Genomic insights into adaptation strategies and microevolutionary forces of novel non-AOA Nitrososphaeria in acid mine drainage ecosystems

The class Nitrososphaeria is best known for ammonia-oxidizing archaea (AOA), yet deeply branching non-AOA lineages remain poorly characterized, leaving a critical gap in our understanding of the group’s early evolution and ecological diversification. Herein, we recovered 44 non-AOA Nitrososphaeria metagenome-assembled genomes (MAGs) from acid mine drainage (AMD) sediments in diverse metal mines, representing two novel genera within the family UBA164, Acidarchaeum and Thermosulfuris . A meta-analysis of 251 AMD-associated metagenomes showed that these potentially thermophilic lineages are globally distributed but typically rare, with local peaks (~6.6%) at sites such as Fankou. Metabolic reconstruction suggested a facultatively anaerobic, mixotrophic lifestyle capable of CO oxidation and sulfur reduction, and extensive acid- and heavy-metal resistance mediated primarily by ether-linked archaeal lipids, ion efflux systems, and enzymatic reduction. Genus-specific traits include dissimilatory sulfate reduction in Thermosulfuris and urea utilization in Acidarchaeum , illuminating distinct ecological niches for them. Population-genomic analyses reveal low homologous recombination and pervasive purifying selection in these non-AOA populations, together with local relaxation of selection and elevated diversity, the former being correlated with geochemical stressors (notably copper), pointing to long-term, geochemically driven adaptation. Overall, these findings provide insights into the biodiversity, ecophysiology, and evolutionary dynamics of non-AOA Nitrososphaeria .