The modeling of dsx protein structure posed a challenge due to the overwhelming presence of loops, owing to which most protein databases only contained the two functional domains of this protein. We modeled these functional domains and sex-specific exons using Phyre2 (39) (protein homology/analogy recognition engine). We performed structural alignment and visualization in PyMOL (2.0.6) (40) and Discovery Studio Visualizer 4.5 (Fig. 4) (41). For this analysis, we used a subset of sequences from 29 species of Lepidoptera, 14 species of Diptera, 10 species of Coleoptera, and 18 species of Hymenoptera (table S1). The reason is that (i) sex-specific sequences were not known from every species for which some dsx sequences were available and (ii) protein structure within genera was often similar; therefore, representative sampling of Dsx protein across insect genera was adequate to study protein structure evolution. We also did not model the protein structure of the Female 2 exon region of Coleoptera and Lepidoptera because these were present only in a few transcripts of a few species, where comparison of homologous regions was not feasible.

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