Nucleotide polymorphism in the RpII215 gene region of the insular species Drosophila guanche: reduced efficacy of weak selection on synonymous variation

An approximately 6.9-kb region encompassing the RpII215 gene was sequenced for 24 individuals of the island endemic species Drosophila guanche. The comparative analysis of synonymous polymorphism and divergence in D. guanche and D. subobscura, two species with pronounced differences in population size, allows contrasting the nearly neutral character of synonymous mutations. In D. guanche, unlike in D. subobscura, (1) the ratio of preferred to unpreferred synonymous changes was similar for polymorphic and fixed changes, (2) the numbers of preferred and unpreferred changes, both polymorphic and fixed, could be explained by the mutational process, and (3) the estimated scaled selection coefficient for unpreferred mutations did not differ significantly from zero. Additionally, the comparative analysis revealed that both the ratio of preferred to unpreferred synonymous changes and the frequency spectrum of unpreferred polymorphic mutations differed significantly between species. All these results indicate that a large fraction of synonymous mutations in the RpII215 gene behave as effectively neutral in D. guanche, whereas they are weakly selected in D. subobscura. The reduced efficacy of selection in the insular species constitutes strong evidence of the nearly neutral character of synonymous mutations and, therefore, of the role of weak selection in maintaining codon bias.     

Synonymous rates at the RpII215 gene of Drosophila: variation among species and across the coding region

The region encompassing the RpII215 gene that encodes the largest component of the RNA polymerase II complex (1889 amino acids) has been sequenced in Drosophila subobscura, D. madeirensis, D. guanche, and D. pseudoobscura. Nonsynonymous divergence estimates (Ka) indicate that this gene has a very low rate of amino acid replacements. Given its low Ka and constitutive expression, synonymous substitution rates are, however, unexpectedly high. Sequence comparisons have allowed the molecular clock hypothesis to be tested. D. guanche is an insular species and it is therefore expected to have a reduced effective size relative to D. subobscura. The significantly higher rate of synonymous substitutions detected in the D. guanche lineage could be explained if synonymous mutations behave as nearly neutral. Significant departure from the molecular clock hypothesis for synonymous and nonsynonymous substitutions was detected when comparing the D. subobscura, D. pseudoobscura, and D. melanogaster lineages. Codon bias and synonymous divergence between D. subobscura and D. melanogaster were negatively correlated across the RpII215 coding region, which indicates that selection coefficients for synonymous mutations vary across the gene. The C-terminal domain (CTD) of the RpII215 protein is structurally and functionally differentiated from the rest of the protein. Synonymous substitution rates were significantly different in both regions, which strongly indicates that synonymous mutations in the CTD and in the non-CTD regions are under detectably different selection coefficients.     

Patterns of selection on synonymous and nonsynonymous variants in Drosophila miranda

We have investigated patterns of within-species polymorphism and between-species divergence for synonymous and nonsynonymous variants at a set of autosomal and X-linked loci of Drosophila miranda. D. pseudoobscura and D. affinis were used for the between-species comparisons. The results suggest the action of purifying selection on nonsynonymous, polymorphic variants. Among synonymous polymorphisms, there is a significant excess of synonymous mutations from preferred to unpreferred codons and of GC to AT mutations. There was no excess of GC to AT mutations among polymorphisms at noncoding sites. This suggests that selection is acting to maintain the use of preferred codons. Indirect evidence suggests that biased gene conversion in favor of GC base pairs may also be operating. The joint intensity of selection and biased gene conversion, in terms of the product of effective population size and the sum of the selection and conversion coefficients, was estimated to be approximately 0.65.     

Inferring Weak Selection from Patterns of Polymorphism and Divergence at ``silent' Sites in Drosophila DNA

Patterns of codon usage and ``silent'''' DNA divergence suggest that natural selection discriminates among synonymous codons in Drosophila. ``Preferred'''' codons are consistently found in higher frequencies within their synonymous families in Drosophila melanogaster genes. This suggests a simple model of silent DNA evolution where natural selection favors mutations from unpreferred to preferred codons (preferred changes). Changes in the opposite direction, from preferred to unpreferred synonymous codons (unpreferred changes), are selected against. Here, selection on synonymous DNA mutations is investigated by comparing the evolutionary dynamics of these two categories of silent DNA changes. Sequences from outgroups are used to determine the direction of synonymous DNA changes within and between D. melanogaster and Drosophila simulans for five genes. Population genetics theory shows that differences in the fitness effect of mutations can be inferred from the comparison of ratios of polymorphism to divergence. Unpreferred changes show a significantly higher ratio of polymorphism to divergence than preferred changes in the D. simulans lineage, confirming the action of selection at silent sites. An excess of unpreferred fixations in 28 genes suggests a relaxation of selection on synonymous mutations in D. melanogaster. Estimates of selection coefficients for synonymous mutations (3.6 <|N(e)s| < 1.3) in D. simulans are consistent with the reduced efficacy of natural selection (|N(e)s| < 1) in the three- to sixfold smaller effective population size of D. melanogaster. Synonymous DNA changes appear to be a prevalent class of weakly selected mutations in Drosophila.     

Positive and negative selection on noncoding DNA in Drosophila simulans

There is now a wealth of evidence that some of the most important regions of the genome are found outside those that encode proteins, and noncoding regions of the genome have been shown to be subject to substantial levels of selective constraint, particularly in Drosophila. Recent work has suggested that these regions may also have been subject to the action of positive selection, with large fractions of noncoding divergence having been driven to fixation by adaptive evolution. However, this work has focused on Drosophila melanogaster, which is thought to have experienced a reduction in effective population size (N(e)), and thus a reduction in the efficacy of selection, compared with its closest relative Drosophila simulans. Here, we examine patterns of evolution at several classes of noncoding DNA in D. simulans and find that all noncoding DNA is subject to the action of negative selection, indicated by reduced levels of polymorphism and divergence and a skew in the frequency spectrum toward rare variants. We find that the signature of negative selection on noncoding DNA and nonsynonymous sites is obscured to some extent by purifying selection acting on preferred to unpreferred synonymous codon mutations. We investigate the extent to which divergence in noncoding DNA is inferred to be the product of positive selection and to what extent these inferences depend on selection on synonymous sites and demography. Based on patterns of polymorphism and divergence for different classes of synonymous substitution, we find the divergence excess inferred in noncoding DNA and nonsynonymous sites in the D. simulans lineage difficult to reconcile with demographic explanations.     

Patterns of mutation and selection at synonymous sites in Drosophila

That natural selection affects molecular evolution at synonymous sites in protein-coding sequences is well established and is thought to predominantly reflect selection for translational efficiency/accuracy mediated through codon bias. However, a recently developed maximum likelihood framework, when applied to 18 coding sequences in 3 species of Drosophila, confirmed an earlier report that the Notch gene in Drosophila melanogaster was evolving under selection in favor of those codons defined as unpreferred in this species. This finding opened the possibility that synonymous sites may be subject to a variety of selective pressures beyond weak selection for increased frequencies of the codons currently defined as "preferred" in D. melanogaster. To further explore patterns of synonymous site evolution in Drosophila in a lineage-specific manner, we expanded the application of the maximum likelihood framework to 8,452 protein coding sequences with well-defined orthology in D. melanogaster, Drosophila sechellia, and Drosophila yakuba. Our analyses reveal intragenomic and interspecific variation in mutational patterns as well as in patterns and intensity of selection on synonymous sites. In D. melanogaster, our results provide little statistical evidence for recent selection on synonymous sites, and Notch remains an outlier. In contrast, in D. sechellia our findings provide evidence in support of selection predominantly in favor of preferred codons. However, there is a small subset of genes in this species that appear to be evolving under selection in favor of unpreferred codons, which indicates that selection on synonymous sites is not limited to the preferential fixation of mutations that enhance the speed or accuracy of translation in this species.     

Patterns of natural selection at the Alcohol dehydrogenase gene of Drosophila americana

Similar outcomes are often observed in species exposed to similar selective regimes, but it is unclear how often the same mechanism of adaptive evolution is followed. Here we present an analysis of selection affecting sequence variation in the Alcohol dehydrogenase (Adh) gene of Drosophila americana, a species endemic to a large climate range that has been colonized by D. melanogaster. Unlike D. melanogaster, there is no evidence of selection on allozymes of ADH across the sampled range. This indicates that if there has been a similar adaptive response to climate in D. americana, it is not within the coding region of Adh. Instead, analyses of a combined dataset containing 86 alleles of Adh reveal purifying selection on the Adh gene, especially within its intron sequences. Frequency spectra of derived unpreferred variants at synonymous sites indicate that these sites are affected by weak purifying selection, but the deviation from neutrality is less drastic than observed for derived variants in noncoding introns. This contrast further supports the notion that noncoding sites in Drosophila are often subject to stronger selection pressures than synonymous sites.     

Sites of P element insertion and structures of P element deletions in the 5'' region of Drosophila melanogaster RpII215.

Several P element insertion and deletion mutations near the 5' end of Drosophila melanogaster RpII215 have been examined by nucleotide sequencing. Two different sites of P element insertion, approximately 90 nucleotides apart, have been detected in this region of the gene. Therefore, including an additional site of P element insertion within the coding region, there are at least three distinct sites of P element insertion at RpII215. Both 5' sites are within a noncoding portion of transcribed sequences. The sequences of four revertants of one P element insertion mutation (D50) indicate that the P element is either precisely deleted or internally deleted to restore RpII215 activity. Partial internal deletions of the P element result in different RpII215 activity levels, which appear to depend on the specific sequences that remain after excision.     

Natural selection on synonymous sites is correlated with gene length and recombination in Drosophila.

Evolutionary analysis of codon bias in Drosophila indicates that synonymous mutations are not neutral, but rather are subject to weak selection at the translation level. Here we show that the effectiveness of natural selection on synonymous sites is strongly correlated with the rate of recombination, in accord with the nearly neutral hypothesis. This correlation, however, is apparent only in genes encoding short proteins. Long coding regions have both a lower codon bias and higher synonymous substitution rates, suggesting that they are affected less efficiently by selection. Therefore, both the length of the coding region and the recombination rate modulate codon bias. In addition, the data indicate that selection coefficients for synonymous mutations must vary by a minimum of one or two orders of magnitude. Two hypotheses are proposed to explain the relationship among the coding region length, the codon bias, and the synonymous divergence and polymorphism levels across the range of recombination rates in Drosophila. The first hypothesis is that selection coefficients on synonymous mutations are inversely related to the total length of the coding region. The second hypothesis proposes that interference among synonymous mutations reduces the efficacy of selection on these mutations. We investigated this second hypothesis by carrying out forward simulations of weakly selected mutations in model populations. These simulations show that even with realistic recombination rates, this interference, which we call the "small-scale" Hill-Robertson effect, can have a moderately strong influence on codon bias.     

Hypervariable noncoding sequences in Saccharomyces cerevisiae

Compared to protein-coding sequences, the evolution of noncoding sequences and the selective constraints placed on these sequences is not well characterized. To compare the evolution of coding and noncoding sequences, we have conducted a survey for DNA polymorphism at five randomly chosen loci among a diverse collection of 81 strains of Saccharomyces cerevisiae. Average rates of both polymorphism and divergence are 40% lower at noncoding sites and 90% lower at nonsynonymous sites in comparison to synonymous sites. Although noncoding and coding sequences show substantial variability in ratios of polymorphism to divergence, two of the loci, MLS1 and PDR10, show a higher rate of polymorphism at noncoding compared to synonymous sites. The high rate of polymorphism is not accompanied by a high rate of divergence and is limited to a few small regions. These hypervariable regions include sites with three segregating bases at a single site and adjacent polymorphic sites. We show that this clustering of polymorphic sites is significantly greater than one would expect on the basis of the spacing between polymorphic fourfold degenerate sites. Although hypervariable noncoding sequences could result from selection on regulatory mutations, they could also result from transient mutational hotspots.     

Selection on codon usage in Drosophila americana

Synonymous codons are not used at random, significantly influencing the base composition of the genome. The selection-mutation-drift model proposes that this bias reflects natural selection in favor of a subset of preferred codons. Previous estimates in Drosophila of the intensity of selective forces involved seem too large to be reconciled with theoretical predictions of the level of codon bias. This probably results from confounding effects of the demographic histories of the species concerned. We have studied three species of the virilis group of Drosophila, which are more likely to satisfy the assumptions of the evolutionary models. We analyzed the patterns of polymorphism and divergence in a sample of 18 genes and applied a new method for estimating the intensity of selection on synonymous mutations based on the frequencies of unpreferred mutations among polymorphic sites. This yielded estimates of selection intensities (N(e)s) of the order of 0.65, which is more compatible with the observed levels of codon bias. Our results support the action of both selection and mutational bias on codon usage bias and suggest that codon usage and genome base composition in the D. americana lineage are in approximate equilibrium. Biased gene conversion may also contribute to the observed patterns.     

In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein

The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s >or= 10(-5)) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster.     

Natural Selection and the Frequency Distributions of ``silent' DNA Polymorphism in Drosophila

In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons. Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction. This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes. The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D. pseudoobscura genes and five alleles of eight D. simulans genes. This result confirms the existence of fitness classes of silent mutations. Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D. pseudoobscura and D. simulans (|N(e)s| & 1). Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution. This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity.     

DNA variability and divergence at the notch locus in Drosophila melanogaster and D. simulans: a case of accelerated synonymous site divergence

DNA diversity in two segments of the Notch locus was surveyed in four populations of Drosophila melanogaster and two of D. simulans. In both species we observed evidence of non-steady-state evolution. In D. simulans we observed a significant excess of intermediate frequency variants in a non-African population. In D. melanogaster we observed a disparity between levels of sequence polymorphism and divergence between one of the Notch regions sequenced and other neutral X chromosome loci. The striking feature of the data is the high level of synonymous site divergence at Notch, which is the highest reported to date. To more thoroughly investigate the pattern of synonymous site evolution between these species, we developed a method for calibrating preferred, unpreferred, and equal synonymous substitutions by the effective (potential) number of such changes. In D. simulans, we find that preferred changes per "site" are evolving significantly faster than unpreferred changes at Notch. In contrast we observe a significantly faster per site substitution rate of unpreferred changes in D. melanogaster at this locus. These results suggest that positive selection, and not simply relaxation of constraint on codon bias, has contributed to the higher levels of unpreferred divergence along the D. melanogaster lineage at Notch.     

Changes in the recombinational environment affect divergence in the yellow gene of Drosophila

The complete coding region of the yellow (y) gene was sequenced in different Drosophila species. In the species of the melanogaster subgroup (D. melanogaster, D. simulans, D. mauritiana, D. yakuba, and D. erecta), this gene is located at the tip of the X chromosome in a region with a strong reduction in recombination rate. In contrast, in D. ananassae (included in the ananassae subgroup of the melanogaster group) and in the obscura group species (D. subobscura, D. madeirensis, D. guanche, and D. pseudoobscura), the y gene is located in regions with normal recombination rates. As predicted by the hitchhiking and background selection models, this change in the recombinational environment affected synonymous divergence in the y-gene-coding region. Estimates of the number of synonymous substitutions per site were much lower between the obscura group species and D. ananassae than between the species of the obscura group and the melanogaster subgroup. In fact, a highly significant increase in the rate of synonymous substitution was detected in all lineages leading to the species of the melanogaster subgroup relative to the D. ananassae lineage. This increase can be explained by a higher fixation rate of mutations from preferred to unpreferred codons (slightly deleterious mutations). The lower codon bias detected in all species of the melanogaster subgroup relative to D. ananassae (or to the obscura group species) would be consistent with this proposal. Therefore, at least in Drosophila, changes in the recombination rate in different lineages might cause deviations of the molecular-clock hypothesis and contribute to the overdispersion of the rate of synonymous substitution. In contrast, the change in the recombinational environment of the y gene has no detectable effect on the rate of amino acid replacement in the Yellow protein.     

Patterns of natural selection at the Alcohol dehydrogenase gene of Drosophila americana

Similar outcomes are often observed in species exposed to similar selective regimes, but it is unclear how often the same mechanism of adaptive evolution is followed. Here we present an analysis of selection affecting sequence variation in the Alcohol dehydrogenase (Adh) gene of Drosophila americana, a species endemic to a large climate range that has been colonized by D. melanogaster. Unlike D. melanogaster, there is no evidence of selection on allozymes of ADH across the sampled range. This indicates that if there has been a similar adaptive response to climate in D. americana, it is not within the coding region of Adh. Instead, analyses of a combined dataset containing 86 alleles of Adh reveal purifying selection on the Adh gene, especially within its intron sequences. Frequency spectra of derived unpreferred variants at synonymous sites indicate that these sites are affected by weak purifying selection, but the deviation from neutrality is less drastic than observed for derived variants in noncoding introns. This contrast further supports the notion that noncoding sites in Drosophila are often subject to stronger selection pressures than synonymous sites.     

The frequency distribution of nucleotide variation in Drosophila simulans.

Patterns of codon bias in Drosophila suggest that silent mutations can be classified into two types: unpreferred (slightly deleterious) and preferred (slightly beneficial). Results of previous analyses of polymorphism and divergence in Drosophila simulans were interpreted as supporting a mutation-selection-drift model in which slightly deleterious, silent mutants make significantly greater contributions to polymorphism than to divergence. Frequencies of unpreferred polymorphisms were inferred to be lower than frequencies of other silent polymorphisms. Here, I analyzed additional D. simulans data to reevaluate the support for these ideas. I found that D. simulans has fixed more unpreferred than preferred mutations, suggesting that this lineage has not been at mutation-selection-drift equilibrium at silent sites. Frequencies of polarized unpreferred polymorphisms are not skewed toward rare alleles. However, frequencies of unpolarized unpreferred codons are lower in high-bias genes than in low-bias genes. This supports the idea that unpreferred codons are borderline deleterious mutations. Purifying selection on silent sites appears to be stronger at twofold-degenerate codons than at fourfold-degenerate codons. Finally, I found that X-linked polymorphisms occur at a higher average frequency than polymorphisms on chromosome arm 3R, even though an average X-linked site is significantly less likely to be polymorphic than an average site on 3R. This result supports a previous analysis of D. simulans indicating different population genetics of X-linked versus autosomal mutations.     

Selection, recombination and demographic history in Drosophila miranda

Selection, recombination, and the demographic history of a species can all have profound effects on genomewide patterns of variability. To assess the impact of these forces in the genome of Drosophila miranda, we examine polymorphism and divergence patterns at 62 loci scattered across the genome. In accordance with recent findings in D. melanogaster, we find that noncoding DNA generally evolves more slowly than synonymous sites, that the distribution of polymorphism frequencies in noncoding DNA is significantly skewed toward rare variants relative to synonymous sites, and that long introns evolve significantly slower than short introns or synonymous sites. These observations suggest that most noncoding DNA is functionally constrained and evolving under purifying selection. However, in contrast to findings in the D. melanogaster species group, we find little evidence of adaptive evolution acting on either coding or noncoding sequences in D. miranda. Levels of linkage disequilibrium (LD) in D. miranda are comparable to those observed in D. melanogaster, but vary considerably among chromosomes. These patterns suggest a significantly lower rate of recombination on autosomes, possibly due to the presence of polymorphic autosomal inversions and/or differences in chromosome sizes. All chromosomes show significant departures from the standard neutral model, including too much heterogeneity in synonymous site polymorphism relative to divergence among loci and a general excess of rare synonymous polymorphisms. These departures from neutral equilibrium expectations are discussed in the context of nonequilibrium models of demography and selection.     

Synonymous codon bias is not caused by mutation bias in G+C-rich genes in humans

It is has been suggested that synonymous codon bias is a consequence of mutation bias in mammals. We tested this hypothesis in humans using single-nucleotide polymorphism data. We found a pattern of polymorphism which was inconsistent with the mutation bias hypothesis in G+C-rich genes. However, the data were consistent with the action of natural selection or biased gene conversion. Similar patterns of polymorphism were also observed in noncoding DNA, suggesting that natural selection or biased gene conversion may affect large tracts of the human genome.