Darwin identified sexual selection as an important evolutionary process resulting from differences among males in their ability to secure mates. In the latter half of the 20th century, it became apparent that females often mate with multiple partners within the same reproductive cycle, leading to the overlap of ejaculates from multiple males and sperm competition for the fertilization of the eggs. Here, I examine how sperm competition has influenced the evolution of Xiphophorus nigrensis, an internally fertilized, livebearing fish with alternative male mating tactics that are dependent upon male size. I find that variation in male tactic is correlated with variation in traits relevant to sperm competition: small males that sneak copulations produce ejaculates with a greater proportion of fertilization-capable sperm (sperm viability) and sperm that is longer-lived following activation compared to large males that court females. Sperm morphology is also divergent between tactics and correlated with sperm performance: smaller males have larger midpieces and midpiece size is positively correlated with sperm velocity and longevity. Social environment also affects ejaculate quality, with sperm velocity rapidly increasing when a small male is exposed to another small male compared to when he is exposed to a large male. Large male ejaculates were invariant across social environments. Next, I demonstrate experimentally that the observed variation in sperm quality has important consequences for the outcome of sperm competition. Males with superior sperm viability sire more offspring, while sperm velocity is negatively associated with sperm competitive ability when sperm are stored within the female prior to fertilization. Finally, I show that sperm competition is likely to have important effects on male reproductive success in the wild by characterizing the genetic mating system of X. nigrensis. I find that 61% of females collected from the Nacimiento Río Choy produce offspring sired by 2-4 males. Paternity is strongly skewed among sires, with an average of 70% of offspring sired by one of the males represented in the brood. These studies illustrate sperm competition can have potent effects on the evolution of animals. / text

Male pregnancy is a complex and energetically costly form of male parental care found exclusively in the fish family Syngathidae, which includes pipefishes, seahorses, and seadragons. The novelty of the male brood pouch raises questions about how this trait evolved and what role it plays in sexual selection. One possibility is that brood pouch evolution was partially shaped by postcopulatory sexual selection. We used the sexually dimorphic Gulf pipefish, Syngnathus scovelli, to test for postcopulatory sexual selection within broods and tradeoffs between successive male pregnancies as a function of female attractiveness. Offspring survivorship within a pregnancy was affected by the size of a male's mate, the number of eggs transferred, and the male's mating history. These results indicate the males invest more resources into broods from large, attractive mates to the detriment of future broods. Next, we investigated the effects of food limitation on male parental care strategies. Our data suggests that male Gulf pipefish sacrifice investment in future reproduction, via somatic growth, in favor of current reproduction. A positive relationship between number of failed eggs and male growth rate in our low-food treatments suggests that males may derive an energetic benefit from unsuccessful eggs in the brood pouch. Finally, we used a paired design, to investigate the effect of perceived female attractiveness on offspring survivorship. We found that, in general, males prefer the largest female available. Within a replicate, we found that the male that preferred their mate more, regardless of stimulus female size, also had higher offspring survivorship in the resulting brood. This result is exciting because it shows not only that cryptic male choice affects offspring survivorship in Gulf pipefish but also that mate preference can have a prolonged effect on an individual’s reproductive success. Ultimately, our data suggests that the evolution of the brood pouch has produced a trait that not only nurtures the offspring but also exert cryptic male choice.

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[Truncated abstract] Despite the costs associated with mating, females of many taxa solicit multiple mates during a single reproductive event (polyandry). Polyandry is clearly adaptive when females gain direct benefits from males at mating. However, polyandry has also been shown to increase female fitness in the absence of direct benefits. Thus, a number of genetic benefit hypotheses have been developed to account for the origin of this behaviour. Although not mutually exclusive, a distinction lays between genetic benefits that propose defense against reproductive failure (nonadditive genetic effects), and those that propose benefits from intrinsic sire effects (additive genetic effects). Nonadditive genetic benefits of polyandry have been documented in a number of species; by soliciting multiple mates females can avoid inbreeding and other forms of incompatibility between parental genotypes. Polyandry may also increase female reproductive success when genetically superior males have greater success in sperm competition, and produce better quality offspring. An inevitable consequence of polyandry is that sperm from rival males will overlap in the female reproductive tract and compete to fertilise the ova. The outcome of sperm competition is typically determined by bias in sperm use by the females, interactions between parental genotypes, and ejaculate characteristics that provide a fertilisation advantage. Thus, sperm competition is recognised as a persuasive force in the evolution of male reproductive traits. Comparative analyses across species, and competitive mating trials within species have suggested that sperm competition can influence the evolution of testis size and sperm production, and both sperm form and sperm function. ... After six generations of selection I observed phenotypic divergence in litter size - litter size increased in the polyandrous lines but not in the monandrous lines. This result was not attributable to inbreeding depression, or environmental/maternal effects associated with mating regime. Genetic benefits associated with polyandry could account for this result if increased litter size were attributable to increased embryo survival. However, males from the polyandrous lineages were subject to sperm competition, and evolved ejaculates with more sperm, suggesting that evolutionary increases in litter size may in part be due to improved male fertility. Finally, Chapter Five is an investigation of the natural variation in levels of polyandry in the wild, and the potential for sperm competition to drive macroevolutionary changes in male reproductive traits among geographically isolated island populations of house mice. I sampled seven island populations of house mice along the coast of Western Australia and, by genotyping pregnant females and their offspring, determined the frequency of multiply sired litters within each population. I applied the frequency of multiple paternity as an index of the risk of sperm competition, and looked for selective responses in testis size and ejaculate traits. I found that the risk of sperm competition predicted testis size across the seven island populations. However, variation in sperm traits was not explained by the risk of sperm competition. I discuss these results in relation to sperm competition theory, and extrinsic factors that influence ejaculate quality.

Sturgeon species (order Acipenseriformes) are prone for interspecific hybridization. Anthropogenic activities in river basins influence sturgeon reproduction by destruction of their natural spawning grounds. Consequently, spawning areas, as well as the time of spawning of sturgeon species overlap and different sturgeon species reproduce concurrently. This increases the probability of meeting of heterospecific gametes and pre-zygotic postcopulatory reproductive barriers, comprising of sperm competition and cryptic female choice, may play an important role in preventing undesirable interspecific hybridization. The goal of this study was to evaluate the role of interspecific sperm competition and cryptic female choice during interspecific hybridization of sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baerii). Reproductive characteristics (fertilization rate and hatching rate) were described in each of experimental and control groups showing similar values for competitive and non-competitive trials. Parentage assignment was performed in hatched larvae using combination of mitochondrial DNA and microsatellite DNA markers. Obtained results revealed higher fertilization success of sterlet spermatozoa, when these competed for fertilization with spermatozoa of Siberian sturgeon. Total reproductive success of starlet spermatozoa was 78.9 % and Siberian sturgeon 21.1 %. Contrary, when spermatozoa did not compete for fertilization, males of analysed species showed equal fertilization success. In the trials, where eggs of both studied species were mixed and fertilized by sperm from each species separately, eggs of any species did not show a tendency to bias fertilization by spermatozoa of conspecific males. Probably, there are no pre-zygotic postcopulatory reproductive barriers that prevent interspecific hybridization of sterlet and Siberian sturgeon at the gametic level.

Sperm phenotype is an essential indicator of the male ejaculate quality and may have a significant impact on male reproductive success. Sperm phenotypes are considerably variable across species but variation is also found among males within species. This thesis examines (1) variation in sperm phenotypes among males in barn swallows (Hirundo rustica), (2) changes in male ejaculate quality with age, (3) relationships between sperm morphology and motility, (4) effects of sperm phenotypes (morphology and motility) on male fertilization success, using a large dataset of 174 observation for 130 males, and (5) the influence of female reproductive environment on sperm motility. From the tested variables, only midpiece length correlated with male age. Older males had shorter midpiece but no relationship between male age and reproductive success was found. Sperm length negatively affected sperm motility and, simultaneosly, relative midpiece length posively correlated with sperm motility. No correlation was found between the male reproductive success and sperm motility, presence of abnormalities, length of outermost tail feathers or age. Males with shorter relative midpiece were more successful in within-pair paternity, and males with shorter sperms but longer relative midpiece were more successful in...

Postcopulatory sexual selection—sperm competition and cryptic female choice—has become a major area of research over the past 40 years. Within this field there are many outstanding questions at every level of analysis, from proximate to ultimate. The fitness consequences for both sexes in the period after copulation and before fertilization are considerable, but are obscured within the female reproductive tract. Our understanding of postcopulatory mechanisms is especially sparse in taxa other than birds and insects. Nearly nothing is known in reptiles except that multiple paternity is common and widespread, and often results from long-term sperm storage across breeding seasons. We present some of the very first data on the determinants of fertilization success in the context of sperm competition in reptiles, a group that accounts for 30% of terrestrial vertebrates. In the first chapter, "Asymmetric gametic isolation between two populations of red-sided garter snakes", we discuss the use of between-population crosses to reveal gametic isolation. The effect of population density and operational sex ratios on mating systems and the speciation process has fueled theoretical debate. We attempted to address these issues using two populations of red-sided garter snakes (Thamnophis sirtalis parietalis) from Manitoba, Canada. Our study populations differ markedly in their density mating aggregations, with a 10-fold difference between them. Using microsatellite markers for paternity analysis of litters produced from within and between population crosses. We found that the population with highest aggregation density, and presumably with the highest level of sexual conflict (i.e., when the evolutionary interests of the sexes differ) over mating, was also the population that exhibited homotypic sperm precedence. The less dense population showed a distinct postcopulatory male-size advantage. We also demonstrated that sperm stored within the female over hibernation can father 20-30% of offspring in a litter. In the second chapter, "Sperm competition and mate-order effects in red-sided garter snakes", we test whether females use mate-order effects to ensure that a larger (fitter) male will sire her offspring. Does that second male should have precedence in sperm competition? We tested for second-male precedence using singly-mated females that mated with a second male. Average proportion of paternity was shared equally among the first (P₁, i.e., proportion of offspring from a litter fathered by the first male to mate) and second males (P₂) to mate, and stored sperm (P[subscript ss]). This may be a case where last male precedence breaks down with more than two males. All females were spring virgins (they had not mated that spring, but may have stored sperm from fall matings); thus sperm stored presumably from fall matings is important in this system. As the interval between matings increased P₁ increased at the expense of P[subscript ss]. As the second male to mate's copulation duration increased, P₁ also increased at the expense of P₂. This last result may indicate female influence over sperm transfer during coerced matings. Copulatory plugs (CPs) are found in many taxa, but the functional significance is debated. Male garter snakes produce a gelatinous copulatory plug during mating that occludes the opening of the female reproductive tract for approximately two days. In chapter three, "Not just a chastity belt: the role of mating plugs in red-sided garter snakes revisited", we experimentally tested the role of the CPs. In snakes, sperm are produced in the testes and delivered through the ductus deferens, and the copulatory plug is thought to be produced by the sexual segment of the kidney and conveyed through the ureter. We manipulated the delivery of the two fluids separately by ligating the ducts. We confirmed that the CP is not formed in ureter-ligated males and that sperm leaks out immediately after copulation. The CP is analogous to a spermatophore. The protein matrix contains most of the sperm which are liberated as the plug dissolves within the female's vaginal pouch. One of the fundamental principles in sperm competition is that increased sperm numbers increase the odds of winning in competitions for fertilization success and males will adjust their ejaculate relative to competition and the quality of his mate. In chapter four, "Sperm depleted males and the unfortunate females who mate with them", we detect significant among-male variation in the number of sperm ejaculated, and that male mate-order reduces sperm numbers. Male sperm numbers drop significantly from one mating to the next, and this has implications for sperm competiveness, as Thamnophis sirtalis exhibits a disassociated reproductive tactic, in that sperm stores are produced outside the breeding season, and thus cannot be replenished after mating. Interestingly, however, the on average the mobility of the sperm increased for a male's second mating. Therefore, increased sperm quality may compensate for reduced numbers in a competitive context. Further, females increase their remating rate when mating with males that are unable to deliver sperm. In chapter five, "Sexual conflict during mating in red-sided garter snakes as evidenced by genital manipulation", we revisited the CP in the context of sexual conflict. Sex-differences in optimal copulation duration can be a source of conflict, as increased copulation duration may be advantageous for males as it delays female remating. Males of many species actively guard females to prevent them from remating, and in some cases males produce copulatory plugs to prevent remating. If precopulatory choice is limited at the time of her first mating, conflict may be especially onerous to a female. The size of the plug is influenced by the copulation duration. We experimentally tested the contribution of male and female control over copulation duration. We ablated the largest basal spine on the male's hemipene and found a reduction in copulation duration and an increase in the variation of plug mass. Further, we anesthetized the female's cloaca and found copulation duration increased, which suggests that males benefit from increased copulation duration while females actively try to reduce copulation duration. Therefore, sexual conflict is manifest in divergent copulation duration optima for males and females. / Graduation date: 2013