- Research article
- Open Access
Codon usage in twelve species of Drosophila
© Vicario et al; licensee BioMed Central Ltd. 2007
Received: 08 May 2007
Accepted: 15 November 2007
Published: 15 November 2007
Codon usage bias (CUB), the uneven use of synonymous codons, is a ubiquitous observation in virtually all organisms examined. The pattern of codon usage is generally similar among closely related species, but differs significantly among distantly related organisms, e.g., bacteria, yeast, and Drosophila. Several explanations for CUB have been offered and some have been supported by observations and experiments, although a thorough understanding of the evolutionary forces (random drift, mutation bias, and selection) and their relative importance remains to be determined. The recently available complete genome DNA sequences of twelve phylogenetically defined species of Drosophila offer a hitherto unprecedented opportunity to examine these problems. We report here the patterns of codon usage in the twelve species and offer insights on possible evolutionary forces involved.
(1) Codon usage is quite stable across 11/12 of the species: G- and especially C-ending codons are used most frequently, thus defining the preferred codons. (2) The only amino acid that changes in preferred codon is Serine with six species of the melanogaster group favoring TCC while the other species, particularly subgenus Drosophila species, favor AGC. (3) D. willistoni is an exception to these generalizations in having a shifted codon usage for seven amino acids toward A/T in the wobble position. (4) Amino acids differ in their contribution to overall CUB, Leu having the greatest and Asp the least. (5) Among two-fold degenerate amino acids, A/G ending amino acids have more selection on codon usage than T/C ending amino acids. (6) Among the different chromosome arms or elements, genes on the non-recombining element F (dot chromosome) have the least CUB, while genes on the element A (X chromosome) have the most. (7) Introns indicate that mutation bias in all species is approximately 2:1, AT:GC, the opposite of codon usage bias. (8) There is also evidence for some overall regional bias in base composition that may influence codon usage.
Overall, these results suggest that natural selection has acted on codon usage in the genus Drosophila, at least often enough to leave a footprint of selection in modern genomes. However, there is evidence in the data that random forces (drift and mutation) have also left patterns in the data, especially in genes under weak selection for codon usage for example genes in regions of low recombination. The documentation of codon usage patterns in each of these twelve genomes also aids in ongoing annotation efforts.
The genetic code is redundant, i.e., more than one triplet sequence of DNA bases codes for the same amino acid. Thus genes and species may use different sets of codons preferentially, the phenomenon of codon usage bias (CUB). This well documented un-evenness of use of synonymous codons may come about by a variety of evolutionary forces, in particular, mutation bias, selection, and genetic drift. Generally, species have a characteristic pattern of codon usage that holds across most genes in the species; notable exceptions are warm-blooded vertebrates where isochores, large stretches of DNA with high AT or GC content , appear to affect synonymous codon usage depending on the characteristic AT/GC content of the isochore in which a gene resides . There is evidence for heterogeneous regional base content in Drosophila (discussed later), although clearly not as strong as in mammals and having much less influence on codon usage.
Drosophila have been well-studied for patterns and processes that lead to CUB, in particular D. melanogaster, and less often, a few other species for which very limited sequence data were available. The newly available complete genome sequences of 12 species of Drosophila[3, 4] provide a hitherto unattainable insight into CUB variation and evolution in a set of phylogenetically well-defined related taxa. In addition, knowing the particular codon usage patterns of different species allows more accurate identification of protein-coding sequences (i.e., annotation). Here we examine the patterns of codon usage in these 12 species based on reliably identifiable protein-coding genes that were available in November 2006. We identify patterns of preferred codons for each individual amino acid in each species, as well as examine variation among genes, amino acids, and chromosome arms for level or intensity of CUB in a species. Evidence is provided as to the causes of CUB by considering population sizes and mutation bias as assessed by base composition of introns and non-recombining genes.
Not all species have the same overall intensity of codon usage bias
Identification of preferred codons for each amino acid for each species
Bootstrap analyses of the data in Figure 2 were done to assess the statistical support for identification of the preferred codon (see Additional File 1 and 2 and ). Overall these analyses indicate strong statistical support of identification of the preferred set of codons for each species. For example, of the 216 highest correlation coefficients in Figure 2A (12 species × 18 redundant amino acids), 209 are supported with a bootstrap value of 95–100%. The other methods give similarly high bootstrap support for the preferred codon (see Additional File 1 and 2).
Two important generalities are clear from Figure 2. First, the preferred set of codons is quite constant across Drosophila. Second, in most cases the preferred codons in all species are those with G and especially C in the third codon position. That is, generally in Drosophila, codon usage is biased toward C/G-ending codons as previously deduced from limited data on fewer species. Exceptions to these two generalities exist, with Dwil being the obvious species with the most exceptions as well as being the species with the least overall CUB (Figure 1). We discuss Dwil in more detail later.
Amino acids contribute differently to a gene's CUB
D. willistoni: the outlier
Summary of preferred codons for each amino acid for 11 species, with differences noted for D. willistoni.
Dpse about equal TCC/AGC preference; Dper, and subgenus Drosophila prefers AGC
10% highest RSCU identifies ACG in Dmoj and Dvir; ACA in Dgri
10% highest RSCU identifies CCG in Dvir
10% highest RSCU identifies CAT in Dgri
Very weak bias, methods differ somewhat
10% highest RSCU weakly identifies TAT in subgenus Drosophila
10% highest RSCU identifies no preference in subgenus Drosophila
10% highest RCSU weakly identifies ATT in subgenus Drosophila
Amino acids vary in sensitivity to selection for codon usage
Mutation bias and codon usage
Mutation bias can affect codon usage especially when selection is weak. In order to assess the pattern and strength of mutation bias, we identify regions of the genome thought to have the least selective constraint, wherein base composition at equilibrium should reflect mutation bias. We identified the intron sequences from the 6,698 CDSs with homologues across all 12 species as described in the Materials and Methods. In order to increase the likelihood of examining selectively neutral DNA sequences, we removed embedded transposable elements as well as 50 bp on each end of the introns in order to avoid constrained splicing signals. We also compared all introns to a subset of introns between 100 bp and 2000 bp (after removal of 50 bp at each end) in order to decrease the probability of including unidentified embedded CDS and control elements more likely to be present in very long introns.
Base composition (in percent GC) of introns and at the third position of four-fold degenerate amino acids.
All 6,698 homologues
Total intron length (Mb)
Average intron length (bp)
No. introns: 100/2000a
Intron GC% (Wt.)b
Intron GC% (Ave.)c
Intron GC% (Ave. 100/2000)d
GC% 4-fold AAe
33 F homologues
Intron GC% (Wt.)b
Intron GC% (Ave.)c
Intron GC% (Ave 100/2000)d
GC% 4-fold AAe
We present two ways of summarizing the intron base composition data, a weighted mean provided by simply concatenating all introns and calculating base composition, and an unweighted average counting each intron equally regardless of size. Regardless of ways of calculating base content of introns, it is clear that introns are relatively high in A/T, between 59 and 65% across all 12 species. This implies that for all 12 species, contrary to codon usage bias, mutation bias is toward A/T. This observation is consistent with the previous observation based on fewer genes mainly from D. melanogaster [e.g., [8, 9]] and is now shown to be more general for the genus Drosophila. (For reasons that will become clear in the next section, we separated genes on the fourth chromosome of Dmel in Table 2.)
Also note that when the data are trimmed to avoid very large introns (>2000 bp), the A/T content consistently increases by about one percent across all species (Table 2), implying that there may be some small degree of unidentified embedded CDSs or control elements in the longer introns. This is consistent with Comeron and Kreitman  indicating that higher recombination rate expected in genes with longer introns enhances selection on base composition.
Non-recombining genes have different codon usage
Most species of Drosophila have a "dot" chromosome, the 4th in Dmel, also known as element F [11, 12]. Of particular interest is the fact that genes on this chromosome very rarely recombine compared to all the other chromosomes [13, 14]. Among the 6,698 CDSs homologous across all 12 species, 33 are known to be on the F element in Dmel, and by chromosome arm homology, may be assumed to be on this element in the other species. Among the 12 Drosophila species with completed genome sequences, Dwil is again different in this regard in lacking a dot chromosome . There is good evidence that in Dwil the genes on the F element have been incorporated into one of the other elements, in particular element E . The importance of comparing non-recombining genes to recombining genes is that the effectiveness of selection at individual loci (nucleotide sites in this case) is positively correlated with recombination rate, the well-known Hill-Robertson effect . Codon usage bias has been shown to positively correlate with rate of recombination in Drosophila[18–22] and that genes on the (nearly) non-recombining dot chromosome of Dmel are particularly low in codon usage bias [18, 23].
Alternative codon usage
Given that mutation bias in Drosophila is generally toward A/T while CUB is generally toward G/C, the two forces, selection and mutation bias, are opposed. Also note that when selection is nonexistent or very weak, mutation bias will cause CUB but the pattern will be opposite that caused by selection. Thus non-directional measures of unevenness of codon usage such as ENC will identify genes/amino acids that have CUB due to both preferred GC in the wobble position (due largely to selection in Drosophila) and AT bias (due largely to mutation bias in Drosophila). Genes/amino acids with completely even codon usage would occur only when the mutation bias toward A/T is exactly balanced by selection toward G/C. The directional measure CAI does not have this property as it measures deviation from usage of a set of pre-defined preferred codons. Thus, unlike ENC, the CAI of genes/amino acids dominated by mutation bias would have a lower CAI than those with completely even usage of codons.
It is of considerable interest to note that the upper eight species in Figure 7, the evident linear association between ENC and CAI breaks down at the very lowest end of CAI on the left in these graphs. Genes at this end actually have somewhat lower ENCs resulting in a downward "hook" in this range of the relationship. This means they are biased when measured by unevenness of synonymous codon usage (ENC), but the direction of bias is away from usage of the optimal codon as defined by the reference set used to calculate CAI. As noted by the color differences, these genes tend to be those located on the non-recombining element F. This is as expected if mutation bias is dominating these genes so that they are biased (as measured by ENC) due to mutation bias toward A/T, opposite to usage of the optimal set of codons rich in G/C. The dotted line in each graph in Figure 7 are for completely equal usage of all codons, when mutation bias to A/T is exactly balanced by selection to G/C; thus one interpretation is that genes to the left of the dotted lines are dominated by mutation while those to the right are dominated by selection. [Note that this interpretation would only hold for cases as in Drosophila where mutation bias to A/T is opposite to selection for G/C, and thus this approach may not be generally applied to other organisms where this is not true.]
The four lower species in Figure 7 do not display much evidence for the downward hook associated with the other species, at least not associated with genes known to be on element F in Dmel. This is expected for Dwil, without a separate element F. The other species (Dmoj, Dvir, and Dgri) that do not exhibit this downward hook associated with element F genes are in subgenus Drosophila. Consistent with other evidence below, we speculate this is due to the fact that there has not been complete conservation of linkage of Dmel F element genes in subgenus Drosophila. If at least some genes that are found on element F in Dmel have now become incorporated in other chromosome arms, they would not be expected to produce the downward hook in Figure 7 evident in other species.
In the Additional File 1 and 2 we present graphs similar to those in Figure 7 identifying other genes with the alternative codon usage pattern. It is likely that many of these reside in other regions of the genome that have unusually low recombination such as near telomeres and centromeres.
Another way to identify alternative codon usage on the non-recombining F element is to compare the GC content at third positions of four-fold degenerate amino acids. Table 2 shows these data. An increase of 20–30% in A/T at these positions compared to the total data set is evident for most species. For Dwil, there is much less difference (6%) for these genes that have now become incorporated into normally recombining chromosome arms.
Finally we note the added evidence of the dominance of mutation bias on base composition of element F by examining the base composition of introns on this element compared to introns on all other elements. Table 2 shows that the A/T content of F element introns is consistently higher than introns on other elements. We suspect that the base content in non-recombining introns reflects most accurately the actual mutation bias which, fairly consistently across species, has a mean of 32% G/C, 68% A/T. This implies that introns in genes not on element F are still under some selective constraints (as indicated by a 35–41% G/C content) even after removing 50 bp at each end and eliminating predicted TE sequences. Note again that Dwil is the exception in that there is no difference in base composition between introns in genes that are on the F element in the other species and introns on all other elements (Table 2). This implies that the incorporation of these sequences into "normally" recombining chromosome arms subjects them to the same evolutionary forces as all other sequences.
Also evident in Table 2 is the fact that the F element genes in Dvir and Dgri have fairly high GC% at four-fold degenerate sites. This is consistent with the previous observation (Figure 7) indicating that there has not been complete conservation of synteny between Dmel and these two species in another subgenus diverged for about 50 million years.
Regional patterns of base composition and its effect on codon usage
Linear regressions of GC content of introns on coding sequences of the same gene for each species.a
Stability and change in codon usage in Drosophila
As with many organisms, there is a characteristic pattern of codon usage in the genus Drosophila, at least for 11/12 species with complete sequenced genomes. With the exception of serine, all species have the same preferred codon for all amino acids (Figure 2 and Table 1). Codon usage for serine has a clear phylogenetic component in that all members of the melanogaster group have a different favored codon from all the other species. All favored codons in 11/12 species have either C or G in the third codon position.
D. willistoni is clearly the most different of all the species with regard to codon usage and is a clear exception to the generalities just expressed. The unusual codon usage in this species was first pointed out by  and has since been confirmed to be similar in all species in the lineage leading to Dwil including the saltans group [31–33]. Thus the shift in codon usage likely occurred ancestrally in the lineage leading to all extant members of this willistoni-saltans lineage. Generally, the change in codon usage in Dwil (Table 1) has been toward higher usage of T instead of C in the preferred codons (Arg, Leu, Gly, Asn, Asp, and Ile) or A for C (Pro). This results in a lower overall codon usage bias in this species as can be seen in Figures 1 and 5. But, interestingly not all amino acids shift in preferred codon, although there does seem to be an overall decrease in magnitude of CUB in Dwil even for those amino acids for which there is not a qualitative change in preferred codon.
This shift in codon usage could be due to a number of factors: (a) a quantitative increase in the level of mutation bias toward A/T, (b) a small effective population size so that the relative balance between mutation bias and selection has shifted toward the former even if the magnitude of mutation bias has not changed, and (c) change in the relative abundance of isoaccepting tRNAs so that the level and pattern of selection mediated by tRNA has shifted. There is some evidence that the magnitude of mutation bias toward A/T has increased in Dwil compared to the other species as assessed by intron base composition (Table 2). However, this magnitude of change in mutation bias by itself seems incapable of explaining changes in codon usage in Dwil . Further, if we assume mutation bias is best reflected by base composition of the non-recombining introns of element F with a mean AT of 68% in 11/12 species, Dwil AT content for these same introns as well as all introns is marginally lower (66.5%) arguing that neither the pattern nor strength of mutation bias in Dwil is significantly different from other species. With regard to relaxed selection due to small population size, today Dwil has a very large distribution with dense populations [20, 35, 36] and its contemporary population size is undoubtedly very large. It is possible, however, that this may represent a relatively recent expansion since the Pleistocene and that the willistoni lineage went through a bottleneck that continues to affect patterns of molecular evolution. Consistent with this idea is the fact that Dwil has a higher level of non-synonymous polymorphisms than other species , implying relaxed selection. But again, this would seem to be insufficient to account for greater shifts in codon usage for some amino acids and not others. That the selective pressure for codon usage in the willistoni/saltans lineage has shifted, possibly due to a change in tRNA pools, is still a strong likelihood as argued in more detail in .
Intensity of CUB and population sizes
As can be seen in Figure 1, there is considerable variation in level of CUB among the 12 species. Overall, this pattern is reasonably consistent with what can be inferred from the effective population sizes of the species, with selection for codon usage being more effective in larger populations; this is especially relevant as it has been shown that the level of selection on codon usage is very close to the border of when selection or drift dominate, l2Nesl ≅ 1 [38–42]. Dpse and Dper are widespread species in the western half of N. America and considerable data exist indicating high levels of gene flow among populations [e.g., [43, 44]] consistent with a large Ne. Also consistent with a large Ne for Dpse is that it has a higher level of DNA plymorphism than Dmel or Dsim [20, 45]. On the other hand, Dgri is an Hawaiian endemic with presumably a relatively small population and has the least degree of CUB (with the exception of Dwil). As first pointed out by  and now more thoroughly confirmed [; Akashi et al. in preparation], Dsim has greater CUB than Dmel, again consistent with Dsim having greater molecular variation  indicating a larger Ne than Dmel. Presumably, the relatively recent "out of Africa" history of the domestic populations of Dmel affected its historical effective population size, the effect of which can still be seen in the DNA patterns of variation. Dana has a history similar to Dmel in being a fairly recently domesticated species associated with human refuse. Among the other members of the melanogaster group, Dsec has the least CUB consistent with a small population size for an island endemic.
Given that codon usage in Drosophila favors G and C in the wobble position of codons (Figure 2; Table 1) while mutation bias is toward A and T (Table 2), it is difficult to escape the conclusion that non-random synonymous codon usage is due, at least to a large degree, to selection. This is not to imply that selection need necessarily always act on synonymous codons nor equally on all genes. Figure 7 indicates a range of selection on genes with a minority having codon usage more indicative of mutation bias. Rather selection has acted on most genes over evolutionary time such that the footprint of selection is still evident in the newly available complete genome sequences. Also, as noted above, given that the selection coefficients associated with synonymous codon usage are near to the point where population size is crucial, given the inevitable fluctuations in populations over long periods of time, it seems very likely that the dominance of selection and mutation/drift has also fluctuated. In fact, one can see in the non-recombining element F genes, strong evidence that mutation and drift dominate. Given that we can document the expectations of mutation/drift dominance in non-recombining genes, makes it all the more evident that the patterns of DNA variation in "normally" recombining genes not on element F cannot solely be due to mutation/drift.
Also, we (Table 3) and others have noted that there may well be regional base composition effects that can affect codon usage. The precise quantitative interaction between factors influencing regional base composition and codon usage remains to be determined. However, the data presently available indicate that the influence exists, but it is not great.
But the level of selection must be weak as pointed out above, and different species, genes, and amino acids are differentially affected by selection. This explains the differences in Figures 2A and 2B. Figure 2A indicates that selection is acting reasonably consistently across all 12 species when considered on the entire gene level, yet Figure 2B indicates more complexity when considering individual amino acids for which the balance between selection and mutation bias may differ among species.
Nature of selection
Two, interdependent, factors for selection on synonymous codon usage are speed and accuracy of translation. In unicellular organism (i.e., bacteria and yeast) it is well established that more highly expressed genes have greater CUB than lowly expressed genes and that the codons preferred are optimally translated by the most abundant isoaccepting tRNAs [46–49]. For Drosophila, similar observations have been made [e.g., [6–8]]. For example, there is a good correlation between level of expression and CUB for Dmel and Dpse for which microarray expression data are available . In addition, experimental evidence indicates higher level of expression of alleles with optimal codons compared to non-optimal codons . Finally, the relative abundances of isoaccepting tRNA in Dmel correlate with the preferred codons [7, 52].
With regard to accuracy (avoidance of misincorporation of the wrong amino acid), it is known that synonymous codons can vary by as much as ten-fold in rates of misincorporation . Akashi  presented evidence for selection for accuracy in Drosophila by showing that conserved amino acids among species have higher CUB than amino acids free to vary; the implication is that conserved amino acids are more crucial to protein function than those that vary among species. In this regard, it is interesting that among 2-fold redundant amino acids, Cys has the highest overall contribution to CUB and the second most overall all 18 amino acids (Figure 4) as well as being among the most "sensitive" to increasing gene CUB (Figure 5). Given its importance in three-dimensional structure of proteins by forming disulfide bridges, misincorporation at a Cys site should be strongly selected against. Leu has the greatest contribution on average to overall CUB (Figure 4). Leu is the most abundant hydrophobic amino acid  and thus may on average be more constrained against misincorporation than most amino acids. It is less clear why Lys and Gln are among the most sensitive two-fold degenerate amino acids (Figure 5), although generally (with the exception of Cys), A/G two-fold redundant amino acids are more biased in codon usage than C/T two-fold redundant amino acids.
Another potential explanation for different amino acids varying in intensity of CUB is that it is related to the number of different isoaccepting tRNAs for each amino acid. One possibility is that amino acids with one or very few tRNAs translating it are more prone to CUB than amino acids with several isoaccepting tRNAs. A comparison of the numbers of different isoaccepting tRNAs for each amino acid given in White et al.  and which amino acids contribute most or least to CUB does not obviously support this speculation.
The relative lack of codon bias for Asp can be more clearly associated with tRNA pools. Asp has both the least contribution of amino acids to the total CUB of a gene (Figure 4) as well as being the least sensitive (Figure 5). At least in Dmel (the only species studied for relative abundance of isoaccepting tRNAs), Asp is unique among amino acids as it is the only one for which the most abundant isoaccepting tRNA changes among developmental stages, with the most abundant tRNA in the larval stage optimally translating CAC and other stages optimally translating CAU . Vicario  showed that genes with maximum expression at different developmental stages preferred Asp codon usage that matches the tRNA levels. The fact that genes expressed at different stages have selection for different Asp codons explains why this amino acid has relatively low contribution, on average, to overall gene CUB as well as why, when averaged over all genes (as done in Figure 5), Asp appears to respond slowly to increasing overall CUB.
If subsequent information such as levels of gene expression and tRNA pools becomes available for all 12 species considered here, then what we have referred to as "preferred" codons, may eventually become considered "optimal" codons sensu .
Heterogeneity among chromosome arms (elements)
As noted in Figure 6, genes on the different chromosomal arms of Drosophila, or elements, have different codon usage. We discussed above the role of recombination and its lack in element F in many species of Drosophila and how this affects on codon usage. Less clear is why element A, an X chromosome in all species, has more biased CDS than other elements. This is true not only in Drosophila, but also in Caenorhabditis . Two factors have been suggested, the hemizygosity of X in males and dosage compensation [34, 50]. Exactly the role of these factors is unknown, although the evidence is that, whatever the factor(s) affecting X chromosome codon usage they are still weak. This is most evident in Dpse where an arm, element D, that is an autosome in many Drosophila has become part of a metacentric X chromosome. The codon usage bias in element D in Dpse is indistinguishable from other autosome arms (Figure 6), implying that not enough time has elapsed for this element to evolve a true X pattern of codon usage, although the incorporation of this element into the X likely occurred at least 10 million years ago . It will be of considerable interest to observe what has occurred on element D in Dwil that evidently experienced a similar fusion of D and A elements around 25 million years ago ; this awaits syntenic assignments for this species.
Here we have presented largely descriptive aspects of codon usage in Drosophila based on the newly available complete genome sequences. It can be anticipated that with further analyses of these genomes, as well as acquisition of relevant data such as from microarray expression experiments for all 12 species, we will gain increased insights into the evolution of codon usage and its causes. Synonymous mutation, the basis of the evolution of codon usage, are clearly the kind of "nearly neutral" mutations that likely play a large role in molecular evolution . Examination of codon usage and its evolution provides insights into the dynamics of this crucial class of mutations that have been fundamental in molding sequence patterns of genomes.
Materials and methods
Complete (or nearly complete) genomic DNA sequences for twelve Drosophila species were announced by  and . The species have a well-documented phylogenetic relationship as noted in Figure 1 of , and this figure can be consulted for details such as times of lineage splitting, chromosome composition, etc. The genus Drosophila is split into two major subgenera, Sophophora and Drosophila. Here, "Drosophila" will refer to the genus; the subgenus will be referred to as "subgenus Drosophila". These subgenera split from each other approximately 50 million years ago, and the available genomes are from 9 Sophophora and 3 subgenus Drosophila species. For ease of communication, we will use the following abbreviations to refer to the 12 species: D. melanogaster, Dmel; D. simulans, Dsim; D sechellia, Dsec; D. yakuba, Dyak; D. erecta, Dere; D. ananassae, Dana; D. pseudoobscura, Dpse; D. persimilis, Dper; D. willistoni, Dwil; D. mojavensis, Dmoj; D. virilis, Dvir; and D. grimshawi, Dgri.
Comparative Assembly Freeze 1 (CAF1) of all 12 genomes was downloaded from . The final gene annotations for coding sequences (as of November 16, 2006) were available from . We confined our analysis to the 6,698 protein-coding sequences (CDSs) that at this time have been identified as being homologous across all 12 species as defined in . From these alignments we removed all codons from poorly aligned regions or those with insertion/deletions. This assured that we only compared codons for which there is strong evidence of homology across all 12 species. The D. melanogaster genome sequence was based on the Flybase release 4.3. There are 221 transcripts corresponding to 89 genes identified in the fourth chromosome of D. melanogaster. Thirty three of them were found in the 6,698 homologous CDS set.
Codon usage analysis
Raw counts of the number of times a codon is used for each amino acid is the basic data. Various ways of summarizing codon usage have been proposed and used here.
Relative synonymous codon usage, RSCU
The most straight forward way to measure codon usage bias is simply deviation from even usage. The relative synonymous codon usage (RSCU) statistic is calculated by dividing the observed usage of a codon by that expected if all codons were used equally frequently . Thus an RSCU of 1 indicates a codon is used as expected by random (even) usage, RSCU > 1 indicates a codon used more frequently than expected randomly, and RSCU < 1 indicates a codon used less frequently than random.
Effective number of codons, ENC
Another measure of CUB is "effective number of codons"  which we abbreviate ENC. This is also a measure of the unevenness of use of codons across all amino acids in a protein and is estimated by 2 + 9(1/F2) + (1/F3) + 5(1/F4) + 3(1/F6) where Fi (i = 1,3, 4, 6). The value of F can be interpreted as the average "homozygosity" or probability of two randomly chosen codons for an amino acid being identical for the i-fold degenerate codon groups. If all codons for each amino acid are used equally (completely random usage), ENC will be 61; the other extreme would be if a single codon is used for each amino acid yielding an ENC of 20. Because the magnitude or strength of codon usage bias is negatively correlated with ENC, when correlating level of CUB with ENC, we use negative ENC. In its original formulation , ENC referred to the average "homozygosity" across a whole protein-coding sequence. Moriyama and Powell  modified it to be applicable to each amino acid, X, in a protein, called ENC-X. The maximum possible ENC-X is 2, 3, 4 or 6 depending on the degeneracy of the particular amino acid; thus to normalize codon bias among amino acids, ENC-X is scaled to range from 0 (no bias) to 1 (maximum bias); this is abbreviated sENC-X. The sum of sENC-X across all amino acids is denoted sENC for the protein. Note that sENC is positively correlated with the degree of CUB.
Codon adaptation index, CAI
ENC, in its various formulations, and RSCU are non-directional measures of CUB being simply measures of unevenness. Codon adaptation index, CAI, was devised by Sharp and Li  as a directional measure of codon usage relative to a set of pre-defined reference optimal codons for a species. The identification of the reference set for each species analyzed here was done by examining the genes with the lowest ENC (highest bias) and accumulating at least 100 codons for each amino acid; depending on species, this involved between 12 and 20 most biased genes. Once the reference set is defined, a gene's CAI is simply CAIobs/CAImax where CAIobs is the geometric mean of observed RSCU across all amino acids of a protein and CAImax is the geometric mean of the maximum RSCU for each amino acid in the reference set and is the maximum possible given the particular amino acid composition of the protein being considered. Thus, CAI measures deviation from the optimum codon usage pattern defined for that species, 0 being furthest from the optimal set (no optimal codons used), and 1 indicating only usage of optimal codons.
Intron base composition
Intron sequences were identified from each of the 12 species' genomes based on the gene model GFF3 files available from the AAAwiki site cited above. In order to exclude possible transposable element (TE) sequences embedded in introns, we used the TE annotations based on the BLASTEr/tblastx analysis by Quesneville et al.  (GFF3 files available at ). All 12 genomes were first double masked against their BDGP TE and PILER-DF annotations. Next all introns from the 6,698 homologous gene set were extracted from each genome. Finally 50 bp each at the start and end regions of each intron were excluded to remove possible sites under splicing restrictions. This last step limited our intron analysis for those longer than 200 bp (100 bp after removal). In order to further exclude long introns potentially with embedded CDSs or with misidentified exons, we also examined introns that are longer than 100 bp and shorter than 2000 bp (after removing possible TE sequences and 50 bp from each end). The GC content of each intron was calculated and their unweighted average (disregarding intron length) was obtained from each genome. We also calculated the weighted GC content from the cumulative GC content from all introns concatenated. Further details of the intron data including the GC contents obtained with and without length limitations are available in the Additional File 1 and 2. For correlation analysis of GC contents between introns and CDS of the same gene (Table 2 and Additional File 1 and 2, only the first intron from each gene was used for the convenience.
We thank several reviewers for helpful comments on the work presented here. The work was supported by NIH grant RO1 GM077533.
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