A pre-LECA origin of giant viruses as revealed by polymerase-based time tree

• Shared polymerases provide a direct evolutionary link for NCLDV and eukaryotes. • Fossil-calibrated dating robustly places the NCLDV origin before LECA. • Findings support prolonged co-evolution of NCLDVs and hosts during eukaryogenesis. The viral phylum Nucleocytoviricota (NCLDV) infects a wide range of eukaryotic hosts and exhibits genome complexities and virion sizes comparable to prokaryotes, blurring the boundaries between cellular life and viral entities. Despite significant advances from large-scale metagenomic surveys and identification of endogenous viral elements that expand our understanding of NCLDV’s genomic diversity and host range, their evolutionary origin remains contentious. Here, we utilize DNA-directed DNA polymerase (DNAP) and RNA polymerase (RNAP), conserved markers across eukaryotes, prokaryotes, and NCLDVs, to leverage abundant eukaryotic fossils for dating the origins of NCLDVs. Phylogenetic analyses showed that NCLDV’s DNAP and RNAP consistently form deep branches separate from their eukaryotic homologs. Molecular dating analyses further indicated that both DNAP and RNAP in NCLDV originated before the emergence of last eukaryotic common ancestor (LECA), findings which are robust across various dating settings. Notably, the estimated origin based on RNAP was older compared to DNAP, underscoring the need to identify additional orthologues shared with eukaryotes. Collectively, our findings represent the first attempt, to the best of our knowledge, to establish a reliable temporal framework for NCLDV evolution, supporting a pre-LECA origin of ancestral NCLDVs and suggesting a prolonged co-evolutionary history with their (proto-)eukaryotic hosts during eukaryogenesis.