Insertions in universal proteins confirm that Nanohaloarchaea belong to DPANN-Archaea and suggest several new major archaeal clades.

The phylogenetic position of Nanohaloarchaea has been debated, these nanosized archaea being alternatively proposed as sister group to Haloarchaea, members of the DPANN-Archaea, or sister group to Methanocellales. Screening a set of universal proteins, we identified four insertions located at critical locations in three ribosomal proteins and one RNA polymerase subunit that support the branching of Nanohaloarchaea as sister group to Aenigmarchaea within DPANN cluster II (sensu Dombrowski et al., 2020). Insertion analyses and phylogeny of the monomeric primase specific to DPANN-Archaea confirm the existence of a robust clade grouping Undinarchaea, Naiadarchaea and DPANN cluster II, that we propose to call Nanostetteria. Our insertion analysis also supports including Altiarchaea within DPANN-Archaea and suggest a new clades that has not been recovered in phylogenetic analyses, one grouping DPANN-Archaea with Stygia (Hadarchaea and relative) and an even large one grouping these lineages with Acherontia (Thermococci and relatives). The insertion defining this larger clade, present in the ribosomal protein uS7, is also present at the same position in Thaumarchaea, Korarchaea and a subgroup of Asgardarchaea. Whereas the insertion in Thaumarchaea is certainly due to an independent event, we discuss alternative hypotheses that can explain those present in Korarchaea and Asgardarchaea. Finally, we noticed several cases of MAGs misannotations, indicating that insertion analysis can be useful to identify protein with misleading affiliations. The existence of insertions in otherwise highly conserved universal proteins involved in translation or transcription could partly explain the high rate of protein evolution in some archaeal lineage, especially in DPANN-Archaea.