Gradual enrichment of ¹⁵N with humification of diets in a below-ground food web: Relationship between ¹⁵N and diet age determined using ¹⁴C

1. Stable nitrogen (N) isotope has been widely used to disentangle food webs and to infer trophic positions of organisms based on an assumption that the stepwise enrichment occurs along trophic levels. The enrichment of ¹⁵N in soil organisms with diet humification has also been reported, but the underlying mechanism has not been fully examined. 2. To examine the effect of diet humification on ¹⁵N, we estimated the stable N isotope ratios and diet ages of earthworms and termites. These organisms feed on organic matter with various degrees of humification, ranging from undecomposed plant materials to humified organic matter (soil organic matter), in a gallery forest and a savanna in the Ivory Coast. We defined diet age as the time elapsed since carbon (C) in the diet of earthworms and termites was fixed from atmospheric CO₂ by photosynthesis; it was estimated by comparing the radiocarbon (¹⁴C) content of these organisms to atmospheric ¹⁴CO₂ records. 3. Stable N isotope ratios increased along the humification gradient of diets, and values for earthworms and termites varied from 1.8‰ to 9.9‰ and from -1.5‰ to 15.9‰, respectively. Epigeic (litter-feeding) earthworms had younger diet ages (2-4 years), whereas endogeic (soil-feeding) earthworms generally exhibited older diet ages (5-9 years). Grass-feeding termites had young diet ages (2 years), and wood/soil-feeding termites had the oldest diet ages (c. 50 years). Soil-feeding termites were similar in diet age (7-12 years) to wood feeders (8-11 years), with the exception of one species (18-21 years) that consumes large-diameter wood. 4. A significant positive relationship was found between diet ages and stable N isotope ratios of the two groups in the savanna. This relationship held in the gallery forest when termites feeding on woody tissues were not considered. These results show that the stable N isotope ratios of organisms can increase with diet age, unless C in the diet has been stored as organic matter, such as woody tissue, that is able to age without being subject to humification processes. 5. Given that above-ground food webs are often sustained directly by material and energy flow from below-ground food webs, in addition to trophic interactions, gradual enrichment of ¹⁵N with the humification of below-ground diets should be considered when interpreting stable N isotope ratios of terrestrial food webs.