Within trophic level shifts in collagen–carbonate stable carbon isotope spacing are propagated by diet and digestive physiology in large mammal herbivores

Stable carbon isotope analyses of vertebrate hard tissues such as bones, teeth, and tusks provide information about animal diets in ecological, archeological, and paleontological contexts. There is debate about how carbon isotope compositions of collagen and apatite carbonate differ in terms of their relationship to diet, and to each other. We evaluated relationships between δ¹³Ccollagen and δ¹³Ccarbonate among free-ranging southern African mammals to test predictions about the influences of dietary and physiological differences between species. Whereas the slopes of δ¹³Ccollagen-δ¹³Ccarbonate relationships among carnivores are ≤1, herbivore δ¹³Ccollagen increases with increasing dietary δ¹³C at a slower rate than does δ¹³Ccarbonate, resulting in regression slopes >1. This outcome is consistent with predictions that herbivore δ¹³Ccollagen is biased against low protein diet components (¹³C-enriched C₄ grasses in these environments), and δ¹³Ccarbonate is ¹³C-enriched due to release of ¹³C-depleted methane as a by-product of microbial fermentation in the digestive tract. As methane emission is constrained by plant secondary metabolites in browse, the latter effect becomes more pronounced with higher levels of C₄ grass in the diet. Increases in δ¹³Ccarbonate are also larger in ruminants than nonruminants. Accordingly, we show that Δ¹³Ccollagen-carbonate spacing is not constant within herbivores, but increases by up to 5 ‰ across species with different diets and physiologies. Such large variation, often assumed to be negligible within trophic levels, clearly cannot be ignored in carbon isotope-based diet reconstructions.