Ortholog identification was performed between the gene models annotated in Danaus plexippus, Bombyx mori, and Drosophila melanogaster. The D. plexippus sequences are from an assembly obtained from an individual that belonged to a monarch population found in Irapuato, Mexico. 1-to-1 orthologs were delineated as part of an analysis devised to determining the proportion of gene models with homology support in other Lepidoptera and D. melanogaster, and in relation to a previously published gene annotation of D. plexippus. Additionally, information from 1-to-1 orthologs was used in the process of contig anchoring to the D. plexippus chromosomes and in the examination of the level of macro and microsynteny conservation in relation to B. mori and D. melanogaster.

One-to-one identification between the collection of protein-coding gene models that are part of the gene annotation OGS1_DpMex of D. plexippus and those in B. mori and D. melanogaster was done with OrthoFinder v2.2.6 under the settings –S diamond –M msa, using protein models retrieved either from NCBI or lepBase. The genomic coordinates of orthologs were established according to DpMex_v1 for D. plexippus (10.5281/zenodo.4470132) and GCF_000151625.1_ASM15162v1 for B. mori. As the assembly sequences of B. mori are not associated with particular chromosomes, we ultimately used a second-party chromosomal-based gene coordinates (http://sgp.dna.affrc.go.jp/ComprehensiveGeneSet/files/GenesetA.chromosome.gbrowse.gff.zip).