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.     

Duplication, Selection and Gene Conversion in a Drosophila mojavensis Female Reproductive Protein Family

Protein components of the Drosophila male ejaculate, several of which evolve rapidly, are critical modulators of reproductive success. Recent studies of female reproductive tract proteins indicate they also are extremely divergent between species, suggesting that reproductive molecules may coevolve between the sexes. Our current understanding of intersexual coevolution, however, is severely limited by the paucity of genetic and evolutionary studies on the female molecules involved. Physiological evidence of ejaculate–female coadaptation, paired with a promiscuous mating system, makes Drosophila mojavensis an exciting model system in which to study the evolution of reproductive proteins. Here we explore the evolutionary dynamics of a five-paralog gene family of female reproductive proteases within populations of D. mojavensis and throughout the repleta species group. We show that the proteins have experienced ongoing gene duplication and adaptive evolution and further exhibit dynamic patterns of pseudogenation, copy number variation, gene conversion, and selection within geographically isolated populations of D. mojavensis. The integration of these patterns in a single gene family has never before been documented in a reproductive protein.     

Gene duplication and adaptive evolution of digestive proteases in Drosophila arizonae female reproductive tracts

It frequently has been postulated that intersexual coevolution between the male ejaculate and the female reproductive tract is a driving force in the rapid evolution of reproductive proteins. The dearth of research on female tracts, however, presents a major obstacle to empirical tests of this hypothesis. Here, we employ a comparative EST approach to identify 241 candidate female reproductive proteins in Drosophila arizonae, a repleta group species in which physiological ejaculate-female coevolution has been documented. Thirty-one of these proteins exhibit elevated amino acid substitution rates, making them candidates for molecular coevolution with the male ejaculate. Strikingly, we also discovered 12 unique digestive proteases whose expression is specific to the D. arizonae lower female reproductive tract. These enzymes belong to classes most commonly found in the gastrointestinal tracts of a diverse array of organisms. We show that these proteases are associated with recent, lineage-specific gene duplications in the Drosophila repleta species group, and exhibit strong signatures of positive selection. Observation of adaptive evolution in several female reproductive tract proteins indicates they are active players in the evolution of reproductive tract interactions. Additionally, pervasive gene duplication, adaptive evolution, and rapid acquisition of a novel digestive function by the female reproductive tract points to a novel coevolutionary mechanism of ejaculate-female interaction.     

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.     

Impacts of floral gender and whole-plant gender on floral evolution in Ecballium elaterium (Cucurbitaceae)

Investigation of gender specialization in plants has led to several theories on the evolution of sexual dimorphism: reproductive compensation, based on enhanced reproductive efficiency with gender specialization (flowers should be larger on dioecious plants); Bateman's Principle, based on sex-specific selection (display for pollinator attraction in males and seed set in females); and intersexual floral mimicry, based on mimicry of a reward-providing gender by a non-reward providing gender (reduced dimorphism in dioecious plants due to increased spatial separation of male and female flowers). These theories were evaluated in Ecballium elaterium, which contains two subspecies, elaterium (monoecious) and dioicum (dioecious). Our results show that flowers of the dioecious subspecies are larger and allocate more to reproductive organs than do flowers of the monoecious subspecies. Both subspecies are sexually dimorphic (male flowers larger than female flowers). Variance in flower size among populations is greater in the dioecious subspecies. Finally, there is sufficient genetic variation to enable ongoing response to selection; genetic correlation constraints on independent response of female and male flowers may be stronger in the monoecious subspecies. Our findings provide support for aspects of all three theories, suggesting that the evolution of floral dimorphism is based on a complex interplay of factors.     

No accelerated rate of protein evolution in male-biased Drosophila pseudoobscura genes

Sexually dimorphic traits are often subject to diversifying selection. Genes with a male-biased gene expression also are probably affected by sexual selection and have a high rate of protein evolution. We used SAGE to measure sex-biased gene expression in Drosophila pseudoobscura. Consistent with previous results from D. melanogaster, a larger number of genes were male biased (402 genes) than female biased (138 genes). About 34% of the genes changed the sex-related expression pattern between D. melanogaster and D. pseudoobscura. Combining gene expression with protein divergence between both species, we observed a striking difference in the rate of evolution for genes with a male-biased gene expression in one species only. Contrary to expectations, D. pseudoobscura genes in this category showed no accelerated rate of protein evolution, while D. melanogaster genes did. If sexual selection is driving molecular evolution of male-biased genes, our data imply a radically different selection regime in D. pseudoobscura.     

Signatures of selection and sex-specific expression variation of a novel duplicate during the evolution of the Drosophila desaturase gene family

The tempo and mode of evolution of loci with a large effect on adaptation and reproductive isolation will influence the rate of evolutionary divergence and speciation. Desaturase loci are involved in key biochemical changes in long-chain fatty acids. In insects, these have been shown to influence adaptation to starvation or desiccation resistance and in some cases act as important pheromones. The desaturase gene family of Drosophila is known to have evolved by gene duplication and diversification, and at least one locus shows rapid evolution of sex-specific expression variation. Here, we examine the evolution of the gene family in species representing the Drosophila phylogeny. We find that the family includes more loci than have been previously described. Most are represented as single-copy loci, but we also find additional examples of duplications in loci which influence pheromone blends. Most loci show patterns of variation associated with purifying selection, but there are strong signatures of diversifying selection in new duplicates. In the case of a new duplicate of desat1 in the obscura group species, we show that strong selection on the coding sequence is associated with the evolution of sex-specific expression variation. It seems likely that both sexual selection and ecological adaptation have influenced the evolution of this gene family in Drosophila.     

GENETIC DRIFT IN ANTAGONISTIC GENES LEADS TO DIVERGENCE IN SEX‐SPECIFIC FITNESS BETWEEN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER

Males and females differ in their reproductive roles and as a consequence are often under diverging selection pressures on shared phenotypic traits. Theory predicts that divergent selection can favor the invasion of sexually antagonistic alleles, which increase the fitness of one sex at the detriment of the other. Sexual antagonism can be subsequently resolved through the evolution of sex‐specific gene expression, allowing the sexes to diverge phenotypically. Although sexual dimorphism is very common, recent evidence also shows that antagonistic genetic variation continues to segregate in populations of many organisms. Here we present empirical data on the interaction between sexual antagonism and genetic drift in populations that have independently evolved under standardized conditions. We demonstrate that small experimental populations of Drosophila melanogaster have diverged in male and female fitness, with some populations showing high male, but low female fitness while other populations show the reverse pattern. The between‐population patterns are consistent with the differentiation in reproductive fitness being driven by genetic drift in sexually antagonistic alleles. We discuss the implications of our results with respect to the maintenance of antagonistic variation in subdivided populations and consider the wider implications of drift in fitness‐related genes.     

Speciation in Progress? A Continuum of Reproductive Isolation in <Emphasis Type="Italic">Drosophila Bipectinata</Emphasis>

Incipient species in the early stages of divergence can provide crucial information about the genetic basis of reproductive isolation and the evolutionary forces that promote speciation. In this report, we describe two subspecies of Drosophila bipectinata that show a continuum of reproductive isolation. Crosses between strains of the same subspecies produce fully fertile offspring. At the same time, each subspecies harbors extensive variation for the degree of reproductive isolation from the other subspecies. The percentage of fertile hybrid males varies from 0 to 90%, depending on the origin of parental strains, indicating that the genes responsible for hybrid sterility are not fixed within either subspecies, or even within local populations. Reproductive isolation is non-transitive, so that the extent of hybrid sterility depends on the particular combination of strains. The two subspecies show little or no evidence of genetic differentiation at three nuclear loci, suggesting that they diverged very recently or continue to experience significant levels of gene flow. A hybrid zone between the two subspecies may exist in New Guinea and Northeastern Australia.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

WHAT USE IS AN INFERTILE SPERM? A COMPARATIVE STUDY OF SPERM‐HETEROMORPHIC DROSOPHILA

Sperm size and number are important determinants of male reproductive success. The genus Drosophila exhibits a remarkable diversity of sperm production strategies, including the production of multiple sperm morphs by individual males, a phenomenon called sperm heteromorphism. Sperm-heteromorphic Drosophila species in the obscura group produce large numbers of infertile "parasperm" in addition to fertile eusperm. Parasperm have been hypothesized to perform a number of roles in place of fertilization, predominantly focused on their potential function in postcopulatory sexual selection. However, the evolutionary significance of parasperm remains unknown. Here we measured several male and female morphological, behavioral, and life-history traits in 13 obscura group species to test competing hypotheses of parasperm function using comparative methods. We found that parasperm size was unrelated to female reproductive tract morphology but was negatively related to our two indices of sperm competition, suggesting that postcopulatory sexual selection may indeed have shaped the evolution of parasperm. We also found abundant coevolution between male and female reproductive traits. Some of these relationships have been found in both sperm-monomorphic and sperm-heteromorphic taxa, but others are dissimilar. We discuss the significance of our results to the evolution of reproductive traits and the elusive function of Drosophila parasperm.     

Male genotype influences female reproductive investment in Drosophila melanogaster

In many species, males can influence the amount of resources their mates invest in reproduction. Two favoured hypotheses for this observation are that females assess male quality during courtship or copulation and alter their investment in offspring accordingly, or that males manipulate females to invest heavily in offspring produced soon after mating. Here, we examined whether there is genetic variation for males to influence female short-term reproductive investment in Drosophila melanogaster, a species with strong sexual selection and substantial sexual conflict. We measured the fecundity and egg size of females mated to males from multiple isofemale lines collected from populations around the globe. Although these traits were not strongly influenced by the male's population of origin, we found that 22 per cent of the variation in female short-term reproductive investment was attributable to the genotype of her mate. This is the first direct evidence that male D. melanogaster vary genetically in their proximate influence on female fecundity, egg size and overall reproductive investment.     

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.     

SALIVARY ANDROGEN-BINDING PROTEIN (ABP) MEDIATES SEXUAL ISOLATION IN MUS MUSCULUS

We wanted to determine whether the microevolution of the mouse salivary androgen-binding protein (ABP) Alpha subunit gene (Abpa) could mediate sexual selection and thereby have a potential role in maintaining gene pool integrity where radiating mouse subspecies make secondary contact. This hypothesis is based upon previous work in this laboratory, which has shown that each subspecies apparently has its own allele and that these alleles have a 25-fold excess of nonsynonymous/synonymous base substitutions compared to an average protein under purifying selection. We provide direct evidence for ABP-assortative mate selection in a laboratory setting: Mus musculus domesticus and M. m. musculus female mice recognize and discriminate between the territories of male mice that essentially differ solely in their Abpa genotype and, when the males are present, the female prefers to mate with the one of her own ABP type. The observation that females could differentiate between the territories of the two males when those mice were absent suggests that the males marked their territories with ABP. In this study, we also detected ABP on the pelts of male mice and in their environment. It is likely that the animals apply the protein to their pelts by licking and that it is then deposited in their surroundings. We suggest that females of the two subspecies are able to discriminate between males of those subspecies on the basis of this protein molecule. Mouse salivary ABP might present a worthwhile system with which to study a prezygotic isolation mechanism in a mammal.     

Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, we have little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. In the present study, we estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and show that male mate discrimination accounts for the majority of the current isolation between these species. We further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. Our findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process.     

Functional and morphological diversity of sperm in Drosophila

Unlike mammals, where the males produce huge quantities of tiny spermatozoa, insects, and Drosophila in particular, exhibit a wide range of reproductive strategies. Sperm gigantism in Drosophila deviates from the rules that normally govern anisogamy, i.e. differences in the size and quantity of male and female gametes. Sperm gigantism has driven anatomical, physiological and cytological adaptations that affect the correlated evolution of the male and female reproductive systems, and has led to the evolution of a new structure, the roller, located between the testis and the seminal vesicle, and to sperm coiling to form pellets. The diversification of sperm strategy is investigated in the light of sexual selection processes that occur in the female genital tract after copulation. These processes, which bias paternity, result from interactions either between spermatozoa from different males, or between the spermatozoa and the environment within the female reproductive tract. In Drosophila, increased sperm size does not confer any reproductive advantage on the male. The evolution of sperm gigantism does not seem to be attributable to competition between spermatozoa from different males, as has been shown to occur in some vertebrate species. Alternative mechanisms, such as interactions between spermatozoa and the female reproductive system, are therefore currently viewed as being more likely explanations. In particular, the impact of sperm size on female reproductive physiology is being investigated to find out whether having large spermatozoa increases the likelihood of male reproductive success. Correlated adaptations of the spermatozoa and female storage organs also seem to be a major factor in determining sperm success, and their role in male-female conflicts is discussed briefly.     

Sex in Drosophila mauritiana: a very high level of amino acid polymorphism in a male reproductive protein gene, Acp26Aa

Many genes pertaining to male reproductive functions have been shown to evolve rapidly between species, and evidence increasingly suggest the influence of positive Darwinian selection. The accessory gland protein gene (Acp26Aa) of Drosophila is one such example. In order to understand the mechanism of selection, it is often helpful to examine the pattern of polymorphism. We report here that the level of amino acid polymorphism in the N-terminal quarter of Acp26Aa is high in Drosophila melanogaster and is unprecedented in its sibling species Drosophila mauritiana. We postulate that (1) this N-terminal segment may play a role in sperm competition, and (2) D. mauritiana may have been under much more intense sexual selection than other species. Both postulates have important ramifications and deserve to be tested rigorously.