Tissue-specific transcriptomics uncovers novel craniofacial genes underlying jaw divergence in specialist pupfishes

The regulation of gene expression is one of the key evolutionary processes driving phenotypic divergence among species. Here, we investigate the tissue-specific gene expression of a non-model adaptive radiation of Cyprinodon pupfishes, characterized by their divergent dietary niches and exceptionally fast rates of craniofacial evolution. By comparing tissue-specific gene expression in the most morphologically divergent skeletal structure, the oral jaws, with the relatively morphologically conserved caudal tail region, we identified genes that were differentially expressed exclusively in the developing jaws of each of the three trophic specialists at hatching (8 dpf) and not in any other species. We then assessed their overlap (as transcriptionally-regulated genes) with adaptive regulatory variants identified in previous genomic studies. Our analysis identified pycr3 and atp8a1 as the most promising for craniofacial evolution in the scale-eaters, both genes with no known previous craniofacial function. We functionally confirmed the craniofacial expression of these genes by in situ mRNA hybridization chain reaction and demonstrated their species-specific expression in branchial and muscle tissues between sister species of this young radiation. Our work underscores the power of integrating tissue-specific transcriptomics with speciation genomics to identify novel craniofacial candidate genes controlling divergent morphogenesis in a natural ‘evolutionary mutant’ system.