Effects of shrub expansion on methane emissions from temperate wetlands and their regulatory mechanisms

• Shrub expansion reduces wetland CH 4 emissions. • Shrub expansion represses key genes involved in the acetate pathway for methane production and upregulates genes related to methane oxidation. • The synergistic interaction between soil physicochemical properties and oxidative genes plays a key role in regulating CH₄ emissions. • The bacterial communities exhibited greater sensitivity to shrub expansion than fungal communities. Climate warming and human activities have led to widespread expansion of shrubs in many wetlands, altering the distribution patterns of native vegetation and disrupting C cycling. Although the effects of shrub expansion on soil microbial communities and methane (CH 4 ) emissions have been extensively studied, the specific microbial-mediated pathways involved in methane cycling remain unexplored. The static chamber method was used to investigate the characteristics of CH 4 emission flux changes under different levels of shrub expansion. Additionally, metagenomics technology was employed to assess the effects of different shrub expansion levels on soil microbial community composition, function, and diversity (bacteria and fungi), as well as the methane metabolic pathways mediated by these communities. Shrub expansion in wetlands was categorized into four classes based on shrub coverage. We found that methane flux decreased significantly with increasing shrub expansion, with cumulative emissions under extensive expansion conditions being only 28 % of those under no expansion conditions. The peak emissions on August 15 under no expansion conditions were 2–3 times higher than those in shrub-expanded plots. The partial least squares path model (PLS-PM, GOF = 0.731) indicated that shrub expansion enhanced soil physicochemical properties (β = 0.865), which inhibited methanogenic genes (β = −0.617) and activated methane oxidation pathways (total effect β = 0.728). Methane-oxidizing genes contributed the most to CH 4 reduction, accounting for 72.4 % of the pathway effect. This was primarily manifested as inhibition of key genes involved in the acetate pathway for methane production (comB and hdrA) and upregulation of methane-oxidizing-related genes (mmoB and DAK). Shrub expansion significantly increased soil ammonium nitrogen content while reducing soil moisture content. Although bacterial α diversity remained unchanged, the fungal Chao1 index significantly increased. Additionally, MI and HI treatments significantly altered bacterial community structure, while fungal communities remained relatively stable. The relative abundance of Verrucomicrobia initially increased with shrub expansion but decreased at higher levels, while Ascomycota, Basidiomycota, and Mucoromycota showed significant increases. The study suggests that the reduction in CH 4 emissions caused by shrub expansion is primarily regulated by a synergistic pathway involving the combined effects of soil physicochemical properties and oxidative microbial genes. Furthermore, bacterial communities are more sensitive to shrub expansion than fungal communities. These results highlight the complex interactions between aboveground vegetation dynamics, soil microbial communities, greenhouse gas fluxes, and environmental factors. However, this CH 4 reduction likely reflects hydrological degradation and wetland desiccation, which may increase CO 2 emissions from oxidized peat and compromise long-term carbon sequestration, underscoring the need to prevent shrub encroachment for wetland conservation.