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Abstract Changes in gene expression underlie most phenotypic differences among closely related species. While previous studies in model systems have identified conserved genes and pathways involved in craniofacial evolution, less is known about gene expression differences associated with craniofacial divergence in rapidly evolving species. Here, we investigate craniofacial-specific gene expression in a nascent adaptive radiation of Cyprinodon pupfishes endemic to San Salvador Island, Bahamas, which includes 3 trophic specialists with highly divergent craniofacial morphologies (two scale-eaters and a molluscivore) derived from an ancestral Caribbean-wide generalist. We compared gene expression in the most morphologically divergent craniofacial region with the relatively conserved caudal region across 5 Cyprinodon species and 9 populations. We focused on the hatchling stage, the earliest developmental stage at which craniofacial differences among species are evident. Our approach revealed a large proportion of differentially expressed genes (DEGs) found exclusively in the craniofacial region of the specialists only. By intersecting these specialist-specific craniofacial-exclusive genes with genomic regions harboring fixed single-nucleotide variants under selection in the specialists, we identified 14 candidate genes. We confirmed novel craniofacial expression for 2 of these candidates, pycr3 and atp8a1, genes not previously associated with craniofacial development or function, in hatchlings using in situ mRNA hybridization and observed species-specific differences in the pharyngeal arches and craniofacial muscles, respectively. Our findings demonstrate how an “evolutionary mutant” model can reveal novel gene expression patterns, highlighting the power of integrating tissue-species transcriptomics with speciation genomics to identify novel regulators of craniofacial evolution. 

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage usingin situhybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.