BACKGROUND
animal mitochondrial dna  evolution contrasts with nuclear evolution.
mitochondrial and nuclear genomes differ in many ways, such as total length, ploidy level, mode of inheritance, recombination rate, presence of introns, percentage of non-coding dna, effective population size, and repair mechanisms, e.g.
<cit> .
among these particularities, hypermutability is one of the most striking features of animal mitochondria: the mtdna mutation rate is typically one order of magnitude higher than the nuclear one  <cit> .
the question of the origin and evolution of such high mutation rates is still open and debated  <cit> ; and see  <cit>  for exceptions.
this high mutation rate is one of the reasons why mtdna is a very popular marker for biodiversity studies.
for example, it has been massively used to investigate intraspecific to intra-ordinal evolutionary relationship, and disentangle rapid speciation events in phylogenetic studies .
in recent years, mtdna has been used to identify species using a standardized portion of the cytochrome oxydase i gene , according to the so-called dna barcoding approach  <cit> .
the high mtdna mutation rate, however, is also the source of frequent homoplasy, i.e., phylogenetic incongruence between sites of the molecule because of multiple mutations at the same site.
homoplasy complicates the use of mtdna in phylogenetic  <cit>  and even population genetic  <cit>  studies.
we recently reported several results related to mtdna mutation dynamics in mammals.
first, we showed that mtdna substitution rates are extremely variable between species: they differ by two orders of magnitude between slow-evolving and fast-evolving mammalian lineages  <cit> , definitively rejecting the famous "2% per site per million year" calibration, which should not be generally trusted.
we proposed that mutation rate variations are possibly linked to species longevity through the action of natural selection: too high a mutation rate would be deleterious in long-lived mammals because it could result in premature aging due to the accumulation of somatic mtdna mutations  <cit> , see also  <cit> .
secondly, we found that within-species cytochrome b  nucleotide diversity is correlated with the mitochondrial mutation rate, as expected, but not with any life-history or ecological variable potentially related to population size, including body size, geographic range and conservation status  <cit> .
this surprising pattern was apparently not explained by selective effects  <cit>  , contrary to results obtained at the metazoa level  <cit> , especially invertebrates  <cit> .
we therefore proposed that the lack of correlation between mtdna diversity and potential indicators of population size in mammals is due to strong demographic stochasticity.
at any rate, the mutation rate is the best predictor we have of mitochondrial genetic diversity across mammalian species.
overall, these results highlight the importance of mutation rate variations in shaping mtdna biodiversity patterns in mammals, and suggest that mutational effects should be carefully taken into account when analysing such data.
in this study, we extend our analysis of the evolutionary dynamics of mtdna through the bird/mammal comparison.
our objective is dual.
first, we want to check whether the mammalian results are specific to this group, or have any degree of generality.
secondly, we want to make use of the genetical and physiological specificities of birds to test various evolutionary hypothesis raised by previous studies.
birds are the perfect candidate to challenge the results obtained in mammals: like mammals, they include charismatic, well studied species, in which a large amount of genetic and biological data are available.
being relatively large, warm blooded vertebrates, birds are comparable to mammals in terms of physiology, ecology, and life-history.
birds, furthermore, show a number of peculiarities potentially relevant to mitochondrial evolution and diversity.
the first reason why birds are appropriate for a comparative approach is the availability of direct population size estimates obtained through global population surveys, especially for north american species  <cit> .
birds therefore provide the opportunity to directly test the relationship between population size and mitochondrial diversity, whereas in mammals we had to rely on ecological and life history traits, plausibly, but only supposedly, correlated to population size.
we will therefore check whether the lack of relationship between mtdna diversity and species abundance we reported in mammals is confirmed in birds, or was due to inappropriate measure of the effective population size.
birds, secondly, present a genetic peculiarity: female is the heterogametic sex.
because it is strictly maternally transmitted, the avian mitochondrial genome is thus in full genetic linkage with the female-specific w chromosome  <cit> .
berlin et al.
<cit>  proposed that the mtdna diversity could therefore be reduced by hill-robertson interference: selective effects applying to loci linked to the w will affect mtdna through hitch-hiking.
this clever hypothesis has provoked some comments and reactions  <cit> .
hickey  <cit>  questioned one of the most important arguments of berlin et al.
<cit> , namely the lower synonymous diversity in bird than in mammal mtdna, arguing that mutation rate could be a confounding effect.
an accurate estimation of mtdna substitution rate variations in birds appears necessary to correctly interpret the patterns of mtdna diversity in the bird/mammal comparison.
birds, finally, have quite high metabolic rates –  <dig>  to  <dig>  times higher than mammals of similar sizes  <cit>  -probably because of the high energetic demand of the flying locomotion.
paradoxically, birds species are strikingly long-lived as compared to their mammalian counterparts; on average, birds live three times longer than mammals of similar sizes  <cit> .
birds can be characterized as long-lived homeotherms, a specificity they share with bats  <cit> .
these peculiarities are useful to understand further the impact of metabolic rate and life-span on mtdna substitution rates.
three main hypotheses have been proposed to explain mtdna substitution rate variations in mammals: the generation time hypothesis  <cit> , the metabolic rate hypothesis  <cit> , and the longevity hypothesis  <cit> , presented above.
the classical approach to test these hypotheses in a comparative framework is to correlate substitution rate variations with the relevant life history traits  <cit> : female sexual maturity, basal mass-specific metabolic rate , and maximum longevity.
however, these life-history traits are strongly correlated with each other, so that the respective contributions of the three variables are difficult to disentangle.
the contrast between the avian and mammalian physiologies provides a unique opportunity to discriminate between two of these competing models, namely the longevity and the metabolic rate hypotheses.
according to the longevity hypothesis, birds should present, on average, lower mtdna neutral substitution rates than mammals, while the reverse pattern is expected under the metabolic rate hypothesis.
to test these predictions and challenge the results we obtained in mammals, we propose to accurately estimate the lineage-specific neutral substitution rate variation of mtdna in birds using the phylogenetic framework developed by  <cit> .
using this dataset, we want to check the rough 2%/site/myr calibration.
this calibration, originally estimated with an rflp analysis on mammals by brown et al.
<cit> , was generalized to birds by a study in geese  <cit> .
this calibration rapidly became a standard in ornithology studies, probably because of the scarcity of the fossil record .
recently, the reliability of this calibration was debated in various studies either supporting  <cit>  or rejecting it  <cit> .
we will also ask which of metabolic rate and longevity is the best predictor of mutation rate variations in warm-blooded vertebrates.
finally, we will estimate the mitochondrial genetic diversity  on a wide taxonomic range in birds to test for the neutrality assumption and investigate the link between population size, mutation rate, genetic hitch-hiking, and mtdna diversity.
RESULTS
substitution rate variation and its determinants
to infer the substitution rate variation of a large number of bird species, we need to estimate divergence dates, and the numbers of nucleotide substitutions which occurred during these divergences .
the divergence dates are obtained thanks to paleontologic calibration points, which are mostly available for relatively old divergences.
nucleotide substitution numbers, however, must be estimated using little-divergent sequences because of mutational saturation.
to resolve this methodological problem, we take advantage of the decoupled non-synonymous  and synonymous evolutionary dynamics.
we introduced a 3-step method  <cit> , in which we first define groups of sequences showing limited divergences, then estimate the relative species-specific substitution rate at third codon positions within groups, and finally assign a divergence for each of these groups using amino acid sequences.
at step  <dig> and  <dig>  molecular and fossil information are combined thanks to a sophisticated bayesian method  <cit> .
using this procedure, we analysed a cytb dataset of  <dig> species, which represent ~15% of the total living species of birds.
the average cytb third codon position substitution rate was  <dig>  substitution/site/myr, and the median was  <dig>  substitution/site/myr.
these substitution rates are estimated per lineage, per third-codons position.
to make them roughly comparable to the popular 2% of divergence per site per myr, these numbers must be multiplied by two  and divided by three .
doing this would yield an average divergence of  <dig> % per myr , which is slightly less than the popular 2%.
the distribution across species shows a large variation: the  <dig> % and  <dig> % percentiles are  <dig>  and  <dig>  substitution/site/myr, respectively .
thus, even if we consider the 5% most extreme substitution rate values as outliers, the cytb third codon position substitution rate shows a thirty-fold range of variation across bird lineages.
we quantified the effect of taxonomy on substitution rate variation using a hierarchical anova considering super-order, order, family and genus with random effects.
the order level explains 46%  of the variance in substitution rates across species.
considering only order with more than  <dig> species, passerines are the fastest evolving order  and anseriformes the slowest one .
compared with mammals, birds globally evolve  <dig>  times slower on average , and show a range of variation less extreme .
we correlated the species-specific substitution rate to life-history traits in order to discriminate between the competing explanatory hypotheses.
a smaller amount of life-history data are available in birds than in mammals, especially for female sexual maturity, which is documented in  <dig> species only.
we thus focused on the effects of body mass and maximum longevity using the  <dig> species in which these two variables are available.
in agreement with both the metabolism and the longevity hypotheses, the two life-history traits are negatively linked to the substitution rate.
body mass appears to be a better predictor of the substitution rate  than maximum longevity, which also shows a strong level of correlation .
the distribution of  maximum longevity is strongly bimodal in birds, passerines being substantially more short-lived than other birds .
we separated passerines from non-passerine and re-performed the same analyses in the two subsets separately.
the results are shown in table  <dig>  surprisingly, the third-codon position substitution rate is positively correlated to body mass and maximum longevity in passerines birds.
in non-passerines birds, results are similar to the global analysis: body mass best explains substitution rate variations, and removes the effect of longevity in a two-way anova .
this result is not consistent with mammals  <cit> .
in order to check whether these results were robust to the removal of phylogenetic effects, we applied the phylogenetic contrast method  <cit>  to regress phylogeny out of the analyses.
in this analysis, we used the chronogramme obtained with amino-acid cytochrome b sequences.
the phylogenetic control did not qualitatively change the results although most p-values were decreased .
finally, we jointly analysed the mammalian and bird datasets , including class  as an explanatory variable.
the two-factor model  revealed that the bird vs. mammal status has a strong effect on mtdna substitution rate when body mass is taken into account and a significant but weaker effect as far as longevity is concerned .
there is, moreover, a significant interaction between body mass and class , but not between longevity and class .
determination of mtdna diversity in birds
bird cytb polymorphism sequence datasets were retrieved from the polymorphix database  <cit> .
the synonymous  and non-synonymous  levels of diversity were computed for the  <dig> species in which more than four sequences were available.
the mean per-site synonymous nucleotide diversity was  <dig>  ±  <dig> .
the most variable species was shelley's greenbul , and two species showed no synonymous variations .
to test the relationship between population size and mtdna diversity, we first correlated nucleotide polymorphism and body mass, taken as a proxy of population size, as in nabholz et al.
<cit>  and berlin et al.
<cit> .
πs and body mass were negatively correlated but, contrary to berlin et al.
<cit> , this relationship was not significant .
secondly, we applied a more direct analysis using population size estimates for north american breeding birds through the north american landbird conservation plan  <cit> .
such data were available for  <dig> strictly north american species of our data set.
we found no correlation between direct population size estimate and cytb synonymous diversity .
these results confirm those obtained in mammals  <cit> , here using direct estimations of population sizes instead of life-history and ecological proxies.
the dataset, however, is much smaller in the present analysis.
we tested the taxonomic effect on πs using one-way anovas on order and family level.
we found that neither the family nor the order level had a significant effect.
the sole taxonomic variable having a significant effect on mtdna diversity is the passerine/non-passerine status, passerines being more diverse than other birds .
we compared the distribution of πs between birds and mammals.
similarly to berlin et al.
<cit> , we found that birds have a lower average synonymous diversity than mammals , but this difference is largely reduced when we controlled for the mutation rate .
interestingly, we detected no significant difference of allozyme heterozygosity between mammals  and birds  using the nevo et al.
<cit>  data set .
as in mammals, we found a positive and significant correlation between species-specific cytb third-codon substitution rate and cytb synonymous diversity per genus .
these analyses confirm that the mutation rate is a major determinant of mtdna polymorphism in birds as well as in mammals.
finally, we compared the ratio of non-synonymous to synonymous changes within  and between species  in birds and mammals – these ratio were computed when an outgroup was available.
the two groups showed a similar dn/ds , but the pn/ps ratio was higher in birds than in mammals , as previously reported by berlin et al.
<cit> .
discussion
mtdna substitution rates – support for the longevity hypothesis
we showed that the mtdna mutation rate, measured through the neutrally-evolving third codon positions of cytb, is highly variable between bird lineages.
the molecular clock hypothesis does not apply to bird mtdna evolution, fast-evolving species being thirty times more rapid than slow-evolving ones.
the method we applied is particularly prone to reveal the actual amplitude of substitution rate variation: by overcoming the problem of mutational saturation, it allowed us to use an extensive dataset encompassing the whole taxonomic diversity of birds.
this study, therefore, provides a synthetic view of substitution rate variations across bird lineages, bringing an important result in the field of molecular dating, in which the rough approximation of 2% of substitution per million of years is still debated  <cit> .
our analysis suggests that the molecular clock assumption should be avoided as far as bird mtdna is concerned.
the conclusions of pereira and baker  <cit>  , and some studies done at smaller scale  are here confirmed by cytochrome b third codon positions at wide taxonomic scale.
although the average rate in birds is close to this value, the 2% per site per million year calibration is a very bad summary of the whole picture: the rate is essentially  higher than 2% in passerines, and  lower in non-passerines .
users of mtdna as a tools for inferring divergence dates should imperatively use statistical phylogenetic methods accounting for substitution rate variation across lineages, the so-called clock-relaxed methods  <cit> .
several interesting results were revealed thanks to the comparison with mammals.
first, birds show a more narrow range of variation of life-history traits than mammals.
for example body mass varies from  <dig>  g to  <dig>  kg in the bird dataset, whereas it varies from  <dig>  g to  <dig> tones in mammals.
this result confirms the general influence of life-history traits on neutral mtdna substitution rate in birds and mammals.
this result is important because such a relationship was not reported in every taxonomic group  <cit> , and even not by some studies in mammals  <cit> .
secondly, the neutral mtdna substitution rate is lower in birds than in mammals, and this difference increases if body mass is regressed out of the analysis .
for example, birds evolve four times slower than mammals as far as small species  are concerned.
this result is in agreement with the longevity hypothesis, but not with the metabolic rate hypothesis.
according to the latter model, birds should show a higher average mutation rate than mammals, because of their higher mass-specific metabolic rate and their lower average body mass  <cit> .
during the course of evolution, birds have acquired adaptations to manage their high mass-specific metabolic rate, including an increased of reactive oxygen species  protection and lower ros production  <cit> .
in the context of the mitochondrial theory of aging, which postulates that ros production as a byproduct of mitochondrial respiration contributes to aging, these adaptations could be interpreted as a response to the elevated longevity of birds  <cit> .
the reduced rate of ros production could explain the lower average mutation rate of birds mtdna, as compared to mammals .
it is surprising, therefore, to find a predominant influence of body mass, not longevity, in the within-birds analysis.
passerines show a positive relationship between longevity  and substitution rate, which is quite surprising.
we have no valid explanation for this relationship so far, perhaps because it is not biologically relevant – this relationship is the single result that was not recovered when we used other methods of substitution rate estimation .
within non-passerines, which are essentially long-lived species, body mass is still the variable best explaining substitution rate variations .
using dna-dna hybridization data , mooers and harvey  <cit>  found no support for the metabolic rate hypothesis, but reported a significant generation time effect.
in this context, it would be interesting to enlarge the female sexual maturity data set, in order to test the generation time hypothesis with our extended mitochondrial dataset.
determination of mtdna diversity in birds
the neutral genetic diversity primarily depends on effective population size and mutation rate.
because population sizes appear so variable in time and between species, variations in genetic diversity across species have been frequently interpreted in demographic terms, e.g.
<cit> .
recently, the effect of population size on mtdna diversity has been challenged  <cit> .
the large variations in mutation rates we report in birds suggest that mutational effects should be carefully taken into account in studying mtdna polymorphism in this group, as well as in mammals  <cit> .
in general, the relationship between population size and mtdna diversity is difficult to evaluate because of the scarcity of population size estimates for wild species.
life-history traits, like body mass, have therefore been used as proxy of population size  <cit> .
birds are an exception: thanks to the broad interest of the scientific community in bird ecology and systematics, direct estimates of species effective sizes are available for north american species  <cit> .
we correlated these population size estimates to synonymous mtdna diversity and found no significant relationship.
the data set is rather small , but we note that this relationship should theoretically be strong, given the wide range of population sizes .
when using body mass as an indicator of population size, thus increasing much the data set, we found a negative correlation with cytb synonymous diversity, but this result was not significant.
berlin et al.
<cit>  report a significant relationship between πs and body mass in birds using genus averages.
if we apply the same method, we also find a significant relationship , but the effect is removed when the passerine/non-passerine status is taken into account .
this result is in agreement with our previous mammalian analysis, in which the weak effect of body mass was removed by a taxonomic control  <cit> .
so similarly to mammals, the mitochondrial genetic diversity is essentially uncorrelated to life-history traits in birds, and when it is so, it is most likely via the influence of mutation rate, not population size.
mitochondrial selective regime in birds
to explain the lack of relationship between mtdna diversity and proxies of population size in mammals, nabholz et al.
<cit>  invoked demographic stochasticity, while bazin et al.
<cit>  invoked recurrent hitchhiking effects for other species having larger expected population sizes .
what about birds?
to address this question we computed the neutrality index .
ni <  <dig> indicates positive selection, while ni >  <dig> indicates purifying selection.
the average ni in birds is significantly higher than in mammals  and only three species of birds show a ni <  <dig> .
the high ni values in birds are due to their high pn/ps ratios -dn/ds ratios are similar to those of mammals.
this result indicates that mtdna evolution in birds is mainly governed by purifying selection, as previously reported  <cit> .
it is also indicative of a strong level of constraint acting on bird cytochrome b sequences, which is known as the avian constraint hypothesis  <cit> .
like mammals, birds appear to have population sizes low enough to belong to the "drift domain", rather than the "draft domain" sensu gillespie  <cit>  .
in the absence of a significant impact of positive selection, we have to invoke strong demographic stochasticity to explain the lack of relationship between population size and mtdna diversity.
mutation rate is expected to be a major determinant of within-species genetic diversity whether populations are at mutation-drift equilibrium  or not  <cit> .
consistently, we report a significantly positive relationship between cytb diversity and substitution rate , again paralleling the mammalian situation.
in a recent paper, berlin et al.
<cit>  proposed an alternative hypothesis to explain mtdna polymorphism patterns in birds.
they suggested that hill-robertson effects due to complete association with the w chromosome could explain the lower synonymous mitochondrial diversity and the higher pn/ps observed in birds, as compared to mammals.
this should also explain the lack of correlation between neutral polymorphism and population size.
outwardly, we confirm this hypothesis by showing that the lower synonymous mitochondrial diversity is not found for nuclear allozymes.
but this picture is complicated by the globally lower mtdna substitution rate in birds than in mammals.
controlling for mutation rates actually removes the major part of the difference between birds and mammals.
to quantify the effect of mutation rate variation on synonymous mtdna diversity we combined the bird and mammal polymorphism and neutral substitution rate datasets and performed a multiple regression of substitution rate and bird/mammal status  on synonymous mtdna diversity.
this model explains 21% of πs variation .
the result also shows that the mutation rate has a strong effect on πs  and considerably reduces the effect of class .
this analysis indicates that a subtential part of the difference between birds and mammalian πs is explained by their distinct mutation rates .
the difference in mutation rates, however, cannot explain the higher level of mitochondrial pn/ps found in birds.
birds and mammals have apparently comparable effective population sizes, as suggested by their similar average allozymic heterozygosity.
the higher pn/ps ratio in birds may therefore be due to hill-robertson interferences between the mitochondrial and w chromosomes, as suggested by berlin et al.
<cit> , although this effect has only a weak influence on absolute levels of πs, when mutation rate is controlled for.
CONCLUSIONS
in this work, we achieved a comprehensive comparison of mitochondrial substitution rate variation and mitochondrial diversity between birds and mammals.
cytb neutral substitution rates are, on average,  <dig>  times slower in birds than in mammals.
among-lineages variability, although substantial, is also lower in birds than in mammals.
this analysis corroborates the longevity hypothesis about mutation rate determination  <cit> .
despite their higher metabolic rate, birds undergo a lower mitochondrial mutation rate, probably because of the beneficial effect of reduced oxidative damage in long-lived species  <cit> .
we suggest that the low mtdna mutation rate of birds might explain their exceptional mass-specific longevity.
as for mammals, we report no evidence of adaptive selection on cytb sequence evolution, but still no population size effect, even when using direct estimates of population size available for north american species.
we thus invoke the same demographic instability explanation as previously proposed for mammals  <cit> , although selective interferences with the w chromosome  <cit>  could contribute to the noisy relationship between mtdna diversity and population size.
we also confirm the importance of mutation rate as a determinant of mtdna diversity – the lower mutation rates in birds almost fully explain their lower synonymous diversity.
this study confirms and extends the message of caution expressed by nabholz et al.
<cit>  about the usage of mtdna as a molecular marker of biodiversity in vertebrates:  mtdna diversity is not related to species abundance;  mtdna greatly departs the molecular clock hypothesis.
the 2% per site per million year calibration  has no degree of generality, and should not be used for dating purposes in the absence of fossil data.

 animal mitochondrial dna evolution contrasts with nuclear evolution. mitochondrial and nuclear genomes differ in many ways, such as total length, ploidy level, mode of inheritance, recombination rate, presence of introns, percentage of non-coding dna, effective population size, and repair mechanisms, e.g.. among these particularities, hypermutability is one of the most striking features of animal mitochondria: the mtdna mutation rate is typically one order of magnitude higher than the nuclear one. the question of the origin and evolution of such high mutation rates is still open and debated; and see for exceptions. this high mutation rate is one of the reasons why mtdna is a very popular marker for biodiversity studies. for example, it has been massively used to investigate intraspecific to intra-ordinal evolutionary relationship, and disentangle rapid speciation events in phylogenetic studies. in recent years, mtdna has been used to identify species using a standardized portion of the cytochrome oxydase i gene, according to the so-called dna barcoding approach. the high mtdna mutation rate, however, is also the source of frequent homoplasy, i.e., phylogenetic incongruence between sites of the molecule because of multiple mutations at the same site. homoplasy complicates the use of mtdna in phylogenetic and even population genetic studies. we recently reported several results related to mtdna mutation dynamics in mammals. first, we showed that mtdna substitution rates are extremely variable between species: they differ by two orders of magnitude between slow-evolving and fast-evolving mammalian lineages, definitively rejecting the famous "2% per site per million year" calibration, which should not be generally trusted. we proposed that mutation rate variations are possibly linked to species longevity through the action of natural selection: too high a mutation rate would be deleterious in long-lived mammals because it could result in premature aging due to the accumulation of somatic mtdna mutations, see also. secondly, we found that within-species cytochrome b nucleotide diversity is correlated with the mitochondrial mutation rate, as expected, but not with any life-history or ecological variable potentially related to population size, including body size, geographic range and conservation status. this surprising pattern was apparently not explained by selective effects, contrary to results obtained at the metazoa level, especially invertebrates. we therefore proposed that the lack of correlation between mtdna diversity and potential indicators of population size in mammals is due to strong demographic stochasticity. at any rate, the mutation rate is the best predictor we have of mitochondrial genetic diversity across mammalian species. overall, these results highlight the importance of mutation rate variations in shaping mtdna biodiversity patterns in mammals, and suggest that mutational effects should be carefully taken into account when analysing such data. in this study, we extend our analysis of the evolutionary dynamics of mtdna through the bird/mammal comparison. our objective is dual. first, we want to check whether the mammalian results are specific to this group, or have any degree of generality. secondly, we want to make use of the genetical and physiological specificities of birds to test various evolutionary hypothesis raised by previous studies. birds are the perfect candidate to challenge the results obtained in mammals: like mammals, they include charismatic, well studied species, in which a large amount of genetic and biological data are available. being relatively large, warm blooded vertebrates, birds are comparable to mammals in terms of physiology, ecology, and life-history. birds, furthermore, show a number of peculiarities potentially relevant to mitochondrial evolution and diversity. the first reason why birds are appropriate for a comparative approach is the availability of direct population size estimates obtained through global population surveys, especially for north american species. birds therefore provide the opportunity to directly test the relationship between population size and mitochondrial diversity, whereas in mammals we had to rely on ecological and life history traits, plausibly, but only supposedly, correlated to population size. we will therefore check whether the lack of relationship between mtdna diversity and species abundance we reported in mammals is confirmed in birds, or was due to inappropriate measure of the effective population size. birds, secondly, present a genetic peculiarity: female is the heterogametic sex. because it is strictly maternally transmitted, the avian mitochondrial genome is thus in full genetic linkage with the female-specific w chromosome. berlin et al. proposed that the mtdna diversity could therefore be reduced by hill-robertson interference: selective effects applying to loci linked to the w will affect mtdna through hitch-hiking. this clever hypothesis has provoked some comments and reactions. hickey questioned one of the most important arguments of berlin et al., namely the lower synonymous diversity in bird than in mammal mtdna, arguing that mutation rate could be a confounding effect. an accurate estimation of mtdna substitution rate variations in birds appears necessary to correctly interpret the patterns of mtdna diversity in the bird/mammal comparison. birds, finally, have quite high metabolic rates – to times higher than mammals of similar sizes -probably because of the high energetic demand of the flying locomotion. paradoxically, birds species are strikingly long-lived as compared to their mammalian counterparts; on average, birds live three times longer than mammals of similar sizes. birds can be characterized as long-lived homeotherms, a specificity they share with bats. these peculiarities are useful to understand further the impact of metabolic rate and life-span on mtdna substitution rates. three main hypotheses have been proposed to explain mtdna substitution rate variations in mammals: the generation time hypothesis, the metabolic rate hypothesis, and the longevity hypothesis, presented above. the classical approach to test these hypotheses in a comparative framework is to correlate substitution rate variations with the relevant life history traits: female sexual maturity, basal mass-specific metabolic rate, and maximum longevity. however, these life-history traits are strongly correlated with each other, so that the respective contributions of the three variables are difficult to disentangle. the contrast between the avian and mammalian physiologies provides a unique opportunity to discriminate between two of these competing models, namely the longevity and the metabolic rate hypotheses. according to the longevity hypothesis, birds should present, on average, lower mtdna neutral substitution rates than mammals, while the reverse pattern is expected under the metabolic rate hypothesis. to test these predictions and challenge the results we obtained in mammals, we propose to accurately estimate the lineage-specific neutral substitution rate variation of mtdna in birds using the phylogenetic framework developed by. using this dataset, we want to check the rough 2%/site/myr calibration. this calibration, originally estimated with an rflp analysis on mammals by brown et al., was generalized to birds by a study in geese. this calibration rapidly became a standard in ornithology studies, probably because of the scarcity of the fossil record. recently, the reliability of this calibration was debated in various studies either supporting or rejecting it. we will also ask which of metabolic rate and longevity is the best predictor of mutation rate variations in warm-blooded vertebrates. finally, we will estimate the mitochondrial genetic diversity on a wide taxonomic range in birds to test for the neutrality assumption and investigate the link between population size, mutation rate, genetic hitch-hiking, and mtdna diversity.  substitution rate variation and its determinants to infer the substitution rate variation of a large number of bird species, we need to estimate divergence dates, and the numbers of nucleotide substitutions which occurred during these divergences. the divergence dates are obtained thanks to paleontologic calibration points, which are mostly available for relatively old divergences. nucleotide substitution numbers, however, must be estimated using little-divergent sequences because of mutational saturation. to resolve this methodological problem, we take advantage of the decoupled non-synonymous and synonymous evolutionary dynamics. we introduced a 3-step method, in which we first define groups of sequences showing limited divergences, then estimate the relative species-specific substitution rate at third codon positions within groups, and finally assign a divergence for each of these groups using amino acid sequences. at step and molecular and fossil information are combined thanks to a sophisticated bayesian method. using this procedure, we analysed a cytb dataset of species, which represent ~15% of the total living species of birds. the average cytb third codon position substitution rate was substitution/site/myr, and the median was substitution/site/myr. these substitution rates are estimated per lineage, per third-codons position. to make them roughly comparable to the popular 2% of divergence per site per myr, these numbers must be multiplied by two and divided by three. doing this would yield an average divergence of % per myr, which is slightly less than the popular 2%. the distribution across species shows a large variation: the % and % percentiles are and substitution/site/myr, respectively. thus, even if we consider the 5% most extreme substitution rate values as outliers, the cytb third codon position substitution rate shows a thirty-fold range of variation across bird lineages. we quantified the effect of taxonomy on substitution rate variation using a hierarchical anova considering super-order, order, family and genus with random effects. the order level explains 46% of the variance in substitution rates across species. considering only order with more than species, passerines are the fastest evolving order and anseriformes the slowest one. compared with mammals, birds globally evolve times slower on average, and show a range of variation less extreme. we correlated the species-specific substitution rate to life-history traits in order to discriminate between the competing explanatory hypotheses. a smaller amount of life-history data are available in birds than in mammals, especially for female sexual maturity, which is documented in species only. we thus focused on the effects of body mass and maximum longevity using the species in which these two variables are available. in agreement with both the metabolism and the longevity hypotheses, the two life-history traits are negatively linked to the substitution rate. body mass appears to be a better predictor of the substitution rate than maximum longevity, which also shows a strong level of correlation. the distribution of maximum longevity is strongly bimodal in birds, passerines being substantially more short-lived than other birds. we separated passerines from non-passerine and re-performed the same analyses in the two subsets separately. the results are shown in table surprisingly, the third-codon position substitution rate is positively correlated to body mass and maximum longevity in passerines birds. in non-passerines birds, results are similar to the global analysis: body mass best explains substitution rate variations, and removes the effect of longevity in a two-way anova. this result is not consistent with mammals. in order to check whether these results were robust to the removal of phylogenetic effects, we applied the phylogenetic contrast method to regress phylogeny out of the analyses. in this analysis, we used the chronogramme obtained with amino-acid cytochrome b sequences. the phylogenetic control did not qualitatively change the results although most p-values were decreased. finally, we jointly analysed the mammalian and bird datasets, including class as an explanatory variable. the two-factor model revealed that the bird vs. mammal status has a strong effect on mtdna substitution rate when body mass is taken into account and a significant but weaker effect as far as longevity is concerned. there is, moreover, a significant interaction between body mass and class, but not between longevity and class. determination of mtdna diversity in birds bird cytb polymorphism sequence datasets were retrieved from the polymorphix database. the synonymous and non-synonymous levels of diversity were computed for the species in which more than four sequences were available. the mean per-site synonymous nucleotide diversity was ±. the most variable species was shelley's greenbul, and two species showed no synonymous variations. to test the relationship between population size and mtdna diversity, we first correlated nucleotide polymorphism and body mass, taken as a proxy of population size, as in nabholz et al. and berlin et al.. πs and body mass were negatively correlated but, contrary to berlin et al., this relationship was not significant. secondly, we applied a more direct analysis using population size estimates for north american breeding birds through the north american landbird conservation plan. such data were available for strictly north american species of our data set. we found no correlation between direct population size estimate and cytb synonymous diversity. these results confirm those obtained in mammals, here using direct estimations of population sizes instead of life-history and ecological proxies. the dataset, however, is much smaller in the present analysis. we tested the taxonomic effect on πs using one-way anovas on order and family level. we found that neither the family nor the order level had a significant effect. the sole taxonomic variable having a significant effect on mtdna diversity is the passerine/non-passerine status, passerines being more diverse than other birds. we compared the distribution of πs between birds and mammals. similarly to berlin et al., we found that birds have a lower average synonymous diversity than mammals, but this difference is largely reduced when we controlled for the mutation rate. interestingly, we detected no significant difference of allozyme heterozygosity between mammals and birds using the nevo et al. data set. as in mammals, we found a positive and significant correlation between species-specific cytb third-codon substitution rate and cytb synonymous diversity per genus. these analyses confirm that the mutation rate is a major determinant of mtdna polymorphism in birds as well as in mammals. finally, we compared the ratio of non-synonymous to synonymous changes within and between species in birds and mammals – these ratio were computed when an outgroup was available. the two groups showed a similar dn/ds, but the pn/ps ratio was higher in birds than in mammals, as previously reported by berlin et al.. discussion mtdna substitution rates – support for the longevity hypothesis we showed that the mtdna mutation rate, measured through the neutrally-evolving third codon positions of cytb, is highly variable between bird lineages. the molecular clock hypothesis does not apply to bird mtdna evolution, fast-evolving species being thirty times more rapid than slow-evolving ones. the method we applied is particularly prone to reveal the actual amplitude of substitution rate variation: by overcoming the problem of mutational saturation, it allowed us to use an extensive dataset encompassing the whole taxonomic diversity of birds. this study, therefore, provides a synthetic view of substitution rate variations across bird lineages, bringing an important result in the field of molecular dating, in which the rough approximation of 2% of substitution per million of years is still debated. our analysis suggests that the molecular clock assumption should be avoided as far as bird mtdna is concerned. the conclusions of pereira and baker, and some studies done at smaller scale are here confirmed by cytochrome b third codon positions at wide taxonomic scale. although the average rate in birds is close to this value, the 2% per site per million year calibration is a very bad summary of the whole picture: the rate is essentially higher than 2% in passerines, and lower in non-passerines. users of mtdna as a tools for inferring divergence dates should imperatively use statistical phylogenetic methods accounting for substitution rate variation across lineages, the so-called clock-relaxed methods. several interesting results were revealed thanks to the comparison with mammals. first, birds show a more narrow range of variation of life-history traits than mammals. for example body mass varies from g to kg in the bird dataset, whereas it varies from g to tones in mammals. this result confirms the general influence of life-history traits on neutral mtdna substitution rate in birds and mammals. this result is important because such a relationship was not reported in every taxonomic group, and even not by some studies in mammals. secondly, the neutral mtdna substitution rate is lower in birds than in mammals, and this difference increases if body mass is regressed out of the analysis. for example, birds evolve four times slower than mammals as far as small species are concerned. this result is in agreement with the longevity hypothesis, but not with the metabolic rate hypothesis. according to the latter model, birds should show a higher average mutation rate than mammals, because of their higher mass-specific metabolic rate and their lower average body mass. during the course of evolution, birds have acquired adaptations to manage their high mass-specific metabolic rate, including an increased of reactive oxygen species protection and lower ros production. in the context of the mitochondrial theory of aging, which postulates that ros production as a byproduct of mitochondrial respiration contributes to aging, these adaptations could be interpreted as a response to the elevated longevity of birds. the reduced rate of ros production could explain the lower average mutation rate of birds mtdna, as compared to mammals. it is surprising, therefore, to find a predominant influence of body mass, not longevity, in the within-birds analysis. passerines show a positive relationship between longevity and substitution rate, which is quite surprising. we have no valid explanation for this relationship so far, perhaps because it is not biologically relevant – this relationship is the single result that was not recovered when we used other methods of substitution rate estimation. within non-passerines, which are essentially long-lived species, body mass is still the variable best explaining substitution rate variations. using dna-dna hybridization data, mooers and harvey found no support for the metabolic rate hypothesis, but reported a significant generation time effect. in this context, it would be interesting to enlarge the female sexual maturity data set, in order to test the generation time hypothesis with our extended mitochondrial dataset. determination of mtdna diversity in birds the neutral genetic diversity primarily depends on effective population size and mutation rate. because population sizes appear so variable in time and between species, variations in genetic diversity across species have been frequently interpreted in demographic terms, e.g.. recently, the effect of population size on mtdna diversity has been challenged. the large variations in mutation rates we report in birds suggest that mutational effects should be carefully taken into account in studying mtdna polymorphism in this group, as well as in mammals. in general, the relationship between population size and mtdna diversity is difficult to evaluate because of the scarcity of population size estimates for wild species. life-history traits, like body mass, have therefore been used as proxy of population size. birds are an exception: thanks to the broad interest of the scientific community in bird ecology and systematics, direct estimates of species effective sizes are available for north american species. we correlated these population size estimates to synonymous mtdna diversity and found no significant relationship. the data set is rather small, but we note that this relationship should theoretically be strong, given the wide range of population sizes. when using body mass as an indicator of population size, thus increasing much the data set, we found a negative correlation with cytb synonymous diversity, but this result was not significant. berlin et al. report a significant relationship between πs and body mass in birds using genus averages. if we apply the same method, we also find a significant relationship, but the effect is removed when the passerine/non-passerine status is taken into account. this result is in agreement with our previous mammalian analysis, in which the weak effect of body mass was removed by a taxonomic control. so similarly to mammals, the mitochondrial genetic diversity is essentially uncorrelated to life-history traits in birds, and when it is so, it is most likely via the influence of mutation rate, not population size. mitochondrial selective regime in birds to explain the lack of relationship between mtdna diversity and proxies of population size in mammals, nabholz et al. invoked demographic stochasticity, while bazin et al. invoked recurrent hitchhiking effects for other species having larger expected population sizes. what about birds? to address this question we computed the neutrality index. ni < indicates positive selection, while ni > indicates purifying selection. the average ni in birds is significantly higher than in mammals and only three species of birds show a ni <. the high ni values in birds are due to their high pn/ps ratios -dn/ds ratios are similar to those of mammals. this result indicates that mtdna evolution in birds is mainly governed by purifying selection, as previously reported. it is also indicative of a strong level of constraint acting on bird cytochrome b sequences, which is known as the avian constraint hypothesis. like mammals, birds appear to have population sizes low enough to belong to the "drift domain", rather than the "draft domain" sensu gillespie. in the absence of a significant impact of positive selection, we have to invoke strong demographic stochasticity to explain the lack of relationship between population size and mtdna diversity. mutation rate is expected to be a major determinant of within-species genetic diversity whether populations are at mutation-drift equilibrium or not. consistently, we report a significantly positive relationship between cytb diversity and substitution rate, again paralleling the mammalian situation. in a recent paper, berlin et al. proposed an alternative hypothesis to explain mtdna polymorphism patterns in birds. they suggested that hill-robertson effects due to complete association with the w chromosome could explain the lower synonymous mitochondrial diversity and the higher pn/ps observed in birds, as compared to mammals. this should also explain the lack of correlation between neutral polymorphism and population size. outwardly, we confirm this hypothesis by showing that the lower synonymous mitochondrial diversity is not found for nuclear allozymes. but this picture is complicated by the globally lower mtdna substitution rate in birds than in mammals. controlling for mutation rates actually removes the major part of the difference between birds and mammals. to quantify the effect of mutation rate variation on synonymous mtdna diversity we combined the bird and mammal polymorphism and neutral substitution rate datasets and performed a multiple regression of substitution rate and bird/mammal status on synonymous mtdna diversity. this model explains 21% of πs variation. the result also shows that the mutation rate has a strong effect on πs and considerably reduces the effect of class. this analysis indicates that a subtential part of the difference between birds and mammalian πs is explained by their distinct mutation rates. the difference in mutation rates, however, cannot explain the higher level of mitochondrial pn/ps found in birds. birds and mammals have apparently comparable effective population sizes, as suggested by their similar average allozymic heterozygosity. the higher pn/ps ratio in birds may therefore be due to hill-robertson interferences between the mitochondrial and w chromosomes, as suggested by berlin et al., although this effect has only a weak influence on absolute levels of πs, when mutation rate is controlled for.  in this work, we achieved a comprehensive comparison of mitochondrial substitution rate variation and mitochondrial diversity between birds and mammals. cytb neutral substitution rates are, on average, times slower in birds than in mammals. among-lineages variability, although substantial, is also lower in birds than in mammals. this analysis corroborates the longevity hypothesis about mutation rate determination. despite their higher metabolic rate, birds undergo a lower mitochondrial mutation rate, probably because of the beneficial effect of reduced oxidative damage in long-lived species. we suggest that the low mtdna mutation rate of birds might explain their exceptional mass-specific longevity. as for mammals, we report no evidence of adaptive selection on cytb sequence evolution, but still no population size effect, even when using direct estimates of population size available for north american species. we thus invoke the same demographic instability explanation as previously proposed for mammals, although selective interferences with the w chromosome could contribute to the noisy relationship between mtdna diversity and population size. we also confirm the importance of mutation rate as a determinant of mtdna diversity – the lower mutation rates in birds almost fully explain their lower synonymous diversity. this study confirms and extends the message of caution expressed by nabholz et al. about the usage of mtdna as a molecular marker of biodiversity in vertebrates: mtdna diversity is not related to species abundance; mtdna greatly departs the molecular clock hypothesis. the 2% per site per million year calibration has no degree of generality, and should not be used for dating purposes in the absence of fossil data. 
a phylogenetic analysis of cytochrome b third-codon position confirms that the mitochondrial dna mutation rate is quite variable in birds, passerines being the fastest evolving order. this result is in agreement with the longevity hypothesis, and contradicts the hypothesis of a metabolic rate-dependent mutation rate. the mutation rate is the best predictor we have of within-species mitochondrial diversity in birds. mitochondrial dna diversity patterns in birds are strongly influenced by the wide, unexpected variation of mutation rate across species. from a fundamental point of view, these results are strongly consistent with a relationship between species maximal longevity and mitochondrial mutation rate, in agreement with the mitochondrial theory of ageing. direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals. these properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation. birds show no footprint of adaptive selection on cytochrome b evolutionary patterns, but no link between direct estimates of population size and cytochrome b diversity. form an applied point of view, this study reinforces and extends the message of caution previously expressed for mammals: mitochondrial data tell nothing about species population sizes, and strongly depart the molecular clock assumption. several important results were recently reported using mammals as model organisms, including the absence of relationship between mitochondrial dna diversity and life-history or ecological variables, the absence of prominent adaptive selection, contrary to what was found in invertebrates, and the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. we propose to challenge these results thanks to the bird/mammal comparison. it partly explains the differences in mitochondrial dna diversity patterns observed between mammals and birds, previously interpreted as reflecting hill-robertson interferences with the w chromosome. during the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial dna, the most popular marker of molecular biodiversity.
 during the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial dna, the most popular marker of molecular biodiversity. several important results were recently reported using mammals as model organisms, including the absence of relationship between mitochondrial dna diversity and life-history or ecological variables, the absence of prominent adaptive selection, contrary to what was found in invertebrates, and the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. we propose to challenge these results thanks to the bird/mammal comparison. direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals. these properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation.  a phylogenetic analysis of cytochrome b third-codon position confirms that the mitochondrial dna mutation rate is quite variable in birds, passerines being the fastest evolving order. on average, mitochondrial dna evolves slower in birds than in mammals of similar body size. this result is in agreement with the longevity hypothesis, and contradicts the hypothesis of a metabolic rate-dependent mutation rate. birds show no footprint of adaptive selection on cytochrome b evolutionary patterns, but no link between direct estimates of population size and cytochrome b diversity. the mutation rate is the best predictor we have of within-species mitochondrial diversity in birds. it partly explains the differences in mitochondrial dna diversity patterns observed between mammals and birds, previously interpreted as reflecting hill-robertson interferences with the w chromosome.  mitochondrial dna diversity patterns in birds are strongly influenced by the wide, unexpected variation of mutation rate across species. from a fundamental point of view, these results are strongly consistent with a relationship between species maximal longevity and mitochondrial mutation rate, in agreement with the mitochondrial theory of ageing. form an applied point of view, this study reinforces and extends the message of caution previously expressed for mammals: mitochondrial data tell nothing about species population sizes, and strongly depart the molecular clock assumption. 