Identification of the Branching Order within the Kingdom Bamfordvirae

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 several fundamental unresolved issues related to the evolution of Bamfordvirae . These are questions concerning Bamfordvirae taxonomy including the branching order of Nucleocytoviricota and the question of the monophyly of Preplasmiviricota . Here, based on the analyses of the individual core protein phylogenies, supertree, concatenated trees, dendrograms, as well as superdendrogram, we have refined the branching order of major groups within phylum Nucleocytoviricota using the rooting of the entire phylum on the cellular outgroups. These efforts resulted in several major changes in Bamfordvirae phylogeny. In particular, we showed 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 , Mininucleoviridae, 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 gave evidence for polyphyly of the phylum Preplasmiviricota and argued for a transfer of the families Lavidaviridae , Adintoviridae, and Adenoviridae from the phylum Preplasmiviricota into the phylum Nucleocytoviricota . We also argued for the origin of the Nucleocytoviricota from small prokaryotic viruses and gave arguments against the origin of Nucleocytoviricota from the Adintoviridae /Polinton-like viruses. Importance The monophyly of Varidnaviria , consisting of the Bamfordvirae and Helvetiavirae kingdoms, remains a matter of debate. To confirm or refute the monophyly of Varidnaviria , it is important to identify the sources of origin and phylogenetic relationships common to all Varidnaviria genes, such as the FtsK-HerA superfamily ATPases, major and minor capsid proteins. However, such studies are relevant only if the direction of evolution of both kingdoms was firmly established. Thus, our work, by clarifying the phylogenetic relationships within the Bamfordvirae kingdom, provided 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 ).