BACKGROUND
understanding the causes of rate variation in protein evolution is central for many fields including molecular evolution, comparative genomics and structural biology.
a widely accepted principle is that more important proteins evolve more slowly  <cit> .
however, evolutionary rate variation not only exists between different proteins, but also between lineages  <cit> .
this was already observed in the earliest comparative studies, both within  <cit>  and across animal phyla  <cit> .
following the initial observation that drosophilids are fast evolving  <cit> , it was shown that the rate of synonymous substitutions in drosophila melanogaster is approximately two times higher than in rodents and ten times higher than in primates  <cit> .
however, the estimated acceleration seemed to depend on the types of proteins examined, with another study reporting only a 3-fold difference between drosophila and mammalian rates  <cit> .
a study of the relative rates of ribosomal rna evolution in insect lineages showed that there was an episodic substitution rate increase of about 20-fold in the stem lineage of dipterans  <cit> , suggesting that high evolutionary rates may be characteristic of the whole dipteran order.
however, it is currently unclear whether the observed rate accelerations encompassed the whole nuclear genome, or whether they were restricted to certain classes of genes.
in this study, we assess the genome-wide variation of evolutionary rates between insect lineages, by comparing the beetle tribolium castaneum, and the dipterans drosophila melanogaster and anopheles gambiae.
we test  whether there is an acceleration of protein evolution in dipterans that is observable on a genome-wide scale,  whether this acceleration is confined to an episodic burst of changes at the base of the dipteran lineage, and  whether this acceleration affects all genes to a similar extent.
RESULTS
genomic rate estimates confirm an acceleration in dipterans
to estimate genomic evolutionary rates of insect lineages, we formed  <dig> protein clusters derived from cdna or est data from beetle , fly , mosquito , aphid , and human .
concatenation of these orthologous sequences resulted in a single, well-defined alignment of  <dig>  amino acids.
given the known tree topology in figure  <dig> <cit> , branch lengths were estimated using a maximum likelihood method  <cit>  .
the dipteran branches are much longer than the tribolium branch, indicating accelerated evolution in the diptera.
since their last common ancestor, drosophila has accumulated 36%  more amino acid substitutions compared to tribolium, while the corresponding increase for anopheles compared to tribolium is 23% .
a age of geological stage as defined in "a geologic time scale"  <cit>  .
b relative rate = rate/
in order to assign absolute rates of evolution, we combined the sequence distances in figure  <dig> with absolute dates obtained from palaeontological estimates.
based on the oldest coleopteran fossil  <cit>  and the timing of the primary radiation of holometabolous insect orders  <cit> , the divergence of dipterans  from coleopterans  has been estimated to the early permian .
the divergence between the brachycera  and the nematocera  lineages has been estimated to the middle permian   <cit> .
combining these dates with the maximum-likelihood estimates of branch lengths, we obtained the absolute evolutionary rates listed in table  <dig>  this shows that the  <dig> my time interval starting with the separation of coleopterans and dipterans and culminating in the radiation of diptera was characterised by an episodic increase of evolutionary rate, averaging  <dig>  times the mean rate found for the tribolium lineage .
deviations from clock-like evolution
the accuracy of absolute rate estimates depends on the completeness of the known fossil record.
thus, the uncertainty assigned to the dates in table  <dig>  which reflects the uncertainty in the dating of the strata where fossils were found, is an underestimate of the true uncertainty.
to confirm that the dipteran rate acceleration is not an artefact of erroneous palaeontological estimates, we performed additional analyses that directly compared relative evolutionary rates.
to this end, we implemented several models involving global or local molecular clocks  <cit> .
because these models are nested, they can be compared via likelihood-ratio tests  <cit> .
considering the branch length differences in table  <dig>  it is not surprising that the model employed above  fits the data much better than a model with a global clock, which enforces a constant rate of evolution across all lineages .
is the clock assumption violated only at specific  branches?
table  <dig> assigns the strongest rate acceleration to the base of the diptera.
however, an evolutionary model involving a local clock for this branch  still fits the data significantly worse than the model without a clock ; the same is indeed true for all models involving a single local clock .
thus, a burst of accelerated evolution at the base of the dipterans is not sufficient to explain the rate increase found for dipterans as a whole.
a lnc is the maximum-likelihood estimate of the model without a molecular clock.
b df = degrees of freedom
further comparison of increasingly complex models of evolution show that only models involving separate local clocks for the tribolium and drosophila lineages can fit the data as good as the model without a clock assumption .
all other combinations of local clocks between the tribolium, drosophila and anopheles lineages were found to be inferior to the model without the clock .
thus, although the accelerated evolution of dipterans was most pronounced during a burst of changes that occurred at the base of the order , a sustained increase is also detected in the branches separating drosophila from anopheles.
remarkably, the need for a coleopteran local clock also demonstrates that the tribolium lineage has generally evolved much slower than most of the other taxa considered here.
it should be noted that table  <dig> contains multiple statistical comparisons, thereby decreasing the overall specificity of the statistical test.
a conservative strategy to control for this is to divide the p-value cutoff for significance by the number of comparisons , i.e., to use p <dig> =  <dig> / <dig> =  <dig> , or – if accounting for all comparisons in data not shown – p <dig> =  <dig> / <dig> =  <dig> .
this correction does not affect our conclusions.
the dipteran acceleration affects the majority of individual genes
by analysing concatenated amino acid sequences, we have demonstrated accelerated evolution in dipterans, compared to a slower rate in the tribolium lineage.
is this rate change confined to certain genes, or does it extend across the genome as a whole?
to analyse this issue, we calculated branch lengths for  <dig> orthologous groups comprising sequences from tribolium and drosophila, using human as an outgroup.
in figure  <dig>  we compare the amino acid distances of tribolium and drosophila proteins to their last common ancestor.
there is a very strong relationship between these a priori unrelated distances .
when calculated from single gene distances, the mean evolutionary rate of the drosophila lineage is significantly larger than the one of the tribolium lineage .
the corresponding constant of proportionality is  <dig> , in close agreement with the relative rate increase of  <dig>  obtained from the concatenated sequences .
thus, although evolutionary rates among genes of a genome can vary by several orders of magnitude, these rates are nevertheless correlated between species even after more than  <dig> my of independent evolution.
discussion
using  <dig> nuclear transcripts, we find that the dipteran lineage  has experienced an episodic increase in evolutionary rate when compared to the coleopteran lineage .
this rate subsequently dropped in the diversifying dipteran lineage, but remained above average in drosophila.
this is consistent with previous findings from studies of ribosomal rnas  <cit> .
the rate increases found in our genome-scale analysis of protein sequences are lower than those reported for rrnas, with an average increase of only  <dig> -fold during dipteran evolution versus an at least threefold average increase for rrnas  <cit> .
however, our analysis is necessarily biased towards parts of genes that are sufficiently conserved to be detected in the outgroup  and that could be unequivocally aligned.
still, figure  <dig> demonstrates that the averaged rate increase is representative for a large range of genes.
the neutral theory of evolution  <cit>  predicts that the accumulation of molecular changes is only driven by the mutation rate and the degree of purifying selection.
accordingly, genome-wide variation in the rate of protein evolution between species might be caused by physiological and ecological factors that affect the mutation rate , or by differences in the efficiency of selection .
in the case of the rrna comparisons, we could indeed show that there has been a mutational bias towards incorporating more a/t nucleotides than g/c nucleotides at the base of the diptera  <cit> , which has probably caused the episodic acceleration of evolutionary rates.
such a mutational bias is likely to have affected the whole genome.
however, the gc content of the tribolium genes in this study  is very close to that of the drosophila orthologs , suggesting that mutational biases are unlikely to be responsible for the continued acceleration in dipterans.
this interpretation is supported by the fact that it is the tribolium proteins which show a slight bias towards at-rich amino acids when compared to the drosophila orthologs .
we observe a strong correlation between evolutionary rates in the tribolium and the drosophila lineages .
this indicates that the selective forces acting on the majority of these genes have been similar between the two lineages, consistent with a neutral model.
the correlation would be expected to become weaker or even disappear if positive selection would occur episodically among these genes.
our finding that most proteins diverge in a clock-like fashion, with clock speeds that differ among lineages only due to genome-wide effects, suggests that episodic changes are an exception, although such changes are known to occur in some cases  <cit> .
however, we note that the class of fast evolving genes could not be analysed here, as they diverge too fast to allow identification as orthologs in distant comparisons  <cit> .
in a dedicated study of such genes between closely related drosophila lineages, we found that there are indeed a significant number of genes that must have undergone episodic changes in substitution rates on a gene by gene basis  <cit> .
thus, we emphasize that the above conclusions relate only to relatively conserved  genes, while the generalized evolutionary patterns of fast evolving  genes will need further study.
CONCLUSIONS
we reported here the analysis of evolutionary rate variation of a large number of orthologous genes between insect lineages.
variation in the rate of evolution has genome-wide effects, and is correlated between orthologous genes over very long evolutionary time scales.
because drosophila melanogaster is among the best studied animal model organisms, the peculiar evolutionary pattern confirmed here has practical implications.
in blast searches of human sequences against tribolium and drosophila, tribolium sequences are found in 70% of cases to be more similar to human than drosophila genes .
thus, when attempting to link human genes to their drosophila homologs, data from tribolium will be helpful to provide a more conservative reference sequence.
this approach has been used, e.g., to resolve the evolutionary relationship between the drosophila zen gene and human hox <dig> genes  <cit> .
the slowly evolving beetle tribolium castaneum is thus likely to play a significant role for comparisons between phyla.

 understanding the causes of rate variation in protein evolution is central for many fields including molecular evolution, comparative genomics and structural biology. a widely accepted principle is that more important proteins evolve more slowly. however, evolutionary rate variation not only exists between different proteins, but also between lineages. this was already observed in the earliest comparative studies, both within and across animal phyla. following the initial observation that drosophilids are fast evolving, it was shown that the rate of synonymous substitutions in drosophila melanogaster is approximately two times higher than in rodents and ten times higher than in primates. however, the estimated acceleration seemed to depend on the types of proteins examined, with another study reporting only a 3-fold difference between drosophila and mammalian rates. a study of the relative rates of ribosomal rna evolution in insect lineages showed that there was an episodic substitution rate increase of about 20-fold in the stem lineage of dipterans, suggesting that high evolutionary rates may be characteristic of the whole dipteran order. however, it is currently unclear whether the observed rate accelerations encompassed the whole nuclear genome, or whether they were restricted to certain classes of genes. in this study, we assess the genome-wide variation of evolutionary rates between insect lineages, by comparing the beetle tribolium castaneum, and the dipterans drosophila melanogaster and anopheles gambiae. we test whether there is an acceleration of protein evolution in dipterans that is observable on a genome-wide scale, whether this acceleration is confined to an episodic burst of changes at the base of the dipteran lineage, and whether this acceleration affects all genes to a similar extent.  genomic rate estimates confirm an acceleration in dipterans to estimate genomic evolutionary rates of insect lineages, we formed protein clusters derived from cdna or est data from beetle, fly, mosquito, aphid, and human. concatenation of these orthologous sequences resulted in a single, well-defined alignment of amino acids. given the known tree topology in figure, branch lengths were estimated using a maximum likelihood method. the dipteran branches are much longer than the tribolium branch, indicating accelerated evolution in the diptera. since their last common ancestor, drosophila has accumulated 36% more amino acid substitutions compared to tribolium, while the corresponding increase for anopheles compared to tribolium is 23%. a age of geological stage as defined in "a geologic time scale". b relative rate = rate/ in order to assign absolute rates of evolution, we combined the sequence distances in figure with absolute dates obtained from palaeontological estimates. based on the oldest coleopteran fossil and the timing of the primary radiation of holometabolous insect orders, the divergence of dipterans from coleopterans has been estimated to the early permian. the divergence between the brachycera and the nematocera lineages has been estimated to the middle permian. combining these dates with the maximum-likelihood estimates of branch lengths, we obtained the absolute evolutionary rates listed in table this shows that the my time interval starting with the separation of coleopterans and dipterans and culminating in the radiation of diptera was characterised by an episodic increase of evolutionary rate, averaging times the mean rate found for the tribolium lineage. deviations from clock-like evolution the accuracy of absolute rate estimates depends on the completeness of the known fossil record. thus, the uncertainty assigned to the dates in table which reflects the uncertainty in the dating of the strata where fossils were found, is an underestimate of the true uncertainty. to confirm that the dipteran rate acceleration is not an artefact of erroneous palaeontological estimates, we performed additional analyses that directly compared relative evolutionary rates. to this end, we implemented several models involving global or local molecular clocks. because these models are nested, they can be compared via likelihood-ratio tests. considering the branch length differences in table it is not surprising that the model employed above fits the data much better than a model with a global clock, which enforces a constant rate of evolution across all lineages. is the clock assumption violated only at specific branches? table assigns the strongest rate acceleration to the base of the diptera. however, an evolutionary model involving a local clock for this branch still fits the data significantly worse than the model without a clock; the same is indeed true for all models involving a single local clock. thus, a burst of accelerated evolution at the base of the dipterans is not sufficient to explain the rate increase found for dipterans as a whole. a lnc is the maximum-likelihood estimate of the model without a molecular clock. b df = degrees of freedom further comparison of increasingly complex models of evolution show that only models involving separate local clocks for the tribolium and drosophila lineages can fit the data as good as the model without a clock assumption. all other combinations of local clocks between the tribolium, drosophila and anopheles lineages were found to be inferior to the model without the clock. thus, although the accelerated evolution of dipterans was most pronounced during a burst of changes that occurred at the base of the order, a sustained increase is also detected in the branches separating drosophila from anopheles. remarkably, the need for a coleopteran local clock also demonstrates that the tribolium lineage has generally evolved much slower than most of the other taxa considered here. it should be noted that table contains multiple statistical comparisons, thereby decreasing the overall specificity of the statistical test. a conservative strategy to control for this is to divide the p-value cutoff for significance by the number of comparisons, i.e., to use p = / =, or – if accounting for all comparisons in data not shown – p = / =. this correction does not affect our conclusions. the dipteran acceleration affects the majority of individual genes by analysing concatenated amino acid sequences, we have demonstrated accelerated evolution in dipterans, compared to a slower rate in the tribolium lineage. is this rate change confined to certain genes, or does it extend across the genome as a whole? to analyse this issue, we calculated branch lengths for orthologous groups comprising sequences from tribolium and drosophila, using human as an outgroup. in figure we compare the amino acid distances of tribolium and drosophila proteins to their last common ancestor. there is a very strong relationship between these a priori unrelated distances. when calculated from single gene distances, the mean evolutionary rate of the drosophila lineage is significantly larger than the one of the tribolium lineage. the corresponding constant of proportionality is, in close agreement with the relative rate increase of obtained from the concatenated sequences. thus, although evolutionary rates among genes of a genome can vary by several orders of magnitude, these rates are nevertheless correlated between species even after more than my of independent evolution. discussion using nuclear transcripts, we find that the dipteran lineage has experienced an episodic increase in evolutionary rate when compared to the coleopteran lineage. this rate subsequently dropped in the diversifying dipteran lineage, but remained above average in drosophila. this is consistent with previous findings from studies of ribosomal rnas. the rate increases found in our genome-scale analysis of protein sequences are lower than those reported for rrnas, with an average increase of only -fold during dipteran evolution versus an at least threefold average increase for rrnas. however, our analysis is necessarily biased towards parts of genes that are sufficiently conserved to be detected in the outgroup and that could be unequivocally aligned. still, figure demonstrates that the averaged rate increase is representative for a large range of genes. the neutral theory of evolution predicts that the accumulation of molecular changes is only driven by the mutation rate and the degree of purifying selection. accordingly, genome-wide variation in the rate of protein evolution between species might be caused by physiological and ecological factors that affect the mutation rate, or by differences in the efficiency of selection. in the case of the rrna comparisons, we could indeed show that there has been a mutational bias towards incorporating more a/t nucleotides than g/c nucleotides at the base of the diptera, which has probably caused the episodic acceleration of evolutionary rates. such a mutational bias is likely to have affected the whole genome. however, the gc content of the tribolium genes in this study is very close to that of the drosophila orthologs, suggesting that mutational biases are unlikely to be responsible for the continued acceleration in dipterans. this interpretation is supported by the fact that it is the tribolium proteins which show a slight bias towards at-rich amino acids when compared to the drosophila orthologs. we observe a strong correlation between evolutionary rates in the tribolium and the drosophila lineages. this indicates that the selective forces acting on the majority of these genes have been similar between the two lineages, consistent with a neutral model. the correlation would be expected to become weaker or even disappear if positive selection would occur episodically among these genes. our finding that most proteins diverge in a clock-like fashion, with clock speeds that differ among lineages only due to genome-wide effects, suggests that episodic changes are an exception, although such changes are known to occur in some cases. however, we note that the class of fast evolving genes could not be analysed here, as they diverge too fast to allow identification as orthologs in distant comparisons. in a dedicated study of such genes between closely related drosophila lineages, we found that there are indeed a significant number of genes that must have undergone episodic changes in substitution rates on a gene by gene basis. thus, we emphasize that the above conclusions relate only to relatively conserved genes, while the generalized evolutionary patterns of fast evolving genes will need further study.  we reported here the analysis of evolutionary rate variation of a large number of orthologous genes between insect lineages. variation in the rate of evolution has genome-wide effects, and is correlated between orthologous genes over very long evolutionary time scales. because drosophila melanogaster is among the best studied animal model organisms, the peculiar evolutionary pattern confirmed here has practical implications. in blast searches of human sequences against tribolium and drosophila, tribolium sequences are found in 70% of cases to be more similar to human than drosophila genes. thus, when attempting to link human genes to their drosophila homologs, data from tribolium will be helpful to provide a more conservative reference sequence. this approach has been used, e.g., to resolve the evolutionary relationship between the drosophila zen gene and human hox genes. the slowly evolving beetle tribolium castaneum is thus likely to play a significant role for comparisons between phyla. 
when compared to the beetle tribolium castaneum, we find that the stem lineage of flies and mosquitoes has experienced on average a 3-fold increase in the rate of evolution. to answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes. pairwise gene comparisons between drosophila and tribolium show a high correlation between evolutionary rates of orthologous proteins. among the insects analysed so far, it appears that the tribolium genes show the lowest rates of divergence.
 the rate of molecular evolution varies widely between proteins, both within and among lineages. to what extent is this variation influenced by genome-wide, lineage-specific effects? to answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes.  when compared to the beetle tribolium castaneum, we find that the stem lineage of flies and mosquitoes has experienced on average a 3-fold increase in the rate of evolution. pairwise gene comparisons between drosophila and tribolium show a high correlation between evolutionary rates of orthologous proteins.  gene specific divergence rates remain roughly constant over long evolutionary times, modulated by genome-wide, lineage-specific effects. among the insects analysed so far, it appears that the tribolium genes show the lowest rates of divergence. this has the practical consequence that homology searches for human genes yield significantly better matches in tribolium than in drosophila. we therefore suggest that tribolium is better suited for comparisons between phyla than the widely employed dipterans. 