Elucidating the Genetic Basis of Loss of Flight inthe Emu, Dromaius novaehollandiae

Determining the genetic underpinnings of phenotypic diversity isan enduring problem in evolutionary biology. Convergent evolutionof regulatory regions is thought to drive independent losses of flightin the ratites, the group of flightless birds at the base of the aviantree. Comparative genomics, together with functional testing ofenhancer elements, are powerful tools to determine noncodinggenetic variants that may underlie loss of flight. We used a mas-sively parallel reporter assay (MPRA) to test enhancer activity of55 of 67232 pairs of putative enhancers in chicken and emu, becausechicken is capable of powered flight whereas emu is flightless. Wetested this library of enhancers in the forelimb, hindlimb, and flankof chick embryos, and compared activity between paired chickenand emu enhancers. We found multiple examples of elementsaccelerated in emu that show differential activity in chicken fore-limbs and hindlimbs. Strikingly, we also find conserved elementsexhibiting differential activity between the chicken and emu ele-ments. Finally, we report two regulatory regions located near genesupregulated in the emu forelimb relative to the early chick forelimbin which each emu enhancer has a higher activity than the corre-sponding chicken enhancer in chick limb buds. Using multimodalgenomic data (RNA‐seq, ATAC‐seq, phyloACC, MPRA) in chickenand emu, we have identified two loci that may explain reduction ofthe forelimb, and hence loss of flight, in emus.