Intragenomic variation in non-adaptive nucleotide biases causes underestimation of selection on synonymous codon usage

Patterns of non-uniform usage of synonymous codons vary across genes in an organism and between species across all domains of life. This codon usage bias (CUB) is due to a combination of non-adaptive (e.g. mutation biases) and adaptive (e.g. natural selection for translation efficiency/accuracy) evolutionary forces. Most models quantify the effects of mutation bias and selection on CUB assuming uniform mutational and other non-adaptive forces across the genome. However, non-adaptive nucleotide biases can vary within a genome due to processes such as biased gene conversion (BGC), potentially obfuscating signals of selection on codon usage. Moreover, genome-wide estimates of non-adaptive nucleotide biases are lacking for non-model organisms. We combine an unsupervised learning method with a population genetics model of synonymous coding sequence evolution to assess the impact of intragenomic variation in non-adaptive nucleotide bias on quantification of natural selection on synonymous codon usage across 49 Saccharomycotina yeasts. We find that in the absence of a priori information, unsupervised learning can be used to identify genes evolving under different non-adaptive nucleotide biases. We find that the impact of intragenomic variation in non-adaptive nucleotide bias varies widely, even among closely-related species. We show that the overall strength and direction of translational selection can be underestimated by failing to account for intragenomic variation in non-adaptive nucleotide biases. Interestingly, genes falling into clusters identified by machine learning are also physically clustered across chromosomes. Our results indicate the need for more nuanced models of sequence evolution that systematically incorporate the effects of variable non-adaptive nucleotide biases on codon frequencies.     

Variable strength of translational selection among 12 Drosophila species

Codon usage bias in Drosophila melanogaster genes has been attributed to negative selection of those codons whose cellular tRNA abundance restricts rates of mRNA translation. Previous studies, which involved limited numbers of genes, can now be compared against analyses of the entire gene complements of 12 Drosophila species whose genome sequences have become available. Using large numbers (6138) of orthologs represented in all 12 species, we establish that the codon preferences of more closely related species are better correlated. Differences between codon usage biases are attributed, in part, to changes in mutational biases. These biases are apparent from the strong correlation (r = 0.92, P < 0.001) among these genomes' intronic G + C contents and exonic G + C contents at degenerate third codon positions. To perform a cross-species comparison of selection on codon usage, while accounting for changes in mutational biases, we calibrated each genome in turn using the codon usage bias indices of highly expressed ribosomal protein genes. The strength of translational selection was predicted to have varied between species largely according to their phylogeny, with the D. melanogaster group species exhibiting the strongest degree of selection.     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.     

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify “adaptive codon preference,” a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated “preference” largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.     

Comparative Genomics of Trypanosomatid Pathogens using Codon Usage Bias

It is well known that an amino acid can be encoded by more than one codon, called synonymous codons. The preferential use of one particular codon for coding an amino acid is referred to as codon usage bias (CUB). A quantitative analytical method, CUB and a related tool, Codon Adaptative Index have been applied to comparatively study whole genomes of a few pathogenic Trypanosomatid species. This quantitative attempt is of direct help in the comparison of qualitative features like mutational and translational selection. Pathogens of the Leishmania and Trypanosoma genus cause debilitating disease and suffering in human beings and animals. Of these, whole genome sequences are available for only five species. The complete coding sequences (CDS), highly expressed, essential and low expressed genes have all been studied for their CUB signature. The codon usage bias of essential genes and highly expressed genes show distribution similar to codon usage bias of all CDSs in Trypanosomatids. Translational selection is the dominant force selecting the preferred codon, and selection due to mutation is negligible. In contrast to an earlier study done on these pathogens, it is found in this work that CUB and CAI may be used to distinguish the Trypanosomatid genomes at the sub-genus level. Further, CUB may effectively be used as a signature of the species differentiation by using Principal Component Analysis (PCA).

Abbreviations

CUB - Codon Usage Bias, CAI - Codon Adaptative Index, CDS - Coding sequences, t-RNA - Transfer RNA, PCA - Principal Component Analysis.     

Comparative genome analysis of six malarial parasites using codon usage bias based tools

Codon usage bias (CUB) is an omnipresent phenomenon, which occurs in nearly all organisms. Previous studies of codon bias in Plasmodium species were based on a limited dataset. This study uses whole genome datasets for comparative genome analysis of six Plasmodium species using CUB and other related methods for the first time. Codon usage bias, compositional variation in translated amino acid frequency, effective number of codons and optimal codons are analyzed for P.falciparum, P.vivax, P.knowlesi, P.berghei, P.chabaudii and P.yoelli. A plot of effective number of codons versus GC3 shows their differential codon usage pattern arises due to a combination of mutational and translational selection pressure. The increased relative usage of adenine and thymine ending optimal codons in highly expressed genes of P.falciparum is the result of higher composition biased pressure, and usage of guanine and cytosine bases at third codon position can be explained by translational selection pressure acting on them. While higher usage of adenine and thymine bases at third codon position in optimal codons of P.vivax highlights the role of translational selection pressure apart from composition biased mutation pressure in shaping their codon usage pattern. The frequency of those amino acids that are encoded by AT ending codons are significantly high in P.falciparum due to action of high composition biased mutational pressure compared with other Plasmodium species. The CUB variation in the three rodent parasites, P.berghei, P.chabaudii and P.yoelli is strikingly similar to that of P.falciparum. The simian and human malarial parasite, P.knowlesi shows a variation in codon usage bias similar to P.vivax but on closer study there are differences confirmed by the method of Principal Component Analysis (PCA).

Abbreviations

CDS - Coding sequences, GC1 - GC composition at first site of codon, GC2 - GC composition at second site of codon, GC3 - GC composition at third site of codon, Ala - Alanine, Arg - Arginine, Asn - Asparagine, Asp - Aspartic acid, Cys - Cysteine, Gln - Glutamine Glu - Glutamic acid Gly - Glycine His - Histidine Ile - Isoleucine Leu - Leucine Lys - Lysine Met - Methionine Phe - Phenylalanine Pro - Proline Ser - Serine Thr - Threonine Trp - Tryptophan Tyr - Tyrosine Val - Valine.     

Factors influencing synonymous codon and amino acid usage biases in Mimivirus

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.     

Studies on codon usage in Entamoeba histolytica

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.     

Comparative analysis of codon usage patterns in chloroplast genomes of the Asteraceae family

Codon usage bias (CUB) is an important evolutionary feature in a genome and has been widely documented from prokaryotes to eukaryotes. However, the significance of CUB in the Asteraceae family has not been well understood, with no Asteraceae species having been analyzed for this characteristic. Here, we use bioinformatics approaches to comparatively analyze the general patterns and influencing factors of CUB in five Asteraceae chloroplast (cp) genomes. The results indicated that the five genomes had similar codon usage patterns, showing a strong bias towards a high representation of NNA and NNT codons. Neutrality analysis showed that these cp genomes had a narrow GC distribution and no significant correlation was observed between GC₁₂ and GC₃. Parity Rule 2 (PR2) plot analysis revealed that purines were used more frequently than pyrimidines. Effective number of codons (ENc)-plot analysis showed that most genes followed the parabolic line of trajectory, but several genes with low ENc values lying below the expected curve were also observed. Furthermore, correspondence analysis of relative synonymous codon usage (RSCU) yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggested that both natural selection and mutational bias contributed to codon bias, while selection was the major force to shape the codon usage in these Asteraceae cp genomes. Our study, which is the first to investigate codon usage patterns in Asteraceae plastomes, will provide helpful information about codon distribution and variation in these species, and also shed light on the genetic and evolutionary mechanisms of codon biology within this family.     

Rapid divergence of codon usage patterns within the rice genome

Background

Synonymous codon usage varies widely between genomes, and also between genes within genomes. Although there is now a large body of data on variations in codon usage, it is still not clear if the observed patterns reflect the effects of positive Darwinian selection acting at the level of translational efficiency or whether these patterns are due simply to the effects of mutational bias. In this study, we have included both intra-genomic and inter-genomic comparisons of codon usage. This allows us to distinguish more efficiently between the effects of nucleotide bias and translational selection.

Results

We show that there is an extreme degree of heterogeneity in codon usage patterns within the rice genome, and that this heterogeneity is highly correlated with differences in nucleotide content (particularly GC content) between the genes. In contrast to the situation observed within the rice genome, Arabidopsis genes show relatively little variation in both codon usage and nucleotide content. By exploiting a combination of intra-genomic and inter-genomic comparisons, we provide evidence that the differences in codon usage among the rice genes reflect a relatively rapid evolutionary increase in the GC content of some rice genes. We also noted that the degree of codon bias was negatively correlated with gene length.

Conclusion

Our results show that mutational bias can cause a dramatic evolutionary divergence in codon usage patterns within a period of approximately two hundred million years.The heterogeneity of codon usage patterns within the rice genome can be explained by a balance between genome-wide mutational biases and negative selection against these biased mutations. The strength of the negative selection is proportional to the length of the coding sequences. Our results indicate that the large variations in synonymous codon usage are not related to selection acting on the translational efficiency of synonymous codons.

     

Selection intensity on preferred codons correlates with overall codon usage bias in Caenorhabditis remanei

Adaptive codon usage provides evidence of natural selection in one of its most subtle forms: a fitness benefit of one synonymous codon relative to another. Codon usage bias is evident in the coding sequences of a broad array of taxa, reflecting selection for translational efficiency and/or accuracy as well as mutational biases. Here, we quantify the magnitude of selection acting on alternative codons in genes of the nematode Caenorhabditis remanei, an outcrossing relative of the model organism C. elegans, by fitting the expected mutation-selection-drift equilibrium frequency distribution of preferred and unpreferred codon variants to the empirical distribution. This method estimates the intensity of selection on synonymous codons in genes with high codon bias as N(e)s = 0.17, a value significantly greater than zero. In addition, we demonstrate for the first time that estimates of ongoing selection on codon usage among genes, inferred from nucleotide polymorphism data, correlate strongly with long-term patterns of codon usage bias, as measured by the frequency of optimal codons in a gene. From the pattern of polymorphisms in introns, we also infer that these findings do not result from the operation of biased gene conversion toward G or C nucleotides. We therefore conclude that coincident patterns of current and ancient selection are responsible for shaping biased codon usage in the C. remanei genome.     

Genome-wide codon usage profiling of ocular infective Chlamydia trachomatis serovars and drug target identification

Chlamydia trachomatis (C.t) is a Gram-negative obligate intracellular bacteria and is a major causative of infectious blindness and sexually transmitted diseases. Among the varied serovars of this organism, A, B and C are reported as prominent ocular pathogens. Genomic studies of these strains shall aid in deciphering potential drug targets and genomic influence on pathogenesis. Hence, in this study we performed deep statistical profiling of codon usage in these serovars. The overall base composition analysis reveals that these serovars are over biased to AU than GC. Similarly, relative synonymous codon usage also showed preference towards A/U ending codons. Parity Rule 2 analysis inferred unequal distribution of AT and GC, indicative of other unknown factors acting along with mutational pressure to influence codon usage bias (CUB). Moreover, absolute quantification of CUB also revealed lower bias across these serovars. The effect of natural selection on CUB was also confirmed by neutrality plot, reinforcing natural selection under mutational pressure turned to be a pivotal role in shaping the CUB in the strains studied. Correspondence analysis (COA) clarified that, C.t C/TW-3 to show a unique trend in codon usage variation. Host influence analysis on shaping the codon usage pattern also inferred some speculative relativity. In a nutshell, our finding suggests that mutational pressure is the dominating factor in shaping CUB in the strains studied, followed by natural selection. We also propose potential drug targets based on cumulative analysis of strand bias, CUB and human non-homologue screening.     

The Selective Advantage of Synonymous Codon Usage Bias in Salmonella

The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10⁻⁴ per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.     

In Arabidopsis thaliana codon volatility scores reflect GC3 composition rather than selective pressure

ABSTRACT: BACKGROUND: Synonymous codon usage bias has typically been correlated with, and attributed to translational efficiency. However, there are other pressures on genomic sequence composition that can affect codon usage patterns such as mutational biases. This study provides an analysis of the codon usage patterns in Arabidopsis thaliana in relation to gene expression levels, codon volatility, mutational biases and selective pressures. RESULTS: We have performed synonymous codon usage and codon volatility analyses for all genes in the A. thaliana genome. In contrast to reports for species from other kingdoms, we find that neither codon usage nor volatility are correlated with selection pressure (as measured by dN/dS), nor with gene expression levels on a genome wide level. Our results show that codon volatility and usage are not synonymous, rather that they are correlated with the abundance of G and C at the third codon position (GC3). CONCLUSIONS: Our results indicate that while the A. thaliana genome shows evidence for synonymous codon usage bias, this is not related to the expression levels of its constituent genes. Neither codon volatility nor codon usage are correlated with expression levels or selective pressures but, because they are directly related to the composition of G and C at the third codon position, they are the result of mutational bias. Therefore, in A. thaliana codon volatility and usage do not result from selection for translation efficiency or protein functional shift as measured by positive selection.     

Compositional correlation and codon usage studies in Buchnera aphidicola

Compositional distributions in three different codon positions as well as codon usage biases of all available DNA sequences of Buchnera aphidicola genome have been analyzed. It was observed that GC levels among the three codon positions is I>II>III as observed in other extremely high AT rich organisms. B. aphidicola being an AT rich organism is expected to have A and/or T at the third positions of codons. Overall codon usage analyses indicate that A and/or T ending codons are predominant in this organism and some particular amino acids are abundant in the coding region of genes. However, multivariate statistical analysis indicates two major trends in the codon usage variation among the genes; one being strongly correlated with the GC contents at the third synonymous positions of codons, and the other being associated with the expression level of genes. Moreover, codon usage biases of the highly expressed genes are almost identical with the overall codon usage biases of all the genes of this organism. These observations suggest that mutational bias is the main factor in determining the codon usage variation among the genes in B. aphidicola.     

Evolutionary Lability of Context-Dependent Codon Bias in Bacteria

In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes. Received: 6 May 1999 / Accepted: 29 October 1999     

Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias

Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do `prefer' optimal codons. Nucleic Acids Res. 17, 5029–5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525–2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H. influenzae and M. tuberculosis are similar to those of E. coli, implying that though the composition of all three codon positions is governed by a selection-mutation balance, the mutational pressure has little influence in the choice of bases at the first two codon positions, even in organisms with highly biased base compositions.     

New insights into the interplay between codon bias determinants in plants

Codon bias is the non-random use of synonymous codons, a phenomenon that has been observed in species as diverse as bacteria, plants and mammals. The preferential use of particular synonymous codons may reflect neutral mechanisms (e.g. mutational bias, G|C-biased gene conversion, genetic drift) and/or selection for mRNA stability, translational efficiency and accuracy. The extent to which these different factors influence codon usage is unknown, so we dissected the contribution of mutational bias and selection towards codon bias in genes from 15 eudicots, 4 monocots and 2 mosses. We analysed the frequency of mononucleotides, dinucleotides and trinucleotides and investigated whether the compositional genomic background could account for the observed codon usage profiles. Neutral forces such as mutational pressure and G|C-biased gene conversion appeared to underlie most of the observed codon bias, although there was also evidence for the selection of optimal translational efficiency and mRNA folding. Our data confirmed the compositional differences between monocots and dicots, with the former featuring in general a lower background compositional bias but a higher overall codon bias.     

Codon usage in the siliceous sponge Geodia cydonium: highly expressed genes in the simplest multicellular animals prefer C- and G-ending codons

Among a sample of 39 Geodia cydonium (Demospongiae, Porifera) genes, with an average G + C content of 51.2%, extensive structural heterogeneity and considerable variations in synonymous codon usage were found. The G + C content of coding sequences and G + C content at silent codon positions (GC3S) varied from 42.4 to 59.2% and from 35.6 to 76.5%, respectively. Correspondence analysis of 39 genes revealed that putative highly expressed genes preferentially use a limited subset of codons, which were therefore defined as preferred codons in G. cydonium . A total of 22 preferred codons for 18 amino acids with synonyms in codons were identified and they all (with one exception) end with C or G. Among these codons there are also C- and G-ending codons which were previously identified as codons optimal for translation in a variety of eukaryotes, including metazoans and plants. The bias in synonymous codon usage in putative highly expressed G. cydonium genes is moderate, indicating that these genes are not shaped under strong natural selection. We postulate that the preference for C- and G-ending codons was already established in the ancestor of all Metazoa, including also sponges. This ancestor most probably also had a G + C rich genome. The selection toward C- and G-ending codons has been largely conserved throughout eukaryote evolution; exceptions are, for example, mammals for which strong mutational biases caused switches from that rule.     

Selected codon usage bias in members of the class Mollicutes

Mollicutes are parasitic microorganisms mainly characterized by small cell sizes, reduced genomes and great A and T mutational bias. We analyzed the codon usage patterns of the completely sequenced genomes of bacteria that belong to this class. We found that for many organisms not only mutational bias but also selection has a major effect on codon usage. Through a comparative perspective and based on three widely used criteria we were able to classify Mollicutes according to the effect of selection on codon usage. We found conserved optimal codons in many species and study the tRNA gene pool in each genome. Previous results are reinforced by the fact that, when selection is operative, the putative optimal codons found match the respective cognate tRNA. Finally, we trace selection effect backwards to the common ancestor of the class and estimate the phylogenetic inertia associated with this character. We discuss the possible scenarios that explain the observed evolutionary patterns.