We downloaded insect-specific dsx gene sequences from GenBank (http://www.ncbi.nlm.nih.gov/, last accessed December 2017). From the available genomes and individual sequences, we shortlisted sequences from 145 insect species across four holometabolous insect orders (Lepidoptera, Diptera, Hymenoptera, and Coleoptera) based on the availability of and the ability to annotate intron-exon boundaries of this gene (table S1). We used dsx homolog sequences from three species of Isoptera and three species of Hemiptera as outgroups (Fig. 2A). We used NCBI (National Center for Biotechnology Information) BLAST+ to find the location of dsx within genomes of species where the gene was not annotated. We used SAMtools V0.1.19 (33) for extracting dsx exon sequences from genomes. We obtained sex-specific variants of dsx in most of the shortlisted species and used the corresponding exon sequences for further analysis. We performed multiple sequence alignment of dsx exons from insect species within each order and across all orders using the codon aligner PRANK v150803 (34). Note that the numbering of exons in each sequence of dsx depends on the position at which mRNA begins; however, one or more exons in each sequence may be untranslated. Thus, the exact numbering of exons is arbitrary but used here as per scientific convention (table S1). However, their relative positions and the genetic architecture of domains remain conserved across orders (Fig. 2). Thus, the alignment used for further analysis (Figs. 3 and 4) is as per sequence homology.

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