Fireflies were believed to originally evolve their novel bioluminescence as warning signals, which was later adopted in adult mating. Although evolution of bioluminescence has been investigated extensively, the evolution of firefly toxins has not been systematically examined. In this study, we systematically surveyed the presence or absence of firefly toxin lucibufagins (LBGs) in Lampyridae and outgroup species using LC-MS. We collected transcriptomes to reconstruct firefly phylogeny and ancestral states of LBGs. The presence of LBGs in the common ancestor of Lampyrinae is highly supported, but not supported in more ancient nodes. We further examined effects of amino acid substitutions in firefly ATPĪ± on its interactions with LBGs and found that ATPĪ± in LBGs-containing fireflies are moderately insensitive to LBGs, suggesting that target site insensitivity may contribute to LBGs-containing fireflies to deal with their own toxins. Our results suggest that firefly LBGs probably evolved much later than the evolution of bioluminescence in fireflies, implying that firefly bioluminescence probably did not originally evolve as direct warning signals to LBGs.
Evolution of firefly toxins