Ecological and metabolic signatures of biofilm viromes across a national river surveillance network

Benthic biofilms are biogeochemical hotspots in rivers, yet their endogenous viral communities remain poorly resolved at national scales. We analysed 447 biofilm metagenomes from 146 sites within England's River Surveillance Network (RSN), sampled in spring and autumn from 2021 to 2023, integrating viral operational taxonomic units (vOTUs), water-quality gradients, predicted host associations and auxiliary metabolic genes (AMGs). We recovered 38,534 vOTUs, revealing extensive viral novelty, where 93% of the vOTUs could not be assigned to a known family. Viromes were dominated by Caudoviricetes and unclassified lineages and were extremely heterogeneous: no vOTU occurred in more than 8.3% of samples. Season, but not sampling year, structured virome composition (family-level PERMANOVA R2 = 0.050, p = 0.001; year p = 0.08), and within-site turnover between spring and autumn exceeded turnover between years. Bray-Curtis ordination linked virome structure weakly to pH, dissolved organic carbon, conductivity, nitrate-N and dissolved oxygen (envfit R2 ≤ 0.066), while threshold indicator taxa analysis (TITAN) identified vOTUs with abrupt change points along alkalinity, dissolved oxygen, nitrate-N and temperature gradients. Predicted host associations were concentrated in Pseudomonodota, Cyanobacteriota and Bacteroidota, with Microcoleus, Chamaesiphon and Flavobacterium among the dominant host genera. Virus-host networks were modular, and module abundances correlated with water quality variables (dissolved oxygen, pH, nitrate etc.), suggesting that environmental filtering acts partly through host ecology. Predicted virus-host associations formed a sparse network organised around a few highly connected viral lineages. Functional profiling showed that the biofilm virome was dominated by nucleotide metabolism, DNA methylation, phosphate-stress, glycan-modification and photosynthesis-related AMGs, with nrdA/nrdB ribonucleotide reductase as the dominant AMG signal. Few vOTUs (1.2%) were detected as proviruses, although provirus fractions were higher in selected host-linked lineages. Collectively, these results show that river biofilm viromes are seasonally structured ecological communities whose composition, host associations and metabolic potential reflect the physicochemical and biological state of rivers. We propose that biofilm viromes provide a complementary layer of information for understanding river health, microbial ecology and the propagation of anthropogenic pressures in freshwater systems.