Seeing through the eyes of the sabertooth Thylacosmilus atrox (Metatheria, Sparassodonta).

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.

Probing the genomic limits of de-extinction in the Christmas Island rat.

Three principal methods are under discussion as possible pathways to "true" de-extinction; i.e., back-breeding, cloning, and genetic engineering.1,2 Of these, while the latter approach is most likely to apply to the largest number of extinct species, its potential is constrained by the degree to which the extinct species genome can be reconstructed. We explore this question using the extinct Christmas Island rat (Rattus macleari) as a model, an endemic rat species that was driven extinct between 1898 and 1908.3-5 We first re-sequenced its genome to an average of >60× coverage, then mapped it to the reference genomes of different Rattus species. We then explored how evolutionary divergence from the extant reference genome affected the fraction of the Christmas Island rat genome that could be recovered. Our analyses show that even when the extremely high-quality Norway brown rat (R. norvegicus) is used as a reference, nearly 5% of the genome sequence is unrecoverable, with 1,661 genes recovered at lower than 90% completeness, and 26 completely absent. Furthermore, we find the distribution of regions affected is not random, but for example, if 90% completeness is used as the cutoff, genes related to immune response and olfaction are excessively affected. Ultimately, our approach demonstrates the importance of applying similar analyses to candidates for de-extinction through genome editing in order to provide critical baseline information about how representative the edited form would be of the extinct species.

Pleistocene mitogenomes reconstructed from the environmental DNA of permafrost sediments.

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.

Collapse of the mammoth-steppe in central Yukon as revealed by ancient environmental DNA.

The temporal and spatial coarseness of megafaunal fossil records complicates attempts to to disentangle the relative impacts of climate change, ecosystem restructuring, and human activities associated with the Late Quaternary extinctions. Advances in the extraction and identification of ancient DNA that was shed into the environment and preserved for millennia in sediment now provides a way to augment discontinuous palaeontological assemblages. Here, we present a 30,000-year sedimentary ancient DNA (sedaDNA) record derived from loessal permafrost silts in the Klondike region of Yukon, Canada. We observe a substantial turnover in ecosystem composition between 13,500 and 10,000 calendar years ago with the rise of woody shrubs and the disappearance of the mammoth-steppe (steppe-tundra) ecosystem. We also identify a lingering signal of Equus sp. (North American horse) and Mammuthus primigenius (woolly mammoth) at multiple sites persisting thousands of years after their supposed extinction from the fossil record.

Isotope data from amino acids indicate Darwin's ground sloth was not an herbivore.

Fossil sloths are regarded as obligate herbivores for reasons including peculiarities of their craniodental morphology and that all living sloths feed exclusively on plants. We challenge this view based on isotopic analyses of nitrogen of specific amino acids, which show that Darwin's ground sloth Mylodon darwinii was an opportunistic omnivore. This direct evidence of omnivory in an ancient sloth requires reevaluation of the ecological structure of South American Cenozoic mammalian communities, as sloths represented a major component of these ecosystems across the past 34 Myr. Furthermore, by analyzing modern mammals with known diets, we provide a basis for reliable interpretation of nitrogen isotopes of amino acids of fossils. We argue that a widely used equation to determine trophic position is unnecessary, and that the relative isotopic values of the amino acids glutamate and phenylalanine alone permit reliable reconstructions of trophic positions of extant and extinct mammals.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

New radiometric 40Ar-39Ar dates and faunistic analyses refine evolutionary dynamics of Neogene vertebrate assemblages in southern South America.

The vertebrate fossil record of the Pampean Region of Argentina occupies an important place in South American vertebrate paleontology. An abundance of localities has long been the main basis for constructing the chronostratigraphical/geochronological scale for the late Neogene-Quaternary of South America, as well as for understanding major patterns of vertebrate evolution, including the Great American Biotic Interchange. However, few independently-derived dates are available for constraining this record. In this contribution, we present new 40Ar/39Ar dates on escorias (likely the product of meteoric impacts) from the Argentinean Atlantic coast and statistically-based biochronological analyses that help to calibrate Late Miocene-Pliocene Pampean faunal successions. For the type areas of the Montehermosan and Chapadmalalan Ages/Stages, our results delimit their age ranges to 4.7-3.7 Ma and ca. 3.74-3.04 Ma, respectively. Additionally, from Buenos Aires Province, dates of 5.17 Ma and 4.33 Ma were recovered for "Huayquerian" and Montehermosan faunas. This information helps to better calibrate important first appearances of allochthonous taxa in South America, including one of the oldest records for procyonids (7.24-5.95 Ma), cricetids (6.95-5.46 Ma), and tayassuids (> 3.74 Ma, oldest high-confidence record). These results also constrain to ca. 3 Ma the last appearances of the autochthonous sparassodonts, as well as terror birds of large/middle body size in South America. South American faunal turnover during the late Neogene, including Late Pliocene extinctions, is interpreted as a consequence of knock-on effects from global climatic changes and initiation of the icehouse climate regime.

No evidence for widespread island extinctions after Pleistocene hominin arrival.

The arrival of modern humans into previously unoccupied island ecosystems is closely linked to widespread extinction, and a key reason cited for Pleistocene megafauna extinction is anthropogenic overhunting. A common assumption based on late Holocene records is that humans always negatively impact insular biotas, which requires an extrapolation of recent human behavior and technology into the archaeological past. Hominins have been on islands since at least the early Pleistocene and Homo sapiens for at least 50 thousand y (ka). Over such lengthy intervals it is scarcely surprising that significant evolutionary, behavioral, and cultural changes occurred. However, the deep-time link between human arrival and island extinctions has never been explored globally. Here, we examine archaeological and paleontological records of all Pleistocene islands with a documented hominin presence to examine whether humans have always been destructive agents. We show that extinctions at a global level cannot be associated with Pleistocene hominin arrival based on current data and are difficult to disentangle from records of environmental change. It is not until the Holocene that large-scale changes in technology, dispersal, demography, and human behavior visibly affect island ecosystems. The extinction acceleration we are currently experiencing is thus not inherent but rather part of a more recent cultural complex.

Environmental genomics of Late Pleistocene black bears and giant short-faced bears.

Analysis of ancient environmental DNA (eDNA) has revolutionized our ability to describe biological communities in space and time,1-3 by allowing for parallel sequencing of DNA from all trophic levels.4-8 However, because environmental samples contain sparse and fragmented data from multiple individuals, and often contain closely related species,9 the field of ancient eDNA has so far been limited to organellar genomes in its contribution to population and phylogenetic studies.5,6,10,11 This is in contrast to data from fossils12,13 where full-genome studies are routine, despite these being rare and their destruction for sequencing undesirable.14-16 Here, we report the retrieval of three low-coverage (0.03×) environmental genomes from American black bear (Ursus americanus) and a 0.04× environmental genome of the extinct giant short-faced bear (Arctodus simus) from cave sediment samples from northern Mexico dated to 16-14 thousand calibrated years before present (cal kyr BP), which we contextualize with a new high-coverage (26×) and two lower-coverage giant short-faced bear genomes obtained from fossils recovered from Yukon Territory, Canada, which date to ∼22-50 cal kyr BP. We show that the Late Pleistocene black bear population in Mexico is ancestrally related to the present-day Eastern American black bear population, and that the extinct giant short-faced bears present in Mexico were deeply divergent from the earlier Beringian population. Our findings demonstrate the ability to separately analyze genomic-scale DNA sequences of closely related species co-preserved in environmental samples, which brings the use of ancient eDNA into the era of population genomics and phylogenetics.

Assessment of different screening methods for selecting palaeontological bone samples for peptide sequencing.

Ancient proteomics is being applied to samples dating further and further back in time, with many palaeontological specimens providing protein sequence data for phylogenetic analysis as well as protein degradation studies. However, fossils are a precious material and proteomic analysis is destructive and costly. In this paper we consider three different techniques (ATR-FTIR, MALDI-ToF MS and chiral AA analysis) to screen fossil material for potential protein preservation, aiming to maximise the proteomic information recovered and saving costly time consuming analyses which may produce low quality results. It was found that splitting factor and C/P indices from ATR-FTIR were not a reliable indicator of protein survival as they are confounded by secondary mineralisation of the fossil material. Both MALDI-ToF MS and chiral AA analysis results were able to successfully identify samples with surviving proteins, and it is suggested that one or both of these analyses be used for screening palaeontological specimens. SIGNIFICANCE: This study has shown both chiral amino acid analysis and MALDI-ToF MS are reliable screening methods for predicting protein survival in fossils. Both these methods are quick, cheap, minimally destructive (1 mg and 15 mg respectively) and can provide crucial additional information about the endogeneity of the surviving proteins. It is hoped that the use of these screening methods will encourage the examination of a wide range of palaeontological specimens for potential proteomic analysis. This in turn will give us a better understanding of protein survival far back in time and under different environmental conditions.

American mastodon mitochondrial genomes suggest multiple dispersal events in response to Pleistocene climate oscillations.

Pleistocene glacial-interglacial cycles are correlated with dramatic temperature oscillations. Examining how species responded to these natural fluctuations can provide valuable insights into the impacts of present-day anthropogenic climate change. Here we present a phylogeographic study of the extinct American mastodon (Mammut americanum), based on 35 complete mitochondrial genomes. These data reveal the presence of multiple lineages within this species, including two distinct clades from eastern Beringia. Our molecular date estimates suggest that these clades arose at different times, supporting a pattern of repeated northern expansion and local extirpation in response to glacial cycling. Consistent with this hypothesis, we also note lower levels of genetic diversity among northern mastodons than in endemic clades south of the continental ice sheets. The results of our study highlight the complex relationships between population dispersals and climate change, and can provide testable hypotheses for extant species expected to experience substantial biogeographic impacts from rising temperatures.

Ancient Mitogenomes Reveal the Evolutionary History and Biogeography of Sloths.

Living sloths represent two distinct lineages of small-sized mammals that independently evolved arboreality from terrestrial ancestors. The six extant species are the survivors of an evolutionary radiation marked by the extinction of large terrestrial forms at the end of the Quaternary. Until now, sloth evolutionary history has mainly been reconstructed from phylogenetic analyses of morphological characters. Here, we used ancient DNA methods to successfully sequence 10 extinct sloth mitogenomes encompassing all major lineages. This includes the iconic continental ground sloths Megatherium, Megalonyx, Mylodon, and Nothrotheriops and the smaller endemic Caribbean sloths Parocnus and Acratocnus. Phylogenetic analyses identify eight distinct lineages grouped in three well-supported clades, whose interrelationships are markedly incongruent with the currently accepted morphological topology. We show that recently extinct Caribbean sloths have a single origin but comprise two highly divergent lineages that are not directly related to living two-fingered sloths, which instead group with Mylodon. Moreover, living three-fingered sloths do not represent the sister group to all other sloths but are nested within a clade of extinct ground sloths including Megatherium, Megalonyx, and Nothrotheriops. Molecular dating also reveals that the eight newly recognized sloth families all originated between 36 and 28 million years ago (mya). The early divergence of recently extinct Caribbean sloths around 35 mya is consistent with the debated GAARlandia hypothesis postulating the existence at that time of a biogeographic connection between northern South America and the Greater Antilles. This new molecular phylogeny has major implications for reinterpreting sloth morphological evolution, biogeography, and diversification history.

Palaeoproteomics resolves sloth relationships.

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America.

Ancient DNA of the extinct Jamaican monkey Xenothrix reveals extreme insular change within a morphologically conservative radiation.

The insular Caribbean until recently contained a diverse mammal fauna including four endemic platyrrhine primate species, all of which died out during the Holocene. Previous morphological studies have attempted to establish how these primates are related to fossil and extant platyrrhines, whether they represent ancient or recent colonists, and whether they constitute a monophyletic group. These efforts have generated multiple conflicting hypotheses, from close sister-taxon relationships with several different extant platyrrhines to derivation from a stem platyrrhine lineage outside the extant Neotropical radiation. This diversity of opinion reflects the fact that Caribbean primates were morphologically extremely unusual, displaying numerous autapomorphies and apparently derived conditions present across different platyrrhine clades. Here we report ancient DNA data for an extinct Caribbean primate: a limited-coverage entire mitochondrial genome and seven regions of nuclear genome for the most morphologically derived taxon, the Jamaican monkey Xenothrix mcgregori We demonstrate that Xenothrix is part of the existing platyrrhine radiation rather than a late-surviving stem platyrrhine, despite its unusual adaptations, and falls within the species-rich but morphologically conservative titi monkey clade (Callicebinae) as sister to the newly recognized genus Cheracebus These results are not congruent with previous morphology-based hypotheses and suggest that even morphologically conservative lineages can exhibit phenetic plasticity in novel environments like those found on islands. Xenothrix and Cheracebus diverged ca. 11 Ma, but primates have been present in the Caribbean since 17.5-18.5 Ma, indicating that Caribbean primate diversity was generated by multiple over-water colonizations.

A comprehensive genomic history of extinct and living elephants.

Elephantids are the world's most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant's ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

A new genus of horse from Pleistocene North America.

The extinct 'New World stilt-legged', or NWSL, equids constitute a perplexing group of Pleistocene horses endemic to North America. Their slender distal limb bones resemble those of Asiatic asses, such as the Persian onager. Previous palaeogenetic studies, however, have suggested a closer relationship to caballine horses than to Asiatic asses. Here, we report complete mitochondrial and partial nuclear genomes from NWSL equids from across their geographic range. Although multiple NWSL equid species have been named, our palaeogenomic and morphometric analyses support the idea that there was only a single species of middle to late Pleistocene NWSL equid, and demonstrate that it falls outside of crown group Equus. We therefore propose a new genus, Haringtonhippus, for the sole species H. francisci. Our combined genomic and phenomic approach to resolving the systematics of extinct megafauna will allow for an improved understanding of the full extent of the terminal Pleistocene extinction event.

A mitogenomic timetree for Darwin's enigmatic South American mammal Macrauchenia patachonica.

The unusual mix of morphological traits displayed by extinct South American native ungulates (SANUs) confounded both Charles Darwin, who first discovered them, and Richard Owen, who tried to resolve their relationships. Here we report an almost complete mitochondrial genome for the litoptern Macrauchenia. Our dated phylogenetic tree places Macrauchenia as sister to Perissodactyla, but close to the radiation of major lineages within Laurasiatheria. This position is consistent with a divergence estimate of ∼66 Ma (95% credibility interval, 56.64-77.83 Ma) obtained for the split between Macrauchenia and other Panperissodactyla. Combined with their morphological distinctiveness, this evidence supports the positioning of Litopterna (possibly in company with other SANU groups) as a separate order within Laurasiatheria. We also show that, when using strict criteria, extinct taxa marked by deep divergence times and a lack of close living relatives may still be amenable to palaeogenomic analysis through iterative mapping against more distant relatives.

Fossil and genomic evidence constrains the timing of bison arrival in North America.

The arrival of bison in North America marks one of the most successful large-mammal dispersals from Asia within the last million years, yet the timing and nature of this event remain poorly determined. Here, we used a combined paleontological and paleogenomic approach to provide a robust timeline for the entry and subsequent evolution of bison within North America. We characterized two fossil-rich localities in Canada's Yukon and identified the oldest well-constrained bison fossil in North America, a 130,000-y-old steppe bison, Bison cf. priscus We extracted and sequenced mitochondrial genomes from both this bison and from the remains of a recently discovered, ∼120,000-y-old giant long-horned bison, Bison latifrons, from Snowmass, Colorado. We analyzed these and 44 other bison mitogenomes with ages that span the Late Pleistocene, and identified two waves of bison dispersal into North America from Asia, the earliest of which occurred ∼195-135 thousand y ago and preceded the morphological diversification of North American bison, and the second of which occurred during the Late Pleistocene, ∼45-21 thousand y ago. This chronological arc establishes that bison first entered North America during the sea level lowstand accompanying marine isotope stage 6, rejecting earlier records of bison in North America. After their invasion, bison rapidly colonized North America during the last interglaciation, spreading from Alaska through continental North America; they have been continuously resident since then.

The evolutionary and phylogeographic history of woolly mammoths: a comprehensive mitogenomic analysis.

Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

Protein sequences bound to mineral surfaces persist into deep time.

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).