The rich fossil record of the family Equidae (Mammalia: Perissodactyla) over the past 55 MY has made it an icon for the patterns and processes of macroevolution. Despite this, many aspects of equid phylogenetic relationships and taxonomy remain unresolved. Recent genetic analyses of extinct equids have revealed unexpected evolutionary patterns and a need for major revisions at the generic, subgeneric, and species levels. To investigate this issue we examine 35 ancient equid specimens from four geographic regions (South America, Europe, Southwest Asia, and South Africa), of which 22 delivered 87-688 bp of reproducible aDNA mitochondrial sequence. Phylogenetic analyses support a major revision of the recent evolutionary history of equids and reveal two new species, a South American hippidion and a descendant of a basal lineage potentially related to Middle Pleistocene equids. Sequences from specimens assigned to the giant extinct Cape zebra, Equus capensis, formed a separate clade within the modern plain zebra species, a phenotypicically plastic group that also included the extinct quagga. In addition, we revise the currently recognized extinction times for two hemione-related equid groups. However, it is apparent that the current dataset cannot solve all of the taxonomic and phylogenetic questions relevant to the evolution of Equus. In light of these findings, we propose a rapid DNA barcoding approach to evaluate the taxonomic status of the many Late Pleistocene fossil Equidae species that have been described from purely morphological analyses.DNA taxonomy ͉ equid evolution ͉ macroevolution ͉ phylogeny ͉ ancient DNA T he original sequence of horse fossils found in the 1870s by paleontologist Othaniel Charles Marsh, and popularized by Thomas Huxley (1), has been enriched by a large fossil record over the years and has now become one of the most widely known examples of macroevolutionary change (2). The original linear model of gradual modification of fox-sized animals (Hyracothere horses) to the modern forms has been replaced by a more complex tree, showing periods of explosive diversification and branch extinctions over 55 MY (3). The end of the Early Miocene (15-20 MYA) marks a particularly important transition, separating an initial phase of small leafy browsers from a second phase of more diverse animals, exhibiting tremendous body-size plasticity and modifications in tooth morphology (4). This explosive diversification has been accompanied by several stages of geographic extension from North America to the rest of the New and Old Worlds, so that by the end of the Miocene (5 MYA) more than a dozen distinct genera are represented in the fossil record (4) (Astrohippus,

Appendicular skeletons of isotemnid notoungulates are described from Cañ adó n Vaca (Vacan ''subage'', Casamayoran South American Land Mammal ''Age'', ?middle to late Eocene). Simpson documented three of these, Thomashuxleya externa, Anisotemnus distentus, and Pleurostylodon similis, some 70 years ago, in fashioning a composite isotemnid skeleton, but he did not emphasize their differences from one another. We note variation, especially in the forelimb, that appears to be functionally significant as well as phylogenetically informative. For example, the downwardly curved olecranon, ventrally concave bowing of the ulnar shaft, and orthogonally directed articulation of the elbow joint suggest an erect forelimb stance in Thomashuxleya externa, whereas the forelimbs of Anisotemnus distentus and Pleurostylodon similis show indications of a crouching posture, including ventrally convex bowing of the ulnar shaft with a slight upward curvature of the olecranon, and an elbow joint in which the antebrachium rotated obliquely relative to the humerus. Articular facets on the proximal carpals suggest that the manus of Anisotemnus was habitually extended, indicating a plantigrade stance of the forelimb. Although none of these three taxa have associated hindfoot material, all known Vacan notoungulate astragali have shallow trochlea, well-developed and deep grooves for the flexor hallucis longus, which are separated from the trochlea by a fossa that contains a superior astragalar foramen. An isolated notoungulate pes, not referred to any of the three taxa above, appears to be pentadactyl, having a distinctive, divergent tarsometatarsal joint for its hallux. It also has a shallow trochlea, an astragalar foramen, and a flexor groove, indicating limited rotation of the upper ankle joint. Indeed, a survey of known Casamayoran-aged notoungulate astragali indicates that most taxa had limited mobility at the tibioastragalar joint, in stark contrast to post-Eocene faunas in which nearly all the ungulates had greater rotation of the upper ankle joint and were subcursorial, as evidenced by their longer and trochlear articulation and loss of the astragalar foramen. We suggest that the change from ambulatory-to subcursorial-dominated ungulate faunas across the Eocene-Oliogocene boundary mirrors the changes from brachydont to hypsodont faunas over the same time. Decreased temperatures and rainfall resulting in more open habitats may be related to both morphological evolutionary patterns.

The exceedingly rich middle Pleistocene mammalian fauna from the classic Ensenadan Tarija basin in southern Bolivia contains a diversity of medium to large-bodied herbivores consisting of both endemic (†Toxodontia, †Litopterna, Xenarthra) and immigrant (Rodentia, Proboscidea, Perissodactyla, and Artiodactyla) taxa. In order to characterize feeding ecology and niche differences, a suite of morphological characters was measured for each of 13 species of herbivorous mammals from the Pleistocene of Tarija; these were combined with carbon isotopic results from tooth enamel. (The Xenarthra were excluded from this study because they lack tooth enamel.)Several different bivariate and multivariate combinations of characters can be used to characterize the feeding adaptations, niches, and guild composition of the Tarija mammalian herbivores. During the Pleistocene the browsing guild in the Tarija basin is interpreted to include the tapir (Tapirus tarijensis), extinct llama (Palaeolama weddelli), peccary (Tayassusp.), and deer (Hippocamelussp.). The mixed-feeding guild included two horse species (Hippidion principaleandOnohippidium devillei), litoptern (Macrauchenia patachonica), and capybara (Neochoerus tarijensis). The grazing guild included the numerically dominant horse (Equus insulatus), two lamine species (Lama angustimaxillaand cf.Vicugna, provicugna), notoungulate (Toxodon platensis), and gomphothere proboscidean (Cuvieronius hyodon). The grazing guild has the widest range of body sizes relative to the two other guilds. Closely related sympatric species within the Equidae and Camelidae differentiate their niches from one another using a combination of body size, feeding ecology, and probably local habitat. Most of the paleoecological reconstructions resulting from this combined morphological and isotopic analysis corroborate previous studies based primarily on morphology; there are, however, some notable surprises.

We use two approaches to test hypotheses regarding function in a group of extinct mammals (Family Mesotheriidae, Order Notoungulata) that lack any close extant relatives: a principle-derived paradigm method and empirically derived analog method. Metric and discrete morphological traits of mesotheriid postcranial elements are found to be consistent with the morphology predicted by a modified version of Hildebrand's paradigm for scratch diggers. Ratios of in-force to out-force lever arms based on skeletal elements indicate that the mesotheriids examined had limbs modified for high out-forces (i.e., they were “low geared”), consistent with the digging hypothesis. Other mesotheriid characters, such as cleft ungual phalanges, a curved olecranon, and a highly modified pelvis (with extra vertebrae incorporated into the sacrum and fusion between the ischium and the axial skeleton) are regarded as being functionally significant for digging and also occur in a variety of extant diggers. Analog methods indicate that mesotheriids share numerous traits common to a variety of extant diggers. Principal component analyses of postcranial elements indicate that mesotheriids consistently share morphometric space with larger extant fossorial mammals: aardvark, anteaters, wombats, and badger. Likewise, discriminant function analyses categorized mesotheriids as fossorial, though imperfectly analogous to the extant diggers analyzed. Thus, both theory-driven and empirically derived methods of estimating function in these extinct taxa support a digging hypothesis for the mesotheriids examined. Adaptations for digging in both the forelimb and sacropelvic functional complexes of mesotheriids provide independent support for the fossorial hypothesis.