Increasing plant species diversity suppresses free-living N2 fixation in litter and soil of a subtropical karst forest

1. Free-living N₂ fixation (FNF) is an important source of bioavailable nitrogen for forests. Though increasing plant species diversity (PSD) benefits soil nitrogen accumulation, how it impacts FNF rate has not been explored.2. Forty-five plots covering a PSD gradient were selected in a subtropical karst forest, southwest China. FNF rates in leaf litter and soil were measured using acetylene reduction assay calibrated with a ¹⁵N₂ fixation method. Diazotrophic community, litter and soil properties were determined as well.3. The <i>nifH</i> gene abundance was not significantly altered in litter, but was significantly decreased in soil by higher PSD. Diazotrophic community’s Shannon diversity was significantly increased by higher PSD in both litter and soil. Increasing PSD inhibited litter FNF rate via aggravating phosphorus limitation and decreasing the relative abundance of <i>Betaproteobacteria</i> and <i>Deltaproteobacteria</i>. For the mineral soil horizon, increasing PSD suppressed FNF rate via decreasing <i>nifH</i> gene abundance caused by elevated N availability and lowered phosphorus and iron availability, or through decreasing the relative abundance of <i>Deltaproteobacteria</i> but increasing that of <i>Betaproteobacteria</i>.4. Our findings, for the first time, reveal the microbial and abiotic controls on FNF in litter and soil in response to PSD, and hence benefit the prediction of nitrogen input via biological N₂ fixation under changes in PSD.