Rates of nucleotide substitution and mammalian nuclear gene evolution. Approximate and maximum-likelihood methods lead to different conclusions

Rates and patterns of synonymous and nonsynonymous substitutions have important implications for the origin and maintenance of mammalian isochores and the effectiveness of selection at synonymous sites. Previous studies of mammalian nuclear genes largely employed approximate methods to estimate rates of nonsynonymous and synonymous substitutions. Because these methods did not account for major features of DNA sequence evolution such as transition/transversion rate bias and unequal codon usage, they might not have produced reliable results. To evaluate the impact of the estimation method, we analyzed a sample of 82 nuclear genes from the mammalian orders Artiodactyla, Primates, and Rodentia using both approximate and maximum-likelihood methods. Maximum-likelihood analysis indicated that synonymous substitution rates were positively correlated with GC content at the third codon positions, but independent of nonsynonymous substitution rates. Approximate methods, however, indicated that synonymous substitution rates were independent of GC content at the third codon positions, but were positively correlated with nonsynonymous rates. Failure to properly account for transition/transversion rate bias and unequal codon usage appears to have caused substantial biases in approximate estimates of substitution rates.     

Molecular considerations in the evolution of bacterial genes

Summary Synonymous and nonsynonymous substitution rates at the loci encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and outer membrane protein 3A (ompA) were examined in 12 species of enteric bacteria. By examining homologous sequences in species of varying degrees of relatedness and of known phylogenetic relationships, we analyzed the patterns of synonymous and nonsynonymous substitutions within and among these genes. Although both loci accumulate synonymous substitutions at reduced rates due to codon usage bias, portions of thegap andompA reading frames show significant deviation in synonymous substitution rates not attributable to local codon bias. A paucity of synonymous substitutions in portions of theompA gene may reflect selection for a novel mRNA secondary structure. In addition, these studies allow comparisons of homologous protein-coding sequences (gap) in plants, animals, and bacteria, revealing differences in evolutionary constraints on this glycolytic enzyme in these lineages.     

Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory

The nearly neutral theory of molecular evolution predicts larger generation-time effects for synonymous than for nonsynonymous substitutions. This prediction is tested using the sequences of 49 single-copy genes by calculating the average and variance of synonymous and nonsynonymous substitutions in mammalian star phylogenies (rodentia, artiodactyla, and primates). The average pattern of the 49 genes supports the prediction of the nearly neutral theory, with some notable exceptions.The nearly neutral theory also predicts that the variance of the evolutionary rate is larger than the value predicted by the completely neutral theory. This prediction is tested by examining the dispersion index (ratio of the variance to the mean), which is positively correlated with the average substitution number. After weighting by the lineage effects, this correlation almost disappears for nonsynonymous substitutions, but not quite so for synonymous substitutions. After weighting, the dispersion indices of both synonymous and nonsynonymous substitutions still exceed values expected under the simple Poisson process. The results indicate that both the systematic bias in evolutionary rate among the lineages and the episodic type of rate variation are contributing to the large variance. The former is more significant to synonymous substitutions than to nonsynonymous substitutions. Isochore evolution may be similar to synonymous substitutions. The rate and pattern found here are consistent with the nearly neutral theory, such that the relative contributions of drift and selection differ between the two types of substitutions. The results are also consistent with Gillespie's episodic selection theory.     

Substitution rates in Drosophila nuclear genes: implications for translational selection

The relationships between synonymous and nonsynonymous substitution rates and between synonymous rate and codon usage bias are important to our understanding of the roles of mutation and selection in the evolution of Drosophila genes. Previous studies used approximate estimation methods that ignore codon bias. In this study we reexamine those relationships using maximum-likelihood methods to estimate substitution rates, which accommodate the transition/transversion rate bias and codon usage bias. We compiled a sample of homologous DNA sequences at 83 nuclear loci from Drosophila melanogaster and at least one other species of Drosophila. Our analysis was consistent with previous studies in finding that synonymous rates were positively correlated with nonsynonymous rates. Our analysis differed from previous studies, however, in that synonymous rates were unrelated to codon bias. We therefore conducted a simulation study to investigate the differences between approaches. The results suggested that failure to properly account for multiple substitutions at the same site and for biased codon usage by approximate methods can lead to an artifactual correlation between synonymous rate and codon bias. Implications of the results for translational selection are discussed.     

Estimating absolute rates of synonymous and nonsynonymous nucleotide substitution in order to characterize natural selection and date species divergences

The rate of molecular evolution can vary among lineages. Sources of this variation have differential effects on synonymous and nonsynonymous substitution rates. Changes in effective population size or patterns of natural selection will mainly alter nonsynonymous substitution rates. Changes in generation length or mutation rates are likely to have an impact on both synonymous and nonsynonymous substitution rates. By comparing changes in synonymous and nonsynonymous rates, the relative contributions of the driving forces of evolution can be better characterized. Here, we introduce a procedure for estimating the chronological rates of synonymous and nonsynonymous substitutions on the branches of an evolutionary tree. Because the widely used ratio of nonsynonymous and synonymous rates is not designed to detect simultaneous increases or simultaneous decreases in synonymous and nonsynonymous rates, the estimation of these rates rather than their ratio can improve characterization of the evolutionary process. With our Bayesian approach, we analyze cytochrome oxidase subunit I evolution in primates and infer that nonsynonymous rates have a greater tendency to change over time than do synonymous rates. Our analysis of these data also suggests that rates have been positively correlated.     

Synonymous and nonsynonymous rate variation in nuclear genes of mammals

A maximum likelihood approach was used to estimate the synonymous and nonsynonymous substitution rates in 48 nuclear genes from primates, artiodactyls, and rodents. A codon-substitution model was assumed, which accounts for the genetic code structure, transition/transversion bias, and base frequency biases at codon positions. Likelihood ratio tests were applied to test the constancy of nonsynonymous to synonymous rate ratios among branches (evolutionary lineages). It is found that at 22 of the 48 nuclear loci examined, the nonsynonymous/synonymous rate ratio varies significantly across branches of the tree. The result provides strong evidence against a strictly neutral model of molecular evolution. Our likelihood estimates of synonymous and nonsynonymous rates differ considerably from previous results obtained from approximate pairwise sequence comparisons. The differences between the methods are explored by detailed analyses of data from several genes. Transition/transversion rate bias and codon frequency biases are found to have significant effects on the estimation of synonymous and nonsynonymous rates, and approximate methods do not adequately account for those factors. The likelihood approach is preferable, even for pairwise sequence comparison, because more-realistic models about the mutation and substitution processes can be incorporated in the analysis. Received: 17 May 1997 / Accepted: 28 September 1997     

Molecular evolution of the ent-kaurenoic acid oxidase gene in Oryzeae

We surveyed the substitution patterns in the ent-kaurenoic acid oxidase (KAO) gene in 11 species of Oryzeae with an outgroup in the Ehrhartoidaea. The synonymous and non-synonymous substitution rates showed a high positive correlation with each other, but were negatively correlated with codon usage bias and GC content at third codon positions. The substitution rate was heterogenous among lineages. Likelihood-ratio tests showed that the non-synonymous/synonymous rate ratio changed significantly among lineages. Site-specific models provided no evidence for positive selection of particular amino acid sites in any codon of the KAO gene. This finding suggested that the significant rate heterogeneity among some lineages may have been caused by variability in the relaxation of the selective constraint among lineages or by neutral processes.     

Patterns of nucleotide substitution among simultaneously duplicated gene pairs in Arabidopsis thaliana

We characterized rates and patterns of synonymous and nonsynonymous substitution in 242 duplicated gene pairs on chromosomes 2 and 4 of Arabidopsis thaliana. Based on their collinear order along the two chromosomes, the gene pairs were likely duplicated contemporaneously, and therefore comparison of genetic distances among gene pairs provides insights into the distribution of nucleotide substitution rates among plant nuclear genes. Rates of synonymous substitution varied 13.8-fold among the duplicated gene pairs, but 90% of gene pairs differed by less than 2.6-fold. Average nonsynonymous rates were approximately fivefold lower than average synonymous rates; this rate difference is lower than that of previously studied nonplant lineages. The coefficient of variation of rates among genes was 0.65 for nonsynonymous rates and 0.44 for synonymous rates, indicating that synonymous and nonsynonymous rates vary among genes to roughly the same extent. The causes underlying rate variation were explored. Our analyses tentatively suggest an effect of physical location on synonymous substitution rates but no similar effect on nonsynonymous rates. Nonsynonymous substitution rates were negatively correlated with GC content at synonymous third codon positions, and synonymous substitution rates were negatively correlated with codon bias, as observed in other systems. Finally, the 242 gene pairs permitted investigation of the processes underlying divergence between paralogs. We found no evidence of positive selection, little evidence that paralogs evolve at different rates, and no evidence of differential codon usage or third position GC content.     

Correlated rates of synonymous site evolution across plant genomes

Synonymous substitution rates have been shown to vary among evolutionary lineages of both nuclear and organellar genes across a broad range of taxonomic groups. In animals, rate heterogeneity does not appear to be correlated across nuclear and mitochondrial genes. In this paper, we contrast substitution rates in two plant groups and show that grasses evolve more rapidly than palms at synonymous sites in a mitochondrial, a nuclear, and a plastid gene. Furthermore, we show that the relative rates of synonymous substitution between grasses and palms are similar at the three loci. The correlation in synonymous substitution rates across genes is particularly striking because the three genes evolve at very different absolute rates. In contrast, relative rates of nonsynonymous substitution are not conserved among the three genes.      

Synonymous Substitutions in the Xdh Gene of Drosophila: Heterogeneous Distribution along the Coding Region

The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene.     

Molecular Evolution and Nucleotide Sequences of the Maize Plastid Genes for the α Subunit of Cf1 (atpA) and the Proteolipid Subunit of Cf0 (atpH)

The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.     

Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution

An excess of nonsynonymous substitutions over synonymous ones is an important indicator of positive selection at the molecular level. A lineage that underwent Darwinian selection may have a nonsynonymous/synonymous rate ratio (dN/dS) that is different from those of other lineages or greater than one. In this paper, several codon-based likelihood models that allow for variable dN/dS ratios among lineages were developed. They were then used to construct likelihood ratio tests to examine whether the dN/dS ratio is variable among evolutionary lineages, whether the ratio for a few lineages of interest is different from the background ratio for other lineages in the phylogeny, and whether the dN/dS ratio for the lineages of interest is greater than one. The tests were applied to the lysozyme genes of 24 primate species. The dN/dS ratios were found to differ significantly among lineages, indicating that the evolution of primate lysozymes is episodic, which is incompatible with the neutral theory. Maximum- likelihood estimates of parameters suggested that about nine nonsynonymous and zero synonymous nucleotide substitutions occurred in the lineage leading to hominoids, and the dN/dS ratio for that lineage is significantly greater than one. The corresponding estimates for the lineage ancestral to colobine monkeys were nine and one, and the dN/dS ratio for the lineage is not significantly greater than one, although it is significantly higher than the background ratio. The likelihood analysis thus confirmed most, but not all, conclusions Messier and Stewart reached using reconstructed ancestral sequences to estimate synonymous and nonsynonymous rates for different lineages.      

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae)

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.      

Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites

Maximum-likelihood models of codon substitution were used to analyze sperm lysin genes of 25 abalone (HALIOTIS:) species to identify lineages and amino acid sites under diversifying selection. The models used the nonsynonymous/synonymous rate ratio (omega = d(N)/d(S)) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio tests suggested significant variation in selective pressure among lineages. The variable selective pressure provided an explanation for the previous observation that the omega ratio is >1 in comparisons of closely related species and <1 in comparisons of distantly related species. Computer simulations demonstrated that saturation of nonsynonymous substitutions and constraint on lysin structure were unlikely to account for the observed pattern. Lineages linking closely related sympatric species appeared to be under diversifying selection, while lineages separating distantly related species from different geographic locations were associated with low evolutionary rates. The selective pressure indicated by the omega ratio was found to vary greatly among amino acid sites in lysin. Sites under potential diversifying selection were identified. Ancestral lysins were inferred to trace the route of evolution at individual sites and to provide lysin sequences for future laboratory studies.     

Codon Substitution in Evolution and the "Saturation" of Synonymous Changes

A mathematical model for codon substitution is presented, taking into account unequal mutation rates among different nucleotides and purifying selection. This model is constructed by using a 61 X 61 transition probability matrix for the 61 nonterminating codons. Under this model, a computer simulation is conducted to study the numbers of silent (synonymous) and amino acid-altering (nonsynonymous) nucleotide substitutions when the underlying mutation rates among the four kinds of nucleotides are not equal. It is assumed that the substitution rates are constant over evolutionary time, the codon frequencies being in equilibrium, and, thus, the numbers of synonymous and nonsynonymous substitutions both increase linearly with evolutionary time. It is shown that, when the mutation rates are not equal, the estimate of synonymous substitutions obtained by F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner and J. Dodgson's "Percent Corrected Divergence" method increases nonlinearly, although the true number of synonymous substitutions increases linearly. It is, therefore, possible that the "saturation" of synonymous substitutions observed by Perler et al. is due to the inefficiency of their method to detect all synonymous substitutions.     

Protein Evolutionary Rates Correlate with Expression Independently of Synonymous Substitutions in Helicobacter pylori

In free-living microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, both synonymous and nonsynonymous substitution frequencies correlate with expression levels. Here, we have tested the hypothesis that the correlation between amino acid substitution rates and expression is a by-product of selection for codon bias and translational efficiency in highly expressed genes. To this end, we have examined the correlation between protein evolutionary rates and expression in the human gastric pathogen Helicobacter pylori, where the absence of selection on synonymous sites enables the two types of substitutions to be uncoupled. The results revealed a statistically significant negative correlation between expression levels and nonsynonymous substitutions in both H. pylori and E. coli. We also found that neighboring genes located on the same, but not on opposite strands, evolve at significantly more similar rates than random gene pairs, as expected by co-expression of genes located in the same operon. However, the two species differ in that synonymous substitutions show a strand-specific pattern in E. coli, whereas the weak similarity in synonymous substitutions for neighbors in H. pylori is independent of gene orientation. These results suggest a direct influence of expression levels on nonsynonymous substitution frequencies independent of codon bias and selective constraints on synonymous sites. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Rates of nucleotide substitution in Cornaceae (Cornales)-Pattern of variation and underlying causal factors

Identifying causes of genetic divergence is a central goal in evolutionary biology. Although rates of nucleotide substitution vary among taxa and among genes, the causes of this variation tend to be poorly understood. In the present study, we examined the rate and pattern of molecular evolution for five DNA regions over a phylogeny of Cornus, the single genus of Cornaceae. To identify evolutionary mechanisms underlying the molecular variation, we employed Bayesian methods to estimate divergence times and to infer how absolute rates of synonymous and nonsynonymous substitutions and their ratios change over time. We found that the rates vary among genes, lineages, and through time, and differences in mutation rates, selection type and intensity, and possibly genetic drift all contributed to the variation of substitution rates observed among the major lineages of Cornus. We applied independent contrast analysis to explore whether speciation rates are linked to rates of molecular evolution. The results showed no relationships for individual genes, but suggested a possible localized link between species richness and rate of nonsynonymous nucleotide substitution for the combined cpDNA regions. Furthermore, we detected a positive correlation between rates of molecular evolution and morphological change in Cornus. This was particularly pronounced in the dwarf dogwood lineage, in which genome-wide acceleration in both molecular and morphological evolution has likely occurred.     

Rates of Conservative and Radical Nonsynonymous Nucleotide Substitutions in Mammalian Nuclear Genes

To understand the process and mechanism of protein evolution, it is important to know what types of amino acid substitutions are more likely to be under selection and what types are mostly neutral. An amino acid substitution can be classified as either conservative or radical, depending on whether it involves a change in a certain physicochemical property of the amino acid. Assuming Kimura's two-parameter model of nucleotide substitution, I present a method for computing the numbers of conservative and radical nonsynonymous (amino acid altering) nucleotide substitutions per site and estimate these rates for 47 nuclear genes from mammals. The results are as follows. (1) The average radical/conservative rate ratio is 0.81 for charge changes, 0.85 for polarity changes, and 0.49 when both polarity and volume changes are considered. (2) The radical/conservative rate ratio is positively correlated with the nonsynonymous/synonymous rate ratio for charge changes or when both polarity and volume changes are considered. (3) Both the conservative/synonymous rate ratio and the radical/synonymous rate ratio are lower in the rodent lineage than in the primate or artiodactyl lineage, suggesting more intense purifying selection in the rodent lineage, for both conservative and radical nonsynonymous substitutions. (4) Neglecting transition/transversion bias would cause an underestimation of both radical and conservative rates and the ratio thereof. (5) Transversions induce more dramatic genetic alternations than transitions in that transversions produce more amino acid altering changes and among which, more radical changes. Received: 6 April 1999 / Accepted: 16 August 1999     

Substitution rates in hepatitis delta virus

Substitution rates were estimated for the coding and noncoding regions of the hepatitis delta virus (HDV). The estimated rates of synonymous substitution in HDV were lower than the rates of substitution at nonsynonymous sites and in the noncoding region. HDV has lower synonymous substitution rates than the hepatitis C virus, though both are RNA viruses. The relatively low rate of synonymous substitution in HDV may be due to a strong preference of G and C nucleotides at third codon positions. Variation in substitution rate among HDV lineages may be correlated with the clinical development of the HDV-induced hepatitis. The phylogenetic tree inferred for 24 HDV strains reveals similarities between lineages isolated from the same geographic region. Correspondence to: W.-H. Li     

The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias

Genes sequences from Escherichia coli, Salmonella typhimurium, and other members of the Enterobacteriaceae show a negative correlation between the degree of synonymous-codon usage bias and the rate of nucleotide substitution at synonymous sites. In particular, very highly expressed genes have very biased codon usage and accumulate synonymous substitutions very slowly. In contrast, there is little correlation between the degree of codon bias and the rate of protein evolution. It is concluded that both the rate of synonymous substitution and the degree of codon usage bias largely reflect the intensity of selection at the translational level. Because of the high variability among genes in rates of synonymous substitution, separate molecular clocks of synonymous substitution might be required for different genes.