Divergence in cuticular wax profiles generates partial behavioural isolation between leaf beetle populations with different dispersal traits.

Sex pheromones are among the most studied insect mating signals, with their extensive diversity underscoring their crucial role in promoting behavioural isolation during speciation. In Chrysomelidae, cuticular wax (CW), a hydrophobic layer covering the insect cuticle, functions as a mating signal and potentially facilitates behavioural isolation. Male leaf beetles use female CW as a mating signal, and their species-specific profiles prevent heterospecific matings, indicating that divergence in CW profiles may promote reproductive isolation and, hence, contribute to speciation. However, the role of CW as an isolating barrier remains unclear owing to limited knowledge regarding intraspecific divergence in female CW and its coevolution with male preferences. Through chemical analysis and behavioural experiments, we demonstrated that intraspecific divergence in female CW contributes to partial behavioural isolation between leaf beetle populations with different dispersal traits: the flight-capable macropterous and flightless brachypterous forms of Galerucella grisescens (Coleoptera: Chrysomelidae). Our results demonstrated a divergence in female CW and male preferences at the intraspecific level, indicating the potential role of CW as an isolating barrier in Chrysomelidae. Additionally, our findings suggest that diverging dispersal ability can enhance divergence in pheromone communication channels, consistent with previous findings that loss of flight enhances beetle diversification.