Evolutionary dynamics of predicted G-quadruplexes in human and other great apes

G-quadruplexes (G4s) are non-canonical DNA structures that can form at approximately 1% of the human genome. They facilitate genomic instability by increasing point mutations and structural variation. Numerous G4s participate in telomere maintenance and regulating transcription and replication, and evolve under purifying selection. Despite these important functions, G4s have remained under-studied in human and ape genomes due to incomplete assemblies. Here, we conduct a comprehensive analysis of predicted G4s (pG4s) in the recently released, telomere-to-telomere (T2T) genomes of human, bonobo, chimpanzee, gorilla, Bornean orangutan, and Sumatran orangutan. We annotate 41,232–174,442 new pG4s in these T2T compared to previous ape genome assemblies (5%–21% increase). Analyzing inter-species whole-genome alignments, we identify pG4s shared across apes (approximately one-third of all pG4s) and thousands of species-specific pG4s. pG4s accumulate and diverge at rates consistent with divergence times between species, following molecular clock. pG4s shared across apes are enriched and hypomethylated at regulatory regions—enhancers, promoters, UTRs, and origins of replication—suggesting their conserved formation and functions. Species-specific pG4s (constituting 11–27% of all pG4s) are located in regulatory regions, potentially contributing to adaptations, and in repeats, likely driving genome expansions. Our findings illuminate the evolutionary dynamics of G4s, conservation of their role in gene regulation, and their contributions to ape genome evolution. Our study highlights the utility of high-resolution T2T genomes in revealing elusive yet likely functionally relevant genomic features previously hidden by incomplete assemblies.