A model for the evolution of Or67b and comparison with the known evolution of other Or and Ir orthologs in D. melanogaster and specialist drosophilid species. (A) Model for the evolution of Scaptomyza Or67b. The evolution of this Or begins with a shift in the ligand specificity of an ancestral Or67b (a) tuned to Dmel activators, GLVs, and ITCs (neofunctionalization, b). Subsequent gene triplication of Sfla Or67b gave rise to two additional paralogous Or67b genes (Sfla Or67b1, Sfla Or67b2, and Sfla Or67b3; c), each of the paralogs has different but overlapping ITC odorant-receptive ranges (figs. 3 and 4). (B) Evolution of drosophilid orthologs with known ligand specificities (Or22a, Ir75a, Ir75b, top; and Or67b, bottom). The Or22a protein orthologs from D. melanogaster (Dmel Or22a), D. sechellia (Dsec Or22a), D. erecta (Der Or22a), and D. suzukii (Dsuz Or22a) are all strongly activated by species-specific host-derived esters (compounds 1–4; top left; Dekker et al. 2006; Linz et al. 2013; Mansourian et al. 2018; Keesey et al. 2019). The Ir75a protein orthologs from D. melanogaster (Dmel Ir75a), and D. sechellia (Dsec Ir75a) are strongly activated respectively by the acid compounds 5 and 6 (Prieto-Godino et al. 2017). Similarly, the Ir75b protein orthologs from D. melanogaster (Dmel Ir75b) and D. sechellia (Dsec Ir75b) are respectively activated by the acids 7 and 8 (top right; Prieto-Godino et al. 2016). Dmel Or67b and Spal Or67b are strongly activated respectively by acetophenone and cis-3-hexenyl butyrate (compounds 9 and 10), whereas Sfla Or67b copies are activated by ITCs only (bottom, paralog-specific activation by compounds 11–13, abbreviations as in fig. 3). Note that Or22a, Ir75a, and Ir75b orthologs are all divergent but activated by ligands belonging to a single chemical class (whether esters or acids). On the other hand, the ligands of orthologs Or67b from Dmel and Spal are responsive to a variety of chemical classes which include alcohols, aldehydes, and ketones, whereas Sfla Or67b orthologs are responsive to ITCs, an entirely different compound chemical class. Notably, copies of Or22a, Or42b, and Or85d have been evolutionarily lost in S. flava. These Ors are important in mediating attraction to canonical microbe (especially yeast)-associated odors (Goldman-Huertas et al. 2015).