<p>Abstract</p> <p>Background</p> <p>Stable isotope ratios (<sup>13</sup>C/<sup>12</sup>C and <sup>18</sup>O/<sup>16</sup>O) 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 between <it>Equus (Amerhippus) </it>and <it>Hippidion </it>is less understood.</p> <p>Results</p> <p>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 C<sub>3</sub>/C<sub>4 </sub>transitions. Stable isotope data demonstrate changes in C<sub>4 </sub>grass consumption, inter-species dietary partitioning and variation in isotopic niche breadth of mixed feeders with latitudinal gradient.</p> <p>Conclusions</p> <p>The data for <it>Hippidion </it>indicate a preference varying from C<sub>3 </sub>plants to mixed C<sub>3</sub>-C<sub>4 </sub>plants in their diet. <it>Equus (Amerhippus) </it>shows three different patterns of dietary partitioning <it>Equus </it>(<it>A</it>.) <it>neogeus </it>from the province of Buenos Aires indicate a preference for C<sub>3 </sub>plants in the diet. <it>Equus </it>(<it>A</it>.) <it>andium </it>from Ecuador and <it>Equus </it>(<it>A</it>.) <it>insulatus </it>from Bolivia show a preference for to a diet of mixed C<sub>3</sub>-C<sub>4 </sub>plants, while <it>Equus </it>(<it>A</it>.) <it>santaeelenae </it>from La Carolina (sea level of Ecuador) and Brazil are mostly C<sub>4 </sub>feeders. 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 C<sub>3</sub>/C<sub>4 </sub>diet signal and a pure C<sub>4 </sub>signal around 32° S and a change from a mixed diet signal to an exclusively C<sub>3 </sub>signal around 35°S.</p> <p>We found that the horses living at high altitudes and at low to middle latitude still have a C<sub>4 </sub>component in their diet, except the specimens from 4000 m, which have a pure C<sub>3 </sub>diet. The change in altitudinal vegetation gradients during the Pleistocene is one of several possibilities to explain the C<sub>4 </sub>dietary component in horses living at high altitudes. Other alternative explanations imply that the horses fed partially at lower altitudes.</p
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