Pinzoni, L.; Morbiato, E.; Dorsey, O. C.; Hernandez Melo, J.; Devigili, A.; Gasparini, C.; Rosenthal, G.

Avoiding fertilization with genetically incompatible partners, whether too similar or too divergent, is a central challenge for sexually reproducing organisms. Selection can favor mechanisms acting before and after mating, with postmating processes potentially compensating for constraints on premating choice. In the postmating context, female reproductive fluid (FRF) can modulate sperm performance and bias fertilization outcomes, but its contribution to reproductive isolation remains unclear. We tested whether FRF mediates discrimination against heterospecific and related sperm in two naturally hybridizing sister species of swordtails, Xiphophorus birchmanni and X. malinche, that diverge in premating behavior towards heterospecifics. Effects of FRF differed sharply between species. In X. malinche, FRF enhanced the velocity of conspecific sperm relative to heterospecifics, consistent with postmating discrimination against hybridization. In contrast, FRF in X. birchmanni did not favor conspecific sperm. Evidence for inbreeding avoidance was weaker, and we found no indication of a trade-off between discrimination against genetically similar and dissimilar sperm. These results show that female reproductive fluid can serve as a rapidly evolving axis of reproductive isolation through postmating female choice.