Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome

Summary Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 ± 0.11 × 10^–8 per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 × 10^–9 per site per year forCOI and the highest rate is 4.5 × 10^–9 forATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 × 10^–9 per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.     

Synonymous substitution rates in Drosophila: Mitochondrial versus nuclear genes

Synonymous substitution rates in mitochondrial and nuclear genes of Drosophila were compared. To make accurate comparisons, we considered the following: (1) relative synonymous rates, which do not require divergence time estimates, should be used; (2) methods estimating divergence should take into account base composition; (3) only very closely related species should be used to avoid effects of saturation; (4) the heterogeneity of rates should be examined. We modified the methods estimating synonymous substitution numbers to account for base composition bias. By using these methods, we found that mitochondrial genes have 1.7–3.4 times higher synonymous substitution rates than the fastest nuclear genes or 4.5–9.0 times higher rates than the average nuclear genes. The average rate of synonymous transversions was 2.7 (estimated from the melanogaster species subgroup) or 2.9 (estimated from the obscura group) times higher in mitochondrial genes than in nuclear genes. Synonymous transversions in mitochondrial genes occurred at an approximately equivalent rate to those in the fastest nuclear genes. This last result is not consistent with the hypothesis that the difference in turnover rates between mitochondrial and nuclear genomes is the major factor determining higher synonymous substitution rates in mtDNA. We conclude that the difference in synonymous substitution rates is due to a combination of two factors: a higher transitional mutation rate in mtDNA and constraints on nuclear genes due to selection for codon usage. Received: 27 November 1996 / Accepted: 8 May 1997     

Synonymous substitution-rate constants in Escherichia coli and Salmonella typhimurium and their relationship to gene expression and selection pressure

Based on the differences in synonymous codon use between E. coli and S. typhimurium, the synonymous substitution rates can be estimated. In contrast to previous studies on the substitution rates in these two organisms, we use a kinetic model that explicitly takes the selection bias into account. The selection pressure on synonymous codons for a particular amino acid can be calculated from the observed codon bias. This offers a unique opportunity to study systematically the relationship between substitution-rate constants and selection pressure. The results indicate that the codon bias in these organisms is determined by a mutation-selection balance rather than by stabilizing selection. A best fit to the data implies that the mutation rate constant increases about threefold in genes at low expression levels relative to those that are highly expressed.Correspondence to: O.G. Berg     

Contrasting rates of nucleotide substitution in the X-Linked and Y-Linked zinc finger genes

We have sequenced the entire exon (1,180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 by were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.Correspondence to: W.-H. Li     

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.     

Relative rates of nuclear DNA evolution in human and Old World monkey lineages

Rates of substitution were compared between humans and Old World monkeys for sequences in or adjacent to 19 genes. The comparison of 21,299 sites in noncoding regions indicates that the substitution rate is approximately 43% greater in the Old World monkey lineage. However, 83% of the compared sites are in the region of the beta-globin gene family. Outside this region there is no consistent pattern of rate difference between the two lineages. Comparison of the coding regions of 16 genomically dispersed genes, involving 1,592 synonymous sites and 5,275 nonsynonymous sites, showed a faster rate of substitution in the human lineage at the nonsynonymous sites of the prion gene, but otherwise no evidence of rate difference between the two lineages. It is concluded that rate differences between these two lineages may be specific to certain regions of the genome rather than being a general phenomenon. This conclusion needs to be confirmed by comparison of a larger number of genomically dispersed sequences. It is, however, consistent with the results of DNA-DNA hybridization experiments, which show no difference in evolutionary rate between the two lineages.      

Rates of Synonymous Substitution and Base Composition of Nuclear Genes in Drosophila

We compared the rates of synonymous (silent) substitution among various genes in a number of species of Drosophila. First, we found that even for a particular gene, the rate of synonymous substitution varied considerably with Drosophila lineages. Second, we showed a large variation in synonymous substitution rates among nuclear genes in Drosophila. These rates of synonymous substitution were correlated negatively with C content and positively with A content at the third codon positions. Nucleotide sequences were also compared between pseudogenes and their functional homologs. The C content of the pseudogenes was lower than that of the functional genes and the A content of the former was higher than that of the latter. Because the synonymous substitution for functional genes and the nucleotide substitution for pseudogenes are exempted from any selective constraint at the protein level, these observations could be explained by a biased pattern of mutation in the Drosophila nuclear genome. Such a bias in the mutation pattern may affect the molecular clock (local clock) of each nuclear gene of each species. Finally, we obtained the average rates of synonymous substitution for three gene groups in Drosophila; 11.0 x 10(-9), 17.5 x 10(-9) and 27.1 x 10(-9)/site/year.     

Detecting evolutionary rate heterogeneity among mangroves and their close terrestrial relatives

Mangroves form the dominant intertidal ecosystems and differ morphologically and physiologically from their close terrestrial relatives. We investigate the molecular evolutionary pattern of the typical mangrove family, i.e. Rhizophoraceae, and rate heterogeneity for the plastid matK and rbcL genes in different species of the family, as revealed by phylogenetic analyses and relative‐rate tests. Our study documents evolutionary rate heterogeneity in the Rhizophoraceae for the two genes: the mangrove genus Bruguiera has relatively slow substitution rates compared to the terrestrial genus Carallia at both synonymous and non‐synonymous sites in the matK sequences, and the synonymous and non‐synonymous substitution matrices are correlated. However, the rbcL non‐synonymous sites exhibit a high degree of rate heterogeneity among mangroves and related terrestrial groups, and uncoupling of rates with the synonymous sites. Selection is probably an important influence on the rate variation, suggesting further investigation for better understanding of various forces contributing to the rate heterogeneity and molecular adaptation in mangroves.     

Shifts in the evolutionary rate and intensity of purifying selection between two Brassica genomes revealed by analyses of orthologous transposons and relics of a whole genome triplication

Recent sequencing of the Brassica rapa and Brassica oleracea genomes revealed extremely contrasting genomic features such as the abundance and distribution of transposable elements between the two genomes. However, whether and how these structural differentiations may have influenced the evolutionary rates of the two genomes since their split from a common ancestor are unknown. Here, we investigated and compared the rates of nucleotide substitution between two long terminal repeats (LTRs) of individual orthologous LTR‐retrotransposons, the rates of synonymous and non‐synonymous substitution among triplicated genes retained in both genomes from a shared whole genome triplication event, and the rates of genetic recombination estimated/deduced by the comparison of physical and genetic distances along chromosomes and ratios of solo LTRs to intact elements. Overall, LTR sequences and genic sequences showed more rapid nucleotide substitution in B. rapa than in B. oleracea. Synonymous substitution of triplicated genes retained from a shared whole genome triplication was detected at higher rates in B. rapa than in B. oleracea. Interestingly, non‐synonymous substitution was observed at lower rates in the former than in the latter, indicating shifted densities of purifying selection between the two genomes. In addition to evolutionary asymmetry, orthologous genes differentially regulated and/or disrupted by transposable elements between the two genomes were also characterized. Our analyses suggest that local genomic and epigenomic features, such as recombination rates and chromatin dynamics reshaped by independent proliferation of transposable elements and elimination between the two genomes, are perhaps partially the causes and partially the outcomes of the observed inter‐specific asymmetric evolution.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

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.     

Not so different after all: a comparison of methods for detecting amino acid sites under selection

We consider three approaches for estimating the rates of nonsynonymous and synonymous changes at each site in a sequence alignment in order to identify sites under positive or negative selection: (1) a suite of fast likelihood-based "counting methods" that employ either a single most likely ancestral reconstruction, weighting across all possible ancestral reconstructions, or sampling from ancestral reconstructions; (2) a random effects likelihood (REL) approach, which models variation in nonsynonymous and synonymous rates across sites according to a predefined distribution, with the selection pressure at an individual site inferred using an empirical Bayes approach; and (3) a fixed effects likelihood (FEL) method that directly estimates nonsynonymous and synonymous substitution rates at each site. All three methods incorporate flexible models of nucleotide substitution bias and variation in both nonsynonymous and synonymous substitution rates across sites, facilitating the comparison between the methods. We demonstrate that the results obtained using these approaches show broad agreement in levels of Type I and Type II error and in estimates of substitution rates. Counting methods are well suited for large alignments, for which there is high power to detect positive and negative selection, but appear to underestimate the substitution rate. A REL approach, which is more computationally intensive than counting methods, has higher power than counting methods to detect selection in data sets of intermediate size but may suffer from higher rates of false positives for small data sets. A FEL approach appears to capture the pattern of rate variation better than counting methods or random effects models, does not suffer from as many false positives as random effects models for data sets comprising few sequences, and can be efficiently parallelized. Our results suggest that previously reported differences between results obtained by counting methods and random effects models arise due to a combination of the conservative nature of counting-based methods, the failure of current random effects models to allow for variation in synonymous substitution rates, and the naive application of random effects models to extremely sparse data sets. We demonstrate our methods on sequence data from the human immunodeficiency virus type 1 env and pol genes and simulated alignments.     

Reduction of synonymous substitutions in the core protein gene of hepatitis C virus

Molecular evolutionary analyses were carried out to elucidate the phylogenetic relationships, the evolutionary rate, and the divergence times of hepatitis C viruses. Using the nucleotide sequences of the viruses isolated from various locations in the world, we constructed phylogenetic trees. The trees showed that strains isolated from a single location were not necessarily clustered as a group. This suggests that the viruses may be transferred with blood on a worldwide scale. We estimated the evolutionary rates at synonymous and nonsynonymous sites for all genes in the viral genome. We then found that the rate (1.35 × 10^–3 per site per year) at synonymous sites for the C gene was much smaller than those for the other genes (e.g., 6.29 × 10^–3 per site per year for the E gene). This indicates that a special type of functional constraint on synonymous substitutions may exist in the C gene. Because we found an open reading frame (ORF) with the C gene region, the possibility exists that synonymous substitutions for the C gene are constrained by the overlapping ORF whose reading frame is different from that of the C gene. Applying the evolutionary rates to the trees, we also suggest that major groups of hepatitis C viruses diverged from their common ancestor several hundred years ago. Correspondence to: T. Gojobori     

Selective modes determine evolutionary rates,gene compactness and expression patterns in Brassica

It has been well documented that most nuclear protein‐coding genes in organisms can be classified into two categories: positively selected genes (PSGs) and negatively selected genes (NSGs). The characteristics and evolutionary fates of different types of genes, however, have been poorly understood. In this study, the rates of nonsynonymous substitution (K_a) and the rates of synonymous substitution (K_s) were investigated by comparing the orthologs between the two sequenced Brassica species, Brassica rapa and Brassica oleracea, and the evolutionary rates, gene structures, expression patterns, and codon bias were compared between PSGs and NSGs. The resulting data show that PSGs have higher protein evolutionary rates, lower synonymous substitution rates, shorter gene length, fewer exons, higher functional specificity, lower expression level, higher tissue‐specific expression and stronger codon bias than NSGs. Although the quantities and values are different, the relative features of PSGs and NSGs have been largely verified in the model species Arabidopsis. These data suggest that PSGs and NSGs differ not only under selective pressure (K_a/K_s), but also in their evolutionary, structural and functional properties, indicating that selective modes may serve as a determinant factor for measuring evolutionary rates, gene compactness and expression patterns in Brassica.     

Consistent variation in amino-acid substitution rate, despite uniformity of mutation rate: protein evolution in mammals is not neutral

Variation in mutation rate, attributed to differences in both generation time and in metabolic rate, has been invoked under the neutral theory of molecular evolution to account for differences in substitution rate among mammalian lineages. We show that substitution rates at fourfold-degenerate sites and at sites in noncoding regions do not vary between the primate and rodent lineages, implying mutation- rate uniformity. In contrast, the substitution rates at nondegenerate sites vary both within and between lineages. This difference in substitution-rate pattern between the two types of site is incompatible with neutral theory but may result from substitutions occurring by fixation of slightly deleterious mutations. Variation in the rate of protein evolution among mammalian lineages appears to be due more to differences in population fixation rates than to biochemical or physiological differences affecting mutation rates.      

Molecular evolution of the hepatitis B virus genome

The hepatitis B virus (HBV) has a circular DNA genome of about 3,200 base pairs. Economical use of the genome with overlapping reading frames may have led to severe constraints on nucleotide substitutions along the genome and to highly variable rates of substitution among nucleotide sites. Nucleotide sequences from 13 complete HBV genomes were compared to examine such variability of substitution rates among sites and to examine the phylogenetic relationships among the HBV variants. The maximum likelihood method was employed to fit models of DNA sequence evolution that can account for the complexity of the pattern of nucleotide substitution. Comparison of the models suggests that the rates of substitution are different in different genes and codon positions; for example, the third codon position changes at a rate over ten times higher than the second position. Furthermore, substantial variation of substitution rates was detected even after the effects of genes and codon positions were corrected; that is, rates are different at different sites of the same gene or at the same codon position. Such rates after the correction were also found to be positively correlated at adjacent sites, which indicated the existence of conserved and variable domains in the proteins encoded by the viral genome. A multiparameter model validates the earlier finding that the variation in nucleotide conservation is not random around the HBV genome. The test for the existence of a molecular clock suggests that substitution rates are more or less constant among lineages. The phylogenetic relationships among the viral variants were examined. Although the data do not seem to contain sufficient information to resolve the details of the phylogeny, it appears quite certain that the serotypes of the viral variants do not reflect their genetic relatedness. Correspondence to: Z. Yang     

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.     

Population size and molecular evolution on islands

The nearly neutral theory predicts that the rate and pattern of molecular evolution will be influenced by effective population size (Ne), because in small populations more slightly deleterious mutations are expected to drift to fixation. This important prediction has not been widely empirically tested, largely because of the difficulty of comparing rates of molecular evolution in sufficient numbers of independent lineages which differ only in Ne. Island endemic species provide an ideal test of the effect of Ne on molecular evolution because species restricted to islands frequently have smaller Ne than closely related mainland species, and island endemics have arisen from mainland lineages many times in a wide range of taxa. We collated a dataset of 70 phylogenetically independent comparisons between island and mainland taxa, including vertebrates, invertebrates and plants, from 19 different island groups. The rate of molecular evolution in these lineages was estimated by maximum likelihood using two measures: overall substitution rate and the ratio of non-synonymous to synonymous substitution rates. We show that island lineages have significantly higher ratios of non-synonymous to synonymous substitution rates than mainland lineages, as predicted by the nearly neutral theory, although overall substitution rates do not differ significantly.     

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.      

A gradient of silent substitution rate in the human pseudoautosomal region

It has been demonstrated that recombination in the human p-arm pseudoautosomal region (p-PAR) is at least twenty times more frequent than the genomic average of approximately 1 cM/Mb, which may affect substitution patterns and rates in this region. Here I report the analysis of substitution patterns and rates in 10 human, chimpanzee, gorilla, and orangutan genes across the p-PAR. Between species silent divergence in the p-PAR forms a gradient, increasing toward the telomere. The correlation of silent divergence with distance from the p-PAR boundary is highly significant (rho = 0.911, P < 0.001). After exclusion of the CpG dinucleotides this correlation is still significant (rho = 0.89, P < 0.01), thus the substitution rate gradient cannot be explained solely by the differences in the extent of methylation across the p-PAR. Frequent recombination in the PAR may result in a relatively strong effect of biased gene conversion (BGC), which, because of the increased probability of fixation of the G or C nucleotides at (A or T)/(G or C) segregating sites, may affect substitution rates. BGC, however, does not seem to be the factor creating the substitution rate gradient in the p-PAR, because the only gradient is still detactable if only A<-->T and G<-->C substitutions are taken into account (rho = 0.82, P < 0.01). I hypothesize that the substitution rate gradient in the p-PAR is due to the mutagenic effect of recombination, which is very frequent in the distal human p-PAR and might be lower near the p-PAR boundary.