Comammox biogeography subject to anthropogenic interferences along a high-altitude river

•Comammox clade B was widespread and more abundant than clade A along a plateau river.•Freshwater- and drinking water treatment plant-identified taxa dominated the comammox.•Comammox contributed more abundances and activities to nitrifiers at suburban reach.•Higher nitrate, nitrite or antibiotic contents limited comammox niches in nitrifiers.•Lincosamides, quinolones or macrolides were the major antibiotic predictors. The recent discovery of comammox Nitrospira performing complete ammonia oxidation to nitrate has overturned the long-held dogma of two-step nitrification on Earth, yet little is known about the effect of urbanization interference on their distribution. Using gene-centric metagenomics, we provided the first blueprints about comammox community, biogeography, and environmental drivers along a high-elevation (> 2000 m) river flowing through the largest city on the vulnerable Qinghai-Tibetan Plateau. Our study confirmed a wide presence and diversity of yet-uncultured comammox clade B across wet and dry seasons, with average 3.0 and 2.0 times as abundant as clade-A amoA genes in water and sediments, respectively. Species identified from freshwater and drinking water treatment plants dominated the comammox guilds (58∼100%), suggesting this plateau river shared a similar comammox assemblage with the above habitat types. Compared with the urban area harboring more abundant canonical Nitrospira identified in wastewater (average 24%), the upstream suburban reach had a smaller human population but larger proportions of comammox in ammonia-oxidizing prokaryotes (24∼72% of abundances) and Nitrospira sublineages I/II. Higher contents of nitrate and nitrite in water, and antibiotics in water and sediments, may restrain comammox niches in nitrifiers over the urban area. Further random forest analysis revealed that lincosamides and quinolones were the most important antibiotic predictors for the niche differentiations between comammox and canonical nitrifiers in water, while macrolides for those in sediments. Finally, by incubation experiments, we demonstrated higher activity contributions of benthic comammox in the suburban area (36.2∼92.8% of potential ammonia-oxidation rates) than in the urban reach, and that the contribution variation had significant negative relations with macrolides and their major components. Overall, this study highlighted that anthropogenic activities hampered the advantage of riverine complete nitrifiers over the canonical two-step o