Genomes can be seen as notebooks of evolution on successful genetic experiments. Comparing genomes across species allows identifying conserved DNA regions responsible for conserved biological functions, but additional information could be leveraged when also considering phenotypic variance across species. Here, we exemplify such a "cross-species association study" for the gene TRNP1 that is known to be important for brain growth and cortical folding in mice and ferrets.We find that the rate of TRNP1 protein evolution (ω) co-evolves with the rate of cortical folding across mammals and that TRNP1 proteins from species with more cortical folding induce higher proliferation rates in neural stem cells. Furthermore, we compare the activity of putative cis-regulatory elements (CREs) of TRNP1 in a massively parallel reporter assay (MPRA) and identify one CRE that also co-evolves with cortical folding in Old World Monkeys and Apes. Our analyses indicate that coding and regulatory changes in TRNP1 have modulated its activity to adjust cortical folding during mammalian evolution. They also provide a blueprint how cross-species association studies could help to better understand the evolution and the molecular mechanisms of phenotypes.
