Ecological and hydroclimate responses to strengthening of the Hadley circulation in South America during the Late Miocene cooling.

Near-modern ecosystems were established as a result of rapid ecological adaptation and climate change in the Late Miocene. On land, Late Miocene aridification spread in tandem with expansion of open habitats including C4 grassland ecosystems. Proxy records for the central Andes spanning the Late Miocene cooling (LMC) show the reorganization of subtropical ecosystems and hydroclimate in South America between 15 and 35°S. Continental pedogenic carbonates preserved in Neogene basins record a general increase of δ18O and δ13C values from pre-LMC to post-LMC, most robustly occurring in the subtropics (25 to 30°S), suggesting aridification and a shift toward a more C4-plant-dominated ecosystem. These changes are closely tied to the enhancement of the Hadley circulation and moisture divergence away from the subtropics toward the Intertropical Convergence Zone as revealed by climate model simulations with prescribed sea-surface temperatures (SSTs) reflecting different magnitudes of LMC steepening of equator-to-pole temperature gradient and CO2 decline.

The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

Fluvial response to abrupt global warming at the Palaeocene/Eocene boundary.

Climate strongly affects the production of sediment from mountain catchments as well as its transport and deposition within adjacent sedimentary basins. However, identifying climatic influences on basin stratigraphy is complicated by nonlinearities, feedback loops, lag times, buffering and convergence among processes within the sediment routeing system. The Palaeocene/Eocene thermal maximum (PETM) arguably represents the most abrupt and dramatic instance of global warming in the Cenozoic era and has been proposed to be a geologic analogue for anthropogenic climate change. Here we evaluate the fluvial response in western Colorado to the PETM. Concomitant with the carbon isotope excursion marking the PETM we document a basin-wide shift to thick, multistoried, sheets of sandstone characterized by variable channel dimensions, dominance of upper flow regime sedimentary structures, and prevalent crevasse splay deposits. This progradation of coarse-grained lithofacies matches model predictions for rapid increases in sediment flux and discharge, instigated by regional vegetation overturn and enhanced monsoon precipitation. Yet the change in fluvial deposition persisted long after the approximately 200,000-year-long PETM with its increased carbon dioxide levels in the atmosphere, emphasizing the strong role the protracted transmission of catchment responses to distant depositional systems has in constructing large-scale basin stratigraphy. Our results, combined with evidence for increased dissolved loads and terrestrial clay export to world oceans, indicate that the transient hyper-greenhouse climate of the PETM may represent a major geomorphic 'system-clearing event', involving a global mobilization of dissolved and solid sediment loads on Earth's surface.

Controlled feeding trials with ungulates: a new application of in vivo dental molding to assess the abrasive factors of microwear.

Microwear, the quantification of microscopic scratches and pits on the occlusal surfaces of tooth enamel, is commonly used as a paleodietary proxy. For ungulates (hoofed mammals), scratch-dominant microwear distinguishes modern grazers from browsers, presumably as a result of abrasion from grass phytoliths (biogenic silica). However, it is also likely that exogenous grit (i.e. soil, dust) is a contributing factor to these scratch-dominant patterns, which may reflect soil ingestion that varies with feeding height and/or environmental conditions (e.g. dust production in open and/or arid habitats). This study assessed the contribution of exogenous grit to tooth wear by measuring the effects of fine- and medium-grained silica sand on tooth enamel using a novel live-animal tooth-molding technique. It therefore constitutes the first controlled feeding experiment using ungulates and the first in vivo experiment using abrasives of different sizes. Four sheep were fed three diet treatments: (1) a mixture of Garrison and Brome hay (control), (2) hay treated with fine-grained silica sand (180-250 µm) and (3) hay treated with medium-grained silica sand (250-425 µm). We found a significant increase in pit features that was correlated with an increase in grain size of grit, corroborating earlier chewing simulation experiments that produced pits through grit-induced abrasion (i.e. the 'grit effect'). Our results support an interpretation of large silica grains fracturing to create smaller, more abundant angular particles capable of abrasion, with jaw movement defining feature shape (i.e. scratch or pit).

Predictive equations for the estimation of body size in seals and sea lions (Carnivora: Pinnipedia).

Body size plays an important role in pinniped ecology and life history. However, body size data is often absent for historical, archaeological, and fossil specimens. To estimate the body size of pinnipeds (seals, sea lions, and walruses) for today and the past, we used 14 commonly preserved cranial measurements to develop sets of single variable and multivariate predictive equations for pinniped body mass and total length. Principal components analysis (PCA) was used to test whether separate family specific regressions were more appropriate than single predictive equations for Pinnipedia. The influence of phylogeny was tested with phylogenetic independent contrasts (PIC). The accuracy of these regressions was then assessed using a combination of coefficient of determination, percent prediction error, and standard error of estimation. Three different methods of multivariate analysis were examined: bidirectional stepwise model selection using Akaike information criteria; all-subsets model selection using Bayesian information criteria (BIC); and partial least squares regression. The PCA showed clear discrimination between Otariidae (fur seals and sea lions) and Phocidae (earless seals) for the 14 measurements, indicating the need for family-specific regression equations. The PIC analysis found that phylogeny had a minor influence on relationship between morphological variables and body size. The regressions for total length were more accurate than those for body mass, and equations specific to Otariidae were more accurate than those for Phocidae. Of the three multivariate methods, the all-subsets approach required the fewest number of variables to estimate body size accurately. We then used the single variable predictive equations and the all-subsets approach to estimate the body size of two recently extinct pinniped taxa, the Caribbean monk seal (Monachus tropicalis) and the Japanese sea lion (Zalophus japonicus). Body size estimates using single variable regressions generally under or over-estimated body size; however, the all-subset regression produced body size estimates that were close to historically recorded body length for these two species. This indicates that the all-subset regression equations developed in this study can estimate body size accurately.

Whales originated from aquatic artiodactyls in the Eocene epoch of India.

Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link to the ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth. We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids were aquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.

Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases.

Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.

Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: Implications for paleoclimate reconstruction.

Stable oxygen isotope (δ18O) compositions from vertebrate tooth enamel are widely used as biogeochemical proxies for paleoclimate. However, the utility of enamel oxygen isotope values for environmental reconstruction varies among species. Herein, we evaluate the use of stable oxygen isotope compositions from pronghorn (Antilocapra americana Gray, 1866) enamel for reconstructing paleoclimate seasonality, an elusive but important parameter for understanding past ecosystems. We serially sampled the lower third molars of recent adult pronghorn from Wyoming for δ18O in phosphate (δ18OPO4) and compared patterns to interpolated and measured yearly variation in environmental waters as well as from sagebrush leaves, lakes, and rivers (δ18Ow). As expected, the oxygen isotope compositions of phosphate from pronghorn enamel are enriched in 18O relative to environmental waters. For a more direct comparison, we converted δ18Ow values into expected δ18OPO4* values (δ18OW-PO4*). Pronghorn δ18OPO4 values from tooth enamel record nearly the full amplitude of seasonal variation from Wyoming δ18OW-PO4* values. Furthermore, pronghorn enamel δ18OPO4 values are more similar to modeled δ18OW-PO4* values from plant leaf waters than meteoric waters, suggesting that they obtain much of their water from evaporated plant waters. Collectively, our findings establish that seasonality in source water is reliably reflected in pronghorn enamel, providing the basis for exploring changes in the amplitude of seasonality of ancient climates. As a preliminary test, we sampled historical pronghorn specimens (1720 ± 100 AD), which show a mean decrease (a shift to lower values) of 1-2‰ in δ18OPO4 compared to the modern specimens. They also exhibit an increase in the δ18O amplitude, representing an increase in seasonality. We suggest that the cooler mean annual and summer temperatures typical of the 18th century, as well as enhanced periods of drought, drove differences among the modern and historical pronghorn, further establishing pronghorn enamel as excellent sources of paleoclimate proxy data.

Problems with inferring a lack of competition between Rancho La Brea dire wolves and sabertooth cats based on dental enamel.

Van Valkenburgh et al. challenge the conclusions of a recent study by DeSantis et al. that claimed that sabertooth cats and dire wolves did not compete for similar prey.

Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription.

Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-κB activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR.IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-κB nuclear translocation. This heightened transcriptional activity renders CD4+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation.

Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat.

The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors.IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore.

Humans permanently occupied the Andean highlands by at least 7 ka.

High-elevation environments above 2500 metres above sea level (m.a.s.l.) were among the planet's last frontiers of human colonization. Research on the speed and tempo of this colonization process is active and holds implications for understanding rates of genetic, physiological and cultural adaptation in our species. Permanent occupation of high-elevation environments in the Andes Mountains of South America tentatively began with hunter-gatherers around 9 ka according to current archaeological estimates, though the timing is currently debated. Recent observations on the archaeological site of Soro Mik'aya Patjxa (8.0-6.5 ka), located at 3800 m.a.s.l. in the Andean Altiplano, offer an opportunity to independently test hypotheses for early permanent use of the region. This study observes low oxygen (δ18O) and high carbon (δ13C) isotope values in human bone, long travel distances to low-elevation zones, variable age and sex structure in the human population and an absence of non-local lithic materials. These independent lines of evidence converge to support a model of permanent occupation of high elevations and refute logistical and seasonal use models. The results constitute the strongest empirical support to date for permanent human occupation of the Andean highlands by hunter-gatherers before 7 ka.

Aquatic Habits of Cetacean Ancestors: Integrating Bone Microanatomy and Stable Isotopes.

The earliest cetaceans were interpreted as semi-aquatic based on the presence of thickened bones and stable oxygen isotopes in tooth enamel. However, the origin of aquatic behaviors in cetacean relatives (e.g., raoellids, anthracotheres) remains unclear. This study reconstructs the origins of aquatic behaviors based on long bone microanatomy and stable oxygen isotopes of tooth enamel in modern and extinct cetartiodactylans. Our findings are congruent with published accounts that microanatomy can be a reliable indicator of aquatic behaviors in taxa that are obligatorily aquatic, and also highlight that some "semi-aquatic" behaviors (fleeing into the water to escape predation) may have a stronger relationship to bone microanatomy than others (herbivory in near-shore aquatic settings). Bone microanatomy is best considered with other lines of information in the land-to-sea transition of cetaceans, such as stable isotopes. This study extends our understanding of the progression of skeletal phenotypes associated with habitat shifts in the relatives of cetaceans.

Functional implications of variation in tooth spacing and crown size in pinnipedimorpha (mammalia: carnivora).

Pinnipeds (seals, sea lions, and walruses) show variation in tooth morphology that relates to ecology. However, crown size and spacing are two aspects of morphology that have not been quantified in prior studies. We measured these characters for nearly all extant pinnipeds and three fossil taxa and then determined the principal sources of variation in tooth size and spacing using principal components (PCAs) and hierarchical cluster analysis (HCA). PCA and HCA showed that species sorted into three groups: taxa with small crowns and large diastemata, taxa with large crowns and small diastemata, and taxa that fell between these two extremes. We then performed discriminant function analysis (DFA) to determine if tooth morphology correlated with foraging strategy or diet. DFA results indicated weak correlation with diet, and stronger correlation with prey capture strategies. Tooth size and spacing were most strongly correlated with the importance of teeth in prey acquisition, with tooth size decreasing and tooth spacing increasing as teeth become less necessary in capturing food items. Taxa which relied on teeth for filtering prey from the water column or processing larger or tougher food items generally had larger crowns and smaller tooth spacing then taxa which swallowed prey whole. We found the fossil taxa Desmatophoca and Enaliarctos were most similar in tooth morphology to extant otariids, suggesting that both taxa were generalist feeders. This study established the relationship between tooth size and feeding behavior, and provides a new tool to explore the paleoecology of fossil pinnipeds and other aquatic tetrapods.

Cope's rule and the evolution of body size in Pinnipedimorpha (Mammalia: Carnivora).

Cope's rule describes the evolutionary trend for animal lineages to increase in body size over time. In this study, we tested the validity of Cope's rule for a marine mammal clade, the Pinnipedimorpha, which includes the extinct Desmatophocidae, and extant Phocidae (earless seals), Otariidae (fur seals and sea lions), and Odobenidae (walruses). We tested for the presence of Cope's rule by compiling a large dataset of body size data for extant and fossil pinnipeds and then examined how body size evolved through time. We found that there was a positive relationship between geologic age and body size. However, this trend is the result of differences between early assemblages of small-bodied pinnipeds (Oligocene to early Miocene) and later assemblages (middle Miocene to Pliocene) for which species exhibited greater size diversity. No significant differences were found between the number of increases or decreases in body size within Pinnipedimorpha or within specific pinniped clades. This suggests that the pinniped body size increase was driven by passive diversification into vacant niche space, with the common ancestor of Pinnipedimorpha occurring near the minimum adult body size possible for a marine mammal. Based upon the above results, the evolutionary history of pinnipeds does not follow Cope's rule.

Didehydro-cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice.

HIV-1 Tat protein has been shown to have a crucial role in HIV-1-associated neurocognitive disorders (HAND), which includes a group of syndromes ranging from undetectable neurocognitive impairment to dementia. The abuse of psychostimulants, such as cocaine, by HIV infected individuals, may accelerate and intensify neurological damage. On the other hand, exposure to Tat potentiates cocaine-mediated reward mechanisms, which further promotes HAND. Here, we show that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, crosses the blood-brain barrier, cross-neutralizes Tat activity from several HIV-1 clades and decreases Tat uptake by glial cell lines. In addition, dCA potently inhibits Tat mediated dysregulation of IL-1ß, TNF-α and MCP-1, key neuroinflammatory signaling proteins. Importantly, using a mouse model where doxycycline induces Tat expression, we demonstrate that dCA reverses the potentiation of cocaine-mediated reward. Our results suggest that adding a Tat inhibitor, such as dCA, to current antiretroviral therapy may reduce HIV-1-related neuropathogenesis.

Differentiating aquatic mammal habitat and foraging ecology with stable isotopes in tooth enamel.

We analyzed the carbon and oxygen isotope composition of tooth enamel from mammals inhabiting marine and terrestrial ecosystems to determine whether these stable isotopes were robust indicators of foraging and habitat preferences. Consumers were separated into six habitats (offshore, nearshore, kelp beds, estuarine, freshwater, terrestrial). Consumer δ13C values were correlated with the δ13C values of primary producers within each habitat, suggesting that δ13C values of tooth enamel are a viable proxy for foraging zones. Offshore and terrestrial consumer δ13C values were not significantly different, however, indicating that carbon isotope analysis alone is not sufficient to distinguish foraging within these two ecosystems. We propose that oxygen isotopes can be used along with δ13C values to further clarify habitat use. Oxygen isotopes were assessed as an indicator of habitat use. Consumers were grouped into four categories: aquatic-marine, aquatic-estuarine, aquatic-freshwater, and terrestrial. Populations of aquatic taxa had significantly lower standard deviations for δ18O values than those of terrestrial taxa. Mean δ18O values of aquatic taxa were significantly different among groups, but surprisingly, the mean values for freshwater taxa were higher than those for marine taxa. We conclude that variation in δ18O values of mammalian populations is a valid indicator of aquatic habits, but that mean δ18O values should be utilized with caution when trying to discriminate between marine and freshwater habitat use. Together, δ13C and δ18O values serve as valuable tools for identifying foraging and habitat preferences in modern marine and terrestrial ecosystems, and may provide similar information on ancient ecosystems.

Analysis of RNA processing reactions using cell free systems: 3' end cleavage of pre-mRNA substrates in vitro.

The 3' end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3' end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5' cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3' end processing of HIV-1 mRNAs.

Anthracobunids from the middle eocene of India and pakistan are stem perissodactyls.

Anthracobunidae is an Eocene family of large mammals from south Asia that is commonly considered to be part of the radiation that gave rise to elephants (proboscideans) and sea cows (sirenians). We describe a new collection of anthracobunid fossils from Middle Eocene rocks of Indo-Pakistan that more than doubles the number of known anthracobunid fossils and challenges their putative relationships, instead implying that they are stem perissodactyls. Cranial, dental, and postcranial elements allow a revision of species and the recognition of a new anthracobunid genus. Analyses of stable isotopes and long bone geometry together suggest that most anthracobunids fed on land, but spent a considerable amount of time near water. This new evidence expands our understanding of stem perissodactyl diversity and sheds new light on perissodactyl origins.