Phylogenetic analyses of conserved core genes have disentangled most of the ancient relationships in Archaea. However, some groups remain debated, like the DPANN, a deep-branching super-phylum composed of nanosized archaea with reduced genomes. Among these, the Nanohaloarchaea require high-salt concentrations for growth. Their discovery in 2012 was significant because they represent, together with Halobacteria (a Class belonging to Euryarchaeota), the only two described lineages of extreme halophilic archaea. The phylogenetic position of Nanohaloarchaea is highly debated, being alternatively proposed as the sister-lineage of Halobacteria or a member of the DPANN super-phylum. Pinpointing the phylogenetic position of extreme halophilic archaea is important to improve our knowledge of the deep evolutionary history of Archaea and the molecular adaptive processes and evolutionary paths that allowed their emergence. Using comparative genomic approaches, we identified 258 markers carrying a reliable phylogenetic signal. By combining strategies limiting the impact of biases on phylogenetic inference, we showed that Nanohaloarchaea and Halobacteria represent two independent lines that derived from two distinct but related methanogen Class II lineages. This implies that adaptation to high salinity emerged twice independently in Archaea and indicates that emergence of Nanohaloarchaea within DPANN in previous studies is likely the consequence of a tree reconstruction artifact, challenging the existence of this super-phylum.

Methanonatronarchaeia, a newly discovered archaeal lineage of extremely halophilic 19 48 multiple substitutions 7 . This analysis, both by ML and Bayesian approaches including non-49 homogeneous evolutionary models, shows that the clustering of Halobacteria and 50 Methanonatronarchaeia (Fig. 1B-C, red line) was recovered only when the fastest evolving-51 sites are included in the analysis, while the progressive removal of these sites shifted the 52 position of Methanonatronarchaeia away from Halobacteria and to a deeper branching 53 position at the base of the superclass 'Methanotecta' 8 (Fig. 1B-C, green line). This placement 54 is also consistently and robustly recovered when Methanonatronarchaeia were included in 55 two recently published supermatrices comprising a larger number of markers 6 (over 250 56 conserved protein families) or a larger taxonomic sampling of the Methanotecta 9 (including 57 ANME1, Syntrophoarchaeales, Methanoliparia, and a third Methanonatronarchaeia 58 member). In contrast with the dataset of Sorokin et al. 1 , the new placement of59Methanonatronarchaeia was robust to the removal of the fastest-evolving sites for both these 60 supermatrices (Fig. 1D-G).