Temporal dynamics of bacterial community assembly and network complexity under five years of nitrogen addition in a subtropical forest

Abstract Nitrogen (N) deposition is anticipated to alter soil microbial communities. However, the temporal dynamics of bacterial community assembly and network complexity under prolonged N addition remain unclear, particularly in relation to dissolved organic matter (DOM). We implemented a N addition experiment spanning five years and integrated three complementary approaches: the neutral community model (NCM), quantifying assembly processes based on entire-community null model analysis (QPEN), and inferring community assembly mechanisms by phylogenetic-bin-based null model analysis (iCAMP). We further integrated co-occurrence network and DOM analyses to uncover mechanisms shaping bacterial communities and their interactions. All three methods consistently suggested that stochastic processes, especially drift, mainly shaped community assembly. Both iCAMP and QPEN frameworks revealed a common temporal pattern: the first two years of N addition strengthened stochastic processes due to resource pulses (e.g., nitrate and less humified DOM), whereas deterministic processes gained importance in later years as soil conditions stabilized. Network complexity, assessed through co-occurrence metrics, remained stable across treatments and years. Random forest and regression analyses identified DOM quality, particularly its humification index and carbon-to-nitrogen ratio, as key predictors of network complexity (explaining up to 33.7% of variation). Overall, this study demonstrates a temporal transition in bacterial community assembly under prolonged N addition and emphasizes the contribution of DOM substrate quality in sustaining bacterial network stability. The results inform strategies to maintain microbial ecosystem function under sustained N enrichment. Graphical Abstract