Background: C4 grasses were not abundant in North America during the middle Miocene (c. 15 Ma). They did not become abundant until around 7 Ma. One can analyse stable carbon (δ13C) and oxygen (δ18O) isotope values in the enamel of fossil horse teeth to determine the
extent to which horses were eating C4 grasses even during the period before those grasses became abundant.
Questions: In southern California, what proportion of a middle Miocene horse’s diet was made up of C4 grasses? Was the amount enough to influence the size and shape of horse teeth?
Organisms: Eighty-five specimens of five fossil horse species – Acritohippus stylodontus, Archaeohippus mourningi, Merychippus californicus, Scaphohippus intermontanus, and Scaphohippus sumani – from the middle Miocene (c. 16 Ma) of southern California (i.e. Barstow Formation, Cajon Valley Formation, and Temblor Formation).
Methods: To determine if C4 grasses were present in middle Miocene horse diets, we analysed stable carbon (δ13C) and oxygen (δ18O) isotope values from the enamel of the fossils. If the result did indicate C4 foraging at a locality, we modelled the percentage of C4 grasses in equid diets using Stable Isotope Analysis in R (SIAR) v.4.2.2.
Results: Modelled percentage C4 in equid diets was \<20\%. Each formation was statistically significantly different from the others in terms of δ13C values. Barstow specimens had the highest values, those from Cajon Valley the lowest, and those from Temblor were intermediate.
Those results indicate that horses ate C4 grasses within the Barstow and possibly the Temblor Formation but not the Cajon Valley Formation. Within the Barstow sample, Scaphohippus sumani had statistically significantly lower δ13C but statistically significantly higher δ18O values than Acritohippus stylodontus, suggesting a higher proportion of C3 grasses in the diet of Scaphohippus sumani versus a higher proportion of C4 grasses for Acritohippus stylodontus. The latter species also had higher tooth crowns, consistent with a diet richer in C4 grasses. There were no statistically significant differences between species at Cajon Valley for either δ13C or δ18O. The δ13C values for Merychippus californicus suggest that the habitats of the Temblor Formation had a low percentage (\<6\%) of C4 plants.

Conclusions: C4 grasses lived in the mid-Miocene landscape in southern California up to 8 million years before the rapid increase in C4 ecosystems that occurred worldwide about 7 to 5 Ma. Horses foraged these grasses, so hypotheses related to horse morphological evolution must take C4 plants into account.