A skew in poo: Biases in primate fecal isotope analysis and recommendations for standardized sample preparation

Feces are a treasure trove in the study of animal behavior and ecology. Stable carbon and nitrogen isotope analysis allows to assess the dietary niches of elusive primate species and primate breastfeeding behavior. However, some fecal isotope data may unwillingly be biased toward the isotope ratios of undigested plant matter, requiring more consistent sample preparation protocols. We assess the impact of this potential data skew in 114 fecal samples of wild bonobos (Pan paniscus) by measuring the isotope differences (Δ¹³ C, Δ¹⁵ N) between bulk fecal samples containing larger particles (>1 mm) and filtered samples containing only small particles (<1 mm). We assess the influence of fecal carbon and nitrogen content (ΔC:N) and sample donor age (subadult, adult) on the resulting Δ¹³ C, Δ¹⁵ N values (n = 228). Additionally, we measure the isotope ratios in three systematically sieved fecal samples of chimpanzees (Pan troglodytes verus), with particle sizes ranging from 20 μm to 8 mm (n = 30). We found differences in fecal carbon and nitrogen content, with the smaller fecal fraction containing more nitrogen on average. While the Δ¹³ C values were small and not affected by age or ΔC:N, the Δ¹⁵ N values were significantly influenced by fecal ΔC:N, possibly resulting from the differing proportions of undigested plant macroparticles. Significant relationships between carbon stable isotope ratios (δ¹³ C) values and %C in large fecal fractions of both age groups corroborated this assessment. Δ¹⁵ N values were significantly larger in adults than subadults, which should be of concern in isotope studies comparing adult females with infants to assess breastfeeding. We found a random variation of up to 3.0‰ in δ¹³ C and 2.0‰ in nitrogen stable isotope ratios within the chimpanzee fecal samples separated by particle sizes. We show that particle size influences isotope ratios and propose a simple, cost-effective filtration method for primate feces to exclude larger undigested food particles from the analysis, which can easily be adopted by labs worldwide.