Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

9Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic 10Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially 11 ancient component of the eukaryotic virome. However, their origin(s), evolution and 12 potential roles in the emergence of modern eukaryotes remain a subject of intense 13 debate. Since the characterization of the mimivirus in 2003, many big and giant viruses 14 have been discovered at a steady pace, offering a vast material for evolutionary 15 investigations. In parallel, phylogenetic tools are constantly being improved, offering 16 more rigorous approaches for reconstruction of deep evolutionary history of viruses 17 and their hosts. Here we present robust phylogenetic trees of NCLDVs, based on the 8 18 most conserved proteins responsible for virion morphogenesis and informational 19 processes. Our results uncover the evolutionary relationships between different NCLDV 20 families and support the existence of two superclades of NCLDVs, each encompassing 21 several families. We present evidence strongly suggesting that the NCLDV core genes, 22which are involved in both informational processes and virion formation, were acquired 23 vertically from a common ancestor. Among them, the largest subunits of the DNA-24 dependent RNA polymerase were seemingly transferred from two clades of NCLDVs to 25 7 125The core markers share a similar phylogenetic signal 126Using a maximum-likelihood (ML) framework, the monophyly of all known 127 NCLDV families, except the Phycodnaviridae, was obtained with high support in most of 128 the 8 single-protein phylogenetic trees ( Supplementary Figure 1). As often observed in 129 published NCLDV phylogenies 26 , Ascoviridae were however nested within the 130Iridoviridae in most trees. The grouping of the Mimiviridae with related unclassified 131 viruses with smaller genomes often referred to as the "extended Mimiviridae" 21 or more 132 recently the "Mesomimivirinae" 29 , was obtained in five out of the 8 trees. We will refer 133 to this grouping as the "Megavirales" putative order (see Supplementary Information). 134 135The Poxviridae clade consistently formed a long branch and displayed the most 136 unstable position, branching next to various families (see Supplementary Information). 137

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