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Data for stable C and N isotope natural abundances of arbuscular mycorrhizal (AM) fungi are currently sparse, as fungal material is difficult to access for analysis. So far, isotope analyses have been limited to lipid compounds associated with fungal membranes or storage structures (biomarkers), fungal spores and soil hyphae. However, it remains unclear whether any of these components are an ideal substitute for intraradical AM hyphae as the functional nutrient trading organ. Thus, we isolated intraradical hyphae of the AM fungus Rhizophagus irregularis from roots of the grass Festuca ovina and the legume Medicago sativa via an enzymatic and a mechanical approach. In addition, extraradical hyphae were isolated from a sand-soil mix associated with each plant. All three approaches revealed comparable isotope signatures of R. irregularis hyphae. The hyphae were 13C- and 15N-enriched relative to leaves and roots irrespective of the plant partner, while they were enriched only in 15N compared with soil. The 13C enrichment of AM hyphae implies a plant carbohydrate source, whereby the enrichment was likely reduced by an additional plant lipid source. The 15N enrichment indicates the potential of AM fungi to gain nitrogen from an organic source. Our isotope signatures of the investigated AM fungus support recent findings for mycoheterotrophic plants which are suggested to mirror the associated AM fungi isotope composition. Stable isotope natural abundances of intraradical AM hyphae as the functional trading organ for bi-directional carbon-for-mineral nutrient exchanges complement data on spores and membrane biomarkers.

Background and Aims Stable isotope two-source linear mixing models are frequently used to calculate the nutrient-uptake efficiency of carnivorous plants from pooled prey. This study aimed to separate prey into three trophic levels as pooled prey limits statements about the contribution of a specific trophic level to the nutrition of carnivorous plants. Phytoplankton were used as an autotrophic reference for aquatic plants as the lack of suitable reference plants impedes calculation of their efficiency. Methods Terrestrial (Pinguicula) and aquatic (Utricularia) carnivorous plants alongside autotrophic reference plants and potential prey from six sites in Germany and Austria were analysed for their stable isotope natural abundances (δ15N, δ13C). A two-source linear mixing model was applied to calculate the nutrient-uptake efficiency of carnivorous plants from pooled prey. Prey preferences were determined using a Bayesian inference isotope mixing model. Key Results Phytophagous prey represented the main contribution to the nutrition of Pinguicula (approx. 55 %), while higher trophic levels contributed a smaller amount (diverse approx. 27 %, zoophagous approx. 17 %). As well as around 48 % nitrogen, a small proportion of carbon (approx. 9 %) from prey was recovered in the tissue of plants. Aquatic Utricularia australis received 29 % and U. minor 21 % nitrogen from zooplankton when applying phytoplankton as the autotrophic reference. Conclusions The separation of prey animals into trophic levels revealed a major nutritional contribution of lower trophic level prey (phytophagous) for temperate Pinguicula species. Naturally, prey of higher trophic levels (diverse, zoophagous) are rarer, resulting in a smaller chance of being captured. Phytoplankton represents an adequate autotrophic reference for aquatic systems to estimate the contribution of zooplankton-derived nitrogen to the tissue of carnivorous plants. The autonomous firing of Utricularia bladders results in the additional capture of phytoplankton, calling for new aquatic references to determine the nutritional importance of phytoplankton for aquatic carnivorous plants.

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