Tree species diversity enhances dark microbial CO2 fixation rates in soil of a subtropical forest

Tree species diversity (TSD) is crucial for modulating microbial processes and enhancing soil organic carbon (SOC) accumulation in forest ecosystems. However, the role of TSD in regulating dark microbial CO₂ fixation (DMCF), a key microbial pathway contributing to SOC formation, remains largely unexplored. Here, we conducted a¹³C–CO₂ labeling experiment across three CO₂ concentrations (2 %, 5 %, and 15 %) using soils from a gradient of TSD (Shannon index 0.15–3.57) in a subtropical forest. Our results revealed that DMCF rates averaged 0.27 μg C g⁻¹ soil day⁻¹ and 174.39 μg C g⁻¹ MBC day⁻¹, offsetting 0.67 %–6.16 % of total soil CO₂ emissions in a subtropical karst forest. DMCF rates were positively correlated with TSD at both 2 % and 5 % atmospheric CO₂ concentrations. In contrast, this positive relationship disappeared under 15 % CO₂, where DMCF rates peaked at intermediate TSD, suggesting a potential stress-induced decoupling. TSD influenced DMCF through its effects on soil properties, microbial communities, and SOC stability. Microbial biomass carbon and the abundance of gram-positive bacteria were identified as key drivers of DMCF, underscoring the significance of microbial community composition in regulating DMCF rates. These findings highlight the often-overlooked contribution of DMCF to SOC storage in subtropical forests and emphasize the role of TSD in enhancing SOC sequestration. This study provides novel insights into the mechanisms governing DMCF, challenging the assumption that biodiversity consistently enhances ecosystem functions under extreme climatic drivers.