Effect of environmental variations on amino acid δ¹⁵N values in macroalgae

Macroalgae are widely used as indicators of environmental change, yet distinguishing natural physiological variability from environmental stressor-induced biochemical responses remains challenging in dynamic coastal environments. We investigated amino acid (AA) specific nitrogen isotope composition (δ¹⁵N_AA) variations in the macroalgae Ecklonia and Ulva to evaluate eco-physiological responses across contrasting environmental conditions. We assessed perennial Ecklonia and ephemeral Ulva collected in different seasons and combined with cultured Ulva exposed to nitrogen addition under a temperature gradient (12 °C, 20 °C, and 27.5 °C) in laboratory experiments. In field-collected macroalgae, the δ¹⁵N_AA values varied temporally, but overall δ¹⁵N_AA patterns were relatively consistent, characterized by isotopically high glutamic acid (Glu) relative to isotopically low glycine (Gly) and serine (Ser). In contrast, overall δ¹⁵N_AA patterns (i.e., δ¹⁵N_Glu > δ¹⁵N_Gly and δ¹⁵N_Ser) were not comparably detected in cultured Ulva, where the magnitude and direction of the isotopic shifts depended on nitrogen addition and temperature. The δ¹⁵N offset between glutamic acid and phenylalanine was 4.0 ± 1.0 ‰ in Ecklonia, consistent with recalculated reference values (3.9 ± 1.1 ‰) but was lower in field-collected Ulva (1.8 ± 1.1 ‰) and cultured Ulva at 12 °C with N addition (i.e., -5.6 ± 1.5 ‰). These results show that δ¹⁵N_AA metrics were sensitive to N enrichment and temperature in controlled experiments, and exhibited seasonal differences between species in the field, reflecting alterations in glutamic acid-centered nitrogen metabolism, particularly in opportunistic Ulva. Ultimately, this approach enhances our interpretation of food-web baselines by linking species-specific physiological traits to ecological responses in eutrophic and warming coastal ecosystems.