Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen (ϳ11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that ϳ90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.positive selection ͉ accessory glands ͉ seminal fluid ͉ peptide hormones ͉ sexual conflict T he Drosophila male accessory gland is a highly specialized reproductive organ. Its function is to secrete seminal-fluid proteins. Therefore, it may be relatively easy to identify many of the proteins found in seminal fluid by sequencing expressed sequence tags (ESTs) from the accessory gland. Secreted accessory gland proteins (Acps) have diverse and important reproductive roles and interesting patterns of evolutionary change. Acps are transferred along with sperm to the female's reproductive tract and have a variety of effects on the female's reproductive physiology (1). Acps increase the egg-laying rate of mated females by inducing oogenesis (2, 3) and ovulation (4), decrease the female's propensity to remate (5), are required for sperm storage (6, 7), and influence egg hatchability (8, 9). Also, Acps may play a role in cryptic female choice (10), sperm competition (11), and intersexual genomic conflict (12)-three evolutionary scenarios thought to promote the divergence of reproductive proteins. The unique role of Acps has made them the focus of much interest by cell and evolutionary biologists, because they seem to be a currency of chemical communication between males and females (1).Two-dimensional protein electrophoresis has been used to show that male reproductive proteins (including Acps) are twice as diverse as nonreproductive proteins (13), but because the nucleotide sequences encoding these proteins remained unidentified, it was impossible to determine whether positive selection or low constraint on amino acid sequence led to the apparent high divergence of this large class of proteins. Identification of the nucleotide sequences encoding these highly variable proteins will allow for evolutionary inferences of the magnitude of forces affecting their evolution (14) and provide tools for determining the molecular function of the selected gene (2-6, 1...

The progeny of Drosophila females doubly-mated to males from the same and a closely related species are mostly sired by conspecific males. We examined the genetic basis for conspecific mating preference and sperm precedence by using 186 Drosophila lines in which random chromosomal fragments of D. sechellia were introgressed into D. simulans. Sperm competition was measured for each of these lines by crossing ebony D. simulans female with ebony D. simulans males followed by wild-type males from the introgressed lines. Variation in sperm competition (proportion of progeny sired by the second male), mating discrimination (proportion of introgressed males that failed to remate), and male fecundity (proportion of progeny sired by introgressed males) were scored. The introgressed lines exhibited highly significant heterogeneity in the three phenotypes scored, motivating an analysis to locate quantitative trait loci (QTLs) responsible for the differences. Applying composite interval mapping, we found eight QTLs that explain a significant level of variation among introgressed lines in the phenotypes scored. Cytological position overlapped among some QTLs suggesting possible pleiotropic effects. Analysis of the joint effects of simulans/sechellia genetic composition at different QTLs and markers suggests that complex interactions among alleles are partially responsible for interspecific differences in sexual traits.

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp’s), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.