Interspecific variation in the tetradactyl manus of modern tapirs (Perissodactyla: Tapirus) exposed using geometric morphometrics

The distal forelimb (autopodium) of quadrupedal mammals is a key morphological unit involved in locomotion, body support, and interaction with the substrate. The manus of the tapir (Perissodactyla: Tapirus ) is unique within modern perissodactyls, as it retains the plesiomorphic tetradactyl (four‐toed) condition also exhibited by basal equids and rhinoceroses. Tapirs are known to exhibit anatomical mesaxonic symmetry in the manus, although interspecific differences and biomechanical mesaxony have yet to be rigorously tested. Here, we investigate variation in the manus morphology of four modern tapir species (Tapirus indicus , Tapirus bairdii , Tapirus pinchaque , and Tapirus terrestris ) using a geometric morphometric approach. Autopodial bones were laser scanned to capture surface shape and morphology was quantified using 3D‐landmark analysis. Landmarks were aligned using Generalised Procrustes Analysis, with discriminant function and partial least square analyses performed on aligned coordinate data to identify features that significantly separate tapir species. Overall, our results support the previously held hypothesis that T. indicus is morphologically separate from neotropical tapirs; however, previous conclusions regarding function from morphological differences are shown to require reassessment. We find evidence indicating that T. bairdii exhibits reduced reliance on the lateral fifth digit compared to other tapirs. Morphometric assessment of the metacarpophalangeal joint and the morphology of the distal facets of the lunate lend evidence toward high loading on the lateral digits of both the large T. indicus (large body mass) and the small, long limbed T. pinchaque (ground impact). Our results support other recent studies on T. pinchaque , suggesting subtle but important adaptations to a compliant but inclined habitat. In conclusion, we demonstrate further evidence that the modern tapir forelimb is a variable locomotor unit with a range of interspecific features tailored to habitual and biomechanical needs of each species.     

Phylogenetics of Perissodactyla and Tests of the Molecular Clock

Two mitochondrial genes, the protein-coding cytochrome c oxidase subunit II (COII) gene and a portion of the 12S rRNA gene, were used for phylogenetic investigation of the mammalian order Perissodactyla. The primary objective of the study was to utilize the extensive fossil record of perissodactyls for calibrating molecular clocks and comparing estimates of divergence times using both genes and two fossil calibration points. Secondary objectives included clarification of previously unresolved relationships within Tapiridae and comparison of the results of separate and combined analyses of two genes. Analyses included several perissodactyl lineages representing all three families (Tapiridae, Equidae, and Rhinocerotidae), most extant genera, all four species of tapirs, two to four species of rhinoceros, and two species of Equus. The application of a relatively recent fossil calibration point and a relatively ancient calibration point produced greatly different estimates of evolutionary rates and divergence times for both genes, even though a relative rates test did not find significant rate differences among taxa. A likelihood-ratio test, however, rejected a molecular clock for both genes. Neither calibration point produced estimates of divergence times consistent with paleontological evidence over a range of perissodactyl radiations. The combined analysis of both genes produces a well-resolved phylogeny with Perissodactyla that conforms to traditional views of interfamilial relationships and supports monophyly of neotropical tapirs. Combining the data sets increases support for most nodes but decreases the support for a neotropical tapir clade because the COII and 12S rRNA data sets are in conflict for tapir relationships. Received: 6 January 1999 / Accepted: 2 August 1999     

Molecular phylogeny and evolution of the Perissodactyla

The evolution of perissodactyls (rhinoceroses, tapirs, and horses) has been well studied primarily because of their extensive fossil record. Nevertheless, controversy persists regarding relationships of some of the extant taxa, reflecting inconsistencies between molecular and morphological studies. Here we examine the phylogenetic relationships of 16 living perissodactyl species by concatenating two mitochondrial and nine nuclear genes, and we estimate their divergence times using a relaxed Bayesian molecular clock approach. Our analyses recovered the monophyly of the suborders Ceratomorpha and Hippomorpha, and the families Rhinoceratidae, Tapiridae, and Equidae. We supported the early divergence of the Indian rhinoceros in the late Oligocene (26 Mya) relative to the Sumatran and African rhinoceroses, and the split of caballine (domestic horse and Przewalski's wild horse) and noncaballine equids (zebras and African and Asiatic asses) in the Pliocene (4 Mya). An important implication of this study is that Equus asinus, the African wild ass was found to be the sister taxon of Asiatic asses and zebras, diverging from the common ancestor with caballine horses 2 Mya. Rates of chromosome rearrangements were also evaluated in perissodactyls, placing a notably high rate of variation amongst equids, particularly within the zebra clade. The robust phylogenetic results presented here are relevant in terms of understanding the evolutionary history of this highly threatened group of mammals. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1289–1303.     

The Phylogeny and Classification of Tapiromorph Perissodactyls (Mammalia)

Despite an excellent fossil record, the phylogeny of Perissodactyla is not well understood, in terms of both the relationships within Perissodactyla and the position of the Perissodactyla among the orders of mammals. This paper provides a phylogenetic analysis of one major perissodactyl lineage, the Tapiromorpha. This analysis combines a more comprehensive sampling of characters and taxa with rigorous tree-searching methods to create a new hypothesis of tapiromorph relationships. The phylogeny of tapiromorph perissodactyls is analyzed using 45 characters of the skull, postcranial skeleton, and dentition scored for 29 taxa, including three nontapiromorph outgroups. Phylogenetic taxonomic definitions are constructed for suprageneric taxa. According to the results of this analysis, the Chalicotherioidea cannot be unequivocally assigned to the Tapiromorpha, nor can Homogalax or Cardiolophus. Isectolophus , Tapiroidea, and Rhinocerotoidea are unequivocal members of the Tapiromorpha. Heptodon is included in a monophyletic Tapiroidea. Amynodontid rhinocerotoids come out as the sister group to rhinocerotids, and indricotheres do not fall within the Hyracodontidae. The results of this study provide further arguments that tapiromorphs (and putative tapiromorphs) may be important for understanding the ancestral morphology of Perissodactyla.     

Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia)

The complete 12S rRNA gene has been sequenced in 4 Ungulata (hoofed eutherians) and 1 marsupial and compared to 38 available mammalian sequences in order to investigate the molecular evolution of the mitochondrial small-subunit ribosomal RNA molecule. Ungulata were represented by one artiodactyl (the collared peccary, Tayassu tajacu, suborder Suiformes), two perissodactyls (the Grevy's zebra, Equus grevyi, suborder Hippomorpha; the white rhinoceros, Ceratotherium simum, suborder Ceratomorpha), and one hyracoid (the tree hyrax, Dendrohyrax dorsalis). The fifth species was a marsupial, the eastern gray kangaroo (Macropus giganteus). Several transition/transversion biases characterized the pattern of changes between mammalian 12S rRNA molecules. A bias toward transitions was found among 12S rRNA sequences of Ungulata, illustrating the general bias exhibited by ribosomal and protein-encoding genes of the mitochondrial genome. The derivation of a mammalian 12S rRNA secondary structure model from the comparison of 43 eutherian and marsupial sequences evidenced a pronounced bias against transversions in stems. Moreover, transversional compensatory changes were rare events within double-stranded regions of the ribosomal RNA. Evolutionary characteristics of the 12S rRNA were compared with those of the nuclear 18S and 28S rRNAs. From a phylogenetic point of view, transitions, transversions and indels in stems as well as transversional and indels events in loops gave congruent results for comparisons within orders. Some compensatory changes in double-stranded regions and some indels in single-stranded regions also constituted diagnostic events. The 12S rRNA molecule confirmed the monophyly of infraorder Pecora and order Cetacea and demonstrated the monophyly of suborder Suiformes. However, the monophyly of the suborder Ruminantia was not supported, and the branching pattern between Cetacea and the artiodactyl suborders Ruminantia and Suiformes was not established. The monophyly of the order Perissodactyla was evidenced, but the relationships between Artiodactyla, Cetacea, and Perissodactyla remained unresolved. Nevertheless, we found no support for a Perissodactyla + Hyracoidea clade, neither with distance approach, nor with parsimony reconstruction. The 12S rRNA was useful to solve intraordinal relationships among Ungulata, but it seemed to harbor too few informative positions to decipher the bushlike radiation of some Ungulata orders, an event which has most probably occurred in a short span of time between 55 and 70 MYA. Correspondence to: E. Douzery     

Phylogenetic analysis of the perissodactylan family Tapiridae using mitochondrial cytochromec oxidase (COII) sequences

The four extant members of the family Tapiridae have a disjunct, relictual distribution, with three species being Neotropical (Tapirus bairdii, T. terrestris, andT. pinchaque) and one found in Southeast Asia (T. indicus). Little recent work on tapir systematics have appeared, and no molecular studies of this group have been published. A phylogenetic analysis was undertaken using sequences of the mitochondrial cytochromec oxidase subunit II gene (COII) from representatives of the four species of tapirs, as well as a representative outgroup,Equus caballus. Analyses of the COII sequences indicate a close relationship between the two South American species of tapirs,T. terrestris andT. pinchaque, and estimates of divergence dates using rates of COII evolution are compatible with migration of a single tapir lineage into South America following the emergence of the isthmus of Panama, about 3 million years bp. Various methods of analysis, including maximum parsimony, maximum likelihood, and neighbor-joining, provided poorer resolution of other tapir relationship. The COII data suggest that three distinct tapir mitochondrial lineages, a South American (represented byT. terrestris andT. pinchaque), a Central American (represented byT. bairdii), and an Asian (represented byT. indicus) diverged relatively rapidly, 20–30 million years bp. Another goal of this study was to calibrate the rate of COII evolution in a eutherian mammal group which has a good fossil record, such as perissodactyls, to estimate accurately the rate of COII evolution in a nonprimate mammalian group. The rate of COII evolution in equids and tapirs has been relatively constant and, using corrected distances, calibrated to be approximately 0.22% lineage/million years. This rate is three-to fourfold lower than that of hominoid primates.     

Experimentelle Untersuchungen zur optischen Kommunikation bei im Zoo gehaltenen Schabracken- und Flachlandtapiren (Tapirus indicus und Tapirus terrestris)

Until now, unlike their relatives, rhinos and horses tapirs have received considerably less attention in studies about communication. Therefore, it was the aim of this study to test which stimuli contain optical information for tapirs. For this purpose, the reactions of tapirs on optical stimuli (posters with edited tapir silhouettes) were examined. Research visits took place at the zoos of Berlin, Dortmund, Heidelberg, Munich, Nuremberg and Osnabrück during the year 2006. A total of 23 individuals, thereof 8 (5.3) Malayan tapirs (Tapirus indicus) and 15 (5.10) Lowland tapirs (Tapirus terrestris) attended the experiment. The results of the optical test with variously intense edited tapir silhouettes speak for the importance of the white ear rims as a family specific key stimulus. But that effect could not be amplified by adding a greater extent of white to the silhouette. Tapirs of both species reacted most strongly to the normal tapir silhouette followed by a silhouette without proboscis.     

A three‐dimensional morphometric analysis of upper forelimb morphology in the enigmatic tapir (Perissodactyla: Tapirus) hints at subtle variations in locomotor ecology

Forelimb morphology is an indicator for terrestrial locomotor ecology. The limb morphology of the enigmatic tapir (Perissodactyla: Tapirus) has often been compared to that of basal perissodactyls, despite the lack of quantitative studies comparing forelimb variation in modern tapirs. Here, we present a quantitative assessment of tapir upper forelimb osteology using three‐dimensional geometric morphometrics to test whether the four modern tapir species are monomorphic in their forelimb skeleton. The shape of the upper forelimb bones across four species (T. indicus; T. bairdii; T. terrestris; T. pinchaque) was investigated. Bones were laser scanned to capture surface morphology and 3D landmark analysis was used to quantify shape. Discriminant function analyses were performed to reveal features which could be used for interspecific discrimination. Overall our results show that the appendicular skeleton contains notable interspecific differences. We demonstrate that upper forelimb bones can be used to discriminate between species (>91% accuracy), with the scapula proving the most diagnostic bone (100% accuracy). Features that most successfully discriminate between the four species include the placement of the cranial angle of the scapula, depth of the humeral condyle, and the caudal deflection of the olecranon. Previous studies comparing the limbs of T. indicus and T. terrestris are corroborated by our quantitative findings. Moreover, the mountain tapir T. pinchaque consistently exhibited the greatest divergence in morphology from the other three species. Despite previous studies describing tapirs as functionally mediportal in their locomotor style, we find osteological evidence suggesting a spectrum of locomotor adaptations in the tapirs. We conclude that modern tapir forelimbs are neither monomorphic nor are tapirs as conserved in their locomotor habits as previously described. J. Morphol. 277:1469–1485, 2016. © 2016 Wiley Periodicals, Inc.     

Evolutionary Transitions in the Fossil Record of Terrestrial Hoofed Mammals

In the past few decades, many new discoveries have provided numerous transitional fossils that show the evolution of hoofed mammals from their primitive ancestors. We can now document the origin of the odd-toed perissodactyls, their early evolution when horses, brontotheres, rhinoceroses, and tapirs can barely be distinguished, and the subsequent evolution and radiation of these groups into distinctive lineages with many different species and interesting evolutionary transformations through time. Similarly, we can document the evolution of the even-toed artiodactyls from their earliest roots and their great radiation into pigs, peccaries, hippos, camels, and ruminants. We can trace the complex family histories in the camels and giraffes, whose earliest ancestors did not have humps or long necks and looked nothing like the modern descendants. Even the Proboscidea and Sirenia show many transitional fossils linking them to ancient ancestors that look nothing like modern elephants or manatees. All these facts show that creationist attacks on the fossil record of horses and other hoofed mammals are completely erroneous and deceptive. Their critiques of the evidence of hoofed mammal evolution are based entirely on reading trade books and quoting them out of context, not on any firsthand knowledge or training in paleontology or looking at the actual fossils.     

Pregnancy detection in tapirs by direct urinary estrone sulfate analysis

Urinary levels of estrone sulfate (ES), indexed by creatinine (CR), were evaluated by a direct radioimmunoassay of four Malayan (Tapirus indicus) and one Brazillian (Tapirus terrestris) tapir pregnancies. Levels rose above baseline ES values of 20 ± 1.2 ng/mg CR (n = 200) in the Brazilian animal and 25 ± 1.3 ng/mg CR (n = 105) in the Malayan animals at approximately 7 months prior to parturition and continued to rise in both species until just before parturition. Quantitatively, levels rose 10-fold higher in the Brazillian animal than in the Malayan animals through approximately 1 month prior to parturition in both species. These findings indicate that routine urinary monitoring provides an accurate means for detecting pregnancy in tapirs and suggests differences in estrogen excretion patterns between tapir species and with other perissodactyls.     

Higher‐level phylogeny of the insect order Hemiptera: is Auchenorrhyncha really paraphyletic?

The higher‐level phylogeny of the order Hemiptera remains a contentious topic in insect systematics. The controversy is chiefly centred on the unresolved question of whether or not the hemipteran suborder Auchenorrhyncha (including the extant superfamilies Fulgoroidea, Membracoidea, Cicadoidea and Cercopoidea) is a monophyletic lineage. Presented here are the results of a multilocus molecular phylogenetic investigation of relationships among the major hemipteran lineages, designed specifically to address the question of Auchenorrhyncha monophyly in the context of broad taxonomic sampling across Hemiptera. Phylogenetic analyses (maximum parsimony, maximum likelihood and Bayesian inference) were based on DNA nucleotide sequence data from seven gene regions (18S rDNA, 28S rDNA, histone H3, histone 2A, wingless, cytochrome c oxidase I and NADH dehydrogenase subunit 4) generated from 86 in‐group exemplars representing all major lineages of Hemiptera (plus seven out‐group taxa). All combined analyses of these data recover the monophyly of Auchenorrhyncha, and also support the monophyly of each of the following lineages: Hemiptera, Sternorrhyncha, Heteropterodea, Heteroptera, Fulgoroidea, Cicadomorpha, Membracoidea, Cercopoidea and Cicadoidea. Also presented is a review of the major lines of morphological and molecular evidence for and against the monophyly of Auchenorrhyncha.     

Experimentelle Untersuchungen zur akustischen Kommunikation bei im Zoo gehaltenen Schabracken- und Flachlandtapiren (Tapirus indicus und Tapirus terrestris)

Until now, unlike their relatives, rhinos and horses tapirs have received considerably less attention in studies about communication. Therefore, it was the aim of this study to test which stimuli contain acoustical information for tapirs. For this purpose, the reactions of tapirs on acoustical stimuli (playback of different animal voices) were examined. Research visits took place at the zoos of Berlin, Dortmund, Heidelberg, Munich, Nuremberg and Osnabrück during the year 2005. A total of 20 individuals, thereof 8 (4.4) Malayan tapirs (Tapirus indicus) and 12 (4.8) Lowland tapirs (Tapirus terrestris) attended the experiments. The playback experiments showed that tapirs distinguish between the voices of different animal species. The results point to the conclusion that the reactions of the tapirs relate to phylogeny. The most intense interest was taken in their own species followed by the closely related ones.     

Neogastropod phylogenetic relationships based on entire mitochondrial genomes

Background  

The Neogastropoda is a highly diversified group of predatory marine snails (Gastropoda: Caenogastropoda). Traditionally, its monophyly has been widely accepted based on several morphological synapomorphies mostly related with the digestive system. However, recent molecular phylogenetic studies challenged the monophyly of Neogastropoda due to the inclusion of representatives of other caenogastropod lineages (e.g. Littorinimorpha) within the group. Neogastropoda has been classified into up to six superfamilies including Buccinoidea, Muricoidea, Olivoidea, Pseudolivoidea, Conoidea, and Cancellarioidea. Phylogenetic relationships among neogastropod superfamilies remain unresolved.     

Fruit consumption and seed dispersal of Dimorphandra mollis Benth. (Leguminosae) by the lowland tapir in the cerrado of Central Brazil.

Fruit phenology observations and consumption of Dimorphandra mollis (Leguminosae) were analyzed during seven months in an area of cerrado stricto sensu. We analysed 81 fecal samples collected at six different places of lowland tapirs (Tapirus terrestris) in central Brazilian cerrado. In addition, from the feces of five tapirs at the Brasília Zoo to which fruit had been offered, seeds were collected and used in germination tests. The results suggest that the tapir is an important fruit consumer and a potential seed disperser of D. mollis. In the field, however, fruit consumption was found to be very low, probably because of both fruit palatability and the low density of frugivores, especially tapirs. The possibility that the original dispersal agents of D. mollis seeds belonged to the South American Pleistocene megafauna is discussed.     

Stable Isotope Ecology of Extant Tapirs from the Americas

Understanding the ecology of modern and ancient forests can help clarify the evolution of forest dwelling mammals. It is first necessary, however, to elucidate the source and extent of stable isotope variation in forest taxa. Tapirs are of particular interest because they are model organisms for identifying forest environments due to their highly conservative diet and habitat preferences. Here, stable carbon and oxygen isotopic compositions of extant tapirs are quantified to test hypotheses regarding ontogenetic diet shifts, stable isotope variation at the population level, and relationships between stable isotopes and climatic variables. A population of extant tapirs (Tapirus bairdii) demonstrates low δ¹³C variation (～2–3‰) and increased δ¹³C values in late erupting teeth, indicating that juveniles consume ¹³C deplete milk and/or browse in denser forests. Disparate δ¹⁸O values of late erupting teeth are instead reflecting seasonal variation. Extant tapir (T. bairdii, Tapirus pinchaque, Tapirus terrestris) δ¹⁸O values are constrained by climatic and geographic variables. Most notably, δ¹⁸O values of T. bairdii decrease with decreasing precipitation frequency. Tapirus terrestris is typically present in areas with greater precipitation than T. bairdii and δ¹⁸O values instead positively correlate with δ¹³C values. These data indicate that tapirs in wetter areas are getting a larger proportion of their water from leaves experiencing less evaporation in denser canopies, while T. bairdii is interpreted to increase its consumption of water via drinking when present in drier areas. An understanding of extant tapir stable isotope ecology improves ecological interpretations of these elusive mammals both today and in the past. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

Asian Tapirs Are No Elephants When It Comes To Seed Dispersal

The elimination of the largest herbivores (elephants and rhinoceroses) from many forests in tropical East Asia may have severe consequences for plant species that depend on them for seed dispersal. We assessed the capacity of Malayan tapirs Tapirus indicus—the next largest nonruminant herbivore in the region—as a substitute for the lost megafauna in this role by studying their ability to disperse the seeds of nine fleshy‐fruited plants with seeds 5–97 mm in length. We combined information from feeding trials, germination tests, and field telemetry to assess the effect of tapir consumption on seed viability and to estimate how far the seeds would be dispersed. The tapirs (N=8) ingested few seeds. Seed survival through gut passage was moderately high for small‐seeded plants (e.g., 36.9% for Dillenia indica) but very low for medium‐ (e.g., 7.6% for Tamarindus indica) and large‐seeded (e.g., 2.8% for Artocarpus integer) plants. Mean seed gut passage times were long (63–236 h) and only the smallest seeds germinated afterwards. Using movement data from four wild tapirs in Peninsular Malaysia we estimated mean dispersal distances of 917–1287 m (range=22–3289 m) for small‐seeded plants. Malayan tapirs effectively dispersed small‐seeded plants but acted as seed predators for the large‐seeded plants included in our study, suggesting that they cannot replace larger herbivores in seed dispersal. With the absence of elephants and rhinos many megafaunal‐syndrome plants in tropical East Asia are expected to face severe dispersal limitation problems.     

What is a Suiforme (Artiodactyla)?: Contribution of Cranioskeletal and Mitochondrial DNA Data

Suiformes (Artiodactyla) traditionally includes three families: Suidae, Tayassuidae, and Hippopotamidae but the monophyly of this suborder has recently been questioned from molecular data. A maximum parsimony analysis of molecular, morphological, and combined data was performed on the same set of taxa including representatives of the three Artiodactyla suborders (Suiformes, Ruminantia, and Tylopoda) and Perissodactyla as outgroup. Mitochondrial (cytochromeband 12S rRNA) sequence comparisons support the monophyly of Suina (Suidae and Tayassuidae) and Ancodonta (Hippopotamidae) but not the monophyly of Suiformes. Inversely, our preliminary morphological analysis supports the monophyly of Suiformes whereas relationships among the three families are not resolved. The combined data set does not resolve the relationships between Suina, Ancodonta, and Ruminantia. These results are discussed in relation to morphological characters and paleontological data. Some improvements are suggested to clarify the morphological definition of Suiformes and relationships among them.     

Mitochondrial Phylogenomics of Modern and Ancient Equids

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).     

Ancient collagen reveals evolutionary history of the endemic South American ‘ungulates’

Since the late eighteenth century, fossils of bizarre extinct creatures have been described from the Americas, revealing a previously unimagined chapter in the history of mammals. The most bizarre of these are the ‘native’ South American ungulates thought to represent a group of mammals that evolved in relative isolation on South America, but with an uncertain affinity to any particular placental lineage. Many authors have considered them descended from Laurasian ‘condylarths’, which also includes the probable ancestors of perissodactyls and artiodactyls, whereas others have placed them either closer to the uniquely South American xenarthrans (anteaters, armadillos and sloths) or the basal afrotherians (e.g. elephants and hyraxes). These hypotheses have been debated owing to conflicting morphological characteristics and the hitherto inability to retrieve molecular information. Of the ‘native’ South American mammals, only the toxodonts and litopterns persisted until the Late Pleistocene–Early Holocene. Owing to known difficulties in retrieving ancient DNA (aDNA) from specimens from warm climates, this research presents a molecular phylogeny for both Macrauchenia patachonica (Litopterna) and Toxodon platensis (Notoungulata) recovered using proteomics-based (liquid chromatography–tandem mass spectrometry) sequencing analyses of bone collagen. The results place both taxa in a clade that is monophyletic with the perissodactyls, which today are represented by horses, rhinoceroses and tapirs.