Argyrolagus and its closest relatives (Argyrolagidae; Late Eocene or Early Oligocene-Early Pleistocene) are usually regarded as outliers among marsupials, with many peculiar craniodental and postcranial features that differ considerably from those found in other recognized groups of metatherians. In the literature this factor has led to variant interpretations of basic anatomical features that usually present no difficulty in identification, like eye position and location of some neurovascular foramina. To shed light on these and other unresolved morphological issues, we report here on new and previously described cranial material of Argyrolagus using modern methods (mu CT scanning, morphometrics). Argyrolagus was a small taxon (between 60 g to 150 g body mass) with an elongated, projecting rostrum convergently resembling that of the rodents Dipodomys and Sommeromys, or Rhynchocyon and Elephantulus among elephant shrews. Contrary to some reconstructions, but in line with G.G. Simpson's original interpretation, the orbit of Argyrolagus was most likely located in the caudal half of the cranium, and was large (similar to 9 mm) for its projected body size. The origin of the temporalis muscle, also disputed, appears to have been small and lain on the caudal part of the cranium above the external acoustic meatus. Orbital convergence was low while the angle of orbital verticality was high, indicating panoramic vision. Three-dimensional reconstruction and interpretation of the auditory region (including the tympanic membrane, middle ear ossicles, middle ear volume, inner ear) indicates that hearing in Argyrolagus favored low frequencies. This correlates with hearing capabilities of extant small mammals (mostly rodents) that live in open environments and/ or inhabit underground tunnels. Both interpretations seem plausible considering the paleoenvironments in which Argyrolagus is thought to have lived. Other features, like the cranial circulatory system, appear to be a mosaic of primitive (e.g., small internal jugular vein, most cranial return through external jugular) and derived features (e.g., absence of proximal stapedial artery, absence of postglenoid vein, and a venous arrangement analogous to the sinus communicans found in some placentals). Although some authors have wondered whether argyrolagoids were metatherians or even therians, our reinterpretation of salient anatomical features in Argyrolagus is consistent with their closest affinities being metatherian, as exemplified by absence of an independent optic canal, lack of proximal stapedial artery, enhanced pericarotid system, and inflected angle of dentary, although their precise systematic placement remains elusive.

In the past decade, ancient protein sequences have emerged as a valuable source of data for deep-time phylogenetic inference1-4. Still, even though ancient proteins have been reported from the Middle-Late Miocene5,6, the recovery of protein sequences providing subordinal-level phylogenetic insights does not exceed 3.7 million years ago (Pliocene)1. Here, we push this boundary back to 21-24 million years ago (Early Miocene) by retrieving enamel protein sequences of a rhinocerotid (Epiaceratherium sp.; CMNFV59632) from Canada's High Arctic. We recover partial sequences of seven enamel proteins and more than 1,000 peptide-spectrum matches, spanning at least 251 amino acids. Endogeneity is in line with thermal age estimates and is supported by indicators of protein damage, including several spontaneous and irreversible chemical modifications accumulated during prolonged diagenesis. Bayesian tip-dating places the divergence time of CMNFV59632 in the Middle Eocene-Oligocene, coinciding with a phase of high rhinocerotid diversification7. This analysis identifies a later Oligocene divergence for Elasmotheriinae, weakening alternative models suggesting a deep basal split between Elasmotheriinae and Rhinocerotinae8,9. The findings are consistent with hypotheses on the origin of the enigmatic fauna of the Haughton Crater, which, in spite of considerable endemism, has similarity to distant Eurasian faunas10,11. Our findings demonstrate the potential of palaeoproteomics in obtaining phylogenetic information from a specimen that is approximately ten times older than any sample from which endogenous DNA has been obtained so far.

Evolutionary theory predicts that sensory systems should adaptively respond to environmental selection. Different ecological niches should, in theory, then correlate with changes in sensory anatomy in lineages that have undergone extensive radiation. The afrotherian clade Tenrecomorpha, comprising of African potamogalines and Malagasy tenrecines, is of particular interest because of its variety: the clade reportedly includes fossorial, arboreal, semiaquatic, and even echolocating taxa. To investigate their sensory ecology, we provide geometric morphometric analyses of inner ear endocasts of 24 tenrec species. We expand this dataset with 9 iodine-stained specimens to study trigeminal organization. Although tenrecomorphs display cross-taxon differences in sensory structures, our analyses distinguish signals of conflicting strength and direction within the tenrec ear, with no single factor that might explain a substantial portion of observed variation when accounting for phylogeny. This contrasts with prior studies of the tenrec cranial endocast, where sensory ecotype and habitat are strongly associated with shape. Iodine-enhanced scans of the trigeminal nerve align with this, and other studies based on bony anatomy. The disparate patterns of shape evolution in Tenrecomorpha and the contrasts exhibited by the inner ear and trigeminal nerve provide a nuanced portrait of neurosensory adaptation, differing from expectations set by other mammalian groups.

The Sparassodonta were the dominant mammalian predators in South America during much of the Cenozoic. Among them, Thylacosmilus atrox, with its hyperdeveloped upper canines and bizarre anatomy, has drawn considerable attention. This study investigates infraorbital foramen (IOF) size and its implications for the paleoecology of sparassodonts, focusing on Thylacosmilus. Using computed tomography (CT), micro-computed tomography (mu CT), and comparative analyses, we examined the relationship between IOF area (IOFarea) and certain anatomical correlates, including foramen rotundum area (FRarea), several endocranial structures, and skull dimensions. Comparisons were made within Sparassodonta and to marsupials and carnivorans. Our results reveal that greater variation in IOFarea exists among sparassodonts compared to marsupials, with some large borhyaenoids exhibiting disproportionally large IOFarea. Notably, Thylacosmilus displays intrataxon variation in IOFarea. Contrary to previous studies, which stated that Thylacosmilus possessed a relatively small IOF that might have been consistent with scavenging behavior, our findings indicate that IOFarea in Thylacosmilus does not substantially differ from that of the active predators in our sample. This study highlights the anatomical diversity of IOF in sparassodonts and underscores the complexity of making behavioral inferences from partial cranial morphology.

Fossils representing Cretaceous lineages of crown clade birds (Aves) are exceptionally rare but are crucial to elucidating major ecological shifts across early avian divergences. Among the earliest known putative crown birds is Vegavis iaai1-5, a foot-propelled diver from the latest Cretaceous (69.2-68.4 million years ago)6 of Antarctica with controversial phylogenetic affinities2,7-10. Initially recovered by phylogenetic analyses as a stem anatid (ducks and closely related species)1,2,11, Vegavis has since been recovered as a stem member of Anseriformes (waterfowl)7-9, or outside Aves altogether10. Here we report a new, nearly complete skull of Vegavis that provides new insight into its feeding ecology and exhibits morphologies that support placement among waterfowl within crown-group birds. Vegavis has an avian beak (absence of teeth and reduced maxilla) and brain shape (hyperinflated cerebrum and ventrally shifted optic lobes). The temporal fossa is well excavated and expansive, indicating that this bird had hypertrophied jaw musculature. The beak is narrow and pointed, and the mandible lacks retroarticular processes. Together, these features comprise a feeding apparatus unlike that of any other known anseriform but like that of other extant birds that capture prey underwater (for example, grebes and loons). The Cretaceous occurrence of Vegavis, with a feeding ecology unique among known Galloanserae (waterfowl and landfowl), is further indication that the earliest anseriform divergences were marked by evolutionary experiments unrepresented in the extant diversity3,11-13.

Reconstructions of evolutionary history can be restricted by a lack of high-quality reference genomes. To date, only four of the eight species of bears (family Ursidae) have chromosome-level genome assemblies. Here, we present assemblies for three additional species-the sun, sloth, and Andean bears-and use a whole-genome alignment of all bear species and other carnivores to reconstruct the evolution of Ursidae. Multiple divergence dating approaches suggest that the six Ursine bears likely diversified in the last 5 Ma, but that divergence times within Ursinae are significantly impacted by gene tree heterogeneity. Consistent with this, we observe that nearly 50% of gene trees conflict with our highly supported species tree, a pattern driven by a significant early hybridization event within Ursinae. We also find that the karyotype of Ursinae is largely similar to the ancestral karyotype of all bears twenty million years prior. In contrast to this conservation of structure, dozens of chromosomal fissions and fusions associated with LINE/L1 retrotransposons dramatically restructured the genomes of the giant panda and Andean bear. Finally, we leverage these genomes to identify species-specific evidence for positive selection on genes associated with color, diet, and metabolism. One of these genes, TCPN2, has a role in pigmentation and shows a series of amino acid mutations in the polar bear over the last 0.5 Ma. Collectively, these new genomic resources enable improved reconstruction of the complex evolutionary history of bears and clarify how this enigmatic group diversified.

Climate affects habitat, food availability, and the movement and sustainability of all life. In this work, we apply Indigenous and Western scientific methods, including genomics and isotope profiling, on fossils from across Beringia to explore the effect of climate change on horses. We find that Late Pleistocene horses from Alaska and northern Yukon are related to populations from Eurasia and crossed the Bering land bridge multiple times during the last glacial interval. We also find deeply divergent lineages north and south of the American ice sheets that genetically influenced populations across Beringia and into Eurasia. As climate warmed and horses entered the ice-free corridor connecting Beringia and midcontinental America, restricted mobility and food availability impeded population growth. Our combined Western and Indigenous framework offers critical guidance for wildlife conservation amid ongoing climate change.

In this contribution, we investigate two sparassodonts from the Sarmiento Formation (Colhuehuapian Age; Early Miocene) recovered at the Patagonian locality of Sacanana, Chubut Province, Argentina. The first specimen (MACN-Pv CH1911), identified as Sipalocyon externus, is an almost complete cranium with upper dentition. The second specimen (MACN-Pv CH40), referred to Borhyaena macrodonta, consists of a fragmentary rostral portion of the face with associated m3–m4. The cranium of S. externus was studied through µCT-scanning to investigate its internal anatomy and infer paleoecological aspects of olfaction and hearing. In general, the endocranial anatomy and encephalization quotient are like those of other metatherians. We report the presence of an accessory transverse diploic sinus that has not previously been described in marsupials. Elements of the nasal cavity (e.g., turbinals, ossified nasal septum, cribriform plate) of S. externus exhibit features that are widespread among marsupials. Sense of olfaction, as evaluated from the cribriform plate and the 3D models of the olfactory bulbs, appears to have grossly resembled that of the domestic cat, a well-researched therian proxy. The dimensions of the tympanic membrane, as estimated from the preserved ectotympanic, suggest that the optimal hearing frequency of S. externus would have been similar to that of extant carnivorous marsupials. In sum, our study suggests that S. externus was a typical small-bodied (2–3 kg) hypercarnivorous metatherian that primarily relied on vision to hunt its prey, complemented by other sensory capabilities (olfaction and hearing), in a similar manner to small felids.

In the past decade, ancient protein sequences have emerged as a valuable source of data for deep-time phylogenetic inference. Still, the recovery of protein sequences providing novel phylogenetic insights does not exceed 3.7 Ma (Pliocene). Here, we push this boundary back to 21-24 Ma (early Miocene), by retrieving enamel protein sequences of an early-diverging rhinocerotid ( Epiaceratherium sp. - CMNF-59632) from the Canadian High Arctic. We recover partial sequences of seven enamel proteins (AHSG, ALB, AMBN, AMELX, AMTN, ENAM, MMP20) and over 1000 peptide-spectrum matches, spanning over at least 251 amino acids. Authentic endogeneity of these sequences is supported by indicators of protein damage, including several spontaneous and irreversible post-translational modifications accumulated during prolonged diagenesis and reaching near-complete occupancy at many sites. Bayesian tip-dating, across 15 extant and extinct perissodactyl taxa, places the divergence time of CMNF-59632 in the middle Eocene-Oligocene, and identifies a later divergence time for Elasmotheriinae in the Oligocene. The finding weakens alternative models suggesting a deep basal split between Elasmotheriinae and Rhinocerotinae. This divergence time of CMNF-59632 coincides with a phase of high diversification of rhinocerotids, and supports a Eurasian origin of this clade in the late Eocene or Oligocene. The findings are consistent with previous hypotheses on the origin of the enigmatic fauna of the Haughton crater, which, in spite of their considerable degree of endemism, also display similarity to distant Eurasian faunas. Our findings demonstrate the potential of palaeoproteomics in obtaining phylogenetic information from a specimen that is ten times older than any sample from which endogenous DNA has been obtained.

The Huayquerian Stage of the South American chronostratigraphic scheme (named for the Huayquerias del Este, Argentina) was originally based on a poorly known mammal association of six taxa from the Huayquerias Formation. We studied the geology, age and fauna of the Neogene sequence in this area, including the Huayquerias, Tunuyan and Bajada Grande formations. The sequence comprises a monotonous succession of synorogenic epiclastic sediments deposited under arid to semi-arid conditions. Zircon U-Pb dates from 10 tuffaceous levels (7.2-1.6 Ma) place deposition of the Huayquerias Formation during the late Tortonian or Messinian to early Zanclean, the Tunuyan Formation during the Zanclean-Piacenzian, and the Bajada Grande Formation during the Piacenzian-Calabrian. We present 43 and 31 new mammal taxon records for the Huayquerias and Tunuyan formations, respectively. Progressive faunal change was observed along the sequence. The first records of the Chaco tortoise Chelonoidis chilensis and the notoungulate Xotodon major, and the latest records of Interatheriidae and Typotheriopsis (notoungulates), Metacaremys calfucalel, Phtoramys hidalguense and Lagostomus pretrichodactyla (rodents), Chasicotatus ameghinoi and Macroeuphractus morenoi (xenarthrans) are reported. The faunal associations of the Huayquerias and lower Tunuyan formations are highly similar to each other, and to other coeval localities in Argentina. The Macroeuphractus morenoi Assemblage Biozone is proposed as the basis for redefining the Huayquerian Stage, due to the co-occurrence of three taxa with wide geographical distribution in southern South America: Macroeuphractus morenoi, Pseudotypotherium subinsigne and Lagostomus pretrichodactyla. The age of this biozone is constrained at c. 8-5 Ma in its type area.

The GAARlandia hypothesis has produced vigorous debate among biologists regarding whether now-submerged landforms that existed in the Caribbean region during the late Paleogene might have acted as a barrier for marine organisms and as a bridge for terrestrial biotas migrating from South America into the Greater Antilles. This concept derived from the hypothesized emergence history of the Aves Ridge. In the quarter century since GAARlandia was first proposed, new paleontological, geological and geophysical information has greatly extended the database available. Here we reaffirm that GAARlandia was a positive topographic feature from middle Eocene, and was exposed above sea level between late Eocene and early Oligocene when it facilitated biotic colonization of the northern Greater Antilles and their satellite islands, whether as a series of closely spaced islands or as a continuous peninsula projecting from northeastern South America along the crown of the rise.

This contribution contains the three-dimensional models of the inner ear of the hetaxodontid rodents Amblyrhiza, Clidomys and Elasmodontomys from the West Indies.These specimens were analyzed and discussed in : The inner ear of caviomorph rodents: phylogenetic implications and application to extinct West Indian taxa.

Supplemental Material for 'Transverse canal foramen and pericarotid venous network in Metatheria and other mammals (Bulletin of the American Museum of Natural History, no. 462)' - https://digitallibrary.amnh.org/handle/2246/7324

The evolution of mammalian vision is difficult to study because the actual receptor organs-the eyes-are not preserved in the fossil record. Orbital orientation and size are the traditional proxies for inferring aspects of ocular function, such as stereoscopy. Adaptations for good stereopsis have evolved in living predaceous mammals, and it is reasonable to infer that fossil representatives would follow the same pattern. This applies to the sparassodonts, an extinct group of South American hypercarnivores related to marsupials, with one exception. In the sabertooth Thylacosmilus atrox, the bony orbits were notably divergent, like those of a cow or a horse, and thus radically differing from conditions in any other known mammalian predator. Orbital convergence alone, however, does not determine presence of stereopsis; frontation and verticality of the orbits also play a role. We show that the orbits of Thylacosmilus were frontated and verticalized in a way that favored some degree of stereopsis and compensated for limited convergence in orbital orientation. The forcing function behind these morphological tradeoffs was the extraordinary growth of its rootless canines, which affected skull shape in Thylacosmilus in numerous ways, including relative orbital displacement.

Although few nondental features of the osteocranium consistently discriminate marsupials from placentals, the transverse canal foramen (TCF) has been repeatedly offered as a potential synapomorphy of crown- group Marsupialia and their closest allies. To explore this contention appropriately, the TCF needs to be evaluated in relation to the morphofunctional complex of which it is a part, something never previously undertaken in a systematic fashion. This complex, here defined as the pericarotid venous network (PCVN), is assessed using osteological, histological, and ontogenetic information. Although the TCF is usually thought of as a marsupial attribute, some living placentals also express it. What do these clades actually share in regard to this feature, and how do they differ? Our leading hypothesis is that the chief components of the PCVN begin development in the same way in both Marsupialia and Placentalia, but they follow different ontogenetic trajectories in terms of persistence, size, and connections with other elements of the cephalic venous vasculature. Similarities include shared presence of specific emissary and emissarylike veins in the mesocranial region that connect part of the endocranial dural vasculature (cavernous sinus or CS) to the systemic circulation (external and internal jugular veins plus the cerebrospinal venous system). In marsupials the principal pericarotid vessels are the transverse canal vein (TCV) and internal carotid vein (ICV). These veins almost always attain relatively large size during marsupial ontogeny. By contrast, in most placentals their apparent homologs (among others, emissary vein of the sphenoidal foramen and internal carotid venous plexus) evidently slow down or terminate their growth relatively early, and for this reason they play only a proportionally minor role in cephalic drainage in later life. In both clades, these vessels (informally grouped with others in the same region as pericarotid mesocranial distributaries, or PMDs) play a variable role in draining the CS in conjunction with the much larger petrosal sinuses. A pneumatic space within the basisphenoid-called the sphenoid sinus in placentals, transverse basisphenoid sinus (TBS) in marsupials-communicates with PCVN vasculature and should be considered an integral part of the network. The TBS contains red marrow tissues that are active centers of extramedullary hematopoiesis in young stages of some species, although how widespread this function may be in marsupial clades is not yet known. Previous explorations of the marsupial PCVN have been largely limited to determining whether, in any given taxon, a continuous passageway linking the right and left TCFs could be demonstrated running through the basisphenoid ("intramural" condition). It has long been known that a number of species apparently lack this particular passageway, and that the TCFs instead open into the brain-case ("endocranial" condition). Puzzlingly, some species appear to have both passageways, others one or the other, and a few none at all, thus inviting questions about their equivalency and the circumstances under which the CS is actually drained by the TCV. Morphologically, these uncertainties can be resolved by viewing the full TCV as a tripartite entity, consisting of a trunk and rostral and caudal branches. The trunk, or the part that leaves the TCF for the external jugular system, receives the rostral and caudal branches, if both are present, within the body of the basisphenoid. The rostral or intramural branch has little or no direct communication with the endocranium in most investigated species. By contrast, the caudal or endocranial branch is an ordinary emissarium, in that it connects a part of the endocranial system of dural veins with the extracranial circulation. Determining branch routing alone does not adequately capture the scale of morphological variety and function encountered in marsupial PCVN organization. We distinguish five patterns of association between TCVs and other PCVN components. These patterns, based on both histological and osteological criteria, are defined as follows: (1) Simple: only rostral passageway present, caudal passageway absent or reduced to a thread; rostral branch veins form midline confluence within TBS in advance of hypophysis; minimal interaction with CS and its distributaries; rostral and caudal portions of TBS discontinuous. (2) Complex: mostly as in ( 1), except both rostral and caudal branches present and functional; caudal branches communicate with CS/ICV and do not form a confluence; TBS more extensive. (3) Compound: mostly as in (2), except TBS greatly expanded, incorporating most of rostral branch canals, which are correspondingly short. (4) Hybrid: differs from others in that only the pathways for enlarged caudal branches are significant; they originate from the CS/ICV caudal to the position of the hypophysis; rostral branches absent or highly reduced. (5) Indeterminate: transverse foramina, canals, and branches absent or unidentifiable as such, presumably due to vascular involution early in ontogeny. In light of TCV composition, the trunk of the TCV can be considered a mixed-origin vein, maximally receiving both a quasisystemic or emissarylike vessel (rostral branch) that does not originate from endocranial dural vessels, and a true emissarial vessel (caudal branch) that does. Some extant geomyoid rodents and strepsirrhine primates exhibit enlarged venous structures in the mesocranial region; these are briefly surveyed for comparative purposes, but resemblances to conditions in marsupials are superficial and unmistakably interpretable as convergences. Members of the extinct marsupial sister group Sparassodonta sometimes lack detectable TCFs, as do other non-marsupial metatherians in the fossil record. Evidence for the transverse canal and other PCVN components in other therians is briefly outlined. In summary, the development of mesocranial vasculature as outlined in this paper is hypothesized to be basal for therians, but Marsupialia and Placentalia radically differ in the end expression of PMDs in the adult stage. In prenatal stages of both clades, initial differentiation of these distributaries is presumably similar, but, compared to marsupials, in almost all placental groups these vessels are retained in an undeveloped or neotenic state. By contrast, enhanced expression of the TCV trunk and its branches seems to be a genuine novelty characterizing Marsupialia, although one probably present in some other metatherian groups. Accordingly, the transverse foramen, canal, and related features are probably best regarded as an innovation occurring in the marsupial stem, not a synapomorphy of the crown group as previously suggested by some authors.

With their past and current diversities, West Indian caviomorph rodents dominate the terrestrial mammalian fauna of the Caribbean archipelago. Many of these species have recently become extinct, including the emblematic giant forms known as Heptaxodontidae. The higher-level systematics and content of this family have been widely disputed over the last decades (i.e., membership in Cavioidea vs. Chinchilloidea vs. Octodontoidea). Here we analyzed the phylogenetic signal provided by several characters of the caviomorph inner ear to adress the phylogenetic affinities of the West Indian heptaxodontids. For this, we assembled an exhaustive taxonomic sampling (N = 100) of extant North and South American caviomorphs (including representatives of all families) and a wide array of West Indian forms among octodontoid echimyids (extant and extinct capromyines, as well as extinct heteropsomyines), and some heptaxodontid subfossil taxa such as Amblyrhiza, Clidomys, and Elasmodontomys. Geometric morphometrics and comparative phylogenetic methods were employed to explore shape differences of the inner ear and their potential systematic implications. Our results show that: (1) allometry is a major contributor to shape variation in the bony labyrinth; (2) shape variation bears a strong phylogenetic signal, providing diagnostic characters for Caviidae and Erethizontoidea; and (3) Amblyrhiza and Clidomys are morphologically closer to Chinchilloidea with which they have potential phylogenetic affinities. Elasmodontomys remains a problematic taxon as it exhibits inner ear features that are consistent with either Chinchilloidea or Octodontoidea, depending on how the allometric component is evaluated.

It has long been recognized that, among extant mammals, the afrotherian clade Tenrecomorpha contains an exceptional range of sensory specialists in which arboreal, fossorial, semiaquatic and possibly even echolocating species occur within a single clade. Despite their obvious interest in this regard, the sensory apparatus of these animals has not been investigated with modern techniques. Presented here is a geometric morphometric analysis of virtual endocasts of 24 tenrecomorph species ( 69

The debate regarding the evolutionary relationships of the extinct South American native ungulates (SANUs) to the major placental clades Afrotheria and Boreoeutheria is exciting and has profound implications for our understanding of their early diversification and paleobiogeography. Although this controversy has not yet proven resolvable using morphological evidence, paleoproteomic and ancient DNA analyses support that at least some SANUs (i.e., Litopterna and Notoungulata) are members of Boreoeutheria, closely related to Perissodactyla (the Panperissodactyla hypothesis). Here we present a critical assessment of a recently published morphology-based study that claims that: (1) some SANUs (i.e., Notoungulata, Astrapotheria, Pyrotheria, and Xenungulata) represent a monophyletic supraordinal group, the Sudamericungulata, closely related to the Afrotherian hyracoids; and (2) the remaining SANUs (i.e., Litopterna and Didolodontidae, placed in a separate taxon, Panameridiungulata) are boreoeutherian in origin. Because this proposal (hereafter, the Sudamericungulata - Panameridiungulata or S-P hypothesis) is based on an incongruously reduced sample of boreoeutherians (including only a single perissodactyl) and inadequate character sampling restricted to dental and mandibular traits, it cannot be regarded as a satisfactory test of SANU relationships. Moreover, the S-P hypothesis fails to recover monophyletic Boreoeutheria and/or Afrotheria, making it incompatible with all well-established hypotheses of placental diversification. We find that the introduction of molecular constraints forcing the monophyly of Boreoeutheria and Afrotheria produces new trees, all recovering Sudamericungulata and Panameridiungulata nested within Boreoeutheria. These results are consistent with our analyses using a corrected version of the S-P matrix. Although we acknowledge that boreoeutherian affinities have still not been conclusively demonstrated for all nominal SANUs, it is beyond argument that any further credible testing must be based on much more exhaustive surveys than are currently available.

Traditionally, paleontologists have relied on the morphological features of bones and teeth to reconstruct the evolutionary relationships of extinct animals.1 In recent decades, the analysis of ancient DNA recovered from macrofossils has provided a powerful means to evaluate these hypotheses and develop novel phylogenetic models.2 Although a great deal of life history data can be extracted from bones, their scarcity and associated biases limit their information potential. The paleontological record of Beringia3-the unglaciated areas and former land bridge between northeast Eurasia and northwest North America-is relatively robust thanks to its perennially frozen ground favoring fossil preservation.4,5 However, even here, the macrofossil record is significantly lacking in small-bodied fauna (e.g., rodents and birds), whereas questions related to migration and extirpation, even among well-studied taxa, remain crudely resolved. The growing sophistication of ancient environmental DNA (eDNA) methods have allowed for the identification of species within terrestrial/aquatic ecosystems,6-12 in paleodietary reconstructions,13-19 and facilitated genomic reconstructions from cave contexts.8,20-22 Murchie et al.6,23 used a capture enrichment approach to sequence a diverse range of faunal and floral DNA from permafrost silts deposited during the Pleistocene-Holocene transition.24 Here, we expand on their work with the mitogenomic assembly and phylogenetic placement of Equus caballus (caballine horse), Bison priscus (steppe bison), Mammuthus primigenius (woolly mammoth), and Lagopus lagopus (willow ptarmigan) eDNA from multiple permafrost cores spanning the last 40,000 years. We identify a diverse metagenomic spectra of Pleistocene fauna and identify the eDNA co-occurrence of distinct Eurasian and American mitogenomic lineages.