Patterns of DNA sequence variation suggest the recent action of positive selection in the janus-ocnus region of Drosophila simulans

Levels of nucleotide polymorphism in three paralogous Drosophila simulans genes, janusA (janA), janusB (janB), and ocnus (ocn), were surveyed by DNA sequencing. The three genes lie in tandem within a 2.5-kb region of chromosome arm 3R. In a sample of eight alleles from a worldwide distribution we found a significant departure from neutrality by several statistical tests. The most striking feature of this sample was that in a 1.7-kb region containing the janA and janB genes, 30 out of 31 segregating sites contained variants present only once in the sample, and 29 of these unique variants were found in the same allele. A restriction survey of an additional 28 lines of D. simulans revealed strong linkage disequilibrium over the janA-janB region and identified six more alleles matching the rare haplotype. Among the rare alleles, the level of DNA sequence variation was typical for D. simulans autosomal genes and showed no departure from neutrality. In addition, the rare haplotype was more similar to the D. melanogaster sequence, indicating that it was the ancestral form. These results suggest that the derived haplotype has risen to high worldwide frequency relatively recently, most likely as a result of natural selection.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.     

Adaptive evolution of recently duplicated accessory gland protein genes in desert Drosophila

The relationship between animal mating system variation and patterns of protein polymorphism and divergence is poorly understood. Drosophila provides an excellent system for addressing this issue, as there is abundant interspecific mating system variation. For example, compared to D. melanogaster subgroup species, repleta group species have higher remating rates, delayed sexual maturity, and several other interesting differences. We previously showed that accessory gland protein genes (Acp's) of Drosophila mojavensis and D. arizonae evolve more rapidly than Acp's in the D. melanogaster subgroup and that adaptive Acp protein evolution is likely more common in D. mojavensis/D. arizonae than in D. melanogaster/D. simulans. These findings are consistent with the idea that greater postcopulatory selection results in more adaptive evolution of seminal fluid proteins in the repleta group flies. Here we report another interesting evolutionary difference between the repleta group and the D. melanogaster subgroup Acp's. Acp gene duplications are present in D. melanogaster, but their high sequence divergence indicates that the fixation rate of duplicated Acp's has been low in this lineage. Here we report that D. mojavensis and D. arizonae genomes contain several very young duplicated Acp's and that these Acp's have experienced very rapid, adaptive protein divergence. We propose that rapid remating of female desert Drosophila generates selection for continuous diversification of the male Acp complement to improve male fertilization potential. Thus, mating system variation may be associated with adaptive protein divergence as well as with duplication of Acp's in Drosophila.     

Identification of the Anopheles gambiae ATP-binding cassette transporter superfamily genes

The Anopheles gambiae genome sequence has been analyzed to find ATP-binding cassette protein genes based on deduced protein similarity to known family members. A nonredundant collection of 44 putative genes was identified including five genes not detected by the original Anopheles genome project machine annotation. These genes encode at least one member of all the human and Drosophila melanogaster ATP-binding protein subgroups. Like D. melanogaster, A. gambiae has subgroup ABCH genes encoding proteins different from the ABC proteins found in other complex organisms. The largest Anopheles subgroup is the ABCC genes which includes one member that can potentially encode ten different isoforms of the protein by differential splicing. As with Drosophila, the second largest Anopheles group is the ABCG subgroup with 12 genes compared to 15 genes in D. melanogaster, but only 5 genes in the human genome. In contrast, fewer ABCA and ABCB genes were identified in the mosquito genome than in the human or Drosophila genomes. Gene duplication is very evident in the Anopheles ABC genes with two groups of four genes, one group with three genes and three groups with two head to tail duplicated genes. These characteristics argue that the A. gambiae is actively using gene duplication as a mechanism to drive genetic variation in this important gene group.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Molecular population genetics of accessory gland protein genes and testis-expressed genes in Drosophila mojavensis and D. arizonae

Molecular population genetic investigation of Drosophila male reproductive genes has focused primarily on melanogaster subgroup accessory gland protein genes (Acp's). Consistent with observations from male reproductive genes of numerous taxa, Acp's evolve more rapidly than nonreproductive genes. However, within the Drosophila genus, large data sets from additional types of male reproductive genes and from different species groups are lacking. Here we report findings from a molecular population genetics analysis of male reproductive genes of the repleta group species, Drosophila arizonae and D. mojavensis. We find that Acp's have dramatically higher average pairwise Ka/Ks (0.93) than testis-enriched genes (0.19) and previously reported melanogaster subgroup Acp's (0.42). Overall, 10 of 19 Acp's have Ka/Ks > 1 either in nonpolarized analyses or in at least one lineage of polarized analyses. Of the nine Acp's for which outgroup data were available, average Ka/Ks was considerably higher in D. mojavensis (2.08) than in D. arizonae (0.87). Contrasts of polymorphism and divergence suggest that adaptive protein evolution at Acp's is more common in D. mojavensis than in D. arizonae.     

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.     

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.     

Entropy and GC Content in the beta-esterase gene cluster of the Drosophila melanogaster subgroup

We perform spectral entropy and GC content analyses in the beta-esterase gene cluster, including the Est-6 gene and the psiEst-6 putative pseudogene, in seven species of the Drosophila melanogaster species subgroup. psiEst-6 combines features of functional and nonfunctional genes. The spectral entropies show distinctly lower structural ordering for psiEst-6 than for Est-6 in all species studied. Our observations agree with previous results for D. melanogaster and provide additional support to our hypothesis that after the duplication event Est-6 retained the esterase-coding function and its role during copulation, while psiEst-6 lost that function but now operates in conjunction with Est-6 as an intergene. Entropy accumulation is not a completely random process for either gene. Structural entropy is nucleotide dependent. The relative normalized deviations for structural entropy are higher for G than for C nucleotides. The entropy values are similar for Est-6 and psiEst-6 in the case of A and T but are lower for Est-6 in the case of G and C. The GC content in synonymous positions is uniformly higher in Est-6 than in psiEst-6, which agrees with the reduced GC content generally observed in pseudogenes and nonfunctional sequences. The observed differences in entropy and GC content reflect an evolutionary shift associated with the process of pseudogenization and subsequent functional divergence of psiEst-6 and Est-6 after the duplication event.     

The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup

The DNA sequences of the Adh genes of three members of the Drosophila melanogaster species subgroup have been determined. This completes the Adh sequences of the eight species of this subgroup. Two species, D. yakuba and D. teissieri, possess processed Adh pseudogenes. In all of the species of the subgroup, a gene of unknown function, Adhr, is located about 300 bp 3' to Adh. Although this gene is experiencing a higher rate of synonymous substitution than Adh, it is more constrained at the amino acid level. Phylogenetic relationships between all eight members of the melanogaster subgroup have been analyzed using a variety of methods. All analyses suggested that the D. yakuba and D. teissieri pseudogenes have a single common ancestor, rather than evolving independently in each species, and that D. melanogaster is the sister species to D. simulans, D. sechellia, and D. mauritiana. The evolutionary relationships of the latter three species remain equivocal.      

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.     

Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.     

Phylogeny of the sex-determining gene Sex-lethal in insects.

The Sex-lethal (SXL) protein belongs to the family of RNA-binding proteins and is involved in the regulation of pre-mRNA splicing. SXL has undergone an obvious change of function during the evolution of the insect clade. The gene has acquired a pivotal role in the sex-determining pathway of Drosophila, although it does not act as a sex determiner in non-drosophilids. We collected SXL sequences of insect species ranging from the pea aphid (Acyrtho siphom pisum) to Drosophila melanogaster by searching published articles, sequencing cDNAs, and exploiting homology searches in public EST and whole-genome databases. The SXL protein has moderately conserved N- and C-terminal regions and a well-conserved central region including 2 RNA recognition motifs. Our phylogenetic analysis shows that a single orthologue of the Drosophila Sex-lethal (Sxl) gene is present in the genomes of the malaria mosquito Anopheles gambiae, the honeybee Apis mellifera, the silkworm Bombyx mori, and the red flour beetle Tribolium castaneum. The D. melanogaster, D. erecta, and D. pseudoobscura genomes, however, contain 2 paralogous genes, Sxl and CG3056, which are orthologous to the Anopheles, Apis, Bombyx, and Tribolium Sxl. Hence, a duplication in the fly clade generated Sxl and CG3056. Our hypothesis maintains that one of the genes, Sxl, adopted the new function of sex determiner in Drosophila, whereas the other, CG3056, continued to serve some or all of the yet-unknown ancestral functions.