Caliditerrarchaeota, a new sister to Nanohaloarchaeota, provides insights into the evolution of DPANN halophily

The Nanohaloarchaeota are a clade of halophilic symbionts with small cells and genomes. Originally placed within the Euryarchaeota, they are now widely thought to belong to the DPANN archaea. However, the evolution of this clade and its phylogenetic placement within DPANN remain poorly understood. We applied phylogenetic and comparative genomic analyses to assess the evolutionary relationship and genome evolution of the Nanohaloarchaeota and related DPANN lineages. Our phylogenetic analyses resolve Nanohaloarchaeota as a sister group to a phylum-level lineage referred to as EX4484-52, together forming a clade with Aenigmatarchaeota. Representatives of EX4484-52, for which we propose the name Caliditerrarchaeota, have an anaerobic, thermophilic lifestyle but do not appear to be adapted to high salt concentrations. Using gene tree-species tree reconciliations, we investigated the origin of halophily across these archaeal lineages revealing that adaptations to high-salt appear to have evolved on the branch to Nanohaloarchaeota after their divergence from Aenigmatarchaeota and Caliditerrarchaeota. In agreement with recent work, we also identify hallmarks of halophily in another order-level lineage within the Aenigmatarchaeota (Haloaenigmatarchaeaceae) which appears to represent a second independent adaptation of a DPANN clade to halophily. The two halophilic DPANN lineages are inferred to have distinct sets of proteins that enable them to live in environments with high salt levels. Notably, phylogenetic analyses reveal a dominant signal of gene transfers between Haloaenigmatarchaeaceae and Halarchaeoplasmatales, indicating a potential host-symbiont relationship. This work provides the first detailed investigation of the enigmatic Caliditerrarchaeota, and new insights into the evolution of halophilic lifestyles within DPANN.