The Evolution of Reproductive Isolation Beyond a Strong First Barrier in Speciation Between Micro-Allopatric Host Races of a Phytophagous Ladybird Beetle, Henosepilachna diekei.

Reproductive isolation often accumulates unevenly during speciation, with some barriers evolving early and others appearing only after further divergence. In host-associated phytophagous insects, divergent host use can generate strong habitat isolation that evolves early in speciation and often results in sudden shifts towards strong reproductive isolation. However, whether additional sexual or postmating barriers can accumulate after this strong early ecological barrier has evolved remains unclear. In this study, we examined this question in the host-specialised populations of the Southeast-Asian herbivorous ladybird beetle, Henosepilachna diekei. We focused on the Dicliptera-Mikania race pair and compared it with the previously studied Mikania-Leucas race pair, representing two putative levels of host-race differentiation. Combining laboratory crossing and field-cage experiments, we investigated and quantified four potential isolating factors: premating isolation (habitat and sexual isolation), postmating-prehatching isolation (reduced egg hatchability), and postmating-posthatching isolation (F1 hybrid inviability). The Dicliptera-Mikania race pair showed larger genetic differentiation than the sympatric Mikania-Leucas race pair. Both race pairs showed strong habitat isolation, but only the Dicliptera-Mikania race pair showed additional later-acting barriers, including reduced mating success, reduced egg hatchability, and reduced F1 hybrid survival. Our findings suggest that additional reproductive barriers can evolve even in the presence of exceptionally strong habitat isolation during host-race divergence. Rather than replacing the importance of the initial host-associated barrier, late-acting barriers may accumulate and contribute to the stability of reproductive isolation. Consequently, the strength and dynamics of later-acting barriers may depend on the evolutionary trajectory of each host-race pair, reflecting diverse pathways through which speciation can unfold in host-associated insects.