Light outweighs ammonium: irradiance governs early morphological, physiological and isotopic responses of Zostera marina under controlled nutrient additions

Under accelerating coastal eutrophication, disentangling the relative roles of light limitation and nitrogen enrichment is essential for diagnosing seagrass decline. We conducted a fully crossed light × ammonium experiment using intact Zostera marina cores from Guzhenkou Bay (4 irradiance levels: 180, 90.6, 17, 0 μmol photons m-2 s-1; 4 NH4Cl levels: 0, 25, 50, 100 μmol L-1; 5 weeks; 16 units; no within-cell replication). Across traits, irradiance—not NH4Cl within the tested range—governed responses at the level of dominant main effects. Shading sharply reduced shoot density, above- and below-ground biomass (AGB, BGB) and Fv/Fm, with near-complete collapse at darkness. Biomass turnover accelerated under shading (higher above-ground shedding, below-ground mass loss), and below-ground carbon content declined, together indicating a shift from carbon accrual to expenditure. By contrast, NH4Cl enrichment showed minimal main effects on morphology, pigments, Fv/Fm, or antioxidant enzymes, although tissue δ15N decreased with NH4Cl addition, evidencing ammonium assimilation. Antioxidant responses were variable (POD increased with reduced irradiance; SOD, CAT, GSH-Px inconsistent), while MDA did not increase under shading and was occasionally higher under high light, consistent with lower photo-oxidative load at low irradiance. Leaf and rhizome δ13C showed no significant treatment effects and only a weak, non-significant tendency toward less negative values under severe shading. Collectively, the trait suite diagnoses light-driven carbon limitation as the proximal stressor, with nitrogen effects contingent on concentration and context. We recommend tiered monitoring that couples rapid photophysiology (Fv/Fm) with structural (shoot density, AGB/BGB) and integrative biogeochemical indicators (below-ground C; δ-isotopes), and management focused on water clarity to meet species-specific light requirements.