A new method to track seed dispersal and recruitment using 15N isotope enrichment

Seed dispersal has a powerful influence on population dynamics, genetic structuring, evolutionary rates, and community ecology. Yet, patterns of seed dispersal are difficult to measure due to methodological shortcomings in tracking dispersed seeds from sources of interest. Here we introduce a new method to track seed dispersal: stable isotope enrichment. It consists of leaf-feeding plants with sprays of N-15-urea during the flowering stage such that seeds developed after applications are isotopically enriched. We conducted a greenhouse experiment with Solanum americanum and two field experiments with wild Capsicum annuum in southern Arizona, USA, to field-validate the method. First, we show that plants sprayed with N-15-urea reliably produce isotopically enriched progeny, and that delta N-15 (i.e., the isotopic ratio) of seeds and seedlings is a linear function of the N-15-urea concentration sprayed on mothers. We demonstrate that three urea dosages can be used to distinctly enrich plants and unambiguously differentiate their offspring after seeds are dispersed by birds. We found that, with high urea dosages, the resulting delta N-15 values in seedlings are 10(3)-10(4) times higher than the delta N-15 values of normal plants. This feature allows tracking not only where seeds arrive, but in locations where seeds germinate and recruit, because delta N-15 enrichment is detectable in seedlings that have increased in mass by at least two orders of magnitude before fading to normal delta N-15 values. Last, we tested a mixing model to analyze seed samples in bulk. We used the delta N-15 values of batches (i. e., combined seedlings or seeds captured in seed traps) to estimate the number of enriched seeds coming from isotopically enriched plants in the field. We confirm that isotope enrichment, combined with batch-sampling, is a cheap, reliable, and user-friendly method for bulk-processing seeds and is thus excellent for the detection of rare dispersal events. This method could further the study of dispersal biology, including the elusive, but critically important, estimation of long-distance seed dispersal.