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.

Climate change is affecting the distribution of species, sometimes making previously separated species come into secondary contact. For closely related species, this often leads to competition and sometimes also hybridization, which can have negative effects on genetic bio? diversity. Collared flycatcher (Ficedula albicollis) and pied flycatcher (Ficedula hypoleuca) are two closely related species that hybridize where they meet on the Swedish islands Gotland and Öland. Studies from Gotland and the north of Öland have shown that the pied flycatcher is outcompeted and displaced from favourable, high-quality habitat by the collared flycatcher, leading to a decrease in the number of pied flycatchers in deciduous forest. However, this has not been shown for the ecologically important and one of the largest deciduous forests in Sweden, the Mittland forest located in central Öland. Here I use census data collected during two periods separated by 37 years from the Mittland forest to investigate habitat choice and competition between the two species. Although the number of collared flycatcher has greatly increased in the area, I show that the collared flycatcher has not outcompeted the pied flycatcher from favourable habitat. I also show that pied flycatcher does not have a preference for coniferous forest. Lastly, I show that the two species have a near complete overlap of habitat niches. These results indicate that they are in direct competition over the favourable habitat, that there is no equilibrium between the species and the collared flycatcher might still be expanding in the Mittland forest. Allowing more time to pass and further research will show if the pied flycatcher will be displaced from the high-quality habitats in this area or not.