A versatile pipeline to identify convergently lost ancestral conserved fragments associated with convergent evolution of vocal learning.

Molecular convergence in convergently evolved lineages provides valuable insights into the shared genetic basis of converged phenotypes. However, most methods are limited to coding regions, overlooking the potential contribution of regulatory regions. We focused on the independently evolved vocal learning ability in multiple avian lineages, and developed a whole-genome-alignment-free approach to identify genome-wide Convergently Lost Ancestral Conserved fragments (CLACs) in these lineages, encompassing noncoding regions. We discovered 2711 CLACs that are overrepresented in noncoding regions. Proximal genes of these CLACs exhibit significant enrichment in neurological pathways, including glutamate receptor signaling pathway and axon guidance pathway. Moreover, their expression is highly enriched in brain tissues associated with speech formation. Notably, several have known functions in speech and language learning, including ROBO family, SLIT2, GRIN1, and GRIN2B. Additionally, we found significantly enriched motifs in noncoding CLACs, which match binding motifs of transcriptional factors involved in neurogenesis and gene expression regulation in brain. Furthermore, we discovered 19 candidate genes that harbor CLACs in both human and multiple avian vocal learning lineages, suggesting their potential contribution to the independent evolution of vocal learning in both birds and humans.