Background: Stable isotope ratios (¹³C/¹²C and¹⁸O/¹⁶O) in fossil teeth and bone provide key archives for understanding the ecology of extinct horses during the Plio-Pleistocene in South America; however, what happened in areas of sympatry betweenEquus (Amerhippus)andHippidionis less understood.Results: Here, we use stable carbon and oxygen isotopes preserved in 67 fossil tooth and bone samples for seven species of horses from 25 different localities to document the magnitude of the dietary shifts of horses and ancient floral change during the Plio-Pleistocene. Dietary reconstructions inferred from stable isotopes of both genera of horses present in South America document dietary separation and environmental changes in ancient ecosystems, including C₃/C₄transitions. Stable isotope data demonstrate changes in C₄grass consumption, inter-species dietary partitioning and variation in isotopic niche breadth of mixed feeders with latitudinal gradient.Conclusions: The data forHippidionindicate a preference varying from C₃plants to mixed C₃-C₄plants in their diet.Equus (Amerhippus)shows three different patterns of dietary partitioningEquus(A.)neogeusfrom the province of Buenos Aires indicate a preference for C₃plants in the diet.Equus(A.)andiumfrom Ecuador andEquus(A.)insulatusfrom Bolivia show a preference for to a diet of mixed C₃-C₄plants, whileEquus(A.)santaeelenaefrom La Carolina (sea level of Ecuador) and Brazil are mostly C₄feeders. These results confirm that ancient feeding ecology cannot always be inferred from dental morphology. While the carbon isotope composition of horses skeletal material decreased as latitude increased, we found evidence of boundary between a mixed C₃/C₄diet signal and a pure C₄signal around 32° S and a change from a mixed diet signal to an exclusively C₃signal around 35°S.We found that the horses living at high altitudes and at low to middle latitude still have a C₄component in their diet, except the specimens from 4000 m, which have a pure C₃diet. The change in altitudinal vegetation gradients during the Pleistocene is one of several possibilities to explain the C₄dietary component in horses living at high altitudes. Other alternative explanations imply that the horses fed partially at lower altitudes.

The contemporary mammalian communities of South America were defined by the rise of the Isthmus of Panama and by the deep climatic oscillations during the Pleistocene. Gomphotheres are a conspicuous group of North American immigrant mammals that came to South America during the late Pliocene and did not survive the great megafauna extinction approximately twelve thousand years ago. The present study compiles updated data on the phylogeny, systematics and ecology of this group in South America. Both genera reached South America in two independent dispersal events, Cuvieronius spread across the Andean corridor, while Notiomastodon extent along the eastern route. Cuvieronius hyodon is geographically restricted to the Andean Region in Ecuador, Peru, Bolivia, and Chile. Notiomastodon platensis was recorded in Ecuador, Brazil, Uruguay, Paraguay, Chile, and Argentina, especially the Pampean Region.

The Pampean Region contains sedimentary sequences with abundant mammal fossil records, which constitute the chronological outline of the Plio–Pleistocene of South America. These classic localities have been used for more than a century to correlate with other South American regions. Throughout this time, a series of misinterpretations have appeared. To understand the stratigraphic significance of these localities and the geochronological situation of each unit referring to the Pleistocene, a critical historical study of the antecedents was carried out, evaluating the state of each unit. The biostratigraphic studies of the Pampean Region’s mammalian faunas improved the understanding of biogeographic changes taking into account the environmental fluctuations of the Pleistocene.