Postmating prezygotic isolation can reduce gene flow early in divergence, but the reproductive traits and postmating processes that generate these barriers are often challenging to identify. This is especially true in internally fertilizing animals, where fertilization depends on interactions between male ejaculates and the female reproductive tract, and where reduced fertilization can arise through several distinct postmating steps. In this dissertation, I investigate how divergence in interacting reproductive traits translates into postmating prezygotic isolation in Drosophila montana, with a particular focus on sperm length, female sperm storage, and female-mediated postmating processes.

In Chapter I, I test whether divergence in sperm length and seminal receptacle length predicts fertilization success across populations and genotypes. I show that these traits co-diverge and together form a major compatibility axis, such that fertilization success is best predicted by the sperm to seminal receptacle length gap. Fertilization is lowest when sperm are relatively short for a given seminal receptacle. I further show that both traits have substantial additive genetic variance and a polygenic basis, and that their QTL intervals overlap in several genomic regions, identifying candidate regions that may have facilitated coordinated divergence.

In Chapter II, I ask whether this compatibility relationship is fixed or modifiable by developmental condition. I show that seminal receptacle length is condition-dependent in the population with the longest seminal receptacles and sperm, whereas sperm length remains largely insensitive to developmental condition. I further show that female condition can alter fertilization success, both through general female effects and by amplifying sperm to storage mismatch in the cross-direction that already shows the strongest barrier.

In Chapter III, I examine whether divergence in female sperm management helps explain the postmating prezygotic barrier. Using Rhodamine B-labelled ejaculates and organ-specific sperm storage assays across time, I show that sperm management differs between female populations and between storage organs. Heteropopulation effects are female-population dependent, with reduced persistence of heteropopulation sperm in the seminal receptacle in one population and increased early spermathecal occupancy in the other, while complete sperm depletion is generally higher in one female population across the same postmating window regardless of male population origin.

Together, these results show that postmating prezygotic isolation in D. montana is not explained by a single reproductive failure. Instead, it emerges through divergence in interacting reproductive traits and female-mediated postmating processes. More broadly, this dissertation identifies a quantitative speciation phenotype linking sperm and sperm storage divergence to asymmetric reproductive isolation, shows that its expression can be modified by condition-dependence, and helps narrow the postmating mechanisms through which early reproductive barriers arise during speciation.