Investigating how the ALX1 locus influences beak morphology in Darwin’s finches

Abstract

Darwin’s finches are a model for adaptive radiation and niche partitioning, showcasing species that recently diverged to occupy specific niches within the Galapagos archipelago. A previous study using whole genome sequencing of 120 individuals representing Darwin's finch species in Lamichhaney et al. (2015) identified fixed genomic regions between species possessing blunt vs. pointed beaks. Among these, the 240kb ALX1 locus contained the highest fixation of single-nucleotide polymorphisms (SNPs), including both coding and noncoding SNPs. Though ALX1 is implicated in craniofacial development, the extent to which differences in coding and noncoding genetic variants affect avian beak morphology has yet to be elucidated. We utilized Geospiza magnirostris and Geospiza difficilis as finch models given their beaks pertain to extremes on the morphological spectrum. We evaluated the effect of L110P and I209V mutations in the ALX1 coding region using chicken (Gallus gallus) in ovo RCAS-mediated manipulation. Next, we combined assay for transposase-accessible chromatin sequencing (ATAC-seq) from avian species and differential SNP data to identify candidate cis-regulatory elements (CREs) and tested their effects using luciferase reporter assays. Then, we cloned positive enhancers in a variety of finch species to functionally test them and analyze their sequences. Our experiments indicated no overt phenotypic difference between individuals overexpressing ALX1 variants. However, luciferase assays of candidate CREs revealed differential activity of 2 distinct regions: CRE1 and CRE8. Further dissection revealed a core conserved region within CRE8 to be insufficient in driving full expression. Luciferase assays testing activity of both regions in multiple finch species indicated varied functional activity of enhancers across species. Sequence similarity and tree analyses indicated distinct evolutionary pathways pertaining to each region that were specific to individuals rather than species. Motif enrichment analysis suggested the presence of varying transcription factor binding sites between individuals, possibly differentially modulating enhancer activity. Altogether, our work dissects the contribution of coding and noncoding polymorphisms in the ALX1 locus to the adaptive evolution of Darwin's finches highlighting the role of noncoding genetic variation in trait diversification across closely related species.