Effects of X-linkage and sex-biased gene expression on the rate of adaptive protein evolution in Drosophila

Patterns of polymorphism and divergence in Drosophila protein-coding genes suggest that a considerable fraction of amino acid differences between species can be attributed to positive selection and that genes with sex-biased expression, that is, those expressed predominantly in one sex, have especially high rates of adaptive evolution. Previous studies, however, have been restricted to autosomal sex-biased genes and, thus, do not provide a complete picture of the evolutionary forces acting on sex-biased genes across the genome. To determine the effects of X-linkage on sex-biased gene evolution, we surveyed DNA sequence polymorphism and divergence in 45 X-linked genes, including 17 with male-biased expression, 13 with female-biased expression, and 15 with equal expression in the 2 sexes. Using both single- and multilocus tests for selection, we found evidence for adaptive evolution in both groups of sex-biased genes. The signal of adaptive evolution was particularly strong for X-linked male-biased genes. A comparison with data from 91 autosomal genes revealed a "fast-X" effect, in which the rate of adaptive evolution was greater for X-linked than for autosomal genes. This effect was strongest for male-biased genes but could be seen in the other groups as well. A genome-wide analysis of coding sequence divergence that accounted for sex-biased expression also uncovered a fast-X effect for male-biased and unbiased genes, suggesting that recessive beneficial mutations play an important role in adaptation.     

The genomic rate of adaptive amino acid substitution in Drosophila

The proportion of amino acid substitutions driven by adaptive evolution can potentially be estimated from polymorphism and divergence data by an extension of the McDonald-Kreitman test. We have developed a maximum-likelihood method to do this and have applied our method to several data sets from three Drosophila species: D. melanogaster, D. simulans, and D. yakuba. The estimated number of adaptive substitutions per codon is not uniformly distributed among genes, but follows a leptokurtic distribution. However, the proportion of amino acid substitutions fixed by adaptive evolution seems to be remarkably constant across the genome (i.e., the proportion of amino acid substitutions that are adaptive appears to be the same in fast-evolving and slow-evolving genes; fast-evolving genes have higher numbers of both adaptive and neutral substitutions). Our estimates do not seem to be significantly biased by selection on synonymous codon use or by the assumption of independence among sites. Nevertheless, an accurate estimate is hampered by the existence of slightly deleterious mutations and variations in effective population size. The analysis of several Drosophila data sets suggests that approximately 25% +/- 20% of amino acid substitutions were driven by positive selection in the divergence between D. simulans and D. yakuba.     

Molecular evolution and population genetic analysis of candidate female reproductive genes in Drosophila

Molecular analyses in several taxa have consistently shown that genes involved in reproduction are rapidly evolving and subjected to positive selection. The mechanism behind this evolution is not clear, but several proposed hypotheses involve the coevolution between males and females. In Drosophila, several male reproductive proteins (Acps) involved in male-male and male-female interactions show evidence of rapid adaptive evolution. What has been missing from the Drosophila literature is the identification and analysis of female reproductive genes. Recently, an evolutionary expressed sequence tag analysis of Drosophila female reproductive tract genes identified 169 candidate female reproductive genes. Many of these candidate genes still await further molecular analysis and independent verification of positive selection. Our goal was to expand our understanding of the molecular evolution of Drosophila female reproductive genes with a detailed polymorphism and divergence study on seven additional candidate female reproductive genes and a reanalysis of two genes from the above study. We demonstrate that 6 candidate female genes of the 9 genes surveyed show evidence of positive selection using both polymorphism and divergence data. One of these proteins (CG17012) is modeled to reveal that the sites under selection fall around and within the active site of this protease, suggesting potential differences between species. We discuss our results in light of potential function as well as interaction with male reproductive proteins.     

Adaptive protein evolution of X-linked and autosomal genes in Drosophila: implications for faster-X hypotheses

Patterns of sex chromosome and autosome evolution can be used to elucidate the underlying genetic basis of adaptative change. Evolutionary theory predicts that X-linked genes will adapt more rapidly than autosomes if adaptation is limited by the availability of beneficial mutations and if such mutations are recessive. In Drosophila, rates of molecular divergence between species appear to be equivalent between autosomes and the X chromosome. However, molecular divergence contrasts are difficult to interpret because they reflect a composite of adaptive and nonadaptive substitutions between species. Predictions based on faster-X theory also assume that selection is equally effective on the X and autosomes; this might not be true because the effective population sizes of X-linked and autosomal genes systematically differ. Here, population genetic and divergence data from Drosophila melanogaster, Drosophila simulans, and Drosophila yakuba are used to estimate the proportion of adaptive amino acid substitutions occurring in the D. melanogaster lineage. After gene composition and effective population size differences between chromosomes are controlled, X-linked and autosomal genes are shown to have equivalent rates of adaptive divergence with approximately 30% of amino acid substitutions driven by positive selection. The results suggest that adaptation is either unconstrained by a lack of beneficial genetic variation or that beneficial mutations are not recessive and are thus highly visible to natural selection whether on sex chromosomes or on autosomes.     

No accelerated rate of protein evolution in male-biased Drosophila pseudoobscura genes

Sexually dimorphic traits are often subject to diversifying selection. Genes with a male-biased gene expression also are probably affected by sexual selection and have a high rate of protein evolution. We used SAGE to measure sex-biased gene expression in Drosophila pseudoobscura. Consistent with previous results from D. melanogaster, a larger number of genes were male biased (402 genes) than female biased (138 genes). About 34% of the genes changed the sex-related expression pattern between D. melanogaster and D. pseudoobscura. Combining gene expression with protein divergence between both species, we observed a striking difference in the rate of evolution for genes with a male-biased gene expression in one species only. Contrary to expectations, D. pseudoobscura genes in this category showed no accelerated rate of protein evolution, while D. melanogaster genes did. If sexual selection is driving molecular evolution of male-biased genes, our data imply a radically different selection regime in D. pseudoobscura.     

Adaptive protein evolution and regulatory divergence in Drosophila

Two recent studies demonstrated a positive correlation between divergence in gene expression and protein sequence in Drosophila. This correlation could be driven by positive selection or variation in functional constraint. To distinguish between these alternatives, we compared patterns of molecular evolution for 1,862 genes with two previously reported estimates of expression divergence in Drosophila. We found a slight negative trend (nonsignificant) between positive selection on protein sequence and divergence in expression levels between Drosophila melanogaster and Drosophila simulans. Conversely, shifts in expression patterns during Drosophila development showed a positive association with adaptive protein evolution, though as before the relationship was weak and not significant. Overall, we found no strong evidence for an increase in the incidence of positive selection on protein-coding regions in genes with divergent expression in Drosophila, suggesting that the previously reported positive association between protein and regulatory divergence primarily reflects variation in functional constraint.     

Gene regulation divergence is a major contributor to the evolution of Dobzhansky-Muller incompatibilities between species of Drosophila

The Dobzhansky-Muller model denotes incompatible gene interactions between diverging populations/species and is recognized as the basis of postzygotic reproductive isolation. Little is known about the molecular nature of such gene interactions. We have carried out comparative gene expression analyses in the testes of 3 closely related species of the Drosophila melanogaster subgroup and their hybrids (all of which are sterile). We show that in hybrids 1) a higher proportion of male-biased genes (i.e., genes with a higher level of expression in males) are underexpressed (or not expressed) compared with non-sex-biased genes, 2) the majority of the underexpressed genes appear to be under stabilizing selection by virtue of showing similar levels of expression in the parental species, and only a small proportion of genes show signs of directional selection, 3) very few of the misexpressed genes are shared between species pairs, suggesting that there may not be a "common" set of "speciation genes," and 4) expression of non-testes-specific genes is observed in the testes of interspecific hybrids, and the number of such genes is positively correlated with divergence time. These results suggest that gene regulation divergence of sex- and reproduction-related genes is a major contributor to the evolution of Dobzhansky-Muller incompatibilities between species of Drosophila.     

Adaptive loss of an old duplicated gene during incipient speciation

To probe the role of natural selection in species origin, we performed a DNA polymorphism survey of the Drosophila melanogaster desaturase2 (ds2) locus. ds2 is responsible for a cuticular hydrocarbon difference between two behaviorally isolated races--Zimbabwe (Z) and Cosmopolitan (M). The ds2 allele prevalent in the Z populations is functional, while the allele from the M populations harbors a 16-bp deletion upstream of the gene which knocks out its expression. We find a signature of positive selection in the ds2 promoter, but not in the control gene, sas. This signature appears to be confined to the derived M population. We also find that the selection has been recent because the gene retains a signature of a selective sweep evidenced by the departure of Fay and Wu's H test from neutral expectation. We also find that ds2, as well as its duplicate pair ds1, has been maintained in the Drosophila genus for at least 40 Myr without any sign of adaptive change. Taken together with previous molecular genetic evidence, our results suggest that ds2 is one of the genes responsible for adaptive divergence of the Z and M races of D. melanogaster.     

The effects of sex-biased gene expression and X-linkage on rates of adaptive protein sequence evolution in Drosophila

A faster rate of adaptive evolution of X-linked genes compared with autosomal genes may be caused by the fixation of new recessive or partially recessive advantageous mutations (the Faster-X effect). This effect is expected to be largest for mutations that affect only male fitness and absent for mutations that affect only female fitness. We tested these predictions in Drosophila melanogaster by using genes with different levels of sex-biased expression and by estimating the extent of adaptive evolution of non-synonymous mutations from polymorphism and divergence data. We detected both a Faster-X effect and an effect of male-biased gene expression. There was no evidence for a strong association between the two effects—modest levels of male-biased gene expression increased the rate of adaptive evolution on both the autosomes and the X chromosome, but a Faster-X effect occurred for both unbiased genes and female-biased genes. The rate of genetic recombination did not influence the magnitude of the Faster-X effect, ruling out the possibility that it reflects less Hill–Robertson interference for X-linked genes.     

Adaptive gene expression divergence inferred from population genomics

Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.     

Signatures of selection and gene conversion associated with human color vision variation

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.     

Molecular evolution of sex-biased genes in Drosophila

Studies of morphology, interspecific hybridization, protein/DNA sequences, and levels of gene expression have suggested that sex-related characters (particularly those involved in male reproduction) evolve rapidly relative to non-sex-related characters. Here we report a general comparison of evolutionary rates of sex-biased genes using data from cDNA microarray experiments and comparative genomic studies of Drosophila. Comparisons of nonsynonymous/synonymous substitution rates (d(N)/d(S)) between species of the D. melanogaster subgroup revealed that genes with male-biased expression had significantly faster rates of evolution than genes with female-biased or unbiased expression. The difference was caused primarily by a higher d(N) in the male-biased genes. The same pattern was observed for comparisons among more distantly related species. In comparisons between D. melanogaster and D. pseudoobscura, genes with highly biased male expression were significantly more divergent than genes with highly biased female expression. In many cases, orthologs of D. melanogaster male-biased genes could not be identified in D. pseudoobscura through a Blast search. In contrast to the male-biased genes, there was no clear evidence for accelerated rates of evolution in female-biased genes, and most comparisons indicated a reduced rate of evolution in female-biased genes relative to unbiased genes. Male-biased genes did not show an increased ratio of nonsynonymous/synonymous polymorphism within D. melanogaster, and comparisons of polymorphism/divergence ratios suggest that the rapid evolution of male-biased genes is caused by positive selection.     

GENETIC ARCHITECTURE OF SKELETAL EVOLUTION IN EUROPEAN LAKE AND STREAM STICKLEBACK

Advances in genomic techniques are greatly facilitating the study of molecular signatures of selection in diverging natural populations. Connecting these signatures to phenotypes under selection remains challenging, but benefits from dissections of the genetic architecture of adaptive divergence. We here perform quantitative trait locus (QTL) mapping using 488 F₂ individuals and 2011 single nucleotide polymorphisms (SNPs) to explore the genetic architecture of skeletal divergence in a lake‐stream stickleback system from Central Europe. We find QTLs for gill raker, snout, and head length, vertebral number, and the extent of lateral plating (plate number and height). Although two large‐effect loci emerge, QTL effect sizes are generally small. Examining the neighborhood of the QTL‐linked SNPs identifies several genes involved in bone formation, which emerge as strong candidate genes for skeletal evolution. Finally, we use SNP data from the natural source populations to demonstrate that some SNPs linked to QTLs in our cross also exhibit striking allele frequency differences in the wild, suggesting a causal role of these QTLs in adaptive population divergence. Our study paves the way for comparative analyses across other (lake‐stream) stickleback populations, and for functional investigations of the candidate genes.     

Patterns of Nucleotide Diversity at the Regions Encompassing the Drosophila Insulin-Like Peptide (dilp) Genes: Demography vs. Positive Selection in Drosophila melanogaster

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events.     

Rapid evolution through gene duplication and subfunctionalization of the testes-specific alpha4 proteasome subunits in Drosophila

Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms. Evidence suggests that duplicated genes are retained at a much higher rate than originally thought and that functional divergence of gene copies is a major factor promoting their retention in the genome. We find that two Drosophila testes-specific alpha4 proteasome subunit genes (alpha4-t1 and alpha4-t2) have a higher polymorphism within species and are significantly more diverged between species than the somatic alpha4 gene. Our data suggest that following gene duplication, the alpha4-t1 gene experienced relaxed selective constraints, whereas the alpha4-t2 gene experienced positive selection acting on several codons. We report significant heterogeneity in evolutionary rates among all three paralogs at homologous codons, indicating that functional divergence has coincided with genic divergence. Reproductive subfunctionalization may allow for a more rapid evolution of reproductive traits and a greater specialization of testes function. Our data add to the increasing evidence that duplicated genes experience lower selective constraints and in some cases positive selection following duplication. Newly duplicated genes that are freer from selective constraints may provide a mechanism for developing new interactions and a pathway for the evolution of new genes.     

Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family

Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.