The evolution of lepidopteran brain morphology.

Animals live in diverse environments and have evolved to cope with environmental challenges in different ways. How such adaptations shape overall brain morphology is still unclear. Here, we test how two behavioural adaptations - circadian activity pattern and migratory behaviour - are reflected in the brains of moths and butterflies (Lepidoptera). We predicted that circadian activity pattern affects primary sensory regions, whereas migration impacts integrative centres. Using anti-synapsin immunostaining, we generated detailed 3D reconstructions of each species' brain and performed a phylogenetically corrected volumetric analysis. All lepidopteran brains, including early-diverging lineages, share a characteristic layout that differs from the caddisfly (Trichoptera) outgroup. Some brain regions proved highly evolvable - most notably, the anterior optic tubercle varied qualitatively among species. Most regions, however, differed quantitatively, with tissue volumes strongly shaped by phylogeny as well as behavioural traits. While activity pattern predominantly affected primary visual areas, migratory behaviour correlated with significant volume changes in the fan-shaped body, the accessory medulla and parts of the mushroom body. We also identified several small neuropils as evolutionary "hotspots", showing rapid, lineage-specific expansion or reduction. Finally, positive and negative correlations among neuropil volumes reveal coordinated evolution in defined neuropil groups, suggesting functional linkages and constraints beyond anatomically related regions. These findings generate testable hypotheses about poorly studied brain areas and highlight diverse evolutionary dynamics across the lepidopteran phylogeny.