Examining potential drivers of variation in the carbonate-collagen stable carbon isotope offset of modern and Late Pleistocene ungulates from C3-dominant ecosystems

The stable carbon isotope composition (δ¹³C) of mammalian hard tissues offers a powerful tool for understanding the ecology of modern and fossil species. Over the past 40 years, the offset between δ¹³C values measured in structural carbonate and collagen (Δ_ca-co) has been used as an indicator of trophic category. However, several recent studies have demonstrated substantial variation in the Δ_ca-co values of herbivores, raising calls for caution in the use of static Δ_ca-co values to interpret trophic relationships. To better clarify mechanisms driving variation in herbivore Δ_ca-co, we examined the relationship between δ¹³C_carbonate and δ¹³C_collagen in modern and Late Pleistocene ungulates from C₃-dominant ecosystems in the Americas and Eurasia. Factors investigated included diet, digestive physiology, and tissue type. Our results showed considerable variation in herbivore Δ_ca-co, underscoring the need for caution when applying static values to reconstruct trophic position. We found a consistent diet-driven pattern in Δ_ca-co values (grazer > mixed feeder > browser). Identifying this pattern in modern and ancient species adds to growing evidence for the generalizability of the relationship between herbivore diet and Δ_ca-co. In contrast, the fossil data set showed evidence counter to predictions based on physiology: non-ruminants displayed higher Δ_ca-co values and a greater rate of increase in δ¹³C_carbonate relative to δ¹³C_collagen than ruminants. Finally, tissue type substantially influenced Δ_ca-co, with values derived from teeth generally higher and more variable than from bone. This work highlights the potential for herbivore Δ_ca-co to serve as a refined indicator of dietary ecology in modern and ancient ecosystems.