Phenylalanine 15N enrichment likely indicates fungal-derived organic nutrient acquisition in mycoheterotrophic plants across fungal guilds.

Beyond fully mycoheterotrophic plants, many green plants may also obtain carbon from fungal partners. However, bulk stable isotope analyses often lack sufficient resolution in arbuscular mycorrhizal and rhizoctonia-associated orchid systems, limiting inference of fungal-derived organic nutrient acquisition. We measured δ15N values of glutamic acid (δ15NGlu) and phenylalanine (δ15NPhe) in 10 fully mycoheterotrophic species spanning arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic fungal guilds, as well as in three photosynthetic species with contrasting inferred nutritional modes, together with co-occurring autotrophic references and, where available, fungal material. This Glu-Phe framework was originally developed for animal food-web studies, in which trophic position is estimated from strong 15NGlu enrichment (c. 7-8‰ per trophic step) and minimal 15NPhe enrichment (c. 0-1‰). We found that mycoheterotrophic plants invert this pattern, showing minimal 15NGlu enrichment (-1.2 to 3.3‰) but pronounced 15NPhe enrichment (11.2 to 19.6‰) relative to their fungal partners. They also exhibited amino acid δ15N profiles distinct from autotrophs, consistent with uptake of fungal-derived organic nitrogen. To our knowledge, such strong 15NPhe enrichment relative to nutrient sources has not been reported in other organisms. Overall, amino acid δ15N profiles complement bulk δ2H, δ13C, and δ15N values for assessing fungal-derived organic nutrient acquisition in green plants.