Females of many species mate with multiple males in a breeding season. One explanation for this behavior is that it facilitates mate choice after copulation. Female choice that occurs after copulation is detectable because not all the female's mates have equal paternity; it is called cryptic female choice because the process is hidden. For example, females may transport sperm from particular males to storage sites near where fertilization occurs, which could result in paternity biased towards those males. Cryptic female choice can have profound effects on the strength and direction of sexual selection, the evolution of mating systems, and the coevolution of female and male reproductive traits that leads to speciation. However, the cryptic nature of postcopulatory female choice has made it extremely difficult to document. The PIs have developed a method to label live sperm with fluorescent particles so that the fate of the sperm can be observed in vivo. By observing both where sperm are stored and which sperm fertilize eggs, it will be possible to measure the extent of cryptic female choice. The work will be conducted with garter snakes and the methods are likely to be valuable for many species.

The PIs will develop and pilot an integrated inquiry-based lecture, field and laboratory unit appropriate for upper-level high school biology students. This program builds on a successful module that the PIs developed for middle-school students. The PIs also intend to sustain their commitment to undergraduate research opportunities by training at least two undergraduates in PCR, genotyping, and paternity analysis.

Project Report

(NSF IOS-1011727) Postcopulatory sexual selection—sperm competition and cryptic female choice—has become a major area of research over the past 40 years. The fitness consequences for both sexes in the period after copulation and before fertilization are considerable, but are obscured within the female reproductive tract. This is especially true 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 one-third of terrestrial vertebrates. In the manuscript "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 been a rich source of theoretical debate. We attempted to address these issues using two populations of red-sided garter snakes (Thamnophis sirtalis parietalis) from Manitoba, Canada. Although our study populations do not show divergence at neutral makers, they do differ markedly in mating aggregation density, with a 10-fold difference between them. Microsatellite markers were used 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 over mating, was also the population that exhibited homotypic sperm precedence. The less dense population showed a distinct postcopulatory male-size advantage. Notably, we also demonstrated that sperm stored within the females’ oviducts over hibernation can produce 20-30% of the offspring in a litter. Copulatory plugs (CPs) are found in many taxa, but the functional significance of them is still debated. Male red-sided garter snakes produce a gelatinous copulatory plug during mating that occludes the opening of the female reproductive tract for approximately two days. In "The role of copulatory 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, while the copulatory plug is produced by the kidney and conveyed through the ureters. We manipulated the delivery of the two fluids separately by ligating the ducts. We found 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 as the protein matrix contains most of the sperm which are liberated as the plug dissolves within the female’s cloaca. A fundamental principle in sperm competition theory is that with greater numbers of sperm there is an increase in the odds of winning in competitions for fertilization success. In "Intraspecific, intra- and interpopulational variation in sperm traits in red-sided garter snakes", we show significant among-male variation in the number of sperm ejaculated, and that male mate-order affects sperm numbers. Male sperm numbers drop significantly from one mating to the next. This has implications for sperm competitiveness, as this species 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 mobility score of the sperm increased on average for a male’s second mating. Therefore, increased sperm quality may compensate for reduced numbers in a competitive context. We also found significant variation in sperm length at both the intra-, interpopulational, and interspecific level. This is important, because total sperm length is correlated with fertilization success in many species. In "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, this conflict may be especially onerous to a female. 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 and plug mass increased in this treatment group as well. This 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.

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Division of Environmental Biology (DEB)
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George W. Gilchrist
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Oregon State University
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