Nathaniel L. Clark

Pervasive Adaptive Evolution in Primate Seminal Proteins

Seminal fluid proteins show striking effects on reproduction, involving manipulation of female behavior and physiology, mechanisms of sperm competition, and pathogen defense. Strong adaptive pressures are expected for such manifestations of sexual selection and host defense, but the extent of positive selection in seminal fluid proteins from divergent taxa is unknown. We identified adaptive evolution in primate seminal proteins using genomic resources in a tissue-specific study. We found extensive signatures of positive selection when comparing 161 human seminal fluid proteins and 2,858 prostate-expressed genes to those in chimpanzee. Seven of eight outstanding genes yielded statistically significant evidence of positive selection when analyzed in divergent primates. Functional clues were gained through divergent analysis, including several cases of species-specific loss of function in copulatory plug genes, and statistically significant spatial clustering of positively selected sites near the active site of kallikrein 2. This study reveals previously unidentified positive selection in seven primate seminal proteins, and when considered with findings in Drosophila, indicates that extensive positive selection is found in seminal fluid across divergent taxonomic groups.

Coevolution of interacting fertilization proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.

Rapid evolution of reproductive proteins in abalone and Drosophila

Observations from different taxa, including plants, protozoa, insects and mammals, indicate that proteins involved in reproduction evolve rapidly. Several models of adaptive evolution have been proposed to explain this phenomenon, such as sexual conflict, sexual selection, self versus non-self recognition and pathogen resistance. Here we discuss the potential role of sexual conflict in the rapid evolution of reproductive genes in two different animal systems, abalone (Haliotis) and Drosophila. In abalone, we reveal how specific interacting sperm–egg proteins were identified and discuss this identification in the light of models for rapid protein evolution and speciation. For Drosophila, we describe the genomic approaches taken to identify male accessory gland proteins and female reproductive tract proteins. Patterns of protein evolution from both abalone and Drosophila support the predicted patterns of rapid protein evolution driven by sexual conflict. We stress however that other selective pressures may contribute to the rapid evolution that is observed. We conclude that the key to distinguishing between sexual conflict and other mechanisms of protein evolution will be an integration of genetic, experimental and theoretical data.

Proteomics and Comparative Genomic Investigations Reveal Heterogeneity in Evolutionary Rate of Male Reproductive Proteins in Mice (Mus domesticus)

Male reproductive fitness is strongly affected by seminal fluid. In addition to interacting with the female environment, seminal fluid mediates important physiological characteristics of sperm, including capacitation and motility. In mammals, the male reproductive tract shows a striking degree of compartmentalization, with at least six distinct tissue types contributing material that is combined with sperm in an ejaculate. Although studies of whole ejaculates have been undertaken in some species, we lack a comprehensive picture of the specific proteins produced by different accessory tissues. Here, we perform proteomic investigations of six regions of the male reproductive tract in mice -- seminal vesicles, anterior prostate, dorsolateral prostate, ventral prostate, bulbourethral gland, and bulbourethral diverticulum. We identify 766 proteins that could be mapped to 506 unique genes and compare them with a high-quality human seminal fluid data set. We find that Gene Ontology functions of seminal proteins are largely conserved between mice and humans. By placing these data in an evolutionary framework, we show that seminal vesicle proteins have experienced a significantly higher rate of nonsynonymous substitution compared with the genome, which could be the result of adaptive evolution. In contrast, proteins from the other five tissues showed significantly lower nonsynonymous substitution, revealing a previously unappreciated level of evolutionary constraint acting on the majority of male reproductive proteins.

Adaptive Evolution in Rodent Seminal Vesicle Secretion Proteins

Proteins involved in reproductive fitness have evolved unusually rapidly across diverse groups of organisms. These reproductive proteins show unusually high rates of amino acid substitutions, suggesting that the proteins have been subject to positive selection. We sought to identify seminal fluid proteins experiencing adaptive evolution because such proteins are often involved in sperm competition, host immunity to pathogens, and manipulation of female reproductive physiology and behavior. We performed an evolutionary screen of the mouse prostate transcriptome for genes with elevated evolutionary rates between mouse and rat. We observed that secreted rodent prostate proteins evolve approximately twice as fast as nonsecreted proteins, remarkably similar to findings in the primate prostate and in the Drosophila male accessory gland. Our screen led us to identify and characterize a group of seminal vesicle secretion (Svs) proteins and to show that the gene Svs7 is evolving very rapidly, with many amino acid sites under positive selection. Another gene in this group, Svs5, showed evidence of branch-specific selection in the rat. We also found that Svs7 is under selection in primates and, by using three-dimensional models, demonstrated that the same regions have been under selection in both groups. Svs7 has been identified as mouse caltrin, a protein involved in sperm capacitation, the process responsible for the timing of changes in sperm activity and behavior, following ejaculation. We propose that the most likely explanation of the adaptive evolution of Svs7 that we have observed in rodents and primates stems from an important function in sperm competition.

A Novel Method to Detect Proteins Evolving at Correlated Rates: Identifying New Functional Relationships between Coevolving Proteins

Interacting proteins evolve at correlated rates, possibly as the result of evolutionary pressures shared by functional groups and/or coevolution between interacting proteins. This evolutionary signature can be exploited to learn more about protein networks and to infer functional relationships between proteins on a genome-wide scale. Multiple methods have been introduced that detect correlated evolution using amino acid distances. One assumption made by these methods is that the neutral rate of nucleotide substitution is uniform over time; however, this is unlikely and such rate heterogeneity would adversely affect amino acid distance methods. We explored alternative methods that detect correlated rates using protein-coding nucleotide sequences in order to better estimate the rate of nonsynonymous substitution at each branch (d(N)) normalized by the underlying synonymous substitution rate (d(S)). Our novel likelihood method, which was robust to realistic simulation parameters, was tested on Drosophila nuclear pore proteins, which form a complex with well-documented physical interactions. The method revealed significantly correlated evolution between nuclear pore proteins, where members of a stable subcomplex showed stronger correlations compared with those proteins that interact transiently. Furthermore, our likelihood approach was better able to detect correlated evolution among closely related species than previous methods. Hence, these sequence-based methods are a complementary approach for detecting correlated evolution and could be applied genome-wide to provide candidate protein-protein interactions and functional group assignments using just coding sequences.

Diversity-Enhancing Selection Acts on a Female Reproductive Protease Family in Four Subspecies of Drosophila mojavensis

Protein components of the Drosophila male ejaculate are critical modulators of reproductive success, several of which are known to evolve rapidly. Recent evidence of adaptive evolution in female reproductive tract proteins suggests this pattern may reflect sexual selection at the molecular level. Here we explore the evolutionary dynamics of a five-paralog gene family of female reproductive proteases within geographically isolated subspecies of Drosophila mojavensis. Remarkably, four of five paralogs show exceptionally low differentiation between subspecies and unusually structured haplotypes that suggest the retention of old polymorphisms. These gene genealogies are accompanied by deviations from neutrality consistent with diversifying selection. While diversifying selection has been observed among the reproductive molecules of mammals and marine invertebrates, our study provides the first evidence of this selective regime in any Drosophila reproductive protein, male or female.