and reduce the signal of any potential association.
notably, examination of the runx <dig> sequence data from the  <dig> genomes project  <cit>  indicates virtually no intraspecific variation in qa ratio among humans.
this may be due to the fact that humans have undergone a recent population bottleneck and generally show relatively low levels of intraspecific variation compared to other mammals  <cit> .
future studies that better sample the intraspecific diversity of runx <dig> will be able to test the extent to which the low diversity seen among humans applies across mammals.
this work represents a first step in examining the role of runx <dig> in the evolution of facial length across placental mammals.
the obvious next step is to examine possible associations between runx <dig> tandem repeat ratios and prognathism within groups, especially those that show marked variation in facial length, such as the papionines  <cit> .
similarly, it would be interesting to examine variation at the other two functional domains of runx <dig>  <cit>  or in the cis-regulatory regions of the runx <dig> gene across mammals.
moreover, exciting new linkage and heritability studies of craniofacial variation in populations for which we have genetic maps  <cit>  will undoubtedly yield more detailed insights on the genetics of mammalian facial morphology, including new candidate loci for cross-taxa comparisons.
CONCLUSIONS
this study evaluates a simple molecular polymorphism  that is thought to underlie functionally-important anatomical variation .
although previous studies  <cit>  demonstrated a clear, and likely causative, correlation between the qa-ratio of runx <dig> and face length in carnivorans, our analyses, controlling for phylogeny, evolutionary rates, and divergence times, show that the runx <dig> qa ratio is not generally correlated with facial length across placental mammals.

 work on the molecular basis of animal adaptation is providing ample evidence that small genetic changes can underlie striking phenotypic differences, both within and between species. of recent particular interest is the role of short tandemly repeating dna elements as evolutionary ‘tuning knobs’. microsatellites are mutation-prone and often show substantial length variation in repeat number. these repeats are common in mammalian genomes, often occurring within coding exons, potentially translating into expansions and contractions of poly-amino acid stretches. moreover, proteins containing such repeats are often involved in development. thus, the function of a protein can be easily modified via alterations in repeat tract length and such modifications may have less drastic pleiotropic effects than other types of mutation. an example of this is the correlation between specific tandem repeats and variation in midfacial length in carnivorans. variation in this striking morphological trait appears to be causatively associated with variation in coding sequence repeats within the gene runx. runx codes for a transcription factor that plays an important role in mediating osteoblast differentiation and activity. its function is critical for building and repairing bone. the activity of runx is mediated by a few functional domains, one of which consists of a stretch of glutamines followed by a stretch of alanines. for runx the ratio of glutamines to alanines appears to be positively correlated with the transcriptional activity of the protein. runx mutations in humans cause the skeletal disease cleidocranial dysplasia and have been associated with a shortening or protruding of the face and other skeletodental pathologies, some of which correspond to changes in certain clades of nonhuman mammals. in general, work on runx in humans and mice indicates that when runx is up-regulated, bone development is accelerated. interestingly, in comparisons of the modern human and neanderthal genomes, runx is specifically highlighted by green et al. as one of few genes showing a genetic signature of a selective sweep in the modern human lineage. assuming the neanderthal sequence data are valid, this would suggest a potentially important role for runx in human-specific cranial/skeletal features. the qa ratio of runx is correlated with facial length across breeds of dog. specifically, dog breeds and other carnivoran species with a high qa ratio tend to have a relatively long rostrum or "face". furthermore, sears et al. noted that the correlation was stronger among dogs, bears, raccoons and their relatives than among cats, civets, and mongooses, related to the fact that the rostrum in caniforms shows more positive allometry during growth than in feliforms. that is, compared to a cat, a dog shows more growth in the face relative to the braincase during its ontogeny. the possibility that this correlation represents a general mechanism responsible for at least some aspects of skull growth across non-carnivoran mammals has not yet been thoroughly investigated. facial shape and length are highly variable in many mammalian clades, including afrotherians, xenarthrans and euarchontoglires. morphometrically, the skull shows a tremendous amount of continuous variation across mammals, an observation which lends itself to the metaphor of the "tuning knob" as a genetic basis behind this variation. runx specifically the qa ratio, is thus a good candidate for examining the genetic basis of variation in skull proportions across mammals. here we assess the extent to which the correlation between skull shape and runx qa ratio holds true across a variety of non-carnivoran placental mammals. in particular, we focus on xenarthrans, afrotherians, and primates as these orders show marked variations in face length, which are thought to be adaptively associated with diet and ecology, and for these orders we have access to morphometric and genetic data. in analyzing data for placental mammal species representing orders, we find no correlation between runx sequence and face length across our sample as a whole. *see figure for measurement details. when greater than one, the number of individuals is given in parentheses. 1all variables were natural log transformed before analysis. see figure for measurement details. 1see figure for measurement details; * statistically significant; ns not significant.  runx tandem repeat ratios the translated amino acid sequences for the species are provided in the supplementary material. the runx amino acid sequences flanking the tandem repeat region were generally conserved across taxa providing evidence that orthologous rather than paralogous sequences were successfully retrieved. in support of this, all publicly-available mammalian genomes contain a single annotated runx locus, suggesting that this gene has 1: orthology across mammals and has not been subject to duplication. qa ratios calculated from the translated sequences range from to. with the exception of equus, these qa ratios were generally lower than those of carnivorans. in their analysis of carnivora, sears et al. report a lowest value of for the qa ratio in potos flavus, which is close to the range of domesticated dog breeds. glutamine and alanine amino acid counts for non-carnivoran mammals were not markedly different than those of domestic dog breeds, although carnivorans are on the low end of alanine counts. across the mammals included in our dataset, the majority of the qa ratios lie between and. xenarthrans had the lowest variation in qa ratio across sampled species and laurasiatherians had the highest. we make such taxonomic comparisons tentatively, as species within clades are not equally distant to each other and the species sampled here represent only a subset of the extant diversity for each clade. across the mammals tested, both the polyq and polya are variable, but the polyq has a greater range of values than the polya repeat. also, intraspecific variation was detected in two of the four species where multiple individuals were sequenced. although runx is generally conserved across mammals, three of the runx sequences obtained via genome browsers contain distinctive amino acid changes. we cannot conclusively exclude the possibility of errors in the online genome sequence annotations, and this is of particular concern for the horse where there is extensive divergence in the 3’ flanking sequence. the unusually high qa ratio of for equus caballus is mostly due to the highly shortened poly a sequence, and, thus, this result should be viewed with caution. however, there is no a priori reason to suspect sequence or annotation errors for the three-lined squirrel or bottlenose dolphin; in these cases the sequences flanking the unusual amino acids are highly conserved, as are other mammalian sequences. we also note that the flanking sequences for some of the novel species are short, but in these cases we can confirm the sequence from original trace files. the removal of flanking sequence was done when an ambiguous base pair was present in the sequence trace file. sequences were only included in both the alignment and further analysis if the qa region of the sequence contained no ambiguous trace file calls; thus the qa ratio is likely to be correct in these cases, even though the flanking sequence was partially truncated. the polya repeat was often interrupted by a single valine. in fact, the presence of an interrupting amino acid has an interesting phylogenetic distribution, as most afrotherians and xenarthrans exhibit an interrupted polya sequence but laurasiatherians or euarchontoglires do not. regarding the polyq repeat, only microcebus murinus, the grey mouse lemur, has an interrupted glutamine sequence. in further analyses, we considered both the total qa ratio and the qa ratio for uninterrupted sequences only. for example, we scored the total qa ratio for the mouse lemur as in the initial analysis, but we also ran the analyses with a mouse lemur qa ratio of. whether we included total qa ratios or uninterrupted qa ratios, the direction and significance of the correlation results did not change. since the goal of this study is to examine whether the pattern sears et al. describe for carnivorans holds for mammals in general, we took morphological measurements comparable to those described in sears et al.. after adjusting measurements relative to body size by dividing by proxy measures of overall size, dasypus and myrmecophaga had the longest ‘nose’. these taxa, along with sus and tursiops had the longest ‘face’. at the other extreme, callithrix, bradypus and mustela showed the shortest face; callithrixpongo and nomascus had the shortest nose. those species with the longest and shortest rostra described above were not those with the largest and smallest runx qa ratios, especially in xenarthrans and afrotherians. also, we did not recover a significant correlation between qa ratio and indices of rostrum size in non-carnivorans. none of our phylogenetic generalized linear models showed a significant relationship between qa ratios and relative face and nose lengths. this was true regardless of our method of accounting for body size. in fact, closely related pairs of species that differ substantially in relative rostrum size did not show correspondingly different qa ratios. for example, the extraordinarily long-faced myrmecophaga had a slightly lower qa ratio than the short-faced chlamyphorus. similar results were found for primates. the hamadryas baboon and the rhesus macaque have identical qa ratios but the baboons have markedly longer faces. as is the case for the armadillo, anteater, and tenrec examples mentioned above, gibbons have a qa ratio similar to that of orangutans and this also matches the ancestral reconstruction of the ratio at the great ape node. however, gibbons have much shorter faces than the great apes. thus, in contrast to the trend apparent among carnivorans in which long-faced species possess high qa ratios, this correlation does not seem to hold across placental mammals in general, and particularly not among xenarthrans or afrotherians. in order to characterize these observations further, we used the newly developed bayesian comparative method of lartillot and poujol to study the correlation between runx qa ratios and relative face/nose lengths. as a proof of concept example, the method was first applied to the sears et al. carnivoran dataset. using mt-cytb sequences and fossil calibrations to control for phylogeny and divergence times, the bayesian method retrieved the positive correlation between runx qa ratio and relative facial length in carnivora with a posterior probability of. however, similar analyses of our placental dataset using vwf nuclear sequences and fossil calibrations to control for phylogeny and divergence times showed no significant correlation between runx qa ratio and the four relative face/nose size-controlled variables tested. these results confirm the results of the pgls analyses in demonstrating statistical independence between runx qa ratio and facial length at the global level of placentals. moreover, the bayesian method also allowed the joint ancestral reconstruction of both qa and facial length ratios under the brownian assumption. figure 3a illustrates the relative homogeneity of the runx qa ratio in non-carnivoran placentals. the 95% credibility interval for the ancestral placental qa ratio is -, whereas the ancestral carnivoran qa ratio is estimated at a larger value. xenarthrans, afrotherians, and primates showed remarkably comparable ancestral values. also the relatively modest value reconstructed for the laurasiatheria ancestral node suggests that the runx qa ratio has increased in carnivora possibly as a response to selection for increasing facial length in this clade. the bayesian joint reconstruction of relative facial length shows a much greater variability among placentals than the qa ratio. a larger heterogeneity is observed in both xenarthrans and afrotherians with relatively large ancestral facial ratios being inferred for each group. this contrasts with the homogeneity of their runx qa ratios. also, as previously noted, primates are characterized by relatively reduced facial length ratios compared to the other placental groups. this suggests that the relative facial length has been reduced in the ancestral branch leading to living primates. overall, the comparison between the two panels of figure highlights the general lack of correlation between the runx qa ratio and relative facial length in placental mammals. discussion our study provides the first example of using a fully integrative bayesian method for testing a correlation between genetic and morphometric data while controlling for phylogeny, evolutionary rates, and divergence times. our results show that the runx qa ratio is not correlated with facial length across non-carnivoran placental mammals. the runx qa tandem repeat may yet play an important role in influencing variation in face length in these mammals. however, striking changes in prognathism have occurred along several mammalian lineages without associated changes in qa ratio. variation in qa ratio has similarly occurred without corresponding changes to facial length phenotype. another interesting result is the unusually low qa ratio for both choloepus species, the only placental mammals in our sample with a qa ratio below. however, this cannot be the sole factor determining the short face length of sloths as bradypus tridactylus had a qa ratio above. it should be noted, however, that the two-toed and three-toed sloths are not particularly closely related. genetic evidence suggests that bradypus and choloepus evolved an arboreal lifestyle independently and separated more than million years ago. if face length also shortened independently, then it may be that selection has acted differently upon runx in the two-toed than in the three-toed sloth, explaining the higher qa ratio in bradypus. some work suggests that the relationship between tandem repeats and anatomical form might not be linear. however, with our preliminary data set of 1- data points per order, fitting a quadratic or exponential line seems arbitrary and does not markedly improve fit. although the nature of this study is a broad comparison across orders of mammals, future work focusing on deep sampling within individual orders would be worthwhile. in addition, sears et al. noted a weaker qa ratio in carnivorans without positively allometric growth of the face. a larger sample within orders, particularly for primates, that accounts for variation in the degree of positive allometry across species may similarly reveal a stronger correlation within clades characterized by positively allometric growth of the face. another potential problem is the presence of intraspecific variation. although only likely to produce slight variation of the qa ratio for each species, across a phylogeny it will add to the noise within the independent contrasts analysis and reduce the signal of any potential association. notably, examination of the runx sequence data from the genomes project indicates virtually no intraspecific variation in qa ratio among humans. this may be due to the fact that humans have undergone a recent population bottleneck and generally show relatively low levels of intraspecific variation compared to other mammals. future studies that better sample the intraspecific diversity of runx will be able to test the extent to which the low diversity seen among humans applies across mammals. this work represents a first step in examining the role of runx in the evolution of facial length across placental mammals. the obvious next step is to examine possible associations between runx tandem repeat ratios and prognathism within groups, especially those that show marked variation in facial length, such as the papionines. similarly, it would be interesting to examine variation at the other two functional domains of runx or in the cis-regulatory regions of the runx gene across mammals. moreover, exciting new linkage and heritability studies of craniofacial variation in populations for which we have genetic maps will undoubtedly yield more detailed insights on the genetics of mammalian facial morphology, including new candidate loci for cross-taxa comparisons.  this study evaluates a simple molecular polymorphism that is thought to underlie functionally-important anatomical variation. although previous studies demonstrated a clear, and likely causative, correlation between the qa-ratio of runx and face length in carnivorans, our analyses, controlling for phylogeny, evolutionary rates, and divergence times, show that the runx qa ratio is not generally correlated with facial length across placental mammals. 
the genetic basis of variation in facial shape and length represents a possible example of this phenomenon. in this study we examine whether this correlation holds true across placental mammals, particularly those mammals for which facial length is highly variable and related to adaptive behavior and lifestyle. non-carnivoran taxa generally had substantially lower glutamine-alanine ratios than carnivorans, and we found no correlation between runx sequence and face length across placental mammals. runt-related transcription factor, which is involved in osteoblast differentiation, contains a functionally-important tandem repeat of glutamine and alanine amino acids. notably, in domestic breeds of dog, and in carnivorans in general, the ratio of glutamines to alanines is strongly correlated with facial length. we obtained relative facial length measurements and runx sequences for mammalian species representing orders. using both a phylogenetic generalized least squares model and a recently-developed bayesian comparative method, we tested for a correlation between genetic and morphometric data while controlling for phylogeny, evolutionary rates, and divergence times. results of our diverse comparative phylogenetic analyses indicate that qa ratio does not consistently correlate with face length across the mammalian taxa considered. when simple sequence repeats are integrated into functional genes, they can potentially act as evolutionary ‘tuning knobs’, supplying abundant genetic variation with minimal risk of pleiotropic deleterious effects. the ratio of glutamines to alanines in this protein seemingly influences the regulation of bone development.
 when simple sequence repeats are integrated into functional genes, they can potentially act as evolutionary ‘tuning knobs’, supplying abundant genetic variation with minimal risk of pleiotropic deleterious effects. the genetic basis of variation in facial shape and length represents a possible example of this phenomenon. runt-related transcription factor, which is involved in osteoblast differentiation, contains a functionally-important tandem repeat of glutamine and alanine amino acids. the ratio of glutamines to alanines in this protein seemingly influences the regulation of bone development. notably, in domestic breeds of dog, and in carnivorans in general, the ratio of glutamines to alanines is strongly correlated with facial length.  in this study we examine whether this correlation holds true across placental mammals, particularly those mammals for which facial length is highly variable and related to adaptive behavior and lifestyle. we obtained relative facial length measurements and runx sequences for mammalian species representing orders. using both a phylogenetic generalized least squares model and a recently-developed bayesian comparative method, we tested for a correlation between genetic and morphometric data while controlling for phylogeny, evolutionary rates, and divergence times. non-carnivoran taxa generally had substantially lower glutamine-alanine ratios than carnivorans, and we found no correlation between runx sequence and face length across placental mammals.  results of our diverse comparative phylogenetic analyses indicate that qa ratio does not consistently correlate with face length across the mammalian taxa considered. thus, although runx might function as a ‘tuning knob’ modifying face length in carnivorans, this relationship is not conserved across mammals in general. mammalian evolutionprognathismmolecular evolutionprimatesafrotheriaxenarthramorphology 