Marine and freshwater environments support numerous species of teleost fish with a wide and diverse range of reproductive strategies. Despite the considerable interest in fish reproduction, our knowledge regarding ejaculate traits and factors affecting them is limited. Using computer-assisted sperm analysis (CASA) I measured ejaculate traits (sperm swimming speed, motility, path trajectory, longevity and concentration) from sexually mature chinook salmon (Oncorhynchus tshawytscha) activated in freshwater and ovarian fluid. I also looked at these ejaculate traits in relation to measures of male quality (body condition) and investment into reproduction (relative testes mass). Furthermore, I determined the chemical composition of seminal and ovarian fluid and looked at the effect these fluids have on sperm behaviour. A considerable amount of intraspecific variation existed in all ejaculate traits measured, and investment into reproduction (relative testes mass) was dependent on male body condition, as males in better condition had relatively larger testes. However, these males did not have superior quality ejaculates or ejaculates with a higher density of spermatozoa; hence the potential reproductive advantage of having larger relative testes in this species remains unknown and requires further investigation. In addition, a positive relation between sperm longevity and sperm swimming speed was observed defying the expected trade-off between ejaculate traits according to theory. There was also a weak negative trend in our data between body condition and sperm swimming speed, linearity, and longevity. All sperm traits measured were greatly enhanced when activated in a solution containing ovarian fluid (a viscous fluid which is excreted with the egg batch during spawning) from female chinook salmon. Interestingly, sperm swimming speed activated in fresh water only accounted for < 12% of the observed variation in mean sperm swimming speed in ovarian fluid. This result suggests the sperm traits measured in fresh water are not relevant to those same traits measured in ovarian fluid, so caution should be applied when comparing the potential for individual males to fertilize ova when sperm traits are activated in water, especially in studies of sperm competition in an externally fertilising species. Sperm competition between males is known to strongly influence sperm and ejaculate traits, but less is known about female sperm choice after copulation via a process called cryptic female choice (CFC). In CFC, females may have the ability to favour the sperm of one male over another and bias fertilisation accordingly. To test whether ovarian fluid could act as a mechanism of CFC in an externally fertilising fish species, I measured sperm traits from each male activated in the ovarian fluids from different females. I found that mean sperm swimming speed, longevity, and path trajectory differed significantly among males, but most importantly, the pattern of within-male variation in these traits also varied significantly among males in response to different females’ ovarian fluids. This result suggests that ovarian fluid may be a potential mechanism of CFC whereby females differentially enhance the swimming speed of sperm from different males. In addition, I found that sperm longevity was negatively correlated with variation in [Ca²⁺] and [Mg²⁺] concentration in the ovarian fluid, while percent motility increased with increasing concentration of [Mg²⁺]. These observations provide a possible chemical basis for cryptic female choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males and thus their fertilisation success. This finding is particularly exciting, as we may have uncovered a potential mechanism of CFC in an externally fertilising species, which is poorly understood. In addition, results from this study suggest new directions for genetic studies to provide direct evidence for CFC. For example, does sperm selection via ovarian fluid promote favoured genetic combinations that enhance male reproductive success?

Sexual reproduction entails complex co-evolution between the sexes, necessary for successful fertilization, ensuring individual and population-level fitness. Interfertility is the main criterion for species definition and understanding speciation requires detailed studies of reproductive barriers. However, many studies on reproductive barriers are constrained to infer evolutionary processes from patterns. In this thesis, I focus on a hybrid zone between collared and pied flycatchers (Ficedula albicollis and hypoleuca) on the island of Öland, and a trait that is essential for fertilization: sperm. Long-term monitoring of these species, combined with recent advances in molecular tools, allow me to study how complex on-going intersexual and interspecific interactions influence reproductive isolation in this young hybrid zone. I start by exploring the links between pre- and postmating sexual selection within collared flycatchers (paper I and II). I show that secondary sexual characters and indirect mate-choice benefits are tightly linked to physiology (paper I), and that a male’s attractiveness and dominance status dictate which sperm traits are optimal, as a male’s fertilization success depends on an interaction between sperm and display traits (paper II). I then report a source of strong postzygotic isolation between recently diverged collared and pied flycatchers: impaired spermatogenesis resulting in absence of mature sperm cells in hybrid males (paper III). I show however that pied flycatcher females, who are most exposed to hybridization, can mitigate these costs through mechanisms of cryptic female choice impairing heterospecific sperm performance, allowing them to bias paternity towards pure-species offspring (paper IV). Finally, by exploring the testes transcriptomes and sperm proteomes of both species, I highlight the importance of gene and protein regulation mechanisms in facilitating phenotypic divergence between these species (paper V). Thus, my thesis reveals complex interactions between primary and secondary sexual characters in a wild bird and suggests that mechanisms of sexual selection are tightly linked to essential physiological functions. I also show that genetic incompatibilities can evolve rapidly despite low genome-wide levels of divergence but that divergence in regulatory regions and proteins potentially allows fast evolution of molecular mechanisms impairing or preventing costly heterospecific fertilization.

<p>The evolutionary goal of individuals is reproduction and sexual selection favours traits improving reproductive success. When males invest less than females in offspring, males have potentially a higher reproductive rate than females. This typically results in sex-specific reproductive strategies of male-male competition and female choice of mating partner. Under polyandry, sexual selection can continue after copulation as sperm competition and cryptic female choice. This thesis focuses on male and female pre- and post-copulatory reproductive strategies in the promiscuous red junglefowl, <i>Gallus</i> <i>gallus ssp.</i>, and its domestic subspecies the domestic fowl, <i>Gallus gallus</i> <i>domesticus</i>. Males impose high re-mating rates on females, which triggers female resistance in copulations. In addition, when sexual harassment increases, females re-mate at times of day when male mating propensity is lower, to avoid intense sexual harassment. Males allocate sperm supplies differentially according to (i) variation in female polyandry and own competitive ability, (ii) earlier sperm investment in a female, and (iii) female reproductive quality, signalled by female comb size. Males also perform ‘aspermic’ copulations (i.e. copulations with no semen transfer), which inhibit polyandry and in turn reduce sperm competition. In mating opportunities with relatives, males do not avoid inbreeding. However, females avoid inbreeding before copulation through kin recognition and after copulation by selecting against related males’ sperm. These results show that selection on males to re-mate at higher rates than females and copulate indiscriminately according to partner relatedness, trigger counteracting female responses, creating the potential for sexual conflict over fertilisation. Teasing apart pre- and post-copulatory strategies and the contribution of each sex therefore becomes crucial in order to understand the evolution of reproductive strategies and the mechanisms affecting paternity.</p>

The evolutionary goal of individuals is reproduction and sexual selection favours traits improving reproductive success. When males invest less than females in offspring, males have potentially a higher reproductive rate than females. This typically results in sex-specific reproductive strategies of male-male competition and female choice of mating partner. Under polyandry, sexual selection can continue after copulation as sperm competition and cryptic female choice. This thesis focuses on male and female pre- and post-copulatory reproductive strategies in the promiscuous red junglefowl, Gallus gallus ssp., and its domestic subspecies the domestic fowl, Gallus gallus domesticus. Males impose high re-mating rates on females, which triggers female resistance in copulations. In addition, when sexual harassment increases, females re-mate at times of day when male mating propensity is lower, to avoid intense sexual harassment. Males allocate sperm supplies differentially according to (i) variation in female polyandry and own competitive ability, (ii) earlier sperm investment in a female, and (iii) female reproductive quality, signalled by female comb size. Males also perform ‘aspermic’ copulations (i.e. copulations with no semen transfer), which inhibit polyandry and in turn reduce sperm competition. In mating opportunities with relatives, males do not avoid inbreeding. However, females avoid inbreeding before copulation through kin recognition and after copulation by selecting against related males’ sperm. These results show that selection on males to re-mate at higher rates than females and copulate indiscriminately according to partner relatedness, trigger counteracting female responses, creating the potential for sexual conflict over fertilisation. Teasing apart pre- and post-copulatory strategies and the contribution of each sex therefore becomes crucial in order to understand the evolution of reproductive strategies and the mechanisms affecting paternity.

One of the more fascinating Darwinian puzzles in sexual selection is that of copulatory wounding. For those taxa displaying this behavior, and that does not engage in traumatic insemination, the function of wound infliction during copulation remains unknown. In this study, the intention was to evaluate the purpose of copulatory wounding in Drosophila melanogaster and the consequences it might have for male and female fecundity. The present study measured the size and number of wounds that males from different stocks of Darwinian fitness imposed on females, as well as the copulation duration and the resulting fecundity. The number of wounds and the total wound size showed no apparent correlation to fecundity, but a significant difference in variation of the number of wounds inflicted on females was observed between males from two different lines of fitness. Wounded females were also considerably more common than non-wounded females, as were wounds that were paired compared to non-paired copulatory wounds. The results suggests that wounding might be an adaptive male trait and a copulatory courtship interpretable to females, implying a possible case of cryptic female choice. Since most wounds were paired, this implies that a bilaterally symmetrical organ is causing these wounds. Also, wounding was not as important for fecundity as previously thought. / Ett av de mer fascinerande Darwinistiska pusslen inom sexuell selektion är skadande under kopulationen. För de taxa som uppvisar detta beteende, och inte engagerar sig i traumatisk insemination, så är funktionen av kopulationsskador okänd. I denna studie så var avsikten att utvärdera syftet av kopulationsskador hos Drosophila melanogaster och de konsekvenser beteendet kan ha för det resulterande antalet avkomma. Denna studie uppmätte storleken och antalet skador som hanar av olika Darwinistisk fitness vållade honor, samt kopulationstiden och antalet avkomma. Antalet skador och den totala storleken av skador visade ingen signifikant korrelation till antalet avkommor, däremot uppvisades signifikanta variationsskillnader mellan hanar av två olika fitnessnivåer. Skadade honor visade sig också vara betydligt vanligare än icke skadade honor och parade skador var även vanligare än icke parade skador. Resultaten indikerade att kopulationsskador kan vara en adaptiv egenskap hos hanar och en form av uppvaktning som honorna kan tyda, vilket kan antyda ett möjligt fall av ”kryptiskt honligt val”. Eftersom de flesta skador var parade, så antyder detta att ett bilateralt symmetriskt organ orsakar skadorna. Kopulationsskador visade sig heller inte vara så viktiga som man tidigare antagit.

Sexual selection acts on traits that increase the reproductive success of an individual in competition with other individuals of the same sex over reproductive opportunities, through intra-sexual competition and inter-sexual mate choice. Because males benefit more from remating than females, they are subject to more intense sexual selection. Modern genetic tools have shown that females often mate promiscuously, thus prolonging sexual selection after insemination through intra-sexual (sperm competition) and inter-sexual (cryptic female choice) episodes. Despite increasing interest in post-copulatory sexual selection, the implications of sperm competition, cryptic female choice and underpinning mechanisms remain little understood. This thesis adopts an integrated approach to quantify the relative importance of post-copulatory episodes in the operation of sexual selection, elucidate their proximate mechanisms in the red junglefowl Gallus gallus. By combining behavioural observations of replicate groups with paternity data, I show that female promiscuity decreased the total opportunity for sexual selection in a group, but accounts for an unexpectedly large proportion of the variance in male reproductive success. By comparing the operation of sexual selection on multiple male traits, I show that post-copulatory sexual selection reinforced pre-copulatory sexual selection for male social dominance and that female preferred to mate with compatible males. I used experiments to study the mechanisms of post-copulatory sexual selection by studying the effect of seminal fluid in sperm competition and cryptic female choice in relation to male status and relatedness. Following previous work indicating that seminal fluid products influence sperm quality in this species, I tested in vivo whether the seminal fluid of an ejaculate acts differentially towards sperm from the same ejaculate and rival sperm, and found no evidence for this idea. Finally, I show that cryptic female choice can drastically bias the outcome of sperm competition, and that female fowl might bias paternity toward unrelated males.

<p>Multiple mating by females is common in nature. Yet, the evolution and maintenance of polyandry remains a bit of an evolutionary puzzle. It was my aim in this thesis to reach a greater understanding of this phenomenon as well as to investigate the consequences of polyandry on the evolution of reproductive divergence in insects. In an extensive meta analysis addressing the direct effects of multiple mating on female fitness in insects, I found that insects gain from multiple matings in terms of increased lifetime offspring production. In species without nuptial feeding, increased mating rate leads to decreased female lifespan and my results strongly support the existence of an intermediate optimal female mating rate. However, results from an experimental study where I examined the relationship between female fitness and mating rate in the bean weevil (Callosobruchus maculatus) showed that female fitness was maximized at two alternative mating rates, indicating that some species may exhibit a more complex relationship between the costs and benefits of mating. In the meta analysis on species with nuptial feeding, I found only positive effects of increased mating rate and the puzzle is rather what constrains the actual mating rates of females in these groups.</p><p>Sexual selection is a very potent driver of rapid evolutionary change in reproductive characters. Most research has focussed on precopulatory sexual selection, but in promiscuous species sexual selection continues after copulation and variance in male fertilization success gives rise to postcopulatory sexual selection. In this thesis I found that three allopatric populations of the red flour beetle (Tribolium castaneum) have diverged in traits related to reproduction. Male genotype affected all aspects of female reproduction, but more interestingly, males and females interacted in their effect on offspring production and reproductive rate, showing that the divergence was due to the evolution of both male and female reproductive traits.</p><p>When studying postcopulatory sexual selection, sperm competition has been put forward as the main source of variance in fertilization success. The results from a set of double-mating experiments, using the same populations of flour beetles, provided strong evidence that cryptic female choice is also important in generating variance in male fertilization success. I found not only main effects of female genotype on male fertilization success but also male-female interactions which provide more unambiguous evidence for cryptic female choice. Finally, I attempted to uncover which male signals-female receptors are involved in the reproductive divergence observed in the Tribolium populations. In a double-mating experiment I manipulated female perception of two male reproductive signals, copulatory courtship and cuticular hydrocarbons, and the results indicate that, within populations, both signals are sexually selected. However, only male cuticular hydrocarbons seem to be involved in the reproductive divergence between the populations. </p><p>In conclusion, multiple mating by female insects can be understood solely in terms of direct fitness benefits resulting from increased offspring production. I have shown that postcopulatory sexual selection can lead to rapid divergence in reproductive traits related to mating and that cryptic female choice plays an important role in this divergence.</p>

<p>The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle.</p><p>Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression.</p><p>Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.</p>

Multiple mating by females is common in nature. Yet, the evolution and maintenance of polyandry remains a bit of an evolutionary puzzle. It was my aim in this thesis to reach a greater understanding of this phenomenon as well as to investigate the consequences of polyandry on the evolution of reproductive divergence in insects. In an extensive meta analysis addressing the direct effects of multiple mating on female fitness in insects, I found that insects gain from multiple matings in terms of increased lifetime offspring production. In species without nuptial feeding, increased mating rate leads to decreased female lifespan and my results strongly support the existence of an intermediate optimal female mating rate. However, results from an experimental study where I examined the relationship between female fitness and mating rate in the bean weevil (Callosobruchus maculatus) showed that female fitness was maximized at two alternative mating rates, indicating that some species may exhibit a more complex relationship between the costs and benefits of mating. In the meta analysis on species with nuptial feeding, I found only positive effects of increased mating rate and the puzzle is rather what constrains the actual mating rates of females in these groups. Sexual selection is a very potent driver of rapid evolutionary change in reproductive characters. Most research has focussed on precopulatory sexual selection, but in promiscuous species sexual selection continues after copulation and variance in male fertilization success gives rise to postcopulatory sexual selection. In this thesis I found that three allopatric populations of the red flour beetle (Tribolium castaneum) have diverged in traits related to reproduction. Male genotype affected all aspects of female reproduction, but more interestingly, males and females interacted in their effect on offspring production and reproductive rate, showing that the divergence was due to the evolution of both male and female reproductive traits. When studying postcopulatory sexual selection, sperm competition has been put forward as the main source of variance in fertilization success. The results from a set of double-mating experiments, using the same populations of flour beetles, provided strong evidence that cryptic female choice is also important in generating variance in male fertilization success. I found not only main effects of female genotype on male fertilization success but also male-female interactions which provide more unambiguous evidence for cryptic female choice. Finally, I attempted to uncover which male signals-female receptors are involved in the reproductive divergence observed in the Tribolium populations. In a double-mating experiment I manipulated female perception of two male reproductive signals, copulatory courtship and cuticular hydrocarbons, and the results indicate that, within populations, both signals are sexually selected. However, only male cuticular hydrocarbons seem to be involved in the reproductive divergence between the populations. In conclusion, multiple mating by female insects can be understood solely in terms of direct fitness benefits resulting from increased offspring production. I have shown that postcopulatory sexual selection can lead to rapid divergence in reproductive traits related to mating and that cryptic female choice plays an important role in this divergence.

The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle. Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression. Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.