Chemically defended plant tissues present formidable barriers to herbivores. Although mechanisms to resist plant defenses have been identified in ancient herbivorous lineages, adaptations to overcome plant defenses during transitions to herbivory remain relatively unexplored. The fly genus Scaptomyza is nested within the genus Drosophila and includes species that feed on the living tissue of mustard plants (Brassicaceae), yet this lineage is derived from microbe-feeding ancestors. We found that mustard-feeding Scaptomyza species and microbe-feeding Drosophila melanogaster detoxify mustard oils, the primary chemical defenses in the Brassicaceae, using the widely conserved mercapturic acid pathway. This detoxification strategy differs from other specialist herbivores of mustard plants, which possess derived mechanisms to obviate mustard oil formation. To investigate whether mustard feeding is coupled with evolution in the mercapturic acid pathway, we profiled functional and molecular evolutionary changes in the enzyme glutathione S-transferase D1 (GSTD1), which catalyzes the first step of the mercapturic acid pathway and is induced by mustard defense products in Scaptomyza. GSTD1 acquired elevated activity against mustard oils in one mustard-feeding Scaptomyza species in which GstD1 was duplicated. Structural analysis and mutagenesis revealed that substitutions at conserved residues within and near the substrate-binding cleft account for most of this increase in activity against mustard oils. Functional evolution of GSTD1 was coupled with signatures of episodic positive selection in GstD1 after the evolution of herbivory. Overall, we found that preexisting functions of generalized detoxification systems, and their refinement by natural selection, could play a central role in the evolution of herbivory.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.