The evolution of male courtship discrimination is driven by sympatry and age-dependent pheromones between Drosophila athabasca species complex and its congeners.

Our understanding of how males evolve courtship discrimination and the traits they use for species identification is restricted to a few model systems. Theory predicts that males between more sympatric species should evolve stronger courtship discrimination and target female traits for reliable species identification. Thus, traits that increase divergence specifically during sexual maturity should be prime targets of species recognition. We tested this by measuring male courtship discrimination indexes (CDIs) and cuticular hydrocarbons (CHCs) across ten species pairs of Drosophila affinis subgroup. CDIs ranged from 12% to 86% of total sexual isolation, increasing between more sympatric pairs only if they had postzygotic isolation. Principle Component Analysis on CHCs revealed species-specific clustering in both sexes. CHCs between younger and older flies were not correlated and male CDIs were only correlated with older CHCs. While most CHCs showed species x age interactions, only 11(Z)-tricosene (C23:1), 10,14-pentacosadiene (C25:2), and 2-methylhexacosane (C27:0)) were significantly differentiated and became more divergent with age. We then used "rub-off" experiments and direct transfers of these CHCs to show their species-specific repellent and aphrodisiac properties. This suggests that targeting sexually mature female CHCs allows males to distinguish conspecifics from heterospecifics to avoid maladaptive hybridization or signal interference in sympatry.