Phenotypic plasticity in turtle ants has opposing evolutionary consequences for genes and regulatory loci

Phenotypic plasticity is widespread and evolutionarily important, but genomic consequences of new plastic traits remain unclear. Here, we explore patterns of molecular evolution linked to the repeated evolution of Cephalotes turtle ant worker plasticity, in which smaller minor workers and distinct larger soldiers are produced from a single genomic blueprint through developmentally plastic mechanisms. We integrate developmental transcriptomics with comparative genomic approaches to test the relative relationships of selection on genes, selection on regulatory sequences, and the emergence of lineage-specific genes with the repeated evolution of the soldier morph. We find that phenotypic plasticity shields protein-coding genes from selection, whereas it imposes a strong selective constraint on the evolution of gene regulatory loci. The development of a soldier morph disproportionately involves the activity of evolutionarily ancient genes. Moreover, our data link 3 pathways-nutrition via insulin signaling, imaginal disc development, and for the first time Hippo signaling-which allow for the differential development of soldiers and workers from a single genomic background in turtle ants. Taken together, our results provide evidence that plasticity leads to relaxed selection on genes, but imposes selective constraint on regulatory elements, during the repeated evolution of the turtle ant soldier morph.