Low N Deposition Coupled With Climate Warming Promote Soil Asymbiotic N Fixation via Increasing Microbial Specialists in Alpine Grassland

Biological nitrogen fixation (BNF) is a vital process for introducing new N into natural ecosystems, and this process has been demonstrated to be suppressed by high N deposition. However, the net ecological effect and underlying mechanisms of BNF under chronic low-level N deposition, characteristic of terrestrial ecosystems, remain highly uncertain. Given that climate warming is a key environmental change factor concurrent with N deposition, it is important to investigate whether climate warming can change BNF activity and alter the effects of N deposition on BNF. To fill these knowledge gaps, we implemented a decade-long-term manipulation experiment in an alpine grassland ecosystem with 5 treatments, that is, experimental warming (W), low-level N deposition (N_L), high-level N deposition (N_H), combination of climate warming and low-level N deposition (WN_L) and the control (CK). BNF rate was measured by ¹⁵N₂ isotope discrimination. We found that N_L significantly stimulated BNF by 112%, contrasting sharply with the complete suppression under N_H. Climate warming alone increased the BNF rate by 123%, while the WN_L amplified this effect, stimulating BNF by 234%. Structural equation modeling revealed that WN_L selectively favored specific diazotrophic groups (Desulfovibrio), whose proliferation directly drove the observed BNF shifts. In this N-limited alpine grassland, low-level chronic N deposition, especially when combined with warming, fundamentally shifts the diazotrophic community structure by driving a process of niche contraction. This selection process functionally enriches specialized diazotrophs, resulting in a dramatic, positive feedback that significantly promotes the overall biological nitrogen input. Our findings highlight the potential for increased N inputs under realistic future climate scenarios and provide a scientific basis for precision N management in alpine grasslands, both on the Qinghai-Tibetan Plateau and worldwide.