Thylacine-Wolf Convergence MPRA

Understanding the molecular mechanisms underpinning convergent traits can provide insight into the predictability of the evolutionary process. The extinct marsupial carnivore thylacine has been long known to have a very similar morphology to that of eutherian canids, especially in the craniofacial region, despite having diverged \(\approx\)160 million years ago. Previous comparative genomics studies between thylacine and gray wolf identified a set of 339 craniofacial non-coding vertebrate conserved elements that were convergently accelerated in thylacine and wolf. With a massively parallel reporter assay (MPRA), we tested the regulatory potential of 295 of these thylacine and wolf accelerated regions (TWARs) in mouse preosteoblasts and cranial neural crest cells. We compared orthologous sequences from six different taxa – the focal species (thylacine and wolf), outgroup species with non-convergent craniofacial phenotypes (Tasmanian devil and panda) and ancestral reconstructions for marsupial and placental carnivores (Dasyuromorphia and Carnivora). Activity of TWARs was associated with sequence features, as expected, including percentage GC content and presence of binding motifs. Our results included a substantial proportion of TWARs with conserved levels and patterns of regulatory activity across the six mammals. More TWARs with differential activity were observed among placentals than marsupials; we determined however that both thylacine and wolf were driving divergence within their clades. Dense 10bp tiling of our sequences allowed identification of binding motifs associated with gain or loss of activity. Two TWARs displayed convergent regulatory potential in thylacine and wolf, both located near genes implicated in craniofacial development and related pathologies. Additionally we identified neighbouring TWARs – one with reduced activity in wolf, and the other in thylacine – near the neural crest gene \textit{Phox2b}.