Evolution of limb and digit identity genes since the tetrapod ancestor

Natural selection has sculpted tetrapod limbs and digits into a tremendous morphological diversity, but the underlying developmental mechanism remains unclear. This leads to an enduring debate concerning the identity of the three digits of bird wing. Here we use comparative digit transcriptomics across six tetrapod species to examine gene expression signatures of digit evolution. Here we show that the avian wing digits are homologous to reptilian forelimb digits 1,3,4. We find that birds uniquely increase fore-hindlimb transcriptome divergence during development, relaxing constraints and facilitating wing and foot digit specialization. We identify many limb- or digit- identity associated genes that have a biased expression between fore- or hind-limb, or toward specific digits. There are more limb and digit biased genes among the avian lineages than in other tetrapod lineages. Our work reveals ancient regulatory networks that maintain limb and digit molecular identity through persistent differential expression. We also show that this conserved network does not preclude independent digit evolution, especially when developmental constraints are reduced.