Many genes that mediate sexual reproduction, such as those involved in gamete recognition, diverge rapidly, often as a result of adaptive evolution. This widespread phenomenon might have important consequences, such as the establishment of barriers to fertilization that might lead to speciation. Sequence comparisons and functional studies are beginning to show the extent to which the rapid divergence of reproductive proteins is involved in the speciation process.

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...

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.

Mammalian fertilization exhibits species specificity, and the proteins mediating sperm-egg interactions evolve rapidly between species. In this study, we demonstrate that the evolution of seven genes involved in mammalian fertilization is promoted by positive Darwinian selection by using likelihood ratio tests (LRTs). Several of these proteins are sperm proteins that have been implicated in binding the mammalian egg coat zona pellucida glycoproteins, which were shown previously to be subjected to positive selection. Taken together, these represent the major candidates involved in mammalian fertilization, indicating positive selection is pervasive amongst mammalian reproductive proteins. A new LRT is implemented to determine if the d(N)/d(S) ratio is significantly greater than one. This is a more refined test of positive selection than the previous LRTs which only identified if there was a class of sites with a d(N)/d(S) ratio >1 but did not test if that ratio was significantly greater than one.