How sexual selection can drive the evolution of costly sperm ornamentation.

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male-male competition and female choice as discrete processes.

A putative novel role for Eip74EF in male reproduction in promoting sperm elongation at the cost of male fecundity.

Spermatozoa are the most morphologically variable cell type, yet little is known about genes controlling natural variation in sperm shape. Drosophila fruit flies have the longest sperm known, which are evolving under postcopulatory sexual selection, driven by sperm competition and cryptic female choice. Long sperm outcompete short sperm but primarily when females have a long seminal receptacle (SR), the primary sperm storage organ. Thus, the selection on sperm length is mediated by SR length, and the two traits are coevolving across the Drosophila lineage, driven by a genetic correlation and fitness advantage of long sperm and long SR genotypes in both males and females. Ecdysone-induced protein 74EF (Eip74EF) is expressed during postmeiotic stages of spermatogenesis when spermatid elongation occurs, and we found that it is rapidly evolving under positive selection in Drosophila. Hypomorphic knockout of the E74A isoform leads to shorter sperm but does not affect SR length, suggesting that E74A may be involved in promoting spermatid elongation but is not a genetic driver of male-female coevolution. We also found that E74A knockout has opposing effects on fecundity in males and females, with an increase in fecundity for males but a decrease in females, consistent with its documented role in oocyte maturation. Our results suggest a novel function of Eip74EF in spermatogenesis and demonstrates that this gene influences both male and female reproductive success. We speculate on possible roles for E74A in spermatogenesis and male reproductive success.

Costs and benefits of giant sperm and sperm storage organs in Drosophila melanogaster.

In Drosophila, long sperm are favoured in sperm competition based on the length of the female's primary sperm storage organ, the seminal receptacle (SR). This sperm-SR interaction, together with a genetic correlation between the traits, suggests that the coevolution of exaggerated sperm and SR lengths may be driven by Fisherian runaway selection. Here, we explore the costs and benefits of long sperm and SR genotypes, both in the sex that carries them and in the sex that does not. We measured male and female fitness in inbred lines of Drosophila melanogaster derived from four populations previously selected for long sperm, short sperm, long SRs or short SRs. We specifically asked: What are the costs and benefits of long sperm in males and long SRs in females? Furthermore, do genotypes that generate long sperm in males or long SRs in females impose a fitness cost on the opposite sex? Answers to these questions will address whether long sperm are an honest indicator of male fitness, male post-copulatory success is associated with male precopulatory success, female choice benefits females or is costly, and intragenomic conflict could influence evolution of these traits. We found that both sexes have increased longevity in long sperm and long SR genotypes. Males, but not females, from long SR lines had higher fecundity. Our results suggest that sperm-SR coevolution is facilitated by both increased viability and indirect benefits of long sperm and SRs in both sexes.

Low-mobility sperm phenotype in the domestic turkey: Impact on sperm morphometry and early embryonic death.

The sperm mobility assay measures the ability of sperm to swim through a dense layer of Accudenz® , and the sperm mobility phenotype has been shown to predict fertility and other sperm performance traits in roosters and turkeys. In this study, we examined turkey sperm morphometry and rates of early embryonic death associated with high- and low-mobility semen. We also assessed whether the hypo-osmotic stress test, which evaluates the structural integrity of the sperm plasma membrane, may be used as a faster and simpler assay for sperm mobility and viability. We confirmed previous work that found that high-mobility sperm are faster and swim more linearly than low-mobility sperm, and that mobility traits were repeatable within males. In contrast to previous studies, we did not find higher rates of fertility, but low-mobility sperm was associated with higher rates of early embryonic death, though this trend was not significant. High-mobility sperm had longer sperm heads, explained by longer nuclei, despite shorter acrosomes. Although these sperm were faster, midpiece length and flagellum length did not differ between high- and low-mobility sperm. Finally, mobility was not found to be associated with sperm performance in the hypo-osmotic stress test.

Paternal diet affects differential gene expression, but not sperm competition, in sons.

Parental environment can widely influence offspring phenotype, but paternal effects in the absence of parental care remain poorly understood. We asked if protein content in the larval diet of fathers affected paternity success and gene expression in their sons. We found that males reared on high-protein diet had sons that fared better during sperm competition, suggesting that postcopulatory sexual selection is subject to transgenerational paternal effects. Moreover, immune response genes were downregulated in sons of low-protein fathers, while genes involved in metabolic and reproductive processes were upregulated.

Female mediation of competitive fertilization success in Drosophila melanogaster.

How females store and use sperm after remating can generate postcopulatory sexual selection on male ejaculate traits. Variation in ejaculate performance traits generally is thought to be intrinsic to males but is likely to interact with the environment in which sperm compete (e.g., the female reproductive tract). Our understanding of female contributions to competitive fertilization success is limited, however, in part because of the challenges involved in observing events within the reproductive tract of internally fertilizing species while discriminating among sperm from competing males. Here, we used females from crosses among isogenic lines of Drosophila melanogaster, each mated to two genetically standardized males (the first with green- and the second with red-tagged sperm heads) to demonstrate heritable variation in female remating interval, progeny production rate, sperm-storage organ morphology, and a number of sperm performance, storage, and handling traits. We then used multivariate analyses to examine relationships between this female-mediated variation and competitive paternity. In particular, the timing of female ejection of excess second-male and displaced first-male sperm was genetically variable and, by terminating the process of sperm displacement, significantly influenced the relative numbers of sperm from each male competing for fertilization, and consequently biased paternity. Our results demonstrate that females do not simply provide a static arena for sperm competition but rather play an active and pivotal role in postcopulatory processes. Resolving the adaptive significance of genetic variation in female-mediated mechanisms of sperm handling is critical for understanding sexual selection, sexual conflict, and the coevolution of male and female reproductive traits.

Alternative mating tactics in the yellow dung fly: resolving mechanisms of small-male advantage off pasture.

Recent work suggests that the yellow dung fly mating system may include alternative patroller-competitor mating tactics in which large males compete for gravid females on dung, whereas small, non-competitive males search for females at foraging sites. Small males obtain most matings off pasture, yet the behavioural mechanism(s) giving rise to this pattern are unknown. We investigated the male and female behaviours that determine mating success in this environment by conducting field mating experiments and found small males to benefit from several attributes specific to the off-pasture mating environment. First, small males from foraging sites exhibited higher mating propensity, indicating that large males away from dung may be depleted of energy and/or sperm. Second, small males were more discriminating, being significantly less likely to attempt with non-gravid females, which are absent on dung but common off pasture. Third, non-gravid females were generally more likely to actively struggle and reject mating attempts; however, such behaviours occurred disproportionately more often with large males. Female Scathophaga stercoraria thus appear to preferentially mate with small males when off pasture. These findings challenge assumptions about male-female interactions in systems with alternative mating tactics and reveal hidden processes that may influence selection patterns in the field.

An analytical framework for estimating fertilization bias and the fertilization set from multiple sperm-storage organs.

How sperm from competing males are used to fertilize eggs is poorly understood yet has important implications for postcopulatory sexual selection. Sperm may be used in direct proportion to their numerical representation within the fertilization set or with a bias toward one male over another. Previous theoretical treatments have assumed a single sperm-storage organ, but many taxa possess multiple organs or store sperm within multiple regions of the reproductive tract. In Drosophila, females store sperm in two distinct storage organ types: the seminal receptacle (SR) and the paired spermathecae. Here, we expand previous "raffle" models to describe "fertilization bias" independently for sperm within the SR and the spermathecae and estimate the fertilization set based on the relative contribution of sperm from the different sperm-storage organ types. We apply this model to three closely related species to reveal rapid divergence in the fertilization set and the potential for female sperm choice.

Genome-wide polymorphisms show unexpected targets of natural selection.

Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (F(ST)) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

How female × male and male × male interactions influence competitive fertilization in Drosophila melanogaster.

How males and females contribute to joint reproductive success has been a long-standing question in sexual selection. Under postcopulatory sexual selection, paternity success is predicted to derive from complex interactions among females engaging in cryptic female choice and males engaging in sperm competition. Such interactions have been identified as potential sources of genetic variation in sexually selected traits but are also expected to inhibit trait diversification. To date, studies of interactions between females and competing males have focused almost exclusively on genotypes and not phenotypic variation in sexually selected traits. Here, we characterize within- and between-sex interactions in Drosophila melanogaster using isogenic lines with heritable variation in both male and female traits known to influence competitive fertilization. We confirmed, and expanded on, previously reported genotypic interactions within and between the sexes, and showed that several reproductive events, including sperm transfer, female sperm ejection, and sperm storage, were explained by two- and three-way interactions among sex-specific phenotypes. We also documented complex interactions between the lengths of competing males' sperm and the female seminal receptacle, which are known to have experienced rapid female-male co-diversification. Our results highlight the nonindependence of sperm competition and cryptic female choice and demonstrate that complex interactions between the sexes do not limit the ability of multivariate systems to respond to directional sexual selection.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

Size-dependent alternative male mating tactics in the yellow dung fly, Scathophaga stercoraria.

Whenever males can monopolize females and/or resources used by females, the opportunity for sexual selection will be great. The greater the variation among males in reproductive success, the greater the intensity of selection on less competitive males to gain matings through alternative tactics. In the yellow dung fly, Scathophaga stercoraria, males aggressively compete for access to receptive, gravid females on fresh dung. Larger males are better able to acquire mates and to complete copulation successfully and guard the female throughout oviposition. Here we demonstrate that when an alternative resource is present where females aggregate (i.e. apple pomace, where both sexes come to feed), smaller males will redirect their searching for females from dung to the new substrate. In addition, we identify a class of particularly small males on the alternative substrate that appears never to be present searching for females on or around dung. Smaller males were found to have a mating 'advantage' on pomace, in striking contrast to the pattern observed on dung, providing further support for the existence of an alternative male reproductive tactic in this species.

Intraspecific divergence in sperm morphology of the green sea urchin, Strongylocentrotus droebachiensis: implications for selection in broadcast spawners.

BACKGROUND: Sperm morphology can be highly variable among species, but less is known about patterns of population differentiation within species. Most studies of sperm morphometric variation are done in species with internal fertilization, where sexual selection can be mediated by complex mating behavior and the environment of the female reproductive tract. Far less is known about patterns of sperm evolution in broadcast spawners, where reproductive dynamics are largely carried out at the gametic level. We investigated variation in sperm morphology of a broadcast spawner, the green sea urchin (Strongylocentrotus droebachiensis), within and among spawnings of an individual, among individuals within a population, and among populations. We also examined population-level variation between two reproductive seasons for one population. We then compared among-population quantitative genetic divergence (QST) for sperm characters to divergence at neutral microsatellite markers (FST). RESULTS: All sperm traits except total length showed strong patterns of high diversity among populations, as did overall sperm morphology quantified using multivariate analysis. We also found significant differences in almost all traits among individuals in all populations. Head length, axoneme length, and total length had high within-male repeatability across multiple spawnings. Only sperm head width had significant within-population variation across two reproductive seasons. We found signatures of directional selection on head length and head width, with strong selection possibly acting on head length between the Pacific and West Atlantic populations. We also discuss the strengths and limitations of the QST-FST comparison. CONCLUSION: Sperm morphology in S. droebachiensis is highly variable, both among populations and among individuals within populations, and has low variation within an individual across multiple spawnings. Selective pressures acting among populations may differ from those acting within, with directional selection implicated in driving divergence among populations and balancing selection as a possible mechanism for producing variability among males. Sexual selection in broadcast spawners may be mediated by different processes from those acting on internal fertilizers. Selective divergence in sperm head length among populations is associated with ecological differences among populations that may play a large role in mediating sexual selection in this broadcast spawner.

Postmating Female Control: 20 Years of Cryptic Female Choice.

Cryptic female choice (CFC) represents postmating intersexual selection arising from female-driven mechanisms at or after mating that bias sperm use and impact male paternity share. Although biologists began to study CFC relatively late, largely spurred by Eberhard's book published 20 years ago, the field has grown rapidly since then. Here, we review empirical progress to show that numerous female processes offer potential for CFC, from mating through to fertilization, although seldom has CFC been clearly demonstrated. We then evaluate functional implications, and argue that, under some conditions, CFC might have repercussions for female fitness, sexual conflict, and intersexual coevolution, with ramifications for related evolutionary phenomena, such as speciation. We conclude by identifying directions for future research in this rapidly growing field.

Ecological correlates of population genetic structure: a comparative approach using a vertebrate metacommunity.

Identifying ecological factors associated with population genetic differentiation is important for understanding microevolutionary processes and guiding the management of threatened populations. We identified ecological correlates of several population genetic parameters for three interacting species (two garter snakes and an anuran) that occupy a common landscape. Using multiple regression analysis, we found that species interactions were more important in explaining variation in population genetic parameters than habitat and nearest-neighbour characteristics. Effective population size was best explained by census size, while migration was associated with differences in species abundance. In contrast, genetic distance was poorly explained by the ecological correlates that we tested, but geographical distance was prominent in models for all species. We found substantially different population dynamics for the prey species relative to the two predators, characterized by larger effective sizes, lower gene flow and a state of migration-drift equilibrium. We also identified an escarpment formed by a series of block faults that serves as a barrier to dispersal for the predators. Our results suggest that successful landscape-level management should incorporate genetic and ecological data for all relevant species, because even closely associated species can exhibit very different population genetic dynamics on the same landscape.

No inbreeding depression in sperm storage ability or offspring viability in Drosophila melanogaster females.

Mating between relatives usually decreases genetic quality of progeny as deleterious recessive alleles are expressed in inbred individuals. Inbreeding degrades sperm traits but its effects on sperm storage and fate within females are currently unknown. We quantified the relationship between the degrees of inbreeding relevant to natural populations (f=0, 0.25 and 0.50) and the number of sperm inseminated and stored, sperm swimming speed, long-term sperm viability while in storage, pattern of sperm precedence, mating latency, and offspring viability of female Drosophila melanogaster. The use of transgenic flies that have either red or green fluorescent sperm heads allowed us to distinguish two ejaculates in the female reproductive tract and facilitated quantification of sperm storage and use traits. We found no inbreeding depression in either long- or short-term sperm storage ability. The most inbred females exhibited significantly longer mating latency, which could be explained by males preferring to mate with outbred females. On the other hand, as no evidence for cryptic male choice in the form of ejaculate tailoring of sperm number was found, the most inbred females might just be less eager to mate. We also found no evidence that the degree of maternal inbreeding influenced offspring viability. Comparison with a contemporaneous study of male inbreeding consequences for ejaculate quality suggests that inbreeding depression is more severe in males than in females in our study population.

Postcopulatory sexual selection generates speciation phenotypes in Drosophila.

BACKGROUND: Identifying traits that reproductively isolate species, and the selective forces underlying their divergence, is a central goal of evolutionary biology and speciation research. There is growing recognition that postcopulatory sexual selection, which can drive rapid diversification of interacting ejaculate and female reproductive tract traits that mediate sperm competition, may be an engine of speciation. Conspecific sperm precedence (CSP) is a taxonomically widespread form of reproductive isolation, but the selective causes and divergent traits responsible for CSP are poorly understood. RESULTS: To test the hypothesis that postcopulatory sexual selection can generate reproductive isolation, we expressed GFP or RFP in sperm heads of recently diverged sister species, Drosophila simulans and D. mauritiana, to enable detailed resolution of species-specific sperm precedence mechanisms. Between-species divergence in sperm competition traits and mechanisms prompted six a priori predictions regarding mechanisms of CSP and degree of cross asymmetry in reproductive isolation. We resolved four distinct mechanisms of CSP that were highly consistent with predictions. These comprise interactions between multiple sex-specific traits, including two independent mechanisms by which females exert sophisticated control over sperm fate to favor the conspecific male. CONCLUSIONS: Our results confirm that reproductive isolation can quickly arise from diversifying (allopatric) postcopulatory sexual selection. This experimental approach to "speciation phenotypes" illustrates how knowledge of sperm precedence mechanisms can be used to predict the mechanisms and extent of reproductive isolation between populations and species.

Rapid diversification of sperm precedence traits and processes among three sibling Drosophila species.

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice-mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within-population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.