Long-Term Fertilisation Shapes the Distribution and N2O Reduction Potential of Clade I and Clade II N2O Reducers Along the Soil Profile in Rice Paddies.

Long-term fertilisation alters nitrous oxide (N2O) emissions from paddy soils, but its effects on the ecological roles of distinct N2O-reducing clades remain unclear. Here, we investigated Clade I and Clade II N2O reducers using a 20-year field experiment with unfertilized control (CK), chemical fertilizer (NPK), and organic fertilizer (OF) treatments. Clade II dominated the community, accounting for 72.4% of nosZ reads and 83.3% of recovered nosZ-containing draft metagenome-assembled genomes (MAGs), and was enriched in carbohydrate-active enzyme (CAZy) genes. However, Clade I abundance explained more variation in soil N2O reduction potential than Clade II (R2 = 0.38 vs. 0.28). Structural equation modelling showed that fertilisation enhanced soil carbon and nitrogen status, which strongly promoted Clade I abundance (β = 0.90). Clade I fully mediated fertilisation effects on N2O reduction potential, whereas Clade II showed only partial mediation. The two clades also exhibited contrasting vertical distributions: Clade I was relatively uniform throughout the soil profile, while Clade II displayed niche stratification, with different taxa dominating surface, middle, and deep layers. These results suggest distinct ecological strategies of the two N2O-reducing clades and long-term fertilisation differentially influences their distribution and contribution to the N2O sink function of rice paddy soils during the period examined.