Craniofacial-specific transcriptomics uncovers novel genes underlying jaw divergence in dietary specialist pupfishes.

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 three trophic specialists with highly divergent craniofacial morphologies (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 five Cyprinodon species and nine populations. We focused on the hatching 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 fourteen candidate genes. We confirmed novel craniofacial expression for two 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.