Gamete traits influence the variance in reproductive success, the intensity of sexual selection, and the outcome of sexual conflict among congeneric sea urchins

Sea-urchin species differ in susceptibility to sperm limitation and polyspermy, but the influences of gamete traits on reproductive variance, sexual selection, and sexual conflict are unknown. I compared male and female reproductive success of two congeners at natural densities in the sea. The eggs of the species occurring at higher densities, Strongylocentrotus purpuratus, require higher sperm concentrations for fertilization but are more resistant to polyspermy compared to S. franciscanus. Both species show high variance in male fertilization success at all densities and high variance in female success at low densities, but they differ in female variance at high densities, where only S. franciscanus shows high female variance. The intensity of sexual selection based on Bateman gradients is high in males of both species, variable in S. franciscanus females, and low in S. purpuratus females. Strongylocentrotus franciscanus females experience sexual selection at low densities and sexual conflict at high densities. Strongylocentrotus purpuratus may rarely experience sperm limitation and may have evolved to ameliorate sexual conflict. This reduces the variance in female fertilization, providing females with more control over fertilization. Sperm availability influences sexual selection directly by determining sperm-egg encounter probabilities and indirectly through selection on gamete traits that alter reproductive variances.     

The risk of polyspermy in three congeneric sea urchins and its implications for gametic incompatibility and reproductive isolation

Developmental failure caused by excess sperm (polyspermy) is thought to be an important mechanism driving the evolution of gamete-recognition proteins, reproductive isolation, and speciation in marine organisms. However, these theories assume that there is heritable variation in the susceptibility to polyspermy and that this variation is related to the overall affinity between sperm and eggs. These assumptions have not been critically examined. We investigated the relationship between ease of fertilization and susceptibility to polyspermy within and among three congeneric sea urchins. The results from laboratory studies indicate that, both within and among species, individuals and species that produce eggs capable of fertilization at relatively low sperm concentrations are more susceptible to polyspermy, whereas individuals and species producing eggs that require higher concentrations of sperm to be fertilized are more resistant to polyspermy. This relationship sets the stage for selection on gamete traits that depend on sperm availability and for sexual conflict that can influence the evolution of gamete-recognition proteins and eventually lead to reproductive isolation.     

THE QUICK AND THE DEAD? SPERM COMPETITION AND SEXUAL CONFLICT IN SEA

Our view of sperm competition is largely shaped by game-theoretic models based on external fertilizers. External fertilization is of particular interest as it is the ancestral mode of reproduction and as such, relevant to the evolution and maintenance of anisogamy (i.e., large eggs and tiny, numerous sperm). Current game-theoretic models have been invaluable in generating predictions of male responses to sperm competition in a range of internal fertilizers but these models are less relevant to marine broadcast spawners, the most common and archetypal external fertilizers. Broadcast spawners typically have incomplete fertilization due to sperm limitation and/or polyspermy (too many sperm), but the effects of incomplete (<100% fertilization rates) fertilization on game-theoretic predictions are unclear particular with regards to polyspermy. We show that incorporating the effects of sperm concentration on fertilization success changes the predictions of a classic game-theoretic model, dramatically reversing the relationship between sperm competition and the evolutionarily stable sperm release strategy. Furthermore, our results suggest that male and female broadcast spawners are likely to be in conflict at both ends of the sperm environment continuum rather than only in conditions of excess sperm as previously thought. Across the majority of the parameter space we explored, males release either too little to too much sperm for females to achieve complete fertilization. This conflict could result in a coevolutionary race that may have led to the evolution of internal fertilization in marine organisms.     

The relationship between conspecific fertilization success and reproductive isolation among three congeneric sea urchins

Few data are available on the effectiveness of reproductive isolating mechanisms in externally fertilizing taxa. I investigated patterns of conspecific and heterospecific fertilization among three coexisting sea urchin species, Strongylocentrotus droebachiensis, S.franciscanus, and S. purpuratus. In the laboratory, both among and within species, eggs from individual females whose eggs are more easily fertilized by conspecific sperm are also most susceptible to heterospecific fertilization. At one extreme, S. droebachiensis requires an order of magnitude fewer conspecific sperm to fertilize eggs than do the other two species and shows very little distinction between conspecific and heterospecific sperm in no choice experiments. Strongylocentrotus franciscanus has an intermediate susceptibility to fertilization by heterospecific sperm. At the other extreme, S. purpuratus rarely cross-fertilizes. Field observations indicate that S. droebachiensis is often surrounded by heterospecific sea urchins. Genetic analysis of larvae produced during heterospecific spawning events indicate that hybrids are generally produced if male conspecifics are more than 1 m from a spawning female S. droebachiensis. Laboratory cultures indicate that these hybrids suffer high mortality relative to conspecific larvae. Comparisons of reproductive success of S. droebachiensis during single-species and multispecies spawning events indicate that the benefits of producing easily fertilized eggs under conditions of sperm limitation may outweigh the costs of losing some offspring to hybrid fertilization. Patterns of variability in heterospecific fertilization are considered in light of three hypotheses: phylogenetic relatedness, reinforcement selection, and sexual selection.     

Density-dependent sexual selection in external fertilizers: variances in male and female fertilization success along the continuum from sperm limitation to sexual conflict in the sea urchin Strongylocentrotus franciscanus

Sperm competition and female choice are fundamentally driven by gender differences in investment per offspring and are often manifested as differences in variance in reproductive success: males compete and have high variance; most females are mated and have low variance. In marine organisms that broadcast spawn, however, females may encounter either sperm limitation or sperm competition. I measured the fertilization success of male and female Strongylocentrotus franciscanus over a range of population densities using microsatellite markers. Female fertilization success first increased and then decreased with mate density, limited at low density by sperm limitation and at high density by polyspermy. Mate density affected variance in fertilization success in both males and females. In males, the variance in fertilization success increased with mate density. In females, the pattern was more complex. The variance in female success increased similarly to males with increased mate density but then decreased to low levels at intermediate densities, where almost all eggs were fertilized. As density increased further, the female variances again increased as polyspermy lowered average fertilization success. Male and female variances differed only at intermediate densities. At low densities, both sexes may be under selection to increase fertilization success; at intermediate densities, males may compete; and at high densities, both sexes may be under selection to increase success by increasing (males) or decreasing (females) likelihood of fertilization during sexual conflict. Only within a narrow range of densities do patterns of sexual selection mirror those typically noted in internally fertilizing taxa.     

Does egg competition occur in marine broadcast‐spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.     

Density‐dependent patterns of multivariate selection on sperm motility and morphology in a broadcast spawning mussel

Sperm cells exhibit extraordinary phenotypic variation, both among taxa and within individual species, yet our understanding of the adaptive value of sperm trait variation across multiple contexts is incomplete. For species without the opportunity to choose mating partners, such as sessile broadcast spawning invertebrates, fertilization depends on gamete interactions, which in turn can be strongly influenced by local environmental conditions that alter the concentration of sperm and eggs. However, the way in which such environmental factors impact phenotypic selection on functional gamete traits remains unclear in most systems. Here, we analyze patterns of linear and nonlinear multivariate selection under experimentally altered local sperm densities (densities within the capture zone of eggs) on a range of functionally important sperm traits in the broadcast spawning marine mussel, Mytilus galloprovincialis. Specifically, we assay components of sperm motility and morphology across two fertilization environments that simulate either sperm limitation (when there are too few sperm to fertilize all available eggs), or sperm saturation (when there are many more sperm than required for fertilization, and the risk of polyspermy and embryonic failure is heightened). Our findings reveal that the strength, form, and targets of selection on sperm depend on the prevailing fertilization environment. In particular, our analyses revealed multiple significant axes of nonlinear selection on sperm motility traits under sperm limitation, but only significant negative directional selection on flagellum length under sperm saturation. These findings highlight the importance of local sperm densities in driving the adaptation of sperm phenotypes, particularly those related to sperm motility, in broadcast spawning invertebrates.     

Sperm Swimming Velocity Predicts Competitive Fertilization Success in the Green Swordtail Xiphophorus helleri

Sperm competition is expected to favour the evolution of traits that influence the performance of sperm when they compete to fertilize a female''s eggs. While there is considerable evidence that selection favours increases in sperm numbers, much less is known about how sperm quality contributes towards competitive fertilization success. Here, we determine whether variation in sperm quality influences competitive fertilization success in the green swordtail Xiphophorus helleri, a highly promiscuous livebearing fish. We use artificial insemination as a method of controlled sperm delivery and show that sperm swimming velocity is the primary determinant of fertilization success when ejaculates from two males compete to fertilize a female''s eggs. By contrast, we found no evidence that sperm length had any effect on siring success. We also found no evidence that pre- and postcopulatory sexual traits were phenotypically integrated in this species, suggesting that the previous observation that reproductive skew favours males with high mating rates is unlikely to be due to any direct association between sperm quality and male sexual ornamentation.     

RAPID EVOLUTION OF ASYMMETRIC REPRODUCTIVE INCOMPATIBILITIES IN STALK‐EYED FLIES

The steps by which isolated populations acquire reproductive incompatibilities remain poorly understood. One potentially important process is postcopulatory sexual selection because it can generate divergence between populations in traits that influence fertilization success after copulation. Here we present a comprehensive analysis of this form of reproductive isolation by conducting reciprocal crosses between variably diverged populations of stalk‐eyed flies (Teleopsis dalmanni). First, we measure seven types of reproductive incompatibility between copulation and fertilization. We then compare fertilization success to hatching success to quantify hybrid inviability. Finally, we determine if sperm competition acts to reinforce or counteract any incompatibilities. We find evidence for multiple incompatibilities in most crosses, including failure to store sperm after mating, failure of sperm to reach the site of fertilization, failure of sperm to fertilize eggs, and failure of embryos to develop. Local sperm have precedence over foreign sperm, but this effect is due mainly to differences in sperm transfer and reduced hatching success. Crosses between recently diverged populations are asymmetrical with regard to the degree and type of incompatibility. Because sexual conflict in these flies is low, postcopulatory sexual selection, rather than antagonistic coevolution, likely causes incompatibilities due to mismatches between male and female reproductive traits.     

Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

Effects of the volatile anesthetic halothane on fertilization and early development in the sea urchin Lytechinus variegatus: evidence that abnormal development is due to polyspermy

The volatile anesthetic halothane, when present at fertilization, dose-dependently increases the incidence of abnormally developing sea urchin embryos at the first cell division. Microscopic examinations of eggs stained with aceto-orcein or the DNA fluorochrome bisbenzimide and direct observations on isolated sperm aster complexes show that halothane induces polyspermy (multiple sperm entry) when present at fertilization. Experimental evidence suggests that anesthetic-induced polyspermy involves impairment of both the fast (electrically mediated) and slow (morphological) blocks to multiple sperm entry. These observations clearly show that relatively brief exposures to halothane at fertilization cause polyspermy and that this effect is almost certainly responsible for the ensuing abnormal development observed at the first cell division.     

Sperm competition risk generates phenotypic plasticity in ovum fertilizability

Theory predicts that sperm competition will generate sexual conflict that favours increased ovum defences against polyspermy. A recent study on house mice has shown that ovum resistance to fertilization coevolves in response to increased sperm fertilizing capacity. However, the capacity for the female gamete to adjust its fertilizability as a strategic response to sperm competition risk has never, to our knowledge, been studied. We sourced house mice (Mus domesticus) from natural populations that differ in the level of sperm competition and sperm fertilizing capacity, and manipulated the social experience of females during their sexual development to simulate conditions of either a future ‘risk’ or ‘no risk’ of sperm competition. Consistent with coevolutionary predictions, we found lower fertilization rates in ova produced by females from a high sperm competition population compared with ova from a low sperm competition population, indicating that these populations are divergent in the fertilizability of their ova. More importantly, females exposed to a ‘risk’ of sperm competition produced ova that had greater resistance to fertilization than ova produced by females reared in an environment with ‘no risk’. Consequently, we show that variation in sperm competition risk during development generates phenotypic plasticity in ova fertilizability, which allows females to prepare for prevailing conditions during their reproductive life.     

Life-history consequences of investment in free-spawned eggs and their accessory coats

The optimal trade-off between offspring size and number can depend on details of the mode of reproduction or development. In marine organisms, broadcast spawning is widespread, and external coats are a common feature of spawned eggs. Egg jelly coats are thought to influence several aspects of fertilization and early development, including the size of the target for sperm, fertilization efficiency, egg suspension time, polyspermy, embryo survival, and fecundity. These costs and benefits of investment in jelly result in trade-offs that can influence optimal reproductive allocation and the evolution of egg size. I develop an optimization model that sequentially incorporates assumptions about the function of egg coats in fertilization. The model predicts large variation in coat size and limited variation in ovum size under a broad range of conditions. Heterogeneity among spawning events further limits the range of ovum sizes predicted to evolve under sperm limitation. In contrast, variation in larval mortality predicts a broad range of optimal ovum sizes that more closely reflects natural variation among broadcast-spawning invertebrates. By decoupling physical and energetic size, egg coats can enhance fertilization, maintain high fecundity, and buffer the evolution of ovum size from variation in spawning conditions.     

DOES BATEMAN'S PRINCIPLE APPLY TO BROADCAST-SPAWNING ORGANISMS? EGG TRAITS INFLUENCE IN SITU FERTILIZATION RATES AMONG CONGENERIC SEA URCHINS

Evolutionary biologists generally invoke male competition and female choice as mechanisms driving sexual selection. However, in broadcast-spawning organisms sperm may be limiting and females may compete, in the Darwinian sense, for increased mating success. In this study, I investigate how species differences in egg and sperm traits result in different patterns of fertilization among three closely related sea urchins (Strongylocentrotus purpuratus, S. franciscanus, and S. droebachiensis). Field studies demonstrate that all three species achieve similar percentages of eggs fertilized when eggs and sperm are released simultaneously. However, when sperm must disperse before encountering eggs, differences arise among species such that those with the smaller eggs and faster but shorter-lived sperm achieve relatively fewer fertilizations than do species with larger eggs and slower but longer-lived sperm. A field hybridization experiment, field estimates of sperm dispersal, correlations of egg size to field rates of fertilization, laboratory studies of fertilization kinetics, and a simulation model all suggest that it is attributes of the egg (probably egg size) that are responsible for the differences. These patterns of fertilization match the species' patterns of dispersion; species that do well only when sperm and eggs are released in close proximity are more aggregated, species that do relatively well when sperm and eggs are released farther apart are more dispersed. These results are consistent with the notion that eggs of different species are adapted to maximize reproductive success under different degrees of sperm limitation and suggest that male competition and female choice may not be an appropriate dichotomy in broadcast-spawning organisms.     

Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro

The consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre-fertilization co-culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co-culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm-oviduct cell co-culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre-fertilization co-culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co-culture.     

Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species

Ascidians (invertebrate chordates) are very abundant in many marine subtidal areas. They often live in dense multispecies clumps; thus, interspecific competition for space may be intense. Although most noncolonial species are broadcast spawners, their eggs can be fertilized only by sperm of the same species (1). Multiple fertilization is lethal and all animals have evolved blocks to polyspermy. Ascidian eggs block polyspermy by enzymatic (2) and electrical mechanisms (3). Sperm bind to N-acetylglucosamine groups on the vitelline coat (4, 5, 6, 7). Follice cells surrounding the vitelline coat release N-acetylglucosaminidase during egg activation (8), preventing the binding of all sperm but a few (2). I show here that this interaction is not species-specific; sperm from one species can cause glycosidase release from follicle cells of a second species. Furthermore, once glycosidase release has been induced, the subsequent addition of sperm from the egg-producing species fails to fertilize a substantial proportion of these eggs. This leads to the hypothesis that sperm from one species of ascidian can interfere with fertilization of a second species. While intraspecific sperm competition has been well documented in several taxa (9, 10), this is the first record of sperm competition between species, or interspecific sperm competition.     

The early sperm gets the good egg: mating order effects in free spawners

Mating order can have important consequences for the fertilization success of males whose ejaculates compete to fertilize a clutch of eggs. Despite an excellent body of literature on mating-order effects in many animals, they have rarely been considered in marine free-spawning invertebrates, where both sexes release gametes into the water column. In this study, we show that in such organisms, mating order can have profound repercussions for male reproductive success. Using in vitro fertilization for two species of sea urchin, we found that the 'fertilization history' of a clutch of eggs strongly influenced the size distribution of unfertilized eggs, and consequently the likelihood that they will be fertilized. Males that had first access to a batch of eggs enjoyed elevated fertilization success because they had privileged access to the largest and therefore most readily fertilizable eggs within a clutch. By contrast, when a male's sperm were exposed to a batch of unfertilized eggs left over from a previous mating event, fertilization rates were reduced, owing to smaller eggs remaining in egg clutches previously exposed to sperm. Because of this size-dependent fertilization, the fertilization history of eggs also strongly influenced the size distribution of offspring, with first-spawning males producing larger, and therefore fitter, offspring. These findings suggest that when there is variation in egg size, mating order will influence not only the quantity but also the quality of offspring sired by competing males.     

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.     

Sperm velocity and longevity trade off each other and influence fertilization in the sea urchin Lytechinus variegatus

The theoretical prediction that fast sperm should be more effective at fertilizing eggs has never been documented empirically. Interspecific comparisons suggest an inverse relationship between sperm velocity and sperm longevity but this trade-off has never been demonstrated within a species. Here I investigate how sperm velocity and sperm longevity influence the patterns of fertilization in the sea urchin Lytechinus variegatus. In the laboratory I examined 11 male female pairs of sea urchins for variation in sperm velocity and sperm longevity, and determined the correlations of these traits with the percentage of eggs fertilized with serially diluted sperm. Males with faster sperm had higher rates of fertilization than males with slower sperm. Within individual males, as sperm aged they slowed down and showed a reduced percentage activity and lower rates of fertilization. Across males, the average velocity of freshly spawned sperm was inversely related to sperm longevity. These results establish the possibility that sperm traits are adapted for varying conditions along a continuum from sperm limitation to sperm competition.     

Efficiency of gamete usage in nature: sperm storage, fertilization and polyspermy

Gamete production for both males and females can be energetically expensive such that selection should maximize fertilization opportunities while minimizing fertilization costs. In laboratory studies of Drosophila reproduction, however, the failure of eggs to yield adult progeny can be quite high, suggesting that female control over gamete utilization is surprisingly inefficient. We examined gamete utilization in D. pseudoobscura from nature and compared our observations to those for laboratory populations. In natural populations 100% of oviposited eggs effectively produce adult progeny, and fertilization is exclusively monospermic, indicating that in nature, D. pseudoobscura females maintain a very strict control over their reproduction such that gamete usage is extremely efficient. The potential reasons for the inefficient gamete utilization in the laboratory, as well as the potential impact on laboratory studies of sperm competition, sexual conflict, and the evolution of reproductive barriers are discussed. Furthermore, in this sperm-heteromorphic species, our observations show definitively that in nature, as well as in the laboratory, only the long sperm morph participates in fertilization.