Sexual selection has long been framed as a process that ends when copulation is achieved. However, in species with polyandry (multiple mating by females), competition persists after mating inside the female;;s reproductive tract, where sperm from multiple males must then compete to fertilize a female;;s eggs. This post-mating process, known as sperm competition, is thought to be just as strong as the competition to secure a mate. Because sperm competition has only recently been observed, its evolutionary role remains largely unknown. In this dissertation, I use field, laboratory and computational approaches to understand the evolution of sperm competition in two ways: (1) by testing a possible source of variation in sperm competition within species, and (2) by examining how variation in sperm competition results in DNA evolution across species. My study system is the Agelaius clade of New World blackbirds, a group of songbirds with predicted variation in the intensity of sperm competition. In the first half of the dissertation, I explore the factors that affect how intensely sperm competition is experienced in a population. In Chapter 1, I assess the relationship between genetic diversity and extra-pair paternity (EPP, a proxy for sperm competition) in seven continental and one island population of red-winged blackbird (A. phoeniceus). I find that while genetic diversity varies significantly across populations, the population with the lowest amount of genetic diversity exhibits similar rates of EPP as the more diverse populations, providing no support for a relationship between genetic diversity and EPP rate. This result suggests that genetic diversity by itself is not an determining factor in EPP variation. In Chapter 2, I characterize the mating system of the endangered yellow-shouldered blackbird (A. xanthomus) and provide the first evidence that it, too, engages in EPP despite having low genetic diversity. I additionally present a conservation genetics profile of the species, showing that the yellow-shouldered blackbird;;s low effective population size and genetic diversity, both likely due to a recent bottleneck, may be increasing its vulnerability to extinction. I suggest ways in which future management decisions might account for the genetics of a small population. In the second half of the dissertation, I examine whether sperm competition itself can drive the molecular evolution of a species. I focus on the evolutionary patterns of seminal fluid proteins (Sfps), which are transferred with sperm during copulation and are known targets of sperm competition. I describe in Chapter 3 the transcriptomic and proteomic techniques I use to identify protein-coding genes in a non-model organism, presenting the first list of seminal fluid proteins in a songbird. I contrast the protein profile of the blackbird with the protein profile of insect and mammalian Sfps. Finally, in Chapter 4, I use eight of the proteins identified from the list to look for patterns of positive selection on these proteins. Specifically, I test whether Sfps evolve faster in species with mating systems featuring high levels of sperm competition than in species with mating systems featuring low levels of sperm competition. I first compare EPP rates measured from the previous two species with a third species, the tricolored blackbird (A. tricolor), and find that all three experience similar levels of sperm competition. From the catalog of genes derived in Chapter 3, I select, sequence and search for evidence of rapid evolution in six candidate Sfps and two control genes. I find that not only is there no evidence for positive selection in any of these genes, there is strong evidence for purifying selection and furthermore very low levels of diversity within and divergence across species. Reasons for these unexpected preliminary findings could be both microevolutionary or macroevolutionary in nature and warrant larger-scale studies, especially across a broader sample of taxa and across species with greater variation in sperm competition. Taken together, this dissertation describes the relationship between mating systems, sperm competition and post-mating adaptations. By examining the effect of mating system on protein divergence, it links sexual selection with molecular evolution while generating behavioral, genetic, transcriptomic and proteomic resources for future comparative studies.
