The Codon-Degeneracy Model of Molecular Evolution

Mitochondrial genetic codons can be categorized by four patterns of nucleotide-site degeneracy based on varying combinations of twofold- or nondegenerate sites at first codon positions and twofold- or fourfold-degenerate sites at third codon positions. Herein, a model of molecular evolution is introduced that uses these patterns to calculate expected substitution frequencies for each codon position and substitution type relative to overall number of synonymous or nonsynonymous substitutions. Regions of the pocket gopher cytochrome oxidase subunit I (COI) and cytochrome b (cyt-b) genes are analyzed using this model. Chi-square distributions are used to produce relative goodness-of-fit (GF) scores for measuring the difference between substitution frequencies predicted by the codon-degeneracy model (CDM), and frequencies inferred using a well-supported phylogenetic tree of closely related species. The GF scores for expected and observed synonymous (GF_syn= 0.429, p= 0.807) and nonsynonymous (GF_ns= 2.309, p= 0.679) substitution frequencies resulted in a failure to reject the CDM as a null hypothesis for the molecular evolution of COI and cyt-b in pocket gophers. Alternative tree topologies and calculations of transition bias for these data result in higher GF scores. Received: 25 March 1999 / Accepted: 17 September 1999     

Maximum Likelihood Estimation on Large Phylogenies and Analysis of Adaptive Evolution in Human Influenza Virus A

Algorithmic details to obtain maximum likelihood estimates of parameters on a large phylogeny are discussed. On a large tree, an efficient approach is to optimize branch lengths one at a time while updating parameters in the substitution model simultaneously. Codon substitution models that allow for variable nonsynonymous/synonymous rate ratios (ω=d _N/d _S) among sites are used to analyze a data set of human influenza virus type A hemagglutinin (HA) genes. The data set has 349 sequences. Methods for obtaining approximate estimates of branch lengths for codon models are explored, and the estimates are used to test for positive selection and to identify sites under selection. Compared with results obtained from the exact method estimating all parameters by maximum likelihood, the approximate methods produced reliable results. The analysis identified a number of sites in the viral gene under diversifying Darwinian selection and demonstrated the importance of including many sequences in the data in detecting positive selection at individual sites. Received: 25 April 2000 / Accepted: 24 July 2000     

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (d _N) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d _N / d _S) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense. Received: 8 April 1999 / Accepted: 22 June 1999     

Diversifying Selection Governs Sequence Polymorphism in the Major Adhesin Proteins FimA, PapA, and SfaA of Escherichia coli

Fimbriae or pili are essential adherence factors usually found in pathogenic bacteria to aid colonization of host cells. Three major structural pilin genes, fimA, sfaA, and papA, from Escherichia coli natural isolates were examined and nucleotide sequence data revealed elevated levels of both synonymous and nonsynonymous site variation at these loci. Examination of synonymous site variation shows a fivefold increase in fimA sites, relative to the housekeeping gene mdh; and similarly the sfaA and papA genes have increased synonymous sites variation relative to fimA. Nonsynonymous site variation is also elevated at all three loci but, in particular, at the papA locus (k _N= 0.44). The k _N/k _S ratio for the three genes are among the highest yet reported for E. coli genes. Regional variation in nucleotide polymorphism within each of the genes reveal hypervariable segments where nonsynonymous substitutions exceed synonymous substitutions. We propose that at the fimA, papA, and sfaA genes, diversifying selection has brought about the increase levels of polymorphism. Received: 7 August 1997 / Accepted: 8 March 1998     

Synonymous and Nonsynonymous Substitutions in Genes from Gramineae: Intragenic Correlations

In this work, we have investigated the relationships between synonymous and nonsynonymous rates and base composition in coding sequences from Gramineae to analyze the factors underlying the variation in substitutional rates. We have shown that in these genes the rates of nucleotide divergence, both synonymous and nonsynonymous, are, to some extent, dependent on each other and on the base composition. In the first place, the variation in nonsynonymous rate is related to the GC level at the second codon position (the higher the GC₂ level, the higher the amino acid replacement rate). The correlation is especially strong with T₂, the coefficients being significant in the three data sets analyzed. This correlation between nonsynonymous rate and base composition at the second codon position is also detectable at the intragenic level, which implies that the factors that tend to increase the intergenic variance in nonsynonymous rates also affect the intragenic variance. On the other hand, we have shown that the synonymous rate is strongly correlated with the GC₃ level. This correlation is observed both across genes and at the intragenic level. Similarly, the nonsynonymous rate is also affected at the intragenic level by GC₃ level, like the silent rate. In fact, synonymous and nonsynonymous rates exhibit a parallel behavior in relation to GC₃ level, indicating that the intragenic patterns of both silent and amino acid divergence rates are influenced in a similar way by the intragenic variation of GC₃. This result, taken together with the fact that the number of genes displaying intragenic correlation coefficients between synonymous and nonsynonymous rates is not very high, but higher than random expectation (in the three data sets analyzed), strongly suggests that the processes of silent and amino acid replacement divergence are, at least in part, driven by common evolutionary forces in genes from Gramineae. Received: 2 July 1998 / Accepted: 18 April 1999     

Variable Substitution Rates of the 18 Domain Sequences in Artemia Hemoglobin

The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by a gene representing nine successive globin domains which have different sequences and are presumed to have been copied originally from a single-domain gene. Two different polymers exist as the result of a complete duplication of the nine-domain gene, allowing the formation of either homodimers or the heterodimer. The total population size of 18 domains comprising nine corresponding pairs, coupled with the probability that they reflect several hundred million years of evolution in the same lineage, provides a unique model in which the process of gene multiplication can be analyzed. The outcome has important implications for the reliability of local molecular clocks. The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding individual domains are compared between polymers, amino acid substitution fluctuates by a factor of 2.7-fold from lowest to highest. This variation is not obvious at the DNA level: Domain pair identity values fluctuate by 1.3-fold. Identity values are, however, uncorrected for multiple substitutions, and both silent and nonsilent changes are pooled. Therefore, to determine the variability in relative substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96–99) to determine estimates of nonsynonymous (K _A ) and synonymous (K _S ) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (K _S of 56.9%) far exceeded nonsilent substitutions (K _A of 6.7%); however, for corresponding domain pairs, K _A fluctuates by 2.3-fold and K _S by 1.7-fold. The large discrepancies reflected in the expressed protein have accrued within a single lineage and the implication is that divergence dates of different genera based on amino acid sequences, even with well-studied proteins of reasonable size, can be wrong by a factor well in excess of 2. Received: 4 June 1997 / Accepted: 17 December 1997     

The Trouble with Sliding Windows and the Selective Pressure in BRCA1

Sliding-window analysis has widely been used to uncover synonymous (silent, d_S) and nonsynonymous (replacement, d_N) rate variation along the protein sequence and to detect regions of a protein under selective constraint (indicated by d_N<d_S) or positive selection (indicated by d_N>d_S). The approach compares two or more protein-coding genes and plots estimates dˆ _S and dˆ _N from each sliding window along the sequence. Here we demonstrate that the approach produces artifactual trends of synonymous and nonsynonymous rate variation, with greater variation in dˆ _S than in dˆ _N. Such trends are generated even if the true d_S and d_N are constant along the whole protein and different codons are evolving independently. Many published tests of negative and positive selection using sliding windows that we have examined appear to be invalid because they fail to correct for multiple testing. Instead, likelihood ratio tests provide a more rigorous framework for detecting signals of natural selection affecting protein evolution. We demonstrate that a previous finding that a particular region of the BRCA1 gene experienced a synonymous rate reduction driven by purifying selection is likely an artifact of the sliding window analysis. We evaluate various sliding-window analyses in molecular evolution, population genetics, and comparative genomics, and argue that the approach is not generally valid if it is not known a priori that a trend exists and if no correction for multiple testing is applied.     

Molecular Phylogenies and Evolution of crt Genes in Algae

ABSTRACT

The carotenoids constitute the most widespread class of pigments in nature. Most previous work has concentrated on the identification and characterization of their chemical physical properties and bioavailability. In recent years, significant amounts of research have been conducted in an attempt to analyze the genes and the molecular regulation of the genes involved in the biosynthesis of carotenoids. However, it is important not to lose sight of the early evolution of carotenoid biosynthesis. One of the major obstacles in understanding the evolution of the respective enzymes and their patterns of selection is a lack of a well-supported phylogenic analysis. In the present research, a major long-term objective was to provide a clearer picture of the evolutionary history of genes, together with an evaluation of the patterns of selection in algae. These phylogenies will be important in studies characterizing the evolution of algae. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (cyanobacteria, rhofophyta, chlorophyta) have been analyzed. Phylogenetic relationships among protein-coding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae. The analysis also contains an estimation of the rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N), synonymous nucleotide substitution per synonymous site (d_S), and the ratio of nonsynonmous (d_N/d_S) for the test of selection patterns. The phylogenetic trees show that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The d_N values of crt genes in the early pathway are relatively low, while those of the following steps are slightly higher, while the d_N values of crt genes in chlorophyta are higher than those in cyanobacteria. Most of the d_N/d_S values exceed 1. The phylogenetic analysis revealed that lateral gene transfer may have taken place across algal genomes and the d_N values suggest that most of the early crt genes are well conserved compared to the later crt genes. Furthermore, d_N values also revealed that the crt genes of chlorophyta are more evolutionary than cyanobacteria. The amino acids' changes are mostly adaptive evolution under the influence of positive diversity selection.     

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     

Sexual Selection and the Molecular Evolution of ADAM Proteins

Rapid evolution has been identified for many reproductive genes and recent studies have combined phylogenetic tests and information on species mating systems to test sexual selection. Here we examined the molecular evolution of the ADAM gene family, a diverse group of 35 proteins capable of adhesion to and cleavage of other proteins, using sequence data from 25 mammalian genes. Out of the 25 genes analyzed, all those expressed in male reproductive tissue showed evidence of positive selection. Positively selected amino acids within the protein adhesion domain were only found in sperm surface ADAM proteins (ADAMs 1, 2, 3, 4, and 32) suggesting selection driven by male × female interactions. We tested heterogeneity in rates of evolution of the adhesion domain of ADAM proteins by using sequence data from Hominidae and macaques. The use of the branch and branch-site models (PAML) showed evidence of higher d _N/d _S and/or positive selection linked to branches experiencing high postmating selective pressures (chimpanzee and macaque) for Adams 2, 18, and 23. Moreover, we found consistent higher proportion of nonsynonymous relative to synonymous and noncoding sequence substitutions in chimpanzee and/or macaque only for Adams 2, 18, and 23. Our results suggest that lineage-specific sexual selection bouts might have driven the evolution of the adhesion sperm protein surface domains of ADAMs 2 and 18 in primates. Adams 2 and 18 are localized in chromosome 8 of primates and adjacent to each other, so their evolution might have also been influenced by their common genome localization.     

The Nonrandom Location of Synonymous Codons Suggests That Reading Frame-Independent Forces Have Patterned Codon Preferences

Biased codon usage is common in eukaryotic and prokaryotic genes. Evidence from Escherichia, Saccharomyces, and Drosophila indicates that it favors translational efficiency and accuracy. However, to date no functional advantages have been identified in the codon–anticodon interactions involving the most frequently used (preferred) codons. Here we present evidence that forces not related to the individual codon–anticodon interaction may be involved in determining which synonymous codons are preferred or avoided. We show that the ``off-frame' trinucleotide motif preferences inferrable from Drosophila coding regions are often in the same direction as Drosophila's ``in-frame' codon preferences, i.e., its codon usage. The off-frame preferences were inferred from the nonrandomness of the location of confamilial synonymous codons along coding regions—a pattern often described as a context dependence of nucleotide choice at synonymous positions or as codon-pair bias. We relied on randomizations of the location of confamilial codons that do not alter, and cannot be influenced by, the encoded amino acid sequences, codon usage, or base composition of the genes examined. The statistically significant congruency of in-frame and off-frame trinucleotide preferences suggests that the same kind of reading-frame-independent force(s) may also influence synonymous codon choice. These forces may have produced biases in codon usage that then led to the evolution of the translational advantages of these motifs as preferred codons. Under this scenario, tRNA pool size differences between preferred and nonpreferred codons initially were evolved to track the default overrepresentation of codons with preferred motifs. The motif preference hypothesis can explain the structuring of codon preferences and the similarities in the codon usages of distantly related organisms. Received: 10 November 1998 / Accepted: 23 February 1999     

Multiple Substitutions Affect the Phylogenetic Utility of Cytochrome b and 12S rDNA Data: Examining a Rapid Radiation in Leporid (Lagomorpha) Evolution

Partial sequences of two mitochondrial genes, the 12S ribosomal gene (739 bp) and the cytochrome b gene (672 bp), were analyzed in hopes of reconstructing the evolutionary relationships of 11 leporid species, representative of seven genera. However, partial cytochrome b sequences were of little phylogenetic value in this study. A suite of pairwise comparisons between taxa revealed that at the intergeneric level, the cytochrome b gene is saturated at synonymous coding positions due to multiple substitution events. Furthermore, variation at the nonsynonymous positions is limited, rendering the cytochrome b gene of little phylogenetic value for assessing the relationships between leporid genera. If the cytochrome b data are analyzed without accounting for these two classes of nucleotides (i.e., synonymous and nonsynonymous sites), one may incorrectly conclude that signal exists in the cytochrome b data. The mitochondrial 12S rRNA gene, on the other hand, has not experienced excessive saturation at either stem or loop positions. Phylogenies reconstructed from the 12S rDNA data support hypotheses based on fossil evidence that African rock rabbits (Pronolagus) are outside of the main leporid stock and that leporids experienced a rapid radiation. However, the molecular data suggest that this radiation event occurred in the mid-Miocene several millions of years earlier than the Pleistocene dates suggested by paleontological evidence. Received: 23 April 1998 / Accepted: 14 May 1998     

Coding sequence divergence between two closely related plant species: Arabidopsis thaliana and Brassica rapa ssp. pekinensis

To characterize the coding-sequence divergence of closely related genomes, we compared DNA sequence divergence between sequences from a Brassica rapa ssp. pekinensis EST library isolated from flower buds and genomic sequences from Arabidopsis thaliana. The specific objectives were (i) to determine the distribution of and relationship between K _a and K _s, (ii) to identify genes with the lowest and highest K _a:K _s values, and (iii) to evaluate how codon usage has diverged between two closely related species. We found that the distribution of K _a:K _s was unimodal, and that substitution rates were more variable at nonsynonymous than synonymous sites, and detected no evidence that K _a and K _s were positively correlated. Several genes had K _a:K _s values equal to or near zero, as expected for genes that have evolved under strong selective constraint. In contrast, there were no genes with K _a:K _s >1 and thus we found no strong evidence that any of the 218 sequences we analyzed have evolved in response to positive selection. We detected a stronger codon bias but a lower frequency of GC at synonymous sites in A. thaliana than B. rapa. Moreover, there has been a shift in the profile of most commonly used synonymous codons since these two species diverged from one another. This shift in codon usage may have been caused by stronger selection acting on codon usage or by a shift in the direction of mutational bias in the B. rapa phylogenetic lineage.     

Expression Patterns of Duplicate Genes in the Developing Root in Arabidopsis thaliana

Data on gene expression in the development of the root in Arabidopsis thaliana were used to test for expression profile differences among multi-gene families and to examine the extent to which expression differences accompanied coding sequences divergence within families. Significant differences among families were observed on two principal axes, accounting for over 80% of the variance in the expression data. The number of synonymous nucleotide substitutions per synonymous site (d_S) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N) were estimated between the members of two-member families (N=428) and between phylogenetically independent sister pairs (N=190) of sequences within larger families. Ribosomal proteins and a few other proteins were exceptional in showing highly divergent expression patterns in spite of very low levels of amino acid sequence divergence, as indicated by the low d_N relative to d_S. However, the majority of gene duplicates showed relatively high levels of amino acid sequence divergence without appreciable change in expression pattern in the cell types analyzed. Reviewing Editor:Dr. Manyuan Long     

Neutral and Nonneutral Mitochondrial Genetic Variation in Deep-Sea Clams from the Family Vesicomyidae

Nucleotide sequences at two mitochondrial genes from 57 individuals representing eight species of deep-sea clams (Vesicomyidae) were examined for variation consistent with the neutral model of molecular evolution. One gene, cytochrome oxidase subunit I (COI), deviated from the expectations of neutrality by containing an excess of intraspecific nonsynonymous polymorphism. Additionally, one species, Calyptogena kilmeri, showed a significant excess of rare polymorphism specifically at the COI locus. In contrast, a second mitochondrial gene, the large-subunit 16S ribosomal RNA gene (16S), showed little deviation from neutrality either between or within species. Together, COI and 16S show no deviation from neutral expectations by the HKA test, produce congruent phylogenetic relationships between species, and show correlated numbers of fixed differences between species and polymorphism within species. These patterns of both neutral and nonneutral evolution within the mitochondrial genome are most consistent with a model where intraspecific nonsynonymous polymorphism at COI is near neutrality. In addition to examining the forces of molecular evolution, we extend hypotheses about interspecific relationships within this family for geographical locations previously unexamined by molecular methods including habitats near the Middle Atlantic, the Aleutian Trench, and Costa Rica. Received: 10 March 1999 / Accepted: 13 September 1999     

Comparative Genomics of Mitochondrial DNA in Drosophila simulans

The current study compares the nucleotide variation among 22 complete mitochondrial genomes of the three distinct Drosophila simulans haplotypes with intron 1 of the alcohol dehydrogenase-related locus. This is the first study to investigate the sequence variation of multiple complete mitochondrial genomes within distinct mitochondrial haplotypes of a single species. Patterns of variation suggest distinct forces are influencing the evolution of mitochondrial DNA (mtDNA) and autosomal DNA in D. simulans. First, there is little variation within each mtDNA haplotype but strong differentiation among them. In contrast, there is no support for differentiation of the mitochondrial haplotypes at the autosomal locus. Second, there is a significant deficiency of mitochondrial variation in each haplotype relative to the autosomal locus. Third, the ratio of nonsynonymous to synonymous substitutions is not equal in all branches of the well-resolved phylogeny. There is an excess of nonsynonymous substitutions relative to synonymous substitutions within each D. simulans haplotype. This result is similar to that previously observed within the mtDNA of distinct species. A single evolutionary force may be causally linked to the observed patterns of mtDNA variation—a rickettsia-like microorganism, Wolbachia pipientis, which is known to directly influence mitochondrial evolution but have a less direct influence on autosomal loci. Received: 16 September 1999 / Accepted: 14 March 2000     

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     

Invertebrate Species with Nonpelagic Larvae Have Elevated Levels of Nonsynonymous Substitutions and Reduced Nucleotide Diversities

Under a nearly neutral model in which most amino acid substitutions are slightly deleterious, variation in demography, population structure, and other ecological factors among closely related species can potentially modify the effective population size or the selective regime, leading to differences in the rate of nonsynonymous substitution. Ratios of nonsynonymous to synonymous substitutions (d_N/d_S) between species were analyzed in a sea star genus (Patiriella) and a molluscan genus (Littorina), each with diverse modes of reproduction, including multiple lineages with pelagic and nonpelagic larvae. In both genera, lineages with nonpelagic larvae had significantly higher d_N/d_S ratios than lineages with pelagic larvae. The hypothesis that the elevated d_N/d_S ratios in species with nonpelagic larvae was due to reduced effective population size was tested by comparing nucleotide diversities in three genera of gastropod mollusks (Littorina, Crepidula, and Hydrobia), each with several modes of reproduction. Overall, there was a significant (p < 0.05) reduction in nucleotide diversity in species with nonpelagic larvae compared to species with pelagic larvae.     

A revised evolutionary history of hepatitis B virus (HBV)

Previous studies of the evolutionary history of hepatitis B virus (HBV) have been compromised by intergenotype recombination and complex patterns of nucleotide substitution, perhaps caused by differential selection pressures. We examined the phylogenetic distribution of recombination events among human HBV genotypes and found that genotypes A plus D, and genotypes B plus C, had distinct patterns of recombination suggesting differing epidemiological relationships among them. By analyzing the nonoverlapping regions of the viral genome we found strong bootstrap support for some intergenotypic groupings, with evidence of a division between human genotypes A–E from the viruses sampled from apes and human genotype F. However, the earliest events in the divergence of HBV remain uncertain. These uncertainties could not be explained by differential selection pressures, as the ratio of nonsynonymous-to-synonymous substitutions (d _N/d _S) did not vary extensively among lineages and there is no strong evidence for positive selection across the whole tree. Finally, we provide a new estimate of the mean substitution rate in HBV, 4.2 × 10⁻⁵, which suggests that divergence of HBV in humans and apes has occurred only in the last 6000 years.