Graduation date: 2006 / Most vertebrates exhibit seasonality in many life history traits. Such seasonal rhythms are temporally organized via the transduction of environmental cues (e.g., photoperiod, temperature) into appropriate endocrine signals. However, among ectothermic vertebrates that undergo continuous winter dormancy, temperature is the only environmental cue available for synchronizing seasonal rhythms. Most intriguing is that in species where reproduction occurs immediately following spring emergence, the associated changes in neurophysiology and behavior that accompany reproduction likely occur during winter dormancy. The purpose of this dissertation research was to explore the mechanisms by which temperature cues affect the chronobiology and seasonal reproduction of red-sided garter snakes (Thamnophis sirtalis parietalis). Because of their roles in circadian organization and energy balance, melatonin and corticosterone are likely hormonal components of these time-keeping systems. I first characterized the interactions between melatonin and corticosterone to better understand the hormonal mechanisms facilitating temperature-induced reproduction. Melatonin and corticosterone additively inhibit reproductive behavior during the spring mating season. Experimental manipulations with a serotonin receptor antagonist suggest the mechanism underlying these effects involves a serotonin-regulated system. Although melatonin does not influence corticosterone responses to capture stress, capture stress significantly increases melatonin concentrations. To investigate the functional significance of these interactions in regulating temperature-induced reproduction, I measured body temperatures of snakes as well as circadian melatonin and corticosterone cycles during winter dormancy and spring emergence using a combination of field and laboratory experiments. Surprisingly, an increase in body temperature is not necessary for emergence from winter dormancy. Rather, critically low temperatures may serve as a zeitgeber entraining an endogenous circannual cycle that regulates emergence. Decreased environmental temperatures, in the absence of changing photoperiod cues, modulate circadian melatonin and corticosterone rhythms during hibernation. Such temperature-induced changes in hormone rhythms may facilitate seasonal reproductive behavior following spring emergence. Furthermore, a phase-shift in corticosterone rhythms during the mating season may regulate the seasonal transition between reproductive and non-reproductive states in red-sided garter snakes. Such studies investigating the environmental and hormonal mechanisms underlying time-keeping systems may provide valuable insight into the potential impact of environmental perturbations (e.g., climate change) on seasonal rhythms in physiology and behavior.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

Movements of animals presumably reflect their changing needs and the changing availability of necessary resources. In cold climates, snakes often make long seasonal migrations between hibernacula and summer habitats, Finding suitably deep hibernacula with minimal delay could be critical. I hypothesized that such animals should have highly developed navigational ability. By contrast, snakes living in mild climates can hibernate in shallow sites and probably do not migrate; if so, they might be expected to show poorly developed orientation mechanisms!. The objectives of this study were to determine movement patterns and navigational ability of garter snakes (Thamnophis) living in a mild climate and compare them with a congeneric population known to be migratory. From 1986 - 1988 I examined, using mark-recapture, movement behaviour of two populations of garter snakes at Spectacle Lake Provincial Park (SLPP) on Vancouver Island, British Columbia, focusing on several components typically associated with migratory behaviour - distances travelled, population directionality, fidelity to seasonal sites, distinctness of seasonal habitats, and route directness. Thamnophis sirtalis, the common garter snake, is the most widely distributed North American snake species and high latitude populations are migratory. Thamnophis ordinoides, the northwestern garter snake, is restricted to the Pacific northwest and migratory behaviour has never been reported. Both species displayed combinations of traits clearly suggesting nonmigratory behaviour. These included short-distance (< 500 m), random movements, a lack of den fidelity, and variation in the maintenance of specific home ranges between successive years. Home ranges overlapped between individuals, averaged less than 0.3 ha measured over a single active season, and were not clearly distinct from denning areas. Although some directionality of movement was evident, it was likely related to foraging strategy and unlike the typical unidirectional movements undertaken by migrating snakes, Sexual and reproductive differences in any of these traits generally were not significant. The navigational abilities of a migratory population of T. sirtalis from Wood Buffalo National Park (WBNP) in northern Alberta were examined as were those of the nonmigratory populations of snakes from SLPP. Displacement studies were carried out during the active seasons of 1986 - 1988 to determine the level of orientational abilities present in each population and to examine potential orientation cues. Snakes were displaced from their home range and tested in an arena under a variety of conditions, The results demonstrated that T. sirtalis from both SLPP and WBNP possessed advanced navigational abilities. Advanced skills may be absent in T. ordinoides. Thamnophis sirtalis at both study sites demonstrated time-compensated solar orientation as determined by 6 hr phase-delayed tests. Pheromone trails produced by recently copulated females (but not unmated females) also provided an orientation guide for displaced WBNP males, but results from SLPP were less conclusive. Thamnophis ordinoides did not respond in a discernible way to either cue. Navigational skills thus vary relatively little between migrating and nonmigrating populations of the same species but may be poorly developed in completely nonmigratory species. / Graduate

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