Convergent and lineage-specific regulatory evolution revealed by comparative developmental transcriptomics and epigenomics of flightless birds (Palaeognathae)

A fundamental question in evolutionary biology is whether convergent genomic changes underlie convergent phenotypic changes. Flight has been lost repeatedly and convergently multiple times across the tree for modern birds. With a minimum of three convergent losses of flight within the clade, Palaeognathae, containing flightless ratites such as the common ostrich, emu, and greater rhea, and their volant (flight capable) relatives the tinamous, offers a unique opportunity to study the developmental genomics of reduced forelimbs, a major component of loss of flight. To identify genomic signatures of convergent regulatory evolution within the developing ratite wing, we applied RNA-seq and ATAC-seq to fore- and hindlimbs of the rhea, ostrich, emu, Chilean tinamous (Nothoprocta perdicaria), and chicken (Gallus gallus) at stages HH18 and HH25. At both stages, ~10,500 RNA-seq transcripts reveal higher numbers of differentially expressed genes between the fore- and hindlimb of flightless species than in chicken or tinamou. ~363,000 comparative ATAC-seq peaks reveal numerous instances of convergent chromatin closure in regions near genes for proliferation, differentiation and limb development, such as Btg1, Dach1, and Fgfs. Surprisingly, a previously identified enhancer with differential chromatin accessibility in the limbs of chicken and emu at HH18 appears closed in all other species at this stage. Multivariate and phylogenetic analysis of expression and chromatin patterns support transcriptome- and epigenome-wide convergence in flightless species, and suggest significant phylogenetic and developmental signals in these two data types. Taken together, these data support both convergent and lineage-specific evolutionary regulatory re-wiring coincident with the convergent decrease in forelimb size associated with loss of flight.