Duplication and selection on abalone sperm lysin in an allopatric population

While gene duplication is a major source of evolutionary novelty, the importance of this process in reproductive protein evolution has not been widely investigated. Here, we report the first known case of gene duplication of abalone sperm lysin in an allopatric subspecies found in the Eastern Atlantic, Haliotis tuberculata coccinea. Mass spectrometry identified both copies of the lysin protein in testis tissue, and 3-dimensional structural modeling suggests that both proteins remain functional. We also detected positive selection acting on both paralogs after duplication and found evidence of a recent selective sweep. Because H. t. coccinea occurs in geographic isolation from other abalone species, these findings suggest that the evolution of lysin is not driven to create reproductive barriers to unfit hybrid formation with an overlapping species. Instead, sexual selection or sexual conflict acting during abalone fertilization could be responsible for the recent positive selection on this protein. The presence of multiple, rapidly evolving lysin genes in H. tuberculata presents an opportunity to study the early stages of diversification of a protein whose function is well understood.     

Temporally variable selection on proteolysis-related reproductive tract proteins in Drosophila

In order to gain further insight into the processes underlying rapid reproductive protein evolution, we have conducted a population genetic survey of 44 reproductive tract-expressed proteases, protease inhibitors, and targets of proteolysis in Drosophila melanogaster and Drosophila simulans. Our findings suggest that positive selection on this group of genes is temporally heterogeneous, with different patterns of selection inferred using tests sensitive at different time scales. Such variation in the strength and targets of selection through time may be expected under models of sexual conflict and/or host-pathogen interaction. Moreover, available functional information concerning the genes that show evidence of selection suggests that both sexual selection and immune processes have been important in the evolutionary history of this group of molecules.     

Evolutionary expressed sequence tag analysis of Drosophila female reproductive tracts identifies genes subjected to positive selection

Genes whose products are involved in reproduction include some of the fastest-evolving genes found within the genomes of several organisms. Drosophila has long been used to study the function and evolutionary dynamics of genes thought to be involved in sperm competition and sexual conflict, two processes that have been hypothesized to drive the adaptive evolution of reproductive molecules. Several seminal fluid proteins (Acps) made in the Drosophila male reproductive tract show evidence of rapid adaptive evolution. To identify candidate genes in the female reproductive tract that may be involved in female-male interactions and that may thus have been subjected to adaptive evolution, we used an evolutionary bioinformatics approach to analyze sequences from a cDNA library that we have generated from Drosophila female reproductive tracts. We further demonstrate that several of these genes have been subjected to positive selection. Their expression in female reproductive tracts, presence of signal sequences/transmembrane domains, and rapid adaptive evolution indicate that they are prime candidates to encode female reproductive molecules that interact with rapidly evolving male Acps.     

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.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

Selection on the Drosophila seminal fluid protein Acp62F

Sperm competition and sexual conflict are thought to underlie the rapid evolution of reproductive proteins in many taxa. While comparative data are generally consistent with these hypotheses, few manipulative tests have been conducted and those that have provided contradictory results in some cases. Here, we use both comparative and experimental techniques to investigate the evolution of the Drosophila melanogaster seminal fluid protein Acp62F, a protease inhibitor for which extensive functional tests have yielded ambiguous results. Using between‐species sequence comparisons, we show that Acp62F has been subject to recurrent positive selection. In addition, we experimentally evolved populations polymorphic for an Acp62F null allele over eight generations, manipulating the opportunities for natural and sexual selection. We found that the Acp62F null allele increased in frequency in the presence of natural selection, with no effect of sexual selection.     

Sexual conflict does not drive reproductive isolation in experimental populations of Drosophila pseudoobscura

Sexual conflict has been predicted to drive reproductive isolation by generating arbitrary but rapid coevolutionary changes in reproductive traits among allopatric populations. A testable prediction of this proposal is that allopatric populations experiencing different levels of sexual conflict should exhibit different levels of reproductive isolation. We tested this prediction using experimentally evolved populations of the promiscuous Drosophila pseudoobscura. We manipulated sexual conflict by enforcing either monogamy, maintaining natural levels of promiscuity, or elevating promiscuity. Within each treatment, we carried out sympatric and allopatric crosses using replicated populations and examined pre-zygotic (number of mating pairs, mating speed and copulation duration) and post-zygotic (hybrid inviability and sterility) indicators of reproductive isolation. After 50 generations of selection, none of the measures conformed to predictions of sexual conflict driving reproductive isolation. Our results cannot be explained by lack of genetic variation or weak selection and suggest that sexual conflict may not be a widespread engine of speciation.     

Perspective: sexual conflict and sexual selection: chasing away paradigm shifts

Abstract.— Traditional models of sexual selection propose that partner choice increases both average male and average female fitness in a population. Recent theoretical and empirical work, however, has stressed that sexual conflict may be a potent broker of sexual selection. When the fitness interests of males and females diverge, a reproductive strategy that increases the fitness of one sex may decrease the fitness of the other sex. The chase-away hypothesis proposes that sexual conflict promotes sexually antagonistic, rather than mutualistic, coevolution, whereby manipulative reproductive strategies in one sex are counteracted by the evolution of resistance to such strategies in the other sex. In this paper, we consider the criteria necessary to demonstrate the chase-away hypothesis. Specifically, we review sexual conflict with particular emphasis on the chase-away hypothesis; discuss the problems associated with testing the predictions of the chase-away hypothesis and the extent to which these predictions and the predictions of traditional models of sexual selection are mutually exclusive; discuss misconceptions and mismeasures of sexual conflict; and suggest an alternative approach to demonstrate sexual conflict, measure the intensity of sexually antagonistic selection in a population, and elucidate the coevolutionary trajectories of the sexes.     

Adaptive Evolution in an Avian Reproductive Protein: ZP3

Proteins involved in reproduction appear to be evolving adaptively across taxa. This rapid evolution is thought to be the result of forces involved in sexual selection. One of the most often suggested of these forces is sexual conflict involving sperm competition and polyspermy avoidance. Bird species offer a unique opportunity to test this hypothesis since the avian egg coat tolerates physiological polyspermy, or the penetration of multiple sperm during fertilization, without negative effects on later development. Despite this, and the extensive amount of data gathered on sexual selection in birds, there are limited studies on the patterns of evolution of avian reproductive proteins. Here we present an analysis of the pattern of evolution of Zona Pellucida 3 (ZP3), a protein present on the avian egg coat. We found that, across several galliform and a single anseriform species, ZP3 appears to be diverging by positive adaptive evolution. In an exploratory analysis of portions of the gene in Callipepla californica we also found evidence of a selective sweep at the putative sperm binding region of the protein. In sum, ZP3 in birds, like reproductive proteins in other species, appears to be adaptively evolving. This result suggests that polyspermy avoidance is not sufficient to explain positive Darwinian selection in reproductive proteins across taxonomic groups. Clearly, the inclusion of bird species in the study of reproductive proteins across taxa promises to add greatly to the discussion of the factors driving the widespread phenomenon of adaptive evolution in reproductive proteins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

No evidence that experimental manipulation of sexual conflict drives premating reproductive isolation in Drosophila melanogaster

Theoretical models predict that sexual conflict can drive reproductive isolation by decreasing the probability of matings between individuals from allopatric populations. A recent study in dung flies supported this prediction. To test the generality of this finding we used replicate lines of Drosophila melanogaster that had been selected under high, medium and low levels of sexual conflict, in which the females had evolved differences in their level of resistance to male-induced harm. We compared the proportion of virgin pairs that mated by set time points, for flies from the same replicate within each sexual conflict level vs. flies from different replicates within each sexual conflict level. The results did not support the prediction that, in D. melanogaster, sexual conflict drives population divergence via changes in female willingness to mate. The results were unlikely to be explained by differential inbreeding or by a lack of response to sexual conflict.     

The evolution of sperm competition genes: The effect of mating system on levels of genetic variation within and between species

It is widely established that proteins involved in reproduction diverge between species more quickly than other proteins. For male sperm proteins, rapid divergence is believed to be caused by postcopulatory sexual selection and/or sexual conflict. Here, we derive the expected levels of gene diversity within populations and divergence between them for male sperm protein genes evolving by postcopulatory, prezygotic fertility competition, i.e. the function imputed for some sperm and seminal fluid genes. We find that, at the mutation‐selection equilibrium, both gene diversity within species and divergence between them are elevated relative to genes with similar selection coefficients expressed by both sexes. We show that their expected level of diversity is a function of the harmonic mean number of mates per female, which affects the strength of fertility selection stemming from male–male sperm competition. Our predictions provide a null hypothesis for distinguishing between other selective hypotheses accounting for the rapid evolution of male reproductive genes.     

Evidence of adaptive evolution of accessory gland proteins in closely related species of the Drosophila repleta group

Accessory gland proteins (Acps) are part of the seminal fluid of Drosophila species. These proteins have important reproductive functions, being responsible for the proper functioning of several steps of the fertilization process. Acps also contribute indirectly for the reproductive success of males by modulating female behavior. Evidence that Acps participate in sperm competition and sexual conflict includes findings that, on average, Acps have fast evolutionary rates, suggestive of adaptive evolution. This is especially true in species of the Drosophila repleta group. Nevertheless, only in a few occasions have robust statistical tests been used to determine whether observed evolutionary rates are in fact due to positive selection on amino acid substitutions between related species. Here we apply maximum likelihood tests for positive selection on 14 Acps of the D. repleta group. To increase statistical robustness, we use at least 8 sequences, all belonging to species of the Drosophila mulleri complex, for each gene analyzed. We found significant evidence of adaptive evolution for 10 of the tested genes. Among these, the ones with a conserved protein domain had positively selected sites within the functional region of the sequence. We also detected one instance of lineage-specific adaptive evolution in a clade formed by 2 sister species.     

Does reproductive isolation evolve faster in larger populations via sexually antagonistic coevolution?

Sexual conflict over reproductive investment can lead to sexually antagonistic coevolution and reproductive isolation. It has been suggested that, unlike most models of allopatric speciation, the evolution of reproductive isolation through sexually antagonistic coevolution will occur faster in large populations as these harbour greater levels of standing genetic variation, receive larger numbers of mutations and experience more intense sexual selection. We tested this in bruchid beetle populations (Callosobruchus maculatus) by manipulating population size and standing genetic variability in replicated lines derived from founders that had been released from sexual conflict for 90 generations. We found that after 19 generations of reintroduced sexual conflict, none of our treatments had evolved significant overall reproductive isolation among replicate lines. However, as predicted, measures of reproductive isolation tended to be greater among larger populations. We discuss our methodology, arguing that reproductive isolation is best examined by performing a matrix of allopatric and sympatric crosses whereas measurement of divergence requires crosses with a tester line.     

Female postmating immune responses,immune system evolution and immunogenic males

Females in many taxa experience postmating activation of their immune system, independently of any genital trauma or pathogenic attack arising from male‐female genital contact. This response has always been interpreted as a product of natural selection as it either prepares the female immune system for antigens arising from an implanted embryo (in the case of placental mammals), or is a “pre‐emptive strike” against infection or injury acquired during mating. While the first hypothesis has empirical support, the second is not entirely satisfactory. Recently, studies that have experimentally dissected the postmating responses of Drosophila melanogaster females point to a different explanation: male reproductive peptides/proteins that have evolved in response to postmating male‐male competition are directly responsible for activating particular elements of the female immune system. Thus, in a broad sense, males may be said to be immunogenic to females. Here, we discuss a possible direct role of sexual selection/sexual conflict in immune system evolution, in contrast to indirect trade‐offs with other life‐history traits, presenting the available evidence from a range of taxa and proposing ways in which the competing hypotheses could be tested. The major implication of this review is that immune system evolution is not only a product of natural selection but also that sexual selection and potentially sexual conflict enforces a direct selective pressure. This is a significant shift, and will compel researchers studying immune system evolution and ecological immunity to look beyond the forces generated by parasites and pathogens to those generated by the male ejaculate.     

Adaptive Evolution of Gamete-Recognition Proteins in Birds

Gamete-recognition proteins have been shown to evolve by positive selection in diverse organism groups, such as marine invertebrates and mammals, although underlying evolutionary mechanisms driving this rapid divergence are poorly understood. However, several hypotheses have been put forward to explain the observed pattern, including different forms of sexual conflict and sperm competition. Because female gametes require more energy to produce than male gametes, female organisms suffer more when fertilisation goes wrong. One process that results in a failed mammalian fertilisation is polyspermy, when >1 sperm fertilises the egg. However in birds, there is no such sexual conflict because multiple sperm typically bind and fuse with the egg. If sexual conflict driven by polyspermy avoidance is important for the evolution of gamete-recognition proteins in vertebrates, we expect to find positive selection in the genes to be less pronounced in birds. We therefore sequenced six genes (ZP1, ZP2, ZP4, ZPAX, CD9, and Acrosin) encoding gamete-recognition proteins in several bird species to test for positive selection. For comparison, we also analysed ortologous sequences in a set of mammalian species. We found no major differences in the occurrence of adaptive evolution and the strength of selection between bird and mammal orthologs. From this we conclude that polyspermy avoidance does not act as the main underlying evolutionary force shaping the rate of evolution in these genes. We discuss other possible processes that could explain positive selection of gamete-recognition proteins in birds and mammals, such as hybridisation avoidance, cryptic female choice, and postcopulatory sperm competition.     

Shall we dance or shall we fight? Using DNA sequence data to untangle controversies surrounding sexual selection.

Population and evolutionary genetics studies have largely benefitted from advances in DNA manipulation and sequencing, as well as DNA data analysis techniques. Molecular evolution studies of male reproductive genes show a pattern of rapid evolution shaped, in some cases, by an adaptive selective process. Despite the large body of data on male reproductive genes, the female side of the story has remained unexplored. The few cases of female egg receptors analyzed also show rapid evolution. However, to disentangle between competing hypotheses on how selection operates on male x female molecular interaction leading to fertilization, we need to find male and female molecules that are partners in fertilization. A conflict model of sexual selection (similar to a host-parasite model) assumes a male-driven system where females are being forced under suboptimal conditions. This predicts that the amount of divergence at a female receptor depends on the amount of divergence among the male reproductive proteins that it binds (i.e., males are leading). Under a classical model of runaway sexual selection, female protein receptors might be the key to the rapid molecular changes observed in male reproductive proteins and higher divergence should be expected among female receptors than among their respective male binding proteins.     

Molecular evolution and population genetic analysis of candidate female reproductive genes in Drosophila

Molecular analyses in several taxa have consistently shown that genes involved in reproduction are rapidly evolving and subjected to positive selection. The mechanism behind this evolution is not clear, but several proposed hypotheses involve the coevolution between males and females. In Drosophila, several male reproductive proteins (Acps) involved in male-male and male-female interactions show evidence of rapid adaptive evolution. What has been missing from the Drosophila literature is the identification and analysis of female reproductive genes. Recently, an evolutionary expressed sequence tag analysis of Drosophila female reproductive tract genes identified 169 candidate female reproductive genes. Many of these candidate genes still await further molecular analysis and independent verification of positive selection. Our goal was to expand our understanding of the molecular evolution of Drosophila female reproductive genes with a detailed polymorphism and divergence study on seven additional candidate female reproductive genes and a reanalysis of two genes from the above study. We demonstrate that 6 candidate female genes of the 9 genes surveyed show evidence of positive selection using both polymorphism and divergence data. One of these proteins (CG17012) is modeled to reveal that the sites under selection fall around and within the active site of this protease, suggesting potential differences between species. We discuss our results in light of potential function as well as interaction with male reproductive proteins.     

A sterile sperm caste protects brother fertile sperm from female-mediated death in Drosophila pseudoobscura

Spermicide (i.e., female-mediated sperm death) is an understudied but potentially widespread phenomenon that has important ramifications for the study of sexual conflict, postcopulatory sexual selection, and fertility [1, 2]. Males are predicted to evolve adaptations against spermicide, but few antispermicidal mechanisms have been definitively identified. One such adaptation may be the enigmatic infertile sperm morphs or "parasperm" produced by many species, which have been hypothesized to protect their fertile brother "eusperm" from spermicide [2, 3]. Here, we show that female Drosophila pseudoobscura reproductive tracts are spermicidal and that the survival of eusperm after exposure to the female tract is highest when males produce many parasperm. This study clarifies the adaptive significance of infertile sperm castes, which has remained elusive in Drosophila and other taxa despite much recent interest [2-8]. We suggest that spermicide and male countermeasures against it are more common than is appreciated currently and discuss how spermicide could drive the evolution of several key male traits, including sperm size and number.