Effects of Simulated Nitrogen Deposition on Soil nifH- and nirK-Harboring Functional Gene Communities in a Cold-Temperate Coniferous Forest

Long-term atmospheric nitrogen (N) deposition can reshape forest soil N cycling, yet targeted evidence from functional gene bacterial communities remains particularly limited in cold-temperate coniferous forests. Here, we examined nifH- and nirK-harboring bacterial communities along a long-term simulated N-deposition gradient in a Larix gmelinii forest in the Greater Khingan Mountains, northeastern China. Four treatments had been applied since 2012: control (0 kg N ha−1 yr−1), low N (25 kg N ha−1 yr−1), medium N (50 kg N ha−1 yr−1), and high N (75 kg N ha−1 yr−1), with N supplied as NH4NO3. Soil inorganic N increased along the gradient; mean NO3−-N increased from 4.9 mg kg−1 fresh soil in the control to 8.1 mg kg−1 under high N. The nifH community showed mainly richness-related and exploratory biomarker responses, with observed richness highest under low N and no significant OTU-level whole-community separation. The nirK community showed a stronger medium-N-associated pattern: observed richness increased from 241.3 OTUs in the control to 421.3 OTUs under medium N, and OTU-level community structure differed among treatments. Taxonomic and biomarker patterns were also centered on medium N, but interpretation was constrained by the high proportion of unclassified or uncultured nirK sequences. These results indicate that nifH- and nirK-harboring communities responded differently to long-term simulated N deposition, with the clearest response in the nirK marker-gene community under medium N. Because this study is based on functional gene amplicon profiles, the results should be interpreted as community-profile evidence rather than direct evidence of N fixation, complete denitrification, gene expression, or N gas fluxes.