Navya Shukla, Laura E. Cook, Davide M. Vespasiani, Andrew J. Pask, Irene Gallego Romero
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 ≈160 million years ago. Using a massively parallel reporter assay (MPRA), we tested the regulatory potential of a set of previously identified highly conserved craniofacial enhancers that showed convergent sequence acceleration in thylacine and wolf (TWARs). We compared orthologous sequences from six different taxa, including outgroup taxa with non-convergent craniofacial phenotypes (Tasmanian devil and giant panda) and ancestral reconstructions for marsupial and placental carnivores (Dasyuromorphia and Carnivora). Dense 10bp tiling allowed us to thoroughly examine features associated with activity, including percentage GC content and transcription factor binding motifs turnover. We identified 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 was limited to a set of neighbouring TWARs near the neural crest gene Phox2b – one of which exhibited reduced activity in the wolf, and the other in the thylacine. Ultimately our findings suggest that shared gene regulatory potential is not a feature of these convergently accelerated regions in the thylacine and wolf.
Navya Shukla, Laura E. Cook, Davide M. Vespasiani, Andrew J. Pask, Irene Gallego Romero. Dissecting functional regulatory convergence over 160 million years of therian evolution. 2025-07-11. https://doi.org/10.1101/2025.07.10.662670
An episomal barcode reporter assay tested orthologous thylacine-and-wolf-accelerated regions (TWARs), ancestral and outgroup orthologs, mouse ortholog controls, and cell-line controls in MC3T3-E1 mouse preosteoblasts. The library used 10-bp sliding tiles for sequences longer than 170 bp and three biological replicate pDNA/cDNA measurements.
An episomal barcode reporter assay tested orthologous thylacine-and-wolf-accelerated regions (TWARs), ancestral and outgroup orthologs, mouse ortholog controls, and cell-line controls in O9-1 mouse cranial neural crest cells. The library used 10-bp sliding tiles for sequences longer than 170 bp and three biological replicate pDNA/cDNA measurements.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 6 files (ZIP)all_oligos_annotated_nov24.tsvjuly24.count.tsv.gzMPRAcount_activity.RmdMPRAcount_qc.RmdREADME.mdtycnmpra_README.md