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.     

Patterns of synonymous codon usage in Drosophila melanogaster genes with sex-biased expression

The nonrandom use of synonymous codons (codon bias) is a well-established phenomenon in Drosophila. Recent reports suggest that levels of codon bias differ among genes that are differentially expressed between the sexes, with male-expressed genes showing less codon bias than female-expressed genes. To examine the relationship between sex-biased gene expression and level of codon bias on a genomic scale, we surveyed synonymous codon usage in 7276 D. melanogaster genes that were classified as male-, female-, or non-sex-biased in their expression in microarray experiments. We found that male-biased genes have significantly less codon bias than both female- and non-sex-biased genes. This pattern holds for both germline and somatically expressed genes. Furthermore, we find a significantly negative correlation between level of codon bias and degree of sex-biased expression for male-biased genes. In contrast, female-biased genes do not differ from non-sex-biased genes in their level of codon bias and show a significantly positive correlation between codon bias and degree of sex-biased expression. These observations cannot be explained by differences in chromosomal distribution, mutational processes, recombinational environment, gene length, or absolute expression level among genes of the different expression classes. We propose that the observed codon bias differences result from differences in selection at synonymous and/or linked nonsynonymous sites between genes with male- and female-biased expression.     

Molecular evolution of X-linked accessory gland proteins in Drosophila pseudoobscura

In Drosophila melanogaster and Drosophila simulans, positive Darwinian selection drives high rates of evolution of male reproductive genes, and accessory gland proteins (Acps) in particular. Here, we tested whether 13 X-linked male-specific genes, 4 Acps and 9 non-Acps, are under selective forces in the Drosophila pseudoobscura species group, much as those in the D. melanogaster group. We observed a statistically significant correlation in relative rates of nonsynonymous evolution between the two species groups tested. One Acp examined had a higher rate of nonsynonymous substitution than predicted by a neutral model in both species groups, suggesting its divergence was driven by positive Darwinian selection. To further test for the signature of selection, we examined polymorphism of three Acps within D. pseudoobscura. From this test, no Acp individually bore the signature of positive selection, but the 3 Acps together possessed an excess of nonsynonymous differences between species, relative to polymorphism within species. We conclude that faster evolution of Acps in the D. pseudoobscura group appears to be driven by positive selection, as previously suggested in the D. melanogaster group.     

Using comparative genomic data to test for fast-X evolution

Genes may acquire nonsynonymous substitutions more rapidly when X-linked than when autosomal, but evidence for "fast-X evolution" has been elusive. Fast-X evolution could explain the disproportionate contribution of X-linked genes to hybrid sterility and other traits. Here, we use a comparative genomic approach, with sequences of 30-110 genes in four Drosophila species, to test for fast-X evolution. Specifically, the 3L autosome arm in D. melanogaster and D. simulans is homologous to the right arm of the X chromosome in D. pseudoobscura and D. miranda. We executed two paired comparisons to determine how often genes on this chromosome arm exhibit higher rates of nonsynonymous substitution in the D. pseudoobscura species group, as predicted by fast-X evolution. We found a statistically significant pattern consistent with fast-X evolution in one comparison and a similar trend in the other comparison. Variation in functional constraints across genes may have masked the signature of fast-X evolution in some previous studies, and we conclude paired comparisons are more powerful for examining rates of evolution of genes when X-linked over autosomal.     

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

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.     

Variable Rates of Evolution among Drosophila Opsin Genes

DNA sequences and chromosomal locations of four Drosophila pseudoobscura opsin genes were compared with those from Drosophila melanogaster, to determine factors that influence the evolution of multigene families. Although the opsin proteins perform the same primary functions, the comparisons reveal a wide range of evolutionary rates. Amino acid identities for the opsins range from 90% for Rh2 to more than 95% for Rh1 and Rh4. Variation in the rate of synonymous site substitution is especially striking: the major opsin, encoded by the Rh1 locus, differs at only 26.1% of synonymous sites between D. pseudoobscura and D. melanogaster, while the other opsin loci differ by as much as 39.2% at synonymous sites. Rh3 and Rh4 have similar levels of synonymous nucleotide substitution but significantly different amounts of amino acid replacement. This decoupling of nucleotide substitution and amino acid replacement suggests that different selective pressures are acting on these similar genes. There is significant heterogeneity in base composition and codon usage bias among the opsin genes in both species, but there are no consistent relationships between these factors and the rate of evolution of the opsins. In addition to exhibiting variation in evolutionary rates, the opsin loci in these species reveal rearrangements of chromosome elements.     

Synonymous Substitutions in the Xdh Gene of Drosophila: Heterogeneous Distribution along the Coding Region

The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene.     

DNA Sequence Evolution of the Amylase Multigene Family in Drosophila Pseudoobscura

The alpha-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amy1) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amy1 gene of D. pseudoobscura differs from the Amy-p1 gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 +/- 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 +/- 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amy1. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amy1 sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amy1; and that Amy3 is nonfunctional and subject to random genetic drift.     

Substitution rates in Drosophila nuclear genes: implications for translational selection

The relationships between synonymous and nonsynonymous substitution rates and between synonymous rate and codon usage bias are important to our understanding of the roles of mutation and selection in the evolution of Drosophila genes. Previous studies used approximate estimation methods that ignore codon bias. In this study we reexamine those relationships using maximum-likelihood methods to estimate substitution rates, which accommodate the transition/transversion rate bias and codon usage bias. We compiled a sample of homologous DNA sequences at 83 nuclear loci from Drosophila melanogaster and at least one other species of Drosophila. Our analysis was consistent with previous studies in finding that synonymous rates were positively correlated with nonsynonymous rates. Our analysis differed from previous studies, however, in that synonymous rates were unrelated to codon bias. We therefore conducted a simulation study to investigate the differences between approaches. The results suggested that failure to properly account for multiple substitutions at the same site and for biased codon usage by approximate methods can lead to an artifactual correlation between synonymous rate and codon bias. Implications of the results for translational selection are discussed.     

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.     

Slow evolution of sex‐biased genes in the reproductive tissue of the dioecious plant Salix viminalis

The relative rate of evolution for sex‐biased genes has often been used as a measure of the strength of sex‐specific selection. In contrast to studies in a wide variety of animals, far less is known about the molecular evolution of sex‐biased genes in plants, particularly in dioecious angiosperms. Here, we investigate the gene expression patterns and evolution of sex‐biased genes in the dioecious plant Salix viminalis. We observe lower rates of sequence evolution for male‐biased genes expressed in the reproductive tissue compared to unbiased and female‐biased genes. These results could be partially explained by the lower codon usage bias for male‐biased genes leading to elevated rates of synonymous substitutions compared to unbiased genes. However, the stronger haploid selection in the reproductive tissue of plants, together with pollen competition, would also lead to higher levels of purifying selection acting to remove deleterious variation. Future work should focus on the differential evolution of haploid‐ and diploid‐specific genes to understand the selective dynamics acting on these loci.     

Rapid evolution of female-biased, but not male-biased, genes expressed in the avian brain

The powerful pressures of sexual and natural selection associated with species recognition and reproduction are thought to manifest in a faster rate of evolution in sex-biased genes, an effect that has been documented particularly for male-biased genes expressed in the reproductive tract. However, little is known about the rate of evolution for genes involved in sexually dimorphic behaviors, which often form the neurological basis of intrasexual competition and mate choice. We used microarray data, designed to uncover sex-biased expression patterns in embryonic chicken brain, in conjunction with data on the rate of sequence evolution for >4,000 coding regions aligned between chicken and zebra finch in order to study the role of selection in governing the molecular evolution for sex-biased and unbiased genes. Surprisingly, we found that female-biased genes, defined across a range of cutoff values, show a higher rate of functional evolution than both male-biased and unbiased genes. Autosomal male-biased genes evolve at a similar rate as unbiased genes. Sex-specific genomic properties, such as heterogeneity in genomic distribution and GC content, and codon usage bias for sex-biased classes fail to explain this surprising result, suggesting that selective pressures may be acting differently on the male and female brain.     

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

Positive selection drives the evolution of the Acp29AB accessory gland protein in Drosophila.

Nucleotide sequence variation at the Acp29AB gene region has been surveyed in Drosophila melanogaster from Spain (12 lines), Ivory Coast (14 lines), and Malawi (13 lines) and in one line of D. simulans. The approximately 1.7-kb region studied encompasses the Acp29AB gene that codes for a male accessory gland protein and its flanking regions. Seventy-seven nucleotide and 8 length polymorphisms were detected. Nonsynonymous polymorphism was an order of magnitude lower than synonymous polymorphism, but still high relative to other non-sex-related genes. In D. melanogaster variation at this region revealed no major genetic differentiation between East and West African populations, while differentiation was highly significant between the European and the two African populations. Comparison of polymorphism and divergence at synonymous and nonsynonymous sites showed an excess of fixed nonsynonymous changes, which indicates that the evolution of the Acp29AB protein has been driven by directional selection at least after the split of the D. melanogaster and D. simulans lineages. The pattern of variation in extant populations of D. melanogaster favors a scenario where the fixation of advantageous replacement substitutions occurred in the early stages of speciation and balancing selection is maintaining variation in this species.     

Determinants of synonymous and nonsynonymous variability in three species of Drosophila

We estimated the intensity of selection on preferred codons in Drosophila pseudoobscura and D. miranda at X-linked and autosomal loci, using a published data set on sequence variability at 67 loci, by means of an improved method that takes account of demographic effects. We found evidence for stronger selection at X-linked loci, consistent with their higher levels of codon usage bias. The estimates of the strength of selection and mutational bias in favor of unpreferred codons were similar to those found in other species, after taking into account the fact that D. pseudoobscura showed evidence for a recent expansion in population size. We examined correlates of synonymous and nonsynonymous diversity in these species and found no evidence for effects of recurrent selective sweeps on nonsynonymous mutations, which is probably because this set of genes have much higher than average levels of selective constraints. There was evidence for correlated effects of levels of selective constraints on protein sequences and on codon usage, as expected under models of selection for translational accuracy. Our analysis of a published data set on D. melanogaster provided evidence for the effects of selective sweeps of nonsynonymous mutations on linked synonymous diversity, but only in the subset of loci that experienced the highest rates of nonsynonymous substitutions (about one-quarter of the total) and not at more slowly evolving loci. Our correlational analysis of this data set suggested that both selective constraints on protein sequences and recurrent selective sweeps affect the overall level of codon usage.     

Codon Usage Bias and Base Composition of Nuclear Genes in Drosophila

The nuclear genes of Drosophila evolve at various rates. This variation seems to correlate with codon-usage bias. In order to elucidate the determining factors of the various evolutionary rates and codon-usage bias in the Drosophila nuclear genome, we compared patterns of codon-usage bias with base compositions of exons and introns. Our results clearly show the existence of selective constraints at the translational level for synonymous (silent) sites and, on the other hand, the neutrality or near neutrality of long stretches of nucleotide sequence within noncoding regions. These features were found for comparisons among nuclear genes in a particular species (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis) as well as in a particular gene (alcohol dehydrogenase) among different species in the genus Drosophila. The patterns of evolution of synonymous sites in Drosophila are more similar to those in the prokaryotes than they are to those in mammals. If a difference in the level of expression of each gene is a main reason for the difference in the degree of selective constraint, the evolution of synonymous sites of Drosophila genes would be sensitive to the level of expression among genes and would change as the level of expression becomes altered in different species. Our analysis verifies these predictions and also identifies additional selective constraints at the translational level in Drosophila.     

Widespread adaptive evolution of Drosophila genes with sex-biased expression

Many genes in higher eukaryotes show sexually dimorphic expression, and these genes tend to be among the most divergent between species. In most cases, however, it is not known whether this rapid divergence is caused by positive selection or if it is due to a relaxation of selective constraint. To distinguish between these two possibilities, we surveyed DNA sequence polymorphism in 91 Drosophila melanogaster genes with male-, female-, or nonsex-biased expression and determined their divergence from the sister species D. simulans. Using several single- and multilocus statistical tests, we estimated the type and strength of selection influencing the evolution of the proteins encoded by genes of each expression class. Adaptive evolution, as indicated by a relative excess of nonsynonymous divergence between species, was common among the sex-biased genes (both male and female). Male-biased genes, in particular, showed a strong and consistent signal of positive selection, while female-biased genes showed more variation in the type of selection they experience. Genes expressed equally in the two sexes, in contrast, showed no evidence for adaptive evolution between D. melanogaster and D. simulans. This suggests that sexual selection and intersexual coevolution are the major forces driving genetic differentiation between species.     

Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup

Background  

Genes with sex-biased expression often show rapid molecular evolution between species. Previous population genetic and comparative genomic studies of Drosophila melanogaster and D. simulans revealed that male-biased genes have especially high rates of adaptive evolution. To test if this is also the case for other lineages within the melanogaster group, we investigated gene expression in D. ananassae, a species that occurs in structured populations in tropical and subtropical regions. We used custom-made microarrays and published microarray data to characterize the sex-biased expression of 129 D. ananassae genes whose D. melanogaster orthologs had been classified previously as male-biased, female-biased, or unbiased in their expression and had been studied extensively at the population-genetic level. For 43 of these genes we surveyed DNA sequence polymorphism in a natural population of D. ananassae and determined divergence to the sister species D. atripex and D. phaeopleura.     

Variation in synonymous codon use and DNA polymorphism within the Drosophila genome

A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.