Generation-time Effect Research Articles

How to Make a Rodent Giant: Genomic Basis and Tradeoffs of Gigantism in the Capybara, the World's Largest Rodent.

Gigantism results when one lineage within a clade evolves extremely large body size relative to its small-bodied ancestors, a common phenomenon in animals. Theory predicts that the evolution of giants should be constrained by two tradeoffs. First, because body size is negatively correlated with population size, purifying selection is expected to be less efficient in species of large body size, leading to increased mutational load. Second, gigantism is achieved through generating a higher number of cells along with higher rates of cell proliferation, thus increasing the likelihood of cancer. To explore the genetic basis of gigantism in rodents and uncover genomic signatures of gigantism-related tradeoffs, we assembled a draft genome of the capybara (Hydrochoerus hydrochaeris), the world’s largest living rodent. We found that the genome-wide ratio of nonsynonymous to synonymous mutations (ω) is elevated in the capybara relative to other rodents, likely caused by a generation-time effect and consistent with a nearly neutral model of molecular evolution. A genome-wide scan for adaptive protein evolution in the capybara highlighted several genes controlling postnatal bone growth regulation and musculoskeletal development, which are relevant to anatomical and developmental modifications for an increase in overall body size. Capybara-specific gene-family expansions included a putative novel anticancer adaptation that involves T-cell-mediated tumor suppression, offering a potential resolution to the increased cancer risk in this lineage. Our comparative genomic results uncovered the signature of an intragenomic conflict where the evolution of gigantism in the capybara involved selection on genes and pathways that are directly linked to cancer.

Negative correlation between rates of molecular evolution and flowering cycles in temperate woody bamboos revealed by plastid phylogenomics

BackgroundHeterogeneous rates of molecular evolution are universal across the tree of life, posing challenges for phylogenetic inference. The temperate woody bamboos (tribe Arundinarieae, Poaceae) are noted for their extremely slow molecular evolutionary rates, supposedly caused by their mysterious monocarpic reproduction. However, the correlation between substitution rates and flowering cycles has not been formally tested.ResultsHere we present 15 newly sequenced plastid genomes of temperate woody bamboos, including the first genomes ever sequenced from Madagascar representatives. A data matrix of 46 plastid genomes representing all 12 lineages of Arundinarieae was assembled for phylogenetic and molecular evolutionary analyses. We conducted phylogenetic analyses using different sequences (e.g., coding and noncoding) combined with different data partitioning schemes, revealing conflicting relationships involving internodes among several lineages. A great difference in branch lengths were observed among the major lineages, and topological inconsistency could be attributed to long-branch attraction (LBA). Using clock model-fitting by maximum likelihood and Bayesian approaches, we furthermore demonstrated extensive rate variation among these major lineages. Rate accelerations mainly occurred for the isolated lineages with limited species diversification, totaling 11 rate shifts during the tribe’s evolution. Using linear regression analysis, we found a negative correlation between rates of molecular evolution and flowering cycles for Arundinarieae, notwithstanding that the correlation maybe insignificant when taking the phylogenetic structure into account.ConclusionsUsing the temperate woody bamboos as an example, we found further evidence that rate heterogeneity is universal in plants, suggesting that this will pose a challenge for phylogenetic reconstruction of bamboos. The bamboos with longer flowering cycles tend to evolve more slowly than those with shorter flowering cycles, in accordance with a putative generation time effect.

Life history effects on the molecular clock of autosomes and sex chromosomes

One of the foundational results in molecular evolution is that the rate at which neutral substitutions accumulate on a lineage equals the rate at which mutations arise. Traits that affect rates of mutation therefore also affect the phylogenetic "molecular clock." We consider the effects of sex-specific generation times and mutation rates in species with two sexes. In particular, we focus on the effects that the age of onset of male puberty and rates of spermatogenesis have likely had in hominids (great apes), considering a model that approximates features of the mutational process in mammals, birds, and some other vertebrates. As we show, this model can account for a number of seemingly disparate observations: notably, the puzzlingly low X-to-autosome ratios of substitution rates in humans and chimpanzees and differences in rates of autosomal substitutions among hominine lineages (i.e., humans, chimpanzees, and gorillas). The model further suggests how to translate pedigree-based estimates of human mutation rates into split times among extant hominoids (apes), given sex-specific life histories. In so doing, it largely bridges the gap reported between estimates of split times based on fossil and molecular evidence, in particular suggesting that the human-chimpanzee split may have occurred as recently as 6.6 Mya. The model also implies that the "generation time effect" should be stronger in short-lived species, explaining why the generation time has a major influence on yearly substitution rates in mammals but only a subtle one in human pedigrees.

A generation-time effect on the rate of molecular evolution in bacteria.

Molecular evolutionary rate varies significantly among species and a strict global molecular clock has been rejected across the tree of life. Generation time is one primary life-history trait that influences the molecular evolutionary rate. Theory predicts that organisms with shorter generation times evolve faster because of the accumulation of more DNA replication errors per unit time. Although the generation-time effect has been demonstrated consistently in plants and animals, the evidence of its existence in bacteria is lacking. The bacterial phylum Firmicutes offers an excellent system for testing generation-time effect because some of its members can enter a dormant, nonreproductive endospore state in response to harsh environmental conditions. It follows that spore-forming bacteria would--with their longer generation times--evolve more slowly than their nonspore-forming relatives. It is therefore surprising that a previous study found no generation-time effect in Firmicutes. Using a phylogenetic comparative approach and leveraging on a large number of Firmicutes genomes, we found sporulation significantly reduces the genome-wide spontaneous DNA mutation rate and protein evolutionary rate. Contrary to the previous study, our results provide strong evidence that the evolutionary rates of bacteria, like those of plants and animals, are influenced by generation time.

Reduced evolutionary rates in HIV-1 reveal extensive latency periods among replicating lineages.

BackgroundHIV-1 can persist for the duration of a patient’s life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle.ResultsWe developed a novel molecular clock–based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past.ConclusionsThese results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0081-0) contains supplementary material, which is available to authorized users.

The Human Mutation Rate Is Increasing, Even As It Slows

Substitution rates vary between species, and many explanations regarding the causes of this variation have been proposed. Here we consider how new genomic data on the per-generation mutation rate impinge on proposed hypotheses for substitution rate variation in primates. We propose that the generation-time effect as it is usually understood cannot explain the observed rate variation, but instead that selection for decreased somatic mutation rates can. By considering the disparate causes underlying mutation rate changes in recent human history, we also show that the per-generation mutation rate is increasing even as the per-cell-division rate is decreasing.

ABRUPT DECELERATION OF MOLECULAR EVOLUTION LINKED TO THE ORIGIN OF ARBORESCENCE IN FERNS

Molecular rate heterogeneity, whereby rates of molecular evolution vary among groups of organisms, is a well-documented phenomenon. Nonetheless, its causes are poorly understood. For animals, generation time is frequently cited because longer-lived species tend to have slower rates of molecular evolution than their shorter-lived counterparts. Although a similar pattern has been uncovered in flowering plants, using proxies such as growth form, the underlying process has remained elusive. Here, we find a deceleration of molecular evolutionary rate to be coupled with the origin of arborescence in ferns. Phylogenetic branch lengths within the “tree fern” clade are considerably shorter than those of closely related lineages, and our analyses demonstrate that this is due to a significant difference in molecular evolutionary rate. Reconstructions reveal that an abrupt rate deceleration coincided with the evolution of the long-lived tree-like habit at the base of the tree fern clade. This suggests that a generation time effect may well be ubiquitous across the green tree of life, and that the search for a responsible mechanism must focus on characteristics shared by all vascular plants. Discriminating among the possibilities will require contributions from various biological disciplines,but will be necessary for a full appreciation of molecular evolution.

Different fitnesses for in vivo and in vitro evolutions due to the finite generation-time effect

We consider the finite generation-time effect in virus evolution models, introducing differential equations with delay. The suggested approach more adequately describes the evolution in case of growing populations than the popular models of population genetics, especially for the viruses with large number of offspring during one life cycle. Now the mean fitness, as a coefficient for exponential population growth, could not be defined via instant characteristics of the model. For the constant population size the finite generation-time does not affect mean fitness in the steady state. The growing virus population is characterized by different fitness than the population with a constant size.

A Generation Time Effect on the Rate of Molecular Evolution in Invertebrates

The rate of genome evolution varies significantly between species. Evidence is growing that at least some of this variation is associated with species characteristics, such as body size, diversification rate, or population size. One of the strongest correlates of the rate of molecular evolution in vertebrates is generation time (GT): Species with faster generation turnover tend to have higher rates of molecular evolution, presumably because their genomes are copied more frequently and therefore collect more DNA replication errors per unit time. But the GT effect has never been tested for nonvertebrate animals. Here, we present the first general test of the GT effect in invertebrates, using 15 genes from 143 species spread across the major eumetazoan superphyla (including arthropods, nematodes, molluscs, annelids, platyhelminthes, cnidarians, echinoderms, and urochordates). We find significant evidence that rates of molecular evolution are correlated with GT in invertebrates and that this effect applies consistently across genes and taxonomic groups. Furthermore, the GT effect is evident in nonsynonymous substitutions, whereas theory predicts (and most previous evidence has supported) a relationship only in synonymous changes. We discuss both the practical and theoretical implications of these findings.

Mammalian Nonsynonymous Sites Are Not Overdispersed: Comparative Genomic Analysis of Index of Dispersion of Mammalian Proteins

It is often stated that patterns of nonsynonymous rate variation among mammalian lineages are more irregular than expected or overdispersed under the neutral model, whereas synonymous sites conform to the neutral model. Here we reexamined genome-wide patterns of the variance to mean ratio, or index of dispersion (R), of substitutions in proteins from human, mouse, and dog. Contrary to the prevailing notion, we found that the mean index of dispersion for nonsynonymous sites of mammalian proteins is not significantly different from 1. We propose that earlier analyses were biased because the data included disproportionately more protein hormones, which tend to be more dispersed than genes in other functional categories. Synonymous sites exhibit greater degree of dispersion than nonsynonymous sites, although similar to earlier estimates and potentially due to errors associated with correction for multiple hits. Overall, our analysis identifies strong genome-wide generation-time effect and natural selection as important determinants of among-lineage variation of protein evolutionary rates. Furthermore, patterns of lineage-specific selective constraint are consistent with the nearly neutral model of molecular evolution.

Reconsidering the generation time hypothesis based on nuclear ribosomal ITS sequence comparisons in annual and perennial angiosperms

BackgroundDifferences in plant annual/perennial habit are hypothesized to cause a generation time effect on divergence rates. Previous studies that compared rates of divergence for internal transcribed spacer (ITS1 and ITS2) sequences of nuclear ribosomal DNA (nrDNA) in angiosperms have reached contradictory conclusions about whether differences in generation times (or other life history features) are associated with divergence rate heterogeneity. We compared annual/perennial ITS divergence rates using published sequence data, employing sampling criteria to control for possible artifacts that might obscure any actual rate variation caused by annual/perennial differences.ResultsRelative rate tests employing ITS sequences from 16 phylogenetically-independent annual/perennial species pairs rejected rate homogeneity in only a few comparisons, with annuals more frequently exhibiting faster substitution rates. Treating branch length differences categorically (annual faster or perennial faster regardless of magnitude) with a sign test often indicated an excess of annuals with faster substitution rates. Annuals showed an approximately 1.6-fold rate acceleration in nucleotide substitution models for ITS. Relative rates of three nuclear loci and two chloroplast regions for the annual Arabidopsis thaliana compared with two closely related Arabidopsis perennials indicated that divergence was faster for the annual. In contrast, A. thaliana ITS divergence rates were sometimes faster and sometimes slower than the perennial. In simulations, divergence rate differences of at least 3.5-fold were required to reject rate constancy in > 80 % of replicates using a nucleotide substitution model observed for the combination of ITS1 and ITS2. Simulations also showed that categorical treatment of branch length differences detected rate heterogeneity > 80% of the time with a 1.5-fold or greater rate difference.ConclusionAlthough rate homogeneity was not rejected in many comparisons, in cases of significant rate heterogeneity annuals frequently exhibited faster substitution rates. Our results suggest that annual taxa may exhibit a less than 2-fold rate acceleration at ITS. Since the rate difference is small and ITS lacks statistical power to reject rate homogeneity, further studies with greater power will be required to adequately test the hypothesis that annual and perennial plants have heterogeneous substitution rates. Arabidopsis sequence data suggest that relative rate tests based on multiple loci may be able to distinguish a weak acceleration in annual plants. The failure to detect rate heterogeneity with ITS in past studies may be largely a product of low statistical power.

RATES OF MOLECULAR EVOLUTION IN BACTERIA ARE RELATIVELY CONSTANT DESPITE SPORE DORMANCY

Rates of molecular evolution are known to vary considerably among lineages, partially due to differences in life-history traits such as generation time. The generation-time effect has been well documented in some eukaryotes, but its prevalence in prokaryotes is unknown. "Because many species of Firmicute bacteria spend long periods of time as metabolically dormant spores, which could result in fewer DNA substitutions per unit time, they present an excellent system for testing predictions of the molecular clock hypothesis." To test whether spore-forming bacteria evolve more slowly than their non-spore-forming relatives, I used phylogenetic methods to determine if there were differences in rates of amino acid substitution between spore-forming and non-spore-forming lineages of Firmicute bacteria. Although rates of evolution do vary among lineages, I find no evidence for an effect of spore-formation on evolutionary rate and, furthermore, evolutionary rates are similar to those calculated for enteric bacteria. These results support the notion that variation in generation time does not affect evolutionary rates in bacterial lineages.

Characteristics, causes and evolutionary consequences of male-biased mutation

Mutation has traditionally been considered a random process, but this paradigm is challenged by recent evidence of divergence rate heterogeneity in different genomic regions. One facet of mutation rate variation is the propensity for genetic change to correlate with the number of germ cell divisions, reflecting the replication-dependent origin of many mutations. Haldane was the first to connect this association of replication and mutation to the difference in the number of cell divisions in oogenesis (low) and spermatogenesis (usually high), and the resulting sex difference in the rate of mutation. The concept of male-biased mutation has been thoroughly analysed in recent years using an evolutionary approach, in which sequence divergence of autosomes and/or sex chromosomes are compared to allow inference about the relative contribution of mothers and fathers in the accumulation of mutations. For instance, assuming that a neutral sequence is analysed, that rate heterogeneity owing to other factors is cancelled out by the investigation of many loci and that the effect of ancestral polymorphism is properly taken into account, the male-to-female mutation rate ratio, alpham, can be solved from the observed difference in rate of X and Y chromosome divergence. The male mutation bias is positively correlated with the relative excess of cell divisions in the male compared to the female germ line, as evidenced by a generation time effect: in mammals, alpham is estimated at approximately 4-6 in primates, approximately 3 in carnivores and approximately 2 in small rodents. Another life-history correlate is sexual selection: when there is intense sperm competition among males, increased sperm production will be associated with a larger number of mitotic cell divisions in spermatogenesis and hence an increase in alpham. Male-biased mutation has implications for important aspects of evolutionary biology such as mate choice in relation to mutation load, sexual selection and the maintenance of genetic diversity despite strong directional selection, the tendency for a disproportionate large role of the X (Z) chromosome in post-zygotic isolation, and the evolution of sex.

The Chloroplast Genome of Phalaenopsis aphrodite (Orchidaceae): Comparative Analysis of Evolutionary Rate with that of Grasses and Its Phylogenetic Implications

Whether the Amborella/Amborella-Nymphaeales or the grass lineage diverged first within the angiosperms has recently been debated. Central to this issue has been focused on the artifacts that might result from sampling only grasses within the monocots. We therefore sequenced the entire chloroplast genome (cpDNA) of Phalaenopsis aphrodite, Taiwan moth orchid. The cpDNA is a circular molecule of 148,964 bp with a comparatively short single-copy region (11,543 bp) due to the unusual loss and truncation/scattered deletion of certain ndh subunits. An open reading frame, orf91, located in the complementary strand of the rrn23 was reported for the first time. A comparison of nucleotide substitutions between P. aphrodite and the grasses indicates that only the plastid expression genes have a strong positive correlation between nonsynonymous (Ka) and synonymous (Ks) substitutions per site, providing evidence for a generation time effect, mainly across these genes. Among the intron-containing protein-coding genes of the sampled monocots, the Ks of the genes are significantly correlated to transitional substitutions of their introns. We compiled a concatenated 61 protein-coding gene alignment for the available 20 cpDNAs of vascular plants and analyzed the data set using Bayesian inference, maximum parsimony, and neighbor-joining (NJ) methods. The analyses yielded robust support for the Amborella/Amborella-Nymphaeales-basal hypothesis and for the orchid and grasses together being a monophyletic group nested within the remaining angiosperms. However, the NJ analysis using Ka, the first two codon positions, or amino acid sequences, respectively, supports the monocots-basal hypothesis. We demonstrated that these conflicting angiosperm phylogenies are most probably linked to the transitional sites at all codon positions, especially at the third one where the strong base-composition bias and saturation effect take place.

Size of the protein-coding genome and rate of molecular evolution

In diploid populations of size N, there will be 2 Nmu mutations per nucleotide (nt) site (or per locus) per generation (mu stands for mutation rate). If either the population or the coding genome double in size, one expects 4 Nmu mutations. What is important is not the population size per se but the number of genes (coding sites), the two being often interconverted. Here we compared the total physical length of protein-coding genomes (n) with the corresponding absolute rates of synonymous substitution (K(S)), an empirical neutral reference. In the classical occupancy problem and in the coupons collector (CC) problem, n was expressed as the mean rate of change (K(CC)). Despite inherently very low power of the approaches involving averaging of rates, the mode of molecular evolution of the total size phenotype of the coding genome could be evidenced through differences between the genomic estimates of K(CC) [K(CC)=1/(ln n + 0.57721) n] and rate of molecular evolution, K(S). We found that (1) the estimates of n and K(S) are reciprocally correlated across taxa (r=0.812; p<< 0.001); (2) the gamete-cell division hypothesis (Chang et al. Proc Natl Acad Sci USA 91:827-831, 1994) can be confirmed independently in terms of K(CC)/K(S) ratios; (3) the time scale of molecular evolution changes with change in mutation rate, as previously shown by Takahata (Proc Natl Acad Sci USA 87:2419-2423, 1990), Takahata et al. (Genetics 130:925-938, 1992), and Vekemans and Slatkin (Genetics 137:1157-1165, 1994); (4) the generation time and population size (Lynch and Conery, Science 302:1401-1404, 2003) effects left their "signatures" at the level of the size phenotype of the protein-coding genome.

Procedures for reliable estimation of viral fitness from time-series data.

In order to develop a better understanding of the evolutionary dynamics of HIV drug resistance, it is necessary to quantify accurately the in vivo fitness costs of resistance mutations. However, the reliable estimation of such fitness costs is riddled with both theoretical and experimental difficulties. Experimental fitness assays typically suffer from the shortcoming that they are based on in vitro data. Fitness estimates based on the mathematical analysis of in vivo data, however, are often questionable because the underlying assumptions are not fulfilled. In particular, the assumption that the replication rate of the virus population is constant in time is frequently grossly violated. By extending recent work of Marée and colleagues, we present here a new approach that corrects for time-dependent viral replication in time-series data for growth competition of mutants. This approach allows a reliable estimation of the relative replicative capacity (with confidence intervals) of two competing virus variants growing within the same patient, using longitudinal data for the total plasma virus load, the relative frequency of the two variants and the death rate of infected cells. We assess the accuracy of our method using computer-generated data. An implementation of the developed method is freely accessible on the Web (http://www.eco.ethz.ch/fitness.html).

Phylogenetic relationships in Lupinus (Fabaceae: Papilionoideae) based on internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 44 taxa of the genus Lupinus and five outgroup taxa were used for phylogenetic analysis. Lupinus appears as a strongly supported monophyletic genus, which is unambiguously part of the Genisteae. The lupines are distributed into five main clades in general accordance with their geographical origin. In the Old World, almost all the recognized taxonomic units are well resolved. The ITS data reveal an unexpectedly close relationship between the diverse sections Angustifoli and Lutei. The ITS results suggest a geographical division between the western New World lupines and the eastern ones. They also indicate the presence of some moderately to strongly supported groups of taxa, such as the Microcarpi-Pusilli group, the L. spariflorus-L. arizonicus group, the L. mexicanus-L. elegans group in the western New World, and the notable L. multiflorus-L. paraguariensis group in the eastern New World. The latter group strongly suggests that the eastern South American compound- and simple-leaved perennial lupines derive from a common ancestor. However, apart from some exceptions, relationships within the genus still remain largely unresolved based on ITS data. The lack of resolution at the base of the genus is suggestive of a rapid initial radiation of the lupines subsequent to the dispersal of their common ancestor. Relative rate tests demonstrate the presence of rate heterogeneity of ITS sequences within Lupinus. In many pairwise comparisons between taxa, substitution rate inequalities are correlated with the habit (annual, perennial), suggesting some role for the generation time effects in the evolutionary history of lupines.

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.

Ancestral polymorphism of Mhc class II genes in mice: implications for balancing selection and the mammalian molecular clock.

To investigate the evolutionary dynamics at Mhc class II DR genes of mice (genus Mus), we sequenced the peptide binding regions (PBRs) of 41 DRB (= E beta) genes and eight DRA (= E alpha) genes from 15 strains representing eight species. As expected trees of these PBR sequences imply extensive maintenance of ancestral DRB alleles across species. We use a coalescent simulation model to show that the number of interspecific coalescent events (c) observed on these trees was higher than the number expected for neutral genealogies and similar sample sizes and is more consistent with balancing selection that with neutrality. Patterns of ancestral polymorphism in mouse DRB alleles were also used to examine the tempo of synonymous substitution in the PBR of mouse class II genes. Both absolute and relative rate tests on DRA and DRB genes imply increased substitution rates at two- and fourfold degenerate sites of mice and rats relative to primates, and decreased rates for the DRB genes of primates relative to ungulate and carnivore relatives. Thus rates of synonymous substitution at Mhc DR genes in mammals appear to be subject to generation time effects in ways similar to those found at other mammalian genes.

Rates of Nucleotide Substitution in Primates and Rodents and the Generation–Time Effect Hypothesis

DNA sequence data from introns, flanking regions, and the η globin pseudogene region all show a significantly higher rate of nucleotide substitution in the Old World monkey lineage than in the human lineage after the separation of the two lineages, or, in other words, the data support the hominoid rate-slowdown hypothesis. Data from both protein sequences and DNA sequences show that the rate of evolution is significantly higher in the rodent lineage than in the primate lineage. Furthermore, DNA sequences from introns show that the rate of nucleotide substitution is at least two times higher in rodents than in higher primates. The male-to-female ratio of mutation rate is estimated to be between 3 and 6 in higher primates, whereas it is only 2 in mice and rats. These ratios are similar to the corresponding male-to-female ratios of germ cell divisions in higher primates and in rodents, suggesting that errors in DNA replication during germ cell division are the primary source of mutation, or, in other words, mutation is largely DNA replication-dependent. This conclusion provides further support for the generation–time effect hypothesis.