Dissecting functional regulatory convergence over 160 million years of therian evolution

Understanding the molecular mechanisms underpinning convergent traits can provide insight into the predictability of the evolutionary process. The thylacine, an extinct marsupial carnivore, has been long known to have a very similar craniofacial morphology to that of eutherian canids, despite having diverged approximately 160 million years ago. Using a massively parallel reporter assay (MPRA), we test the regulatory potential of a set of previously identified highly conserved craniofacial enhancers that showed convergent sequence acceleration in thylacine and wolf (TWARs). We compare orthologous sequences from six different taxa, including outgroup taxa with nonconvergent craniofacial phenotypes (Tasmanian devil and giant panda) and ancestral reconstructions for marsupial and placental carnivores (Dasyuromorphia and Carnivora). Dense 10 bp tiling allowed us to thoroughly examine features associated with activity, including percentage GC content and transcription factor binding motifs turnover. We identify marked conservation of levels and patterns of regulatory activity across all six taxa, as well as multiple cases of differentially active TWARs within each clade, with both thylacine and wolf driving functional divergence. However, evidence of convergent divergence relevant to craniofacial biology is limited to a TWAR with reduced activity in both the thylacine and wolf near the developmental gene LMO4 Ultimately our findings suggest that shared gene regulatory potential is not a feature of these convergently accelerated regions in the thylacine and wolf.