Lepidopteran prolegs are novel traits, not leg homologs

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. We examined the role of three Hox genes in proleg development in Bicyclus anynana butterflies using CRISPR-Cas9 and discovered that under a partial segment abdominal-A ( abd-A ) knockout, both types of appendages can develop in the same segment, arguing for prolegs being a novel trait, not a leg serial homolog. We also discover that specificity protein (sp) genes do not co-localize with Distal-less (Dll) in prolegs, as they do in legs, and that the proleg gene-regulatory network (GRN) mostly resembles the head-horn GRN, another novel trait in the lepidoptera. We propose that larval prolegs evolved from the co-option of a partial limb GRN into novel embryonic coordinates in the abdomen. One-Sentence Summary The chubby legs of caterpillars are evolutionary innovations