Widespread positive selection in synonymous sites of mammalian genes

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.     

Site-to-site variation of synonymous substitution rates

We develop a new model for studying the molecular evolution of protein-coding DNA sequences. In contrast to existing models, we incorporate the potential for site-to-site heterogeneity of both synonymous and nonsynonymous substitution rates. We demonstrate that within-gene heterogeneity of synonymous substitution rates appears to be common. Using the new family of models, we investigate the utility of a variety of new statistical inference procedures, and we pay particular attention to issues surrounding the detection of sites undergoing positive selection. We discuss how failure to model synonymous rate variation in the model can lead to misidentification of sites as positively selected.     

The effect of tandem substitutions on the correlation between synonymous and nonsynonymous rates in rodents.

Nonsynonymous substitutions in DNA cause amino acid substitutions while synonymous substitutions in DNA leave amino acids unchanged. The cause of the correlation between the substitution rates at nonsynonymous (K(A)) and synonymous (K(S)) sites in mammals is a contentious issue, and one that impacts on many aspects of molecular evolution. Here we use a large set of orthologous mammalian genes to investigate the causes of the K(A)-K(S) correlation in rodents. The strength of the K(A)-K(S) correlation exceeds the neutral theory expectation when substitution rates are estimated using algorithmic methods, but not when substitution rates are estimated by maximum likelihood. Irrespective of this methodological uncertainty the strength of the K(A)-K(S) correlation appears mostly due to tandem substitutions, an excess of which is generated by substitutional nonindependence. Doublet mutations cannot explain the excess of tandem synonymous-nonsynonymous substitutions, and substitution patterns indicate that selection on silent sites is the likely cause. We find no evidence for selection on codon usage. The nature of the relationship between synonymous divergence and base composition is unclear because we find a significant correlation if we use maximum-likelihood methods but not if we use algorithmic methods. Finally, we find that K(S) is reduced at the start of genes, which suggests that selection for RNA structure may affect silent sites in mammalian protein-coding genes.     

A method for detecting positive selection at single amino acid sites

A method was developed for detecting the selective force at single amino acid sites given a multiple alignment of protein-coding sequences. The phylogenetic tree was reconstructed using the number of synonymous substitutions. Then, the neutrality was tested for each codon site using the numbers of synonymous and nonsynonymous changes throughout the phylogenetic tree. Computer simulation showed that this method accurately estimated the numbers of synonymous and nonsynonymous substitutions per site, as long as the substitution number on each branch was relatively small. The false-positive rate for detecting the selective force was generally low. On the other hand, the true-positive rate for detecting the selective force depended on the parameter values. Within the range of parameter values used in the simulation, the true-positive rate increased as the strength of the selective force and the total branch length (namely the total number of synonymous substitutions per site) in the phylogenetic tree increased. In particular, with the relative rate of nonsynonymous substitutions to synonymous substitutions being 5.0, most of the positively selected codon sites were correctly detected when the total branch length in the phylogenetic tree was > or = 2.5. When this method was applied to the human leukocyte antigen (HLA) gene, which included antigen recognition sites (ARSs), positive selection was detected mainly on ARSs. This finding confirmed the effectiveness of the present method with actual data. Moreover, two amino acid sites were newly identified as positively selected in non-ARSs. The three-dimensional structure of the HLA molecule indicated that these sites might be involved in antigen recognition. Positively selected amino acid sites were also identified in the envelope protein of human immunodeficiency virus and the influenza virus hemagglutinin protein. This method may be helpful for predicting functions of amino acid sites in proteins, especially in the present situation, in which sequence data are accumulating at an enormous speed.     

Positive selection drives rapid evolution of certain amino acid residues in an evolutionarily highly conserved interferon-inducible antiviral protein of fishes

Viperin, an evolutionarily highly conserved interferon-inducible multifunctional protein, has previously been reported to exhibit antiviral activity against a wide range of DNA and RNA viruses. Utilizing the complete nucleotide coding sequence data of fish viperin antiviral genes, and employing the maximum likelihood-based codon substitution models, the present study reports the pervasive role of positive selection in the evolution of viperin antiviral protein in fishes. The overall rate of nonsynonymous (dN) to synonymous (dS) substitutions (dN/dS) for the three functional domains of viperin (N-terminal, central domain and C-terminal) were 1.1, 0.12, and 0.24, respectively. Codon-by-codon substitution analyses have revealed that while most of the positively selected sites were located at the N-terminal amphipathic α-helix domain, few amino acid residues at the C-terminal domain were under positive selection. However, none of the sites in the central domain were under positive selection. These results indicate that, although viperin is evolutionarily highly conserved, the three functional domains experienced differential selection pressures. Taken together with the results of previous studies, the present study suggests that the persistent antagonistic nature of surrounding infectious viral pathogens might be the likely cause for such adaptive evolutionary changes of certain amino acids in fish viperin antiviral protein.     

Rapidly evolving mitochondrial genome and directional selection in mitochondrial genes in the parasitic wasp nasonia (hymenoptera: pteromalidae)

We sequenced the nearly complete mtDNA of 3 species of parasitic wasps, Nasonia vitripennis (2 strains), Nasonia giraulti, and Nasonia longicornis, including all 13 protein-coding genes and the 2 rRNAs, and found unusual patterns of mitochondrial evolution. The Nasonia mtDNA has a unique gene order compared with other insect mtDNAs due to multiple rearrangements. The mtDNAs of these wasps also show nucleotide substitution rates over 30 times faster than nuclear protein-coding genes, indicating among the highest substitution rates found in animal mitochondria (normally <10 times faster). A McDonald and Kreitman test shows that the between-species frequency of fixed replacement sites relative to silent sites is significantly higher compared with within-species polymorphisms in 2 mitochondrial genes of Nasonia, atp6 and atp8, indicating directional selection. Consistent with this interpretation, the Ka/Ks (nonsynonymous/synonymous substitution rates) ratios are higher between species than within species. In contrast, cox1 shows a signature of purifying selection for amino acid sequence conservation, although rates of amino acid substitutions are still higher than for comparable insects. The mitochondrial-encoded polypeptides atp6 and atp8 both occur in F0F1ATP synthase of the electron transport chain. Because malfunction in this fundamental protein severely affects fitness, we suggest that the accelerated accumulation of replacements is due to beneficial mutations necessary to compensate mild-deleterious mutations fixed by random genetic drift or Wolbachia sweeps in the fast evolving mitochondria of Nasonia. We further propose that relatively high rates of amino acid substitution in some mitochondrial genes can be driven by a "Compensation-Draft Feedback"; increased fixation of mildly deleterious mutations results in selection for compensatory mutations, which lead to fixation of additional deleterious mutations in nonrecombining mitochondrial genomes, thus accelerating the process of amino acid substitutions.     

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.     

Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers

Silent sites in mammals have classically been assumed to be free from selective pressures. Consequently, the synonymous substitution rate (Ks) is often used as a proxy for the mutation rate. Although accumulating evidence demonstrates that the assumption is not valid, the mechanism by which selection acts remain unclear. Recent work has revealed that the presence of exonic splicing enhancers (ESEs) in coding sequence might influence synonymous evolution. ESEs are predominantly located near intron-exon junctions, which may explain the reduced single-nucleotide polymorphism (SNP) density in these regions. Here we show that synonymous sites in putative ESEs evolve more slowly than the remaining exonic sequence. Differential mutabilities of ESEs do not appear to explain this difference. We observe that substitution frequency at fourfold synonymous sites decreases as one approaches the ends of exons, consistent with the existing SNP data. This gradient is at least in part explained by ESEs being more abundant near junctions. Between-gene variation in Ks is hence partly explained by the proportion of the gene that acts as an ESE. Given the relative abundance of ESEs and the reduced rates of synonymous divergence within them, we estimate that constraints on synonymous evolution within ESEs causes the true mutation rate to be underestimated by not more than approximately 8%. We also find that Ks outside of ESEs is much lower in alternatively spliced exons than in constitutive exons, implying that other causes of selection on synonymous mutations exist. Additionally, selection on ESEs appears to affect nonsynonymous sites and may explain why amino acid usage near intron-exon junctions is nonrandom.     

How to calculate the non-synonymous to synonymous rate ratio of protein-coding genes under the Fisher–Wright mutation–selection framework

First principles of population genetics are used to obtain formulae relating the non-synonymous to synonymous substitution rate ratio to the selection coefficients acting at codon sites in protein-coding genes. Two theoretical cases are discussed and two examples from real data (a chloroplast gene and a virus polymerase) are given. The formulae give much insight into the dynamics of non-synonymous substitutions and may inform the development of methods to detect adaptive evolution.     

Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene.

Several codon-based models for the evolution of protein-coding DNA sequences are developed that account for varying selection intensity among amino acid sites. The "neutral model" assumes two categories of sites at which amino acid replacements are either neutral or deleterious. The "positive-selection model" assumes an additional category of positively selected sites at which nonsynonymous substitutions occur at a higher rate than synonymous ones. This model is also used to identify target sites for positive selection. The models are applied to a data set of the V3 region of the HIV-1 envelope gene, sequenced at different years after the infection of one patient. The results provide strong support for variable selection intensity among amino acid sites The neutral model is rejected in favor of the positive-selection model, indicating the operation of positive selection in the region. Positively selected sites are found in both the V3 region and the flanking regions.     

Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila

The gene for a male ejaculatory protein, Acp26Aa, in four sibling species of the Drosophila melanogaster subgroup has previously been shown to have a nonsynonymous rate (Ka) of nucleotide substitution that is indistinguishable from the synonymous rate (Ks). By examining this gene in two other species of this subgroup, we found that Ka is generally large and can sometimes be more than twice as large as Ks. This suggests that positive selection may be operating at this locus of male reproduction.      

Molecular evolution of nuclear genes in Cupressacea, a group of conifer trees

We surveyed the molecular evolutionary characteristics of 11 nuclear genes from 10 conifer trees belonging to the Taxodioideae, the Cupressoideae, and the Sequoioideae. Comparisons of substitution rates among the lineages indicated that the synonymous substitution rates of the Cupressoideae lineage were higher than those of the Taxodioideae. This result parallels the pattern previously found in plastid genes. Likelihood-ratio tests showed that the nonsynonymous-synonymous rate ratio did not change significantly among lineages. In addition, after adjustments for lineage effects, the dispersion indices of synonymous and nonsynonymous substitutions were considerably reduced, and the latter was close to 1. These results indicated that the acceleration of evolutionary rates in the Cupressoideae lineage occurred in both the nuclear and plastid genomes, and that generally, this lineage effect affected synonymous and nonsynonymous substitutions similarly. We also investigated the relationship of synonymous substitution rates with the nonsynonymous substitution rate, base composition, and codon bias in each lineage. Synonymous substitution rates were positively correlated with nonsynonymous substitution rates and GC content at third codon positions, but synonymous substitution rates were not correlated with codon bias. Finally, we tested the possibility of positive selection at the protein level, using maximum likelihood models, assuming heterogeneous nonsynonymous-synonymous rate ratios among codon (amino acid) sites. Although we did not detect strong evidence of positively selected codon sites, the analysis suggested that significant variation in nonsynonymous-synonymous rate ratio exists among the sites. The most likely sites for action of positive selection were found in the ferredoxin gene, which is an important component of the apparatus for photosynthesis.     

The fitness effects of synonymous mutations in DNA and RNA viruses

Despite being silent with respect to protein sequence, synonymous nucleotide substitutions can be targeted by natural selection directly at the DNA or RNA level. However, there has been no systematic assessment of how frequent this type of selection is. Here, we have constructed 53 single random synonymous substitution mutants of the bacteriophages Qβ and ΦX174 by site-directed mutagenesis and assayed their fitness. Analysis of this mutant collection and of previous studies undertaken with a variety of single-stranded (ss) viruses demonstrates that selection at synonymous sites is stronger in RNA viruses than in DNA viruses. We estimate that this type of selection contributes approximately 18% of the overall mutational fitness effects in ssRNA viruses under our assay conditions and that random synonymous substitutions have a 5% chance of being lethal to the virus, whereas in ssDNA viruses, these figures drop to 1.4% and 0%, respectively. In contrast, the effects of nonsynonymous substitutions appear to be similar in ssRNA and ssDNA viruses.     

Uncorrected nucleotide bias in mtDNA can mimic the effects of positive Darwinian selection

The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.     

蕨类植物rpoC1内含子缺失及其分子进化速率

rpoC1基因编码RNA聚合酶β°亚基蛋白, 在转录过程中与DNA模板结合, 与β亚基形成的β-β°亚基复合体构成RNA合成的催化中心。以rpoC1基因为研究对象, 在贝叶斯因子大于20的条件下, 用HyPhy软件位点模型检测到3个正选择位点和541个负选择位点; 用PAML软件位点模型检测到10个正选择位点, 其中3个位点的后验概率超过99%。此外, 基于最大似然法构建64种蕨类植物的系统发育树, 结合HyPhy软件分析rpoC1基因的转换率、颠换率、转换率/颠换率、同义替换率、非同义替换率以及同义替换率/非同义替换率, 探讨rpoC1基因内含子丢失与分子进化速率的关系。结果表明, rpoC1基因内含子缺失对转换率、颠换率以及非同义替换率有一定影响。     

Codon-substitution models for heterogeneous selection pressure at amino acid sites

Comparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. The nonsynonymous/synonymous rate ratio (omega = d(N)d(S)) is an important indicator of selective pressure at the protein level, with omega = 1 meaning neutral mutations, omega < 1 purifying selection, and omega > 1 diversifying positive selection. Amino acid sites in a protein are expected to be under different selective pressures and have different underlying omega ratios. We develop models that account for heterogeneous omega ratios among amino acid sites and apply them to phylogenetic analyses of protein-coding DNA sequences. These models are useful for testing for adaptive molecular evolution and identifying amino acid sites under diversifying selection. Ten data sets of genes from nuclear, mitochondrial, and viral genomes are analyzed to estimate the distributions of omega among sites. In all data sets analyzed, the selective pressure indicated by the omega ratio is found to be highly heterogeneous among sites. Previously unsuspected Darwinian selection is detected in several genes in which the average omega ratio across sites is <1, but in which some sites are clearly under diversifying selection with omega > 1. Genes undergoing positive selection include the beta-globin gene from vertebrates, mitochondrial protein-coding genes from hominoids, the hemagglutinin (HA) gene from human influenza virus A, and HIV-1 env, vif, and pol genes. Tests for the presence of positively selected sites and their subsequent identification appear quite robust to the specific distributional form assumed for omega and can be achieved using any of several models we implement. However, we encountered difficulties in estimating the precise distribution of omega among sites from real data sets.     

Synonymous substitutions substantially improve evolutionary inference from highly diverged proteins

Codon-and amino acid-substitution models are widely used for the evolutionary analysis of protein-coding DNA sequences. Using codon models, the amounts of both nonsynonymous and synonymous DNA substitutions can be estimated. The ratio of these amounts represents the strength of selective pressure. Using amino acid models, the amount of nonsynonymous substitutions is estimated, but that of synonymous substitutions is ignored. Although amino acid models lose any information regarding synonymous substitutions, they explicitly incorporate the information for amino acid replacement, which is empirically derived from databases. It is often presumed that when the protein-coding sequences are highly divergent, synonymous substitutions might be saturated and the evolutionary analysis may be hampered by synonymous noise. However, there exists no quantitative procedure to verify whether synonymous substitutions can be ignored; therefore, amino acid models have been arbitrarily selected. In this study, we investigate the issue of a statistical comparison between codon-and amino acid-substitution models. For this purpose, we propose a new procedure to transform a 20-dimensional amino acid model to a 61-dimensional codon model. This transformation reveals that amino acid models belong to a subset of the codon models and enables us to test whether synonymous substitutions can be ignored by using the likelihood ratio. Our theoretical results and analyses of real data indicate that synonymous substitutions are very informative and substantially improve evolutionary inference, even when the sequences are highly divergent. Therefore, we note that amino acid models should be adopted only after carefully investigating and discarding the possibility that synonymous substitutions can reveal important evolutionary information.     

Detecting selection in noncoding regions of nucleotide sequences

We present a maximum-likelihood method for examining the selection pressure and detecting positive selection in noncoding regions using multiple aligned DNA sequences. The rate of substitution in noncoding regions relative to the rate of synonymous substitution in coding regions is modeled by a parameter zeta. When a site in a noncoding region is evolving neutrally zeta = 1, while zeta > 1 indicates the action of positive selection, and zeta < 1 suggests negative selection. Using a combined model for the evolution of noncoding and coding regions, we develop two likelihood-ratio tests for the detection of selection in noncoding regions. Data analysis of both simulated and real viral data is presented. Using the new method we show that positive selection in viruses is acting primarily in protein-coding regions and is rare or absent in noncoding regions.     

Selective processes and adaptive evolution of the cytochrome b gene in salamanders of the genus Salamandrella

Sequence analysis of the cytochrome b gene fragment in the salamanders of the genus Salamandrella, Siberian salamander and Schrenk salamander was performed with the purpose to elucidate the effect of natural selection on the evolution of mitochondrial DNA (mtDNA) in these species. It was demonstrated that despite of notable influence of negative selection (expressed as very low dN/dS values), speciation and intraspecific divergence in salamanders was accompanied by the appearance of radical amino acid substitutions, caused by the influence of positive (directional) selection. To examine the evolutionary pattern of synonymous mtDNA sites, distribution of conservative and non-conservative substitutions was analyzed. The rates of conservative and non-conservative substitutions were nearly equal, pointing to neutrality of mutation process at synonymous mtDNA sites of salamanders. Analysis of conservative and non-conservative synonymous substitution distributions in different parts of phylogenetic trees showed that the differences between the synonymous groups compared were statistically significant only in one phylogenetic group of Siberian salamander (haplogroup C) (P = 0.02). In the group of single substitutions, located at terminal phylogenetic branches of Siberian salamanders from group C, increased rate of conservative substitutions was observed. Based on these findings, it was suggested that selective processes could have an influence on the formation of the synonymous substitution profile in the Siberian salamander mtDNA fragment examined.