Reinforcement of speciation by behavioural and reproductive barriers in the Drosophila bipectinata species complex

Speciation mainly depends on different mechanisms involved in reproductive isolation; Understanding such barriers that consistently develop to avoid the fitness cost through reinforcement until the speciation continuum completes helps clarify the role of pre-mating and post-mating isolating mechanisms. The Drosophila bipectinata species complex, comprising four closely related taxa, provides a natural system for studying the interplay of behavioural, sensory, and genetic mechanisms underlying reproductive isolation. In this study, a comprehensive characterization was undertaken on twelve different types of sterile F1 hybrid males among the crosses between four species of Drosophila bipectinata species complex which are experiencing reproductive isolation. Comparing the mating latency and copulation duration among the pure species and hybrids, showed a significant increase in the mating latency of hybrids but the duration of copulation among certain hybrids of Drosophila bipectinata, Drosophila parabipectinata and Drosophila malerkotliana showed no significant reduction in the copulation time and emphasized the strong influence of X chromosome inheritance of hybrid genome. Experimental ablation of antennae demonstrated that male olfactory input is indispensable for heterospecific mating success, consistent with the role of pheromone detection in conspecific recognition, while tarsi ablation revealed striking sex-specific asymmetry: male tarsi removal increased heterospecific success, whereas female tarsi removal abolished mating entirely, highlighting the role of gustatory receptors in female mate choice. Testis dissections further confirmed that post-meiotic defects in hybrids, including failure of spermatid elongation and individualization, these findings demonstrates that reproductive isolation in the D. bipectinata species complex arises from the collective action of multiple barriers, prezygotic isolation mediated by pheromonal and gustatory divergence, and postzygotic sterility driven by spermatogenic failure. Our study underscores how behavioural and physiological divergence interact to reinforce reproductive barriers, providing a comprehensive framework for understanding speciation in Drosophila.