The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, “Megavirales”) include the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, and Mimiviridae, as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki’s Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae, have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki’s Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae. Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome. IMPORTANCE Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches.

Highlights d Abundant, diverse, and active chlamydiae are found in anoxic, deep-marine sediments d Several of the retrieved marine-sediment chlamydial genomes form new clades d These genomes encode features of symbionts, despite a lack of evidence for hosts d Chlamydiaceae did not evolve early and have relatives in marine sediment Authors

The bacterial phylum Chlamydiae, which is so far comprised of obligate symbionts of 1 eukaryotic hosts, are well-known as human and animal pathogens 1-3 . However, the 2 Chlamydiae also include so-called environmental lineages 4-6 that primarily infect microbial 3 eukaryotes 7 . Studying environmental chlamydiae, whose genomes display extended 4 metabolic capabilities compared to their pathogenic relatives 8-10 has provided first insights 5 into the evolution of the pathogenic and obligate intracellular lifestyle that is characteristic 6 for this phylum. Here, we report an unprecedented relative abundance and diversity of 7 novel lineages of the Chlamydiae phylum, representing previously undetected, yet 8 potentially important, community members in deep marine sediments. We discovered that 9 chlamydial lineages dominate the microbial communities in the Arctic Mid-Ocean Ridge 11 , 10 which revealed the dominance of chlamydial lineages at anoxic depths, reaching relative 11 abundances of up to 43% of the bacterial community, and a maximum diversity of 163 12 different species-level taxonomic unit. Using genome-resolved metagenomics, we 13 reconstructed 24 draft chlamydial genomes, thereby dramatically expanding known 14 interspecies genomic diversity in this phylum. Phylogenomic and comparative analyses 15 revealed several deep-branching Chlamydiae clades, including a sister clade of the 16 pathogenic Chlamydiaceae. Altogether, our study provides new insights into the diversity,