Seagrass meadows act as important carbon sinks by fixing atmospheric carbon dioxide (CO₂). However, methane (CH₄) produced in their anoxic sediments can be emitted to the atmosphere through the sediment-water interface, partially offsetting their climate mitigation benefits. At present, quantitative understanding of methanogenic pathways and their spatial heterogeneity in seagrass ecosystems remains limited. In addition, macroalgae invasion has become a widespread environmental issue in seagrass meadows globally, yet its effects on methanogenic pathways and the underlying microbial regulatory mechanisms are poorly understood. Furthermore, most previous assessments of CH₄ offset effects only consider sedimentary organic carbon burial as the baseline carbon sink, neglecting lateral carbon export, which may lead to systematic underestimation of the net climate benefit of seagrass systems. To address these gaps, this study investigated a tropical seagrass meadow and a temperate macroalgae-invaded seagrass system. A combination of ¹³C-labeled substrate incubation experiments, ²²²Rn tracer, and in situ multi-parameter observations was employed to systematically examine methanogenic pathways, CH₄ fluxes, microbial mechanisms, and carbon sink offset effects in seagrass ecosystems. We found that methylotrophic methanogenesis dominates in tropical seagrass meadows, which account for over 90% of global seagrass CH₄ emissions. In temperate seagrass-macroalgae mixed systems, where methylotrophic methanogenesis also dominates. CH₄ flux across the sediment-water interface reached 1590±262μmol m^-2 d^-1 in November, approximately 1.4 times higher than in June and substantially exceeding the global median. Despite lower temperatures in November, bioturbation caused by overwintering swans enhanced porewater exchange and CH₄ release. In addition, macroalgae degradation produces approximately 7 times more CH₄ than an equivalent amount of seagrass, with a tighter coupling between dissolved organic carbon (DOC) and CH₄ production. Under substrate coexistence conditions, a positive priming effect occurs at low loading levels but weakens or even disappears at high loading, indicating a substrate load threshold for priming effects. SHAP analysis reveals that DOC and nutrient stoichiometry jointly regulate the priming effect. Correspondingly, macroalgae addition enriches fermentative bacteria and the metabolically versatile Methanosarcina, driving a shift in methanogenic pathways from methylotrophic to multifunctional; seagrass addition, in contrast, maintains a more stable microbial community. In the mixed seagrass-macroalgae mixed system, porewater exchange dominates sediment carbon export, different carbon species exhibit divergent fates of outwelling versus outgassing, and outwellings of total alkalinity and refractory dissolved organic matter constitute indirect carbon sinks, while greenhouse gas emissions offset approximately 9-12% of the carbon sink benefit.

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