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

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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.