Phylogenetic divergence and adaptation of Nitrososphaeria across lake depths and freshwater ecosystems

Abstract Thaumarchaeota (now the class Nitrososphaeria in the phylum Thermoproteota in GTDB taxonomy) are abundant across marine and soil habitats; however, their genomic diversity and evolutionary history in freshwater environments remain elusive. Here, we reconstructed 17 high-quality metagenome-assembled genomes of Nitrososphaeria from a deep lake and two great rivers, and compared all available genomes between freshwater and marine habitats regarding their phylogenetic positions, relative abundance, and genomic content. We found that freshwater Nitrososphaeria were dominated by the family Nitrosopumilaceae and could be grouped into three distinct clades closely related to the genera Nitrosopumilus, Nitrosoarchaeum, and Nitrosotenuis. The Nitrosopumilus-like clade was exclusively from deep lakes, while the Nitrosoarchaeum-like clade was dominated by species from deep lakes and rivers, and the Nitrosotenuis-like clade was mainly from rivers, deep lakes, and estuaries. Interestingly, there was vertical niche separation between two clades in deep lakes, showing that the Nitrosopumilus-like species dominated shallow layers, whereas the relative abundance of the Nitrosoarchaeum-like clade increased toward deep waters. Phylogenetic clustering patterns in the Nitrosopumilaceae supported at least one freshwater-to-marine and two marine-to-freshwater transitions, the former of which refined the potential terrestrial-to-marine evolutionary path as previously proposed. The occurrence of the two marine-to-freshwater transitions were accompanied by horizontal transfer of the genes involved in nutrition regulation, osmoregulation, and cell motility during their colonization to freshwater habitats. Specifically, the Nitrosopumilus-like clade showed losses of genes encoding flagella assembly and ion transport, whereas the Nitrosoarchaeum-like clade had losses of intact genes involved in urea uptake and utilization and gains of genes encoding osmolarity-mediated mechanosensitive channels. Collectively, our results reveal for the first time the high genomic diversity of the class Nitrososphaeria across freshwater ecosystems and provide novel insights into their adaptive mechanisms and evolutionary histories.