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.     

Nucleotide polymorphism at the RpII215 gene in Drosophila subobscura. Weak selection on synonymous mutations

Nucleotide variation in an 8.1-kb fragment encompassing the RpII215 gene, which encodes the largest subunit of the RNA polymerase II complex, is analyzed in a sample of 11 chromosomes from a natural population of Drosophila subobscura. No amino acid polymorphism was detected among the 157 segregating sites. The observed numbers of preferred and unpreferred derived synonymous mutations can be explained by neutral mutational processes. In contrast, preferred mutations segregate at significantly higher frequency than unpreferred mutations, suggesting the action of natural selection. The polymorphism to divergence ratio is different for preferred and unpreferred changes, in agreement with their beneficial and deleterious effects on fitness, respectively. Preferred and unpreferred codons are nonrandomly distributed in the RpII215 gene, leading to a heterogeneous distribution of polymorphic to fixed synonymous differences across this coding region. This intragenic variation of the polymorphism/divergence ratio cannot be explained by different patterns of gene expression, mutation, or recombination rates, and therefore it indicates that selection coefficients for synonymous mutations can vary extensively across a coding region. The application of nucleotide composition stationarity tests in coding and flanking noncoding regions, assumed to behave neutrally, allows the detection of the action of natural selection when stationarity holds in the noncoding region.     

Natural selection for nucleotide usage at synonymous and nonsynonymous sites in influenza A virus genes

Influenza A virus is one of the best-studied viruses and a model organism for the study of molecular evolution; in particular, much research has focused on detecting natural selection on influenza virus proteins. Here, we study the dynamics of the synonymous and nonsynonymous nucleotide composition of influenza A virus genes. In several genes, the nucleotide frequencies at synonymous positions drift away from the equilibria predicted from the synonymous substitution matrices. We investigate possible reasons for this unexpected behavior by fitting several regression models. Relaxation toward a mutation-selection equilibrium following a host jump fails to explain the dynamics of the synonymous nucleotide composition, even if we allow for slow temporal changes in the substitution matrix. Instead, we find that deep internal branches of the phylogeny show distinct patterns of nucleotide substitution and that these branches strongly influence the dynamics of nucleotide composition, suggesting that the observed trends are at least in part a result of natural selection acting on synonymous sites. Moreover, we find that the dynamics of the nucleotide composition at synonymous and nonsynonymous sites are highly correlated, providing evidence that even nonsynonymous sites can be influenced by selection pressure for nucleotide composition.     

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.     

Direct and correlated responses to artificial selection on developmental time and wing length in Drosophila buzzatii

Abstract.— Developmental time and body size are two positively correlated traits closely related to fitness in many organisms including Drosophila . Previous work suggested that these two traits are involved in a trade-off that may result from a negative genetic correlation between their effects on pre-adult and adult fitness. Here, we examine the evolution of developmental time and body size (indexed by wing length) under artificial selection applied to one or both traits in replicated D. buzzatii populations. Directional changes in both developmental time and wing length indicate the presence of substantial additive genetic variance for both traits. The strongest response to selection for fast development was found in lines selected simultaneously to reduce both developmental time and wing length, probably as an expected consequence of a synergistic effect of indirect selection. When selection was applied in the direction opposite to the putative genetic correlation, that is, large wing length but fast development, no responses were observed for developmental time. Lines selected to reduce both wing length and developmental time diverged slightly faster from the control than lines selected to increase wing length and reduce developmental time. However, wing length did not diverge from the control in lines selected only for fast development. These results suggest a complex genetic basis of the correlation between developmental time and wing length, but are generally consistent with the hypothesis that both traits are related in a trade-off. However, we found that this trade-off may disappear under uncrowded conditions, with fast-developing lines exhibiting a higher pre-adult viability than other lines when tested at high larval density.     

Widespread positive selection in synonymous sites of mammalian genes

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.     

Adjusting for selection on synonymous sites in estimates of evolutionary distance

Evolution at silent sites is often used to estimate the pace of selectively neutral processes or to infer differences in divergence times of genes. However, silent sites are subject to selection in favor of preferred codons, and the strength of such selection varies dramatically across genes. Here, we use the relationship between codon bias and synonymous divergence observed in four species of the genus Saccharomyces to provide a simple correction for selection on silent sites.     

Intragenic Hill-Robertson interference influences selection intensity on synonymous mutations in Drosophila

Natural selection influences synonymous mutations and synonymouscodon usage in many eukaryotes to improve the efficiency oftranslation in highly expressed genes. Recent studies of genecomposition in eukaryotes have shown that codon usage also variesindependently of expression levels, both among genes and atthe intragenic level. Here, we investigate rates of evolution(Ks) and intensity of selection (_s) on synonymous mutationsin two groups of genes that differ greatly in the length oftheir exons, but with equivalent levels of gene expression andrates of crossing-over in Drosophila melanogaster. We estimate_s using patterns of divergence and polymorphism in 50 Drosophilagenes (100 kb of coding sequence) to take into account possiblevariation in mutation trends across the genome, among genesor among codons. We show that genes with long exons exhibithigher Ks and reduced _s compared to genes with short exons.We also show that Ks and _s vary significantly across long exons,with higher Ks and reduced _s in the central region comparedto flanking regions of the same exons, hence indicating thatthe difference between genes with short and long exons can bemostly attributed to the central region of these long exons.Although amino acid composition can also play a significantrole when estimating Ks and _s, our analyses show that the differencesin Ks and _s between genes with short and long exons and acrosslong exons cannot be explained by differences in protein composition.All these results are consistent with the Interference Selection(IS) model that proposes that the Hill-Robertson (HR) effectcaused by many weakly selected mutations has detectable evolutionaryconsequences at the intragenic level in genomes with recombination.Under the IS model, exon size and exon-intron structure influencethe effectiveness of selection, with long exons showing reducedeffectiveness of selection when compared to small exons andthe central region of long exons showing reduced intensity ofselection compared to flanking coding regions. Finally, ourresults further stress the need to consider selection on synonymousmutations and its variation—among and across genes andexons—in studies of protein evolution.     

Joint effects of natural selection and recombination on gene flow between Drosophila ananassae populations

We estimated DNA sequence variation in a 5.7-kb fragment of the furrowed (fw) gene region within and between four populations of Drosophila ananassae; fw is located in a chromosomal region of very low recombination. We analyzed gene flow between these four populations along a latitudinal transect on the Indian subcontinent: two populations from southern, subtropical areas (Hyderabad, India, and Sri Lanka) and two from more temperate zones in the north (Nepal and Burma). Furthermore, we compared the pattern of differentiation at fw with published data from Om(1D), a gene located in a region of normal recombination. While differentiation at Om(1D) shows an isolation-by-distance effect, at fw the pattern of differentiation is quite different such that the frequencies of single nucleotide polymorphisms are homogenized over extended geographic regions (i.e., among the two populations of the northern species range from Burma and Nepal as well as among the two southern populations from India and Sri Lanka), but strongly differentiated between the northern and southern populations. To examine these differences in the patterns of variation and differentiation between the Om(1D) and fw gene regions, we determine the critical values of our previously proposed test of the background selection hypothesis (henceforth called F(ST) test). Using these results, we show that the pattern of differentiation at fw may be inconsistent with the background selection model. The data depart from this model in a direction that is compatible with the occurrence of recent selective sweeps in the northern as well as southern populations.     

Effect of directional selection on spontaneous male recombination in Drosophila ananassae.

Artificial selection was carried out for high and low spontaneous male recombination values in D. ananassae for nine generations by using cu b se marker (second chromosome) and wild stocks which were free from heterozygous chromosome inversions. The mean crossing-over frequency of nine generations was 2.22, 0.70 and 1.20% in high, low and control lines respectively. The values of regression coefficient and realized heritability also indicated that male recombination was affected by selection. However, response to selection was more pronounced in high line as compared to low line. This provides evidence that spontaneous male crossing-over in D. ananassae is under polygenic control.     

Reduced natural selection associated with low recombination in Drosophila melanogaster

Synonymous codons are not used equally in many organisms, and the extent of codon bias varies among loci. Earlier studies have suggested that more highly expressed loci in Drosophila melanogaster are more biased, consistent with findings from several prokaryotes and unicellular eukaryotes that codon bias is partly due to natural selection for translational efficiency. We link this model of varying selection intensity to the population-genetics prediction that the effectiveness of natural selection is decreased under reduced recombination. In analyses of 385 D. melanogaster loci, we find that codon bias is reduced in regions of low recombination (i.e., near centromeres and telomeres and on the fourth chromosome). The effect does not appear to be a linear function of recombination rate; rather, it seems limited to regions with the very lowest levels of recombination. The large majority of the genome apparently experiences recombination at a sufficiently high rate for effective natural selection against suboptimal codons. These findings support models of the Hill-Robertson effect and genetic hitchhiking and are largely consistent with multiple reports of low levels of DNA sequence variation in regions of low recombination.      

Interactions between natural selection, recombination and gene density in the genes of Drosophila

In Drosophila, as in many organisms, natural selection leads to high levels of codon bias in genes that are highly expressed. Thus codon bias is an indicator of the intensity of one kind of selection that is experienced by genes and can be used to assess the impact of other genomic factors on natural selection. Among 13,000 genes in the Drosophila genome, codon bias has a slight positive, and strongly significant, association with recombination--as expected if recombination allows natural selection to act more efficiently when multiple linked sites segregate functional variation. The same reasoning leads to the expectation that the efficiency of selection, and thus average codon bias, should decline with gene density. However, this prediction is not confirmed. Levels of codon bias and gene expression are highest for those genes in an intermediate range of gene density, a pattern that may be the result of a tradeoff between the advantages for gene expression of close gene spacing and disadvantages arising from regulatory conflicts among tightly packed genes. These factors appear to overlay the more subtle effect of linkage among selected sites that gives rise to the association between recombination rate and codon bias.     

Natural selection and ribosomal DNA in Drosophila

Natural populations of Drosophila mercatorum are variable for the number of X-linked 28S ribosomal genes bearing a 5-kilobase insert. A separate polymorphic X-linked gene controls whether 28S repeats bearing the insert are preferentially underreplicated during the formation of polytene tissue. Female flies having at least a third of their 28S genes bearing the insert and lacking the ability to preferentially underreplicate inserted repeats display the abnormal abdomen syndrome. The syndrome is characterized by retention of juvenile abdominal cuticle into the adult, a slowdown in larval developmental time, and an increase in early female fecundity. The life history traits are expressed in nature and provide a basis for strong natural selection. The abnormal abdomen syndrome should be favored whenever the adult age structure is skewed towards young individuals, and field studies confirm this prediction. The closely related species, Drosophila hydei, also bears these inserts and appears to be subject to similar selection. However, D. mercatorum responds to this selection primarily through the allelic variation that controls preferential underreplication, whereas D. hydei responds primarily through adjustment of the proportion of inserted 28S genes. This is interpreted to mean that the evolution of a multigene family arises from the interaction of population-level and DNA-level processes.     

Modes of selection and recombination response in Drosophila melanogaster

A selection experiment for sternopleural bristle number in Drosophila melanogaster was undertaken to analyze the correlated effects on recombination. Replicate lines were subjected to divergent directional selection and to stabilizing selection. Recombination rates for markers on chromosomes 2 (dp-cn-bw) and 3 (se-ss-ro) were compared to those from a control. All lines responded as predicted for bristle number. Lines selected for both increased and decreased bristle number exhibited significantly increased recombination rates. The predicted recombination response from stabilizing selection is suggested by our data, but only one comparison is statistically significant. These results, taken with other studies, support the proposal that genetic recombination enhances individual fitness when populations are experiencing environmental change. Less conclusively, our results suggest that populations undergoing stabilizing selection may respond by reducing their rates of crossing over.     

Efficiency of feedback selection for recombination in Drosophila

A number of theoretical models have been developed in population genetics to explain the evolution of recombination system under natural selection for adaptive traits. Using Drosophila cage populations of large size the effect of selection for resistance to daily temperature fluctuations on the rec-system was studied in this work. It is established that selection for population adaptability to extreme conditions leads during several tens of generations to significant increase in crossing-over rate within various parts of the genome (especially in b-cn and ru-h of chromosomes 2 and 3), as well as to relaxation of exchange interference for adjacent and non-adjacent segments. The Fisher's theoretical prognosis about tightening of linkage in optimal constant conditions was experimentally confirmed for the first time.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

Natural selection on gene expression

Changes in genetic regulation contribute to adaptations in natural populations and influence susceptibility to human diseases. Despite their potential phenotypic importance, the selective pressures acting on regulatory processes in general and gene expression levels in particular are largely unknown. Studies in model organisms suggest that the expression levels of most genes evolve under stabilizing selection, although a few are consistent with adaptive evolution. However, it has been proposed that gene expression levels in primates evolve largely in the absence of selective constraints. In this article, we discuss the microarray-based observations that led to these disparate interpretations. We conclude that in both primates and model organisms, stabilizing selection is likely to be the dominant mode of gene expression evolution. An important implication is that mutations affecting gene expression will often be deleterious and might underlie many human diseases.     

Natural selection at linked sites in humans

Theoretical and empirical work indicates that patterns of neutral polymorphism can be affected by linked, selected mutations. Under background selection, deleterious mutations removed from a population by purifying selection cause a reduction in linked neutral diversity. Under genetic hitchhiking, the rise in frequency and fixation of beneficial mutations also reduces the level of linked neutral polymorphism. Here we review the evidence that levels of neutral polymorphism in humans are affected by selection at linked sites. We then discuss four approaches for distinguishing between background selection and genetic hitchhiking based on (i) the relationship between polymorphism level and recombination rate for neutral loci with high mutation rates, (ii) relative levels of variation on the X chromosome and the autosomes, (iii) the frequency distribution of neutral polymorphisms, and (iv) population-specific patterns of genetic variation. Although the evidence for selection at linked sites in humans is clear, current methods and data do not allow us to clearly assess the relative importance of background selection and genetic hitchhiking in humans. These results contrast with those obtained for Drosophila, where the signals of positive selection are stronger.