Beyond hybrid sterility: Direct selection and prezygotic barriers in the speciation paradigm of Drosophila bipectinata species complex

Speciation usually involves complex interactions between genetic and behavioural barriers, specifically when the gene flow persists. To decipher reproductive isolation in the Drosophila bipectinata species complex (D. bipectinata, D. parabipectinata, D. malerkotliana, D. pseudoananassae), this study explores direct selection on mate preferences and postmating, prezygotic incompatibilities as key speciation drivers. Mating latency, copulation duration, and reproductive assays showed prolonged latencies and shorter copulation durations in heterospecific versus conspecific crosses, indicating strong precopulatory and postcopulatory prezygotic barriers. Heterospecific crosses revealed reduced fecundity and fertility compared to conspecific matings. All hybrid males were sterile due to spermatogenesis defects, while F1 hybrid females exhibited fertility comparable to parental species in backcrosses. Fertile hybrid females, however, showed near-parental fertility, suggesting seminal fluid transfer over sperm. These results highlight that direct selection, plasticity and premating as well postmating incompatibilities, beyond hybrid fitness, play key roles in reproductive isolation, suggesting alternative speciation mechanisms to reinforcement.