Supplementary Material for Palmateer et al., 2026

<b>Article Summary</b>We examined how genes and circuits shape courtship behaviors in <i>Drosophila melanogaster</i> and <i>Drosophila simulans</i>. We focused on the <i>fruitless</i> gene, which is expressed in the nervous system and controls male courtship by encoding male-specific transcription factors (Fru^M). <i>fru-</i>expressing neurons in hybrids closely resembled those in <i>Drosophila melanogaster</i> indicating largely shared neuronal architecture. We identified both shared and species-specific Fru^M target genes, including odorant receptor genes bound by Fru^M in a species-specific manner. A genetic screen in <i>Drosophila melanogaster</i>, where we silenced odorant receptor neurons that co-express <i>fruitless</i>, revealed sensory neuron populations that direct quantitative differences in courtship towards <i>Drosophila simulans</i>. Neuroanatomical mapping shows that odor information is relayed to different higher order brain regions. The results uncover molecular and neural circuit mechanisms underlying species differences in behaviors.