Confounding factors in the isotopic analysis of herbivore fecal samples

Stable isotope analysis is a tool used to provide information on species’ ecology, including diet, trophic interactions, and migration patterns. Among biological tissues, fecal samples have the advantage of being relatively easy to collect, supporting studies on rare, elusive, and threatened species. Nonetheless, fecal samples from mammalian herbivores present some confounding factors for isotopic analysis, such as being divided into pellets with a heterogeneous mix of plant matter, the influence of preservation methods, and their decomposition stage. To assess the effects of these confounding factors, using a forest ungulate (Subulo gouazoubira) as a biological model, we conducted three experiments to test whether (i) small fecal portions are representative of an entire fecal pile, and if (ii) preservation of samples in absolute ethanol and (iii) decomposition stage in the field affect stable carbon and nitrogen isotopic values. Portions (4 mm³) from the same fecal pellet and different pellets within the same fecal pile were found to be representative of the isotopic values from the entire pile. Preserving fecal samples with low-fat content in absolute ethanol over time proved to be an effective method, with no substantial change in isotopic values. However, older samples may present biased results, as decomposition was found to increase nitrogen values over time (adj. R² = 0.15; p < 0.001). Our experimental approach improves the reliability of using herbivore fecal samples in isotopic ecology studies. We recommend, however, replicating these experiments with other species in different environments and urge caution when using older, degraded material.