Understanding the molecular mechanisms underpinning convergent traits can help characterize forces that drive adaptation and provide insight into the predictability of the evolutionary process. The extinct thylacine, or Tasmanian tiger, has been long known to have a very similar morphology to that of the eutherian canids, particularly in the craniofacial region, despite having shared a common ancestor ~160 million years ago. Previous comparative genomics studies between the thylacine and a representative canid, the gray wolf, have found that much of underlying molecular convergence between these species is found in non-coding, regulatory regions of the genome. These analyses identified a set of 339 thylacine-wolf accelerated regions (TWARs), putative craniofacial cis-regulatory elements that have undergone accelerated evolution in both thylacine and wolf lineages.
Using a Massively Parallel Reporter Assay, we functionally characterize the activity of 295 orthologous TWAR sequences from 7 different species—the species of interest (thylacine and wolf), ancestral reconstruction from their clades (Dasyuromorphia and Carnivora), two related outgroup species with different craniofacial phenotypes (Tasmanian devil and panda) and a control species (mouse). We tested their activity in two mouse cell populations involved in craniofacial development. By comparing changes in regulatory activity of orthologous elements, we assess which regions showing a convergent shift in molecular activity also underlie convergent phenotypes. Dense 10bp tiling of our sequence library allows much greater resolution to detect specific transcription factor motif changes driving the observed phenotypic changes.