All Figures, Table 1, and Supplementary materials of the "Identification of branching order within the kingdom Bamfordvirae" manuscript are presented․ Supplementary materials consist of S. Figures, S. Tables, S Alignments (.fas), S. .nwk, S. .treefile, as well as ReadMe.txt files. Abstract The kingdom Bamfordvirae comprises the majority of the realm Varidnaviria and, according to the 2021 release of Virus Taxonomy by the International Committee on Taxonomy of Viruses, consists of the phyla Nucleocytoviricota and Preplasmiviricota. There are a number of fundamental unresolved issues related to the evolution of Bamfordvirae. These are questions concerning Bamfordvirae taxonomy include the branching order of Nucleocytoviricota and the question of the monophyly of Preplasmiviricota. Here, based on the analysis of individual core proteins phylogenies, supertree, concatenated trees, dendrograms, as well as superdendrogram, we refine the branching order of major groups within phylum Nucleocytoviricota using the rooting of the entire phylum on the cellular outgroups. This effort results in several major changes in Bamfordvirae phylogeny. In particular, we show that Nucleocytoviricota consists of two sister clades, consisting of Phycodnaviridae sensu lato on the one hand and Mimiviridae sensu lato, Iridoviridae/Ascoviridae, Marseilleviridae, pithoviruses including Cedratvirus, Solumvirus, Solivirus, and Orpheovirus, mininucleoviruses, Asfarviridae sensu lato, and Poxviridae on the other hand. According to our data, Asfarviridae sensu lato and Poxviridae have likely originated from within the class Megaviricetes. We give evidence for polyphyly of the phylum Preplasmiviricota and argue for a transfer of the families Lavidaviridae, Adintoviridae and Adenoviridae from the phylum Preplasmiviricota in to the phylum Nucleocytoviricota. We also argue for the origin of the Nucleocytoviricota from small prokaryotic viruses and give arguments against the origin of Nucleocytoviricota from the Adintoviridae/Polinton-like viruses. Thus, our work, by clarifying the phylogenetic relationships within the Bamfordvirae kingdom, provides a solid basis for studying the evolutionary relationships of this kingdom with a vast diversity of supposedly related viruses outside of Bamfordvirae, including Helvetiavirae (Varidnaviria).

The corresponding manuscript will be placed on the bioRxiv