Phosphorus Availability Mediates Pathway-Specific Nitrogen Cycling in Stratified Peatland Microbiomes

Abstract Peatland microbiomes regulate nitrogen cycling processes that control nutrient retention and greenhouse gas emissions in these carbon-rich ecosystems. While depth-driven redox gradients are known to structure microbial communities, how physicochemical stratification shapes the functional versus taxonomic organisation of nitrogen-cycling microorganisms remains unclear. Here, we used shotgun metagenomics to characterise nitrogen-cycling gene distributions, taxonomic affiliations, and metagenome-assembled genomes across depth and vegetation gradients in a temperate blanket bog. Depth emerged as the primary structuring factor, creating pronounced functional-taxonomic decoupling. Surface peat (0-20 cm) harboured functionally diverse but taxonomically constrained communities assembled deterministically around nitrification and labile N acquisition, while subsurface peat (20-40 cm) supported taxonomically richer but functionally-simpler communities assembled stochastically and enriched in denitrification and dissimilatory nitrate reduction. Linear mixed-effects models revealed pathway-specific controls on nitrogen cycling. Denitrification increased with depth (β=11.53, p<0.05), whereas organic nitrogen transformation declined (β=−5.81, p<0.05); depth effects on nitrification and nitrogen fixation became non-significant after accounting for environmental variables. Phosphorus emerged as the strongest environmental predictor, regulating nitrification (β=95.40, p<0.01), N fixation (β=128.33, p<0.01), organic nitrogen transformation (β=80.53, p<0.01), and denitrification (β=-109.63, p<0.05), highlighting the importance of P availability in structuring microbial nitrogen cycling. This challenges traditional nitrogen-limitation paradigms in ombrotrophic systems. Metagenome-assembled genomes revealed Pseudomonadota as the dominant nitrogen-cycling lineage, while incomplete denitrification capacity indicated genetic potential for N2O accumulation in subsurface layers. These findings demonstrate that phosphorus availability, rather than nitrogen content alone, regulates microbial nitrogen transformation capacity in peatlands, with implications for predicting nutrient dynamics under altered hydrological and nutrient deposition regimes.