Fossil samples were collected from specimens stored at the following institutions in Argentina: Museo de La Plata, Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" in Buenos Aires and Museo de Ciencias Naturales y Antropológicas "Juan Cornelio Moyano"in Mendoza. Museo de la Escuela Politécnica Nacional of Quito, in Ecuador. The museum specimen number, locality, country, age, skeletal tissue (enamel, bone and dentine) and the altitudinal and latitudinal distribution of each sample are given in Tables 1 and 2. We analyzed 26 samples of Hippidion and 41 carbon and oxygen isotope composition samples of Equus (Amerhippus). Additional 32 samples values (Table 1 and 2) were taken from MacFadden et al. [³¹,³³,³⁶] and MacFadden and Shockey [³⁴].

The samples were finely ground in an agate mortar. The chemical pre-treatment of the samples was carried out as described in Koch et al. [⁵⁸] in order to eliminate secondary carbonate. About 40-50 mg of powdered enamel and bone samples were soaked in 2% NaOCl for three days at room temperature to oxidize organic matter. Residues were rinsed and centrifuged five times with de-ionized water, and then treated with buffered 1M acetic acid for one day to remove diagenetic carbonates. Pre-treatment of the enamel was slightly different because samples were soaked in 2% NaOCl for only one day.

Carbon dioxide was obtained by reacting about 40-50 mg of the treated powder with 100% H₃PO₄ for five hours at 50°C. The carbon dioxide was then isolated cryogenically in a vacuum line. Results are reported as δ = ([R_sample/R_standard]-1 ) × 1000, where R = ¹³C/¹²C or ¹⁸O/¹⁶O, and the standards are PDB for carbon and V-SMOW for oxygen. We have applied the data corrections for calcite from Koch et al. [⁵⁹] to calculate the magnitude of the oxygen isotopic fractionation between apatite CO₂ and H₃PO₄ at 50°C. The analytical variation for repeated analyses was 0,1‰ for δ¹³C and 0,2‰ for δ¹⁸O. For the analysis of phosphate we followed the chemical treatment procedure described by Tudge [⁶⁰], which resulted in the precipitation of the phosphate ions in the form of BiPO₄. CO₂ was obtained by reacting BiPO₄ with BrF₅ as described by Longinelli [⁶¹]. All the samples were run in duplicate and the reported results are the mean of at least two consistent results. The analytical precision for repeated analyses was 0,2‰.

We performed both parametric (t-test) and nonparametric (Wilcoxon Signed-Rank) statistical tests to evaluate δ¹³C and δ¹⁸O differences in Middle and Late Pleistocene populations. SPSS 15.0 software was used for the statistical analysis.

We checked the diagenetic alteration between the different skeletal tissues under the assumption that primary values were similar between different skeletal tissues from one individual. The phosphate oxygen isotope composition is usually considered to be more robust against diagenetic alteration than the carbonate oxygen at least if no bacteria are involved during the alteration processes. We measured both δ¹⁸O _CO3 and δ¹⁸O _PO4 values on the same specimens and obtained a regression between both, and compared those with known isotope equilibrium-relationships for biogenic apatite of modern bones and teeth [⁴⁰,⁴¹]. If samples fall in the range of modern biogenic apatite this would argue for a preservation of primary δ¹⁸O _CO3 and δ¹⁸O _PO4 values, and thus likely also δ¹³C values as they are less easily altered than δ¹⁸O _CO3 values. This was taken as an indicator that even the bone and dentine samples may be reasonably well-preserved and can be interpreted to infer feeding ecology and habitat use of these ancient horses.

Several authors [^42-44] suggest a high correlation between these two phases in some European and North American equids. In South America, the equation obtained for Equus(Amerhippus) from Argentina [⁴⁵] was: δ¹⁸O _CO3 = 16.74 δ¹⁸O _PO4 + 0.64; R² = 0.91. More recently, Sánchez et al. [⁸,³²] also found that a significant relationship existed between the δ¹⁸O results in the carbonate and phosphate apatite phases (enamel, dentine, and bone) when they analyzed the oxygen isotopic composition of gomphotheres from several South American localities. The new data shows that δ¹⁸O _PO4 and δ¹⁸O _CO3 are highly correlated, with the latter being about 8.6 ‰ more positive than the former.

We analyzed the δ¹⁸O _PO4 and δ¹⁸O _CO3 of the 26 samples for Hippidion and 41 samples for Equus (Amerhippus) in this way. The results of δ¹⁸O _PO4 versus V-SMOW standard are reported in tables 1 and 2. The analytical variation for repeated analysis was 0.2‰. Each pair of δ¹⁸O values of teeth and bone belonged to the same individual, generally from the jaw or maxilla, the correlation being: δ¹⁸O _PO4 = 0.9452 δ¹⁸O _CO3 - 10.456; R² = 0.80. We also calculated the correlation between pairs of δ¹⁸O _PO4 - δ¹⁸O _CO3 values for the enamel, dentine, and bone, from the same individual. The results are plotted in Figure 3. The correlations between the different PO₄-CO₃ pairs are:

Enamel δ¹⁸O _PO4 = 2.3371 δ¹⁸O _CO3 - 19.095 R² = 0.97; Dentine δ¹⁸O _PO4 = 1.2999 δ¹⁸O _CO3 - 3.4034 R² = 0.389; and Bone δ¹⁸O _PO4 = 1.1709 δ¹⁸O _CO3 - 6.2038 R² = 0.88. In the first case it appears that the sample might have been modified by diagenesis because the carbonate results among the three skeletal phases (enamel, dentine, and bone) are different. In fact, the range of variation is 2.6‰ in the δ¹⁸O _PO4 and 5.6‰ in the δ¹⁸O _CO3 for the samples from the same specimen. Results of δ¹³C obtained from the bone are similar to those obtained from the dentine and are, in general, equal or more negative than results obtained from the enamel. We observed the same pattern in the δ¹⁸O results as we did in the δ¹³C results. The range of variation between the three skeletal phases (enamel, dentine, and bone) was small and we suppose that they have not been significantly altered by diagenesis. For these reasons, we consider these δ¹³C values as representative of each group of horses.

The carbon isotopic ratio of Equus (Amerhippus) and Hippidion remains indicate significant ecological differences (Figure 4). The Hippidion samples analyzed here are more homogeneous than the Equus (Amerhippus) samples, with a δ¹³C range between -12,9 and -8,0 ‰ (Table 1 and 2).

All the species of Hippidion were almost exclusively C₃ feeders but some individuals from Bolivia and Argentina fall at the lower end of the mixed C₃/C₄ range. For instance, H. principale from the Eastern corridor (at sea level) and H. devillei from the Andes corridor yield similar δ¹³C values suggesting that they ate mainly C₃ plants. The same pattern of dietary partitioning was obtained when comparisons were made between the same taxa at different latitudes (between 22°S and 52°S). From the upper Pliocene (H. devillei from Uquía locality) to the lower Pleistocene (H. principale, from the province of Buenos Aires and H. devillei from the Tarija locality) the dietary partitioning remains similar. The same pattern in dietary partitioning is observed throughout the Middle to Late Pleistocene (Figure 5) showing a predominance of C₃ plants. Also, we did not find differences between H. saldiasi from the Ultima Esperanza in southern Patagonia and the other Hippidion species present at different localities across South America.

Equus species have predominantly been grazers, and as such, carbon isotopic values provide evidence of the C₃ and C₄ grasses. The carbon isotope data indicates that Equus (Amerhippus) shows three different patterns of dietary partitioning. Samples of E. (A.) neogeus from the province of Buenos Aires indicate a preference for C₃ plants in the diet. The samples from Ecuador and Bolivia [E. (A.) andium and E. (A.) insulatus] show a preference for a diet of mixed C₃-C₄ plants, while those from La Carolina (sea level of Ecuador), Bolivia, and Brazil are mostly C₄ feeders.

As mentioned before, a few outliers (e.g. δ¹³C values of 9,2; 6,1 and 5,4‰ from La Carolina) cannot be easily explained. These extremely high δ¹³C values (above 3‰) cannot be explained by consumption of C₄ vegetation, which should impart an upper limit of about 3‰. These outliers could be the result from one of several possibilities, such as individuals living in costal peninsula areas of Ecuador during the time in which C₄ grasses were abundant and may have produced δ¹³C values not observed in the modern ecosystem, or the sample presents taphonomic alteration. Specimen number V.3037 has a high variation between dentine, enamel, and bone (9,2; 1,5 and 6,1‰ respectively). MacFadden et al [³⁶] obtained a δ¹³C value of 1,8‰ for the enamel of the same specimen but obtained 5,4‰ for the enamel of specimen number v.68 from the same locality. A more definitive explanation for these outliers must await the analysis of additional samples from these regions.

The δ¹⁸O _CO3 results of the horse remains differ according to altitudinal and latitudinal distribution. There is a clear difference in the δ¹⁸O values obtained from the populations from the lower plains and those from high elevations. The lowest δ¹⁸O _CO3 values are found in high elevation species from Ecuador and Bolivia [E. (A.) insulatus and E. (A.) andium] and range from 21,3 to 28,3‰ with an average of around 25‰. The H. saldiasi samples from southern Patagonia are also included in this group, their low δ¹⁸O values being caused by the effects of altitude and latitude. The second group is mostly represented by E. (A.) neogeus, H. principale and H. devillei. These species come from the province of Buenos Aires, Brazil, and Bolivia. The distribution values range from 27,7 to 32,1‰ with an average of around 30‰. The highest values come from the Carolina Peninsula in Ecuador, with δ¹⁸O _CO3 maximum of 34,9‰.

As might be expected from ecological theory, finer-scale preliminary results from this study suggest feeding and niche differentiation within coexisting horses. Hippidion and Equus (Amerhippus) are sympatric in the same stratigraphic level in two localities: Tarija (22 °S) in Bolivia and the Pampas in Argentina (38 °S). Data from both localities suggest some difference in dietary partitioning. In a previous study, MacFadden and Shockey [³⁴] presented carbon isotopic results from Tarija herbivores that, based on dental morphology, span the spectrum from presumed browsers (e.g., tapirs) to presumed grazers (e.g., horses) coexisting in the same localities. They suggested than the horses clearly occupied different dietary niches and can be separated using the carbon isotopes and hypsodonty index. Hippidion is the least hypsodont taxon and has the most negative mean δ¹³C value, whereas the relatively hypsodont Equus has the most positive mean δ¹³C value. As has been shown in another article [³⁶], Equus is usually among the most grazing adapted mammalian herbivore in Pleistocene terrestrial ecosystems. This position in the herbivore community is similar at Tarija, whereas Hippidion are more adapted to the browsing end of the spectrum and Equus are primarily C₄ grazers based on their carbon isotopic signature.

Our data suggests that the range of δ¹³C values for H. devillei and H. principale from Tarija falls in the low end of the mixed C₃/C₄ range (-10,8 to -6,6‰), whereas E. (A.) insulatus falls in the higher end (-8,3 to -2,3‰). There is some overlap with at least the two values of -8,3 and -6,6‰ indicating some differences in isotopic niche, with H. devillei consuming less grass than E. (A.) insulatus at this place and time [³⁶].

In the data from Arroyo Tapalque (38 °S), one of the localities in the Pampas, the range of δ¹³C values for H. principale (-11,6 to -9,3‰) and E. (A.) neogeus (-8,5 to -7,9‰) do not overlap, but both are close that they suggest the same pattern of dietary partitioning as in Tarija.

If we look at the morphology, these taxa are very different. Several previous papers based on cranial and limb morphology associate these differences with browsing and grazing diet preference [e.g. [⁴⁶]]. In general, the teeth of Equus (Amerhippus) are more hypsodont than Hippidion, and the enamel patterns are more complicated in Equus (Amerhippus).

Another difference between sympatric species is body sizes. For instance, H. principale and E. (A.) neogeus are sympatric in the Pampas in Argentina. Both have a large body size, adapted to open habitat, but they differ in body mass (460 kg and 378 kg, respectively) [⁴⁷]. The skulls of E. (A.) neogeus are big and show an enlarged preorbital and nasal region. The limb bones are large and robust, but more slender than in the other South American Equus species [³,⁴]. On the other hand, H. principale has a retracted nasal notch might signify some sort of proboscis. The skeleton is large and bulky, and the extremities are robust, mainly the metapodials and phalanges. It is the largest and strongest of the South American hippidiforms. These characteristics are classically associated with dietary and habitat preferences. The morphology indicated that H. principale may have been a browser but was able to live in open grasslands and that E. (A.) neogeus is the most hypsodont horse, and has the relatively straight muzzle characteristic of grazing horses [⁴⁶].

Some of the most fundamental patterns in global biogeography are those that are structured by the latitudinal gradients that extend from pole to equator (Figure 6). The proportion of C₃ to C₄ grasses in most modern ecosystems rises with increasing latitude. C₃ grasses are predominant (>90%) in high-latitude steppes and prairies, whereas C₄ grasses are predominant (>70-90%) in most low-elevation, and equatorial grasslands. The transition between C₃ and C₄ dominance in grasslands occurs at about 40-45° latitude in the Northern Hemisphere [^48-50]. Exceptions to this general rule include grasses at high-elevations or in climates with cool-growing seasons (e.g. the Mediterranean basin), where the grasses are predominantly C₃ regardless of latitude [^50-52], and the occasional C₄ grass species that are found in Arctic regions [⁵³].

MacFadden et al. [³⁶] used the Pleistocene distribution of Equus (Amerhippus) in the Americas to present a general δ¹³C gradient that seems to be symmetrical on either side of the Equator. They found that the isotopic transition between a full C₄ signal and full C₃ signal is observed at about 45°N in the northern hemisphere. Although there are considerably fewer data points in the southern hemisphere they [³⁶] have found a signal of exclusively C₃ feeders at around 35°S (province of Buenos Aires). A similar pattern in the Southern Hemisphere was found by Sánchez et al. [³²] in the distribution of gomphotheres in South America. Samples of gomphotheres from the province of Buenos Aires, at around 39°S latitude show a mean δ¹³C value of -10,8‰, while samples from Chile (from several localities around 35° to 41°S) show a mean δ¹³C value of -12,3‰. This fact would confirm the existence of a latitudinal gradient for the Southern Hemisphere, and places the transition between a full C₃ signal and mixed C₃/C₄ signal around 35° to 41°S.

The new data for the genus Equus (Amerhippus) analyzed in this paper are in agreement with the δ¹³C latitudinal pattern postulated by MacFadden et al. [³⁶], while data for Hippidion also seem to follow a clear pattern. Samples of Hippidion show a boundary between an exclusively C₃ to a mixed C₃/C₄ diet at Tarija (22°S), but in this case there is an effect of altitude combined with latitude. At the highest latitude specimens from Patagonia (52°S), the mean value is -12.2‰. The middle latitude samples, from the province of Buenos Aires (34°S), show δ¹³C values that range between -12,9 and -8,1‰, while the lowest latitude specimens, from Tarija (22°S), show δ¹³C values that range between -6,6 to -0,8‰.

There are also significant differences between the δ¹⁸O _CO3 values for the two genera, even when the range of δ¹⁸O values for high altitude and equatorial samples are taken into account (Table 4). This demonstrates the effects of an altitudinal gradient. The Hippidion specimens from the Andes corridor (Bolivia and northern Argentina) and the Equus (Amerhippus) specimens from Ecuador and Bolivia show the lowest values (between 21,7 and 28,3‰). On the other hand, samples from around 500 m of altitude (Bolivia, Ecuador, Brazil, Peru, and Argentina) present values that correspond with more temperate conditions (27,7 to 32,1‰). The effects of low altitude and latitude on δ¹⁸O _CO3 may also explain the higher value obtained for the samples from La Carolina, Ecuador (sea level, latitude 2°S), where samples showed δ¹⁸O values ranging from 32 to 34,9‰.

We calculated a regression of δ¹⁸O _PO4 values with altitude to quantify the effects of altitude. Bryant et al. [⁴⁴] suggest a high correlation between δ¹⁸O _PO4 and δ¹⁸O _CO3 in Miocene North American equids. We found a good correlation between δ¹⁸O _PO4 values and altitude. The equation obtained was: δ¹⁸O _PO4 = -0.0016 altitude + 20.47 (R² = 0.63). We obtained an altitudinal gradient of -0.16 δ unit/100 meters.

An important point concerns altitudinal gradients. It seems that the horses living at high altitudes, 1866 up to 2780 m above sea level, and at low to middle latitude (2° to 22°S) still have a C₄ component in their diet. This is not surprising at all given the distribution of modern C₄ plants in the central Andes. At present, three C₄ Amaranthaceae species occur at high elevations (>4000 m) where C₄ plants are rarely observed and the altitude record reported for any confirmed C₄ species worldwide is 4800 m for the grass Muhlenbergia peruviana [⁵⁴]. However, this is not true for our specimens from 4000 m, which have a pure C₃ diet. In this central Andean region, a clear altitudinal vegetation gradient is present. Subparamo (2000-3000 m) presents mosaics where shrubs and small trees which alternate with grasslands; and Paramo proper (3000 - 4100 m), is dominated by grasslands and shows patches of woody species which occur only in sheltered locations and along water streams. This altitudinal vegetation gradient changed during the Pleistocene. The treeless vegetation above the upper forest line was most widespread during glacial times, whereas it was limited to small areas on mountain tops during interglacial times [⁵⁵]. The fossil pollen records show that such oscillations in patterns of plant distribution were repeated many times during the Pleistocene Ice Ages [⁵⁶]. During the Last Glacial Maximum, when atmospheric pCO₂ was reduced by some 50%, C₄ plants dominated the Paramo vegetation, while only the highest mountain tops were covered by C₃ grasses because of the low temperatures. Such small patches of C₄-rich vegetation are probably relicts from the last ice age during which paramo vegetation was mainly composed of small tussocks and tufts of C₄ grasses [⁵⁷].

The change in altitudinal vegetation gradients during the Pleistocene is one of several possibilities to explain the C₄ dietary component in horses living at high altitudes. Another alternative explanation might be that the horses partially fed at lower altitudes.

Samples with the same "specimen number" correspond to the same individuals. The bones correspond to the mandibular or maxillary remains that contained the teeth that were analyzed. Plio = Pliocene; Pl = Pleistocene; e = enamel; d = dentine; b = bone; t = tooth (enamel + dentine). Ar = Argentina; Bo = Bolivia; Br = Brazil; Ch = Chile. A = Andean; PL = Plain landscape. ¹ Taken from MacFadden et al. [²²], ² taken from MacFadden and Shockey [²⁷] and ³ taken from MacFadden et al. [²⁰].

Samples with the same "specimen number" correspond to the same individuals. The bones correspond to the mandibular or maxillary remains that contained the teeth that were analyzed. Abbreviations as in Table 1. * taken from MacFadden et al. [²⁵].

A: all Hippidion specimens; B: all Equus (Amerhippus) specimens; C: Hippidion from the Late Pleistocene; D: Hippidion from the Late Pliocene to Early Pleistocene; E: Equus (Amerhippus) from the Late Pleistocene; F: Equus (Amerhippus) from the Middle Pleistocene; G: Equus (Amerhippus) from the plains landscape; H: Equus (Amerhippus) from the mountain corridor; I: Hippidion from the plains landscape and J: Hippidion from the mountain corridor. n: number of samples. SD: standard deviation.

Results of tests performed on twelve possible paired comparisons between two genera of South American equids. The definition of groups is the same as for table 3. p: significance level. p < 0.05 in bold.