For over 30 years a central question in molecular evolution has been whether natural selection plays a substantial role in evolution at the DNA sequence level. Evidence has accumulated over the last decade that adaptive evolution does occur at the protein level, but it has remained unclear how prevalent adaptive evolution is. Here we present a simple method by which the number of adaptive substitutions can be estimated and apply it to data from Drosophila simulans and D. yakuba. We estimate that 45% of all amino-acid substitutions have been fixed by natural selection, and that on average one adaptive substitution occurs every 45 years in these species.

We have attempted to quantify the frequency and effects of slightly deleterious mutations (SDMs), those that have selective effects close to the reciprocal of the effective population size of a species, by comparing the level of selective constraint in protein-coding genes of related species that have different present-day effective population sizes. In our two comparisons, the species with the smaller effective population size showed lower constraint, implying that SDMs had become fixed. The fixation of SDMs was supported by the observation of a higher fraction of radical to conservative amino acid substitutions in species with smaller effective population sizes. The fraction of strongly deleterious mutations (which rarely become fixed) is >70% in most species. Only approximately 10% or fewer of mutations seem to behave as SDMs, but SDMs could comprise a substantial fraction of mutations in protein-coding genes that have a chance of becoming fixed between species.

Approximately two thirds of all knockouts of individual mouse genes give rise to viable fertile mice. These genes have thus been termed 'non-essential' in contrast to 'essential' genes, the knockouts of which result in death or infertility. Although non-essential genes are likely to be under selection that favours sequence conservation [1], it is predicted that they are less subject to such stabilising selection than essential genes, and hence evolve faster [2]. We have addressed this issue by analysing the molecular evolution of 108 non-essential and 67 essential genes that have been sequenced in both mouse and rat. On preliminary analysis, the non-essential genes appeared to be faster evolving than the essential ones. We found, however, that the non-essential class contains a disproportionate number of immune-system genes that may be under directional selection (that is, selection favouring change) because of host-parasite coevolution. After correction for this bias, we found that the rate at which genes evolve does not correlate with the severity of the knockout phenotype. This was corroborated by the finding that, whereas neuron-specific genes have significantly lower rates of change than other genes, essential and non-essential neuronal genes have comparable rates of evolution. Our findings most probably reflect strong selection acting against even very subtle deleterious phenotypes, and indicate that the putative involvement of directional selection in host-parasite coevolution and gene expression within the nervous system explains much more of the variance in rates of gene evolution than does the knockout phenotype.

To investigate mutation-rate variation between autosomes and sex chromosomes in the avian genome, we have analyzed divergence between chicken (Gallus gallus) and turkey (Meleagris galopavo) sequences from 33 autosomal, 28 Z-linked, and 14 W-linked introns with a total ungapped alignment length of approximately 43,000 bp. There are pronounced differences in the mean divergence among autosomes and sex chromosomes (autosomes [A] = 10.08%, Z chromosome = 10.99%, and W chromosome = 5.74%), and we use these data to estimate the male-to-female mutation-rate ratio (alpha(m)) from Z/A, Z/W, and A/W comparisons at 1.71, 2.37, and 2.52, respectively. Because the alpha(m) estimates of the three comparisons do not differ significantly, we find no statistical support for a specific reduction in the Z chromosome mutation rate (Z reduction estimated at 4.89%, P = 0.286). The idea of mutation-rate reduction in the sex chromosome hemizygous in one sex (i.e., X in mammals, Z in birds) has been suggested on the basis of theory on adaptive mutation-rate evolution. If it exists in birds, the effect would, thus, seem to be weak; a preliminary power analysis suggests that it is significantly less than 18%. Because divergence may vary within chromosomal classes as a result of variation in mutation and/or selection, we developed a novel double-bootstrapping method, bootstrapping both by introns and sites from concatenated alignments, to estimate confidence intervals for chromosomal class rates and for alpha(m). The narrowest interval for the alpha(m) estimate is 1.88 to 2.97 from the Z/W comparison. We also estimated alpha(m) using maximum likelihood on data from all three chromosome classes; this method yielded alpha(m) = 2.47 and approximate 95% confidence intervals of 2.27 to 2.68. Our data are broadly consistent with the idea that mutation-rate differences between chromosomal classes can be explained by the male mutation bias alone.

A distinctive feature of the avian genome is the large heterogeneity in the size of chromosomes, which are usually classified into a small number of macrochromosomes and numerous microchromosomes. These chromosome classes show characteristic differences in a number of interrelated features that could potentially affect the rate of sequence evolution, such as GC content, gene density, and recombination rate. We studied the effects of these factors by analyzing patterns of nucleotide substitution in two sets of chicken-turkey sequence alignments. First, in a set of 67 orthologous introns, divergence was significantly higher in microchromosomes (chromosomes 11-38; 11.7% divergence) than in both macrochromosomes (chromosomes 1-5; 9.9% divergence; P = 0.016) and intermediate-sized chromosomes (chromosomes 6-10; 9.5% divergence; P = 0.026). At least part of this difference was due to the higher incidence of CpG sites on microchromosomes. Second, using 155 orthologous coding sequences we noted a similar pattern, in which synonymous substitution rates on microchromosomes (13.1%) were significantly higher than were rates on macrochromosomes (10.3%; P = 0.024). Broadly assuming neutrality of introns and synonymous sites, or constraints on such sequences do not differ between chromosomal classes, these observations imply that microchromosomal genes are exposed to more germ line mutations than those on other chromosomes. We also find that dN/dS ratios for genes located on microchromosomes (average, 0.094) are significantly lower than those of macrochromosomes (average, 0.185; P = 0.025), suggesting that the proteins of genes on microchromosomes are under greater evolutionary constraint.

Most studies of microsatellite evolution utilize long, highly mutable loci, which are unrepresentative of the majority of simple repeats in the human genome. Here we use an unbiased sample of 2,467 microsatellite loci derived from alignments of 5.1 Mb of genomic sequence from human and chimpanzee to investigate the mutation process of tandemly repetitive DNA. The results indicate that the process of microsatellite evolution is highly heterogeneous, exhibiting differences between loci of different lengths and motif sizes and between species. We find a highly significant tendency for human dinucleotide repeats to be longer than their orthologues in chimpanzees, whereas the opposite trend is observed in mononucleotide repeat arrays. Furthermore, the rate of divergence between orthologues is significantly higher at longer loci, which also show significantly greater mutability per repeat number. These observations have important consequences for understanding the molecular mechanisms of microsatellite mutation and for the development of improved measures of genetic distance.T he human genome is composed of 40-50% repetitive DNA, an important class being simple tandem repeats or microsatellite DNA sequences (1). Microsatellites are iterations of short (1-6 bp) sequence motifs, repeat numbers generally being less than 30 (2). They are spread over the genome with an estimated average density of one locus per 2-30 kb, the frequency being dependent on the criteria used for defining a microsatellite locus (1, 3). Similar microsatellite densities have also been documented for other eukaryotic genomes (4). Microsatellites are instrumental as genetic linkage markers in genome mapping projects (5) and have also found widespread use for evolutionary and population genetics studies in many species (6, 7), including humans (8).Microsatellites differ from most other DNA sequences in their high degree of polymorphism, with heterozygosities commonly exceeding 70%. As they generally seem to be free of selective constraints, it is evident that the extensive degree of genetic variability requires a high underlying mutation rate. Estimates of the human genomic mutation rate are in the range of 10 Ϫ4 to 10 Ϫ2 per meiosis (9), several orders of magnitude higher than that of unique DNA sequences (10, 11). It is commonly assumed that microsatellite mutations arise from replication slippage (slipped strand mispairing), a process thought to result in the gain or loss of one or a few repeat units (12)(13)(14). A microsatellite locus may initially evolve from the random occurrence of a few repeat units embedded within unique sequence and subsequently, after several steps of repeat expansion, reach a stage of an appreciable number of repeats. In theory, such expansion can proceed indefinitely in the absence of selection unless there are mechanisms that direct mutations toward contractions (15)(16)(17) or that make loci collapse (e.g., point mutations or large deletions; ref. 18).Based on the assumption that replication slippage is the main cause...

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Several studies of substitution rate variation have indicated that the local mutation rate varies over the mammalian genome. In the present study, we show significant variation in substitution rates within the noncoding part of the human genome using 4.7 Mb of human-chimpanzee pairwise comparisons. Moreover, we find a significant positive covariation of lineage-specific chimpanzee and human local substitution rates, and very similar mean substitution rates down the two lineages. The substitution rate variation is probably not caused by selection or biased gene conversion, and so we conclude that mutation rates vary deterministically across the noncoding nonrepetitive regions of the human genome. We also show that noncoding substitution rates are significantly affected by G+C base composition, partly because the base composition is not at equilibrium.