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.

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.

Ancient proteins resolve the evolutionary history of Darwin's South American ungulates.

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

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.

Fifty thousand years of Arctic vegetation and megafaunal diet.

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25-15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

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.

Evolutionary History of the Nesophontidae, the Last Unplaced Recent Mammal Family.

The mammalian evolutionary tree has lost several major clades through recent human-caused extinctions. This process of historical biodiversity loss has particularly affected tropical island regions such as the Caribbean, an area of great evolutionary diversification but poor molecular preservation. The most enigmatic of the recently extinct endemic Caribbean mammals are the Nesophontidae, a family of morphologically plesiomorphic lipotyphlan insectivores with no consensus on their evolutionary affinities, and which constitute the only major recent mammal clade to lack any molecular information on their phylogenetic placement. Here, we use a palaeogenomic approach to place Nesophontidae within the phylogeny of recent Lipotyphla. We recovered the near-complete mitochondrial genome and sequences for 17 nuclear genes from a ∼750-year-old Hispaniolan Nesophontes specimen, and identify a divergence from their closest living relatives, the Solenodontidae, more than 40 million years ago. Nesophontidae is thus an older distinct lineage than many extant mammalian orders, highlighting not only the role of island systems as "museums" of diversity that preserve ancient lineages, but also the major human-caused loss of evolutionary history.

American mastodon extirpation in the Arctic and Subarctic predates human colonization and terminal Pleistocene climate change.

Existing radiocarbon ((14)C) dates on American mastodon (Mammut americanum) fossils from eastern Beringia (Alaska and Yukon) have been interpreted as evidence they inhabited the Arctic and Subarctic during Pleistocene full-glacial times (∼ 18,000 (14)C years B.P.). However, this chronology is inconsistent with inferred habitat preferences of mastodons and correlative paleoecological evidence. To establish a last appearance date (LAD) for M. americanum regionally, we obtained 53 new (14)C dates on 36 fossils, including specimens with previously published dates. Using collagen ultrafiltration and single amino acid (hydroxyproline) methods, these specimens consistently date to beyond or near the ∼ 50,000 y B.P. limit of (14)C dating. Some erroneously "young" (14)C dates are due to contamination by exogenous carbon from natural sources and conservation treatments used in museums. We suggest mastodons inhabited the high latitudes only during warm intervals, particularly the Last Interglacial [Marine Isotope Stage (MIS) 5] when boreal forests existed regionally. Our (14)C dataset suggests that mastodons were extirpated from eastern Beringia during the MIS 4 glacial interval (∼ 75,000 y ago), following the ecological shift from boreal forest to steppe tundra. Mastodons thereafter became restricted to areas south of the continental ice sheets, where they suffered complete extinction ∼ 10,000 (14)C years B.P. Mastodons were already absent from eastern Beringia several tens of millennia before the first humans crossed the Bering Isthmus or the onset of climate changes during the terminal Pleistocene. Local extirpations of mastodons and other megafaunal populations in eastern Beringia were asynchrononous and independent of their final extinction south of the continental ice sheets.

Genomic Data from Extinct North American Camelops Revise Camel Evolutionary History.

Recent advances in paleogenomic technologies have enabled an increasingly detailed understanding of the evolutionary relationships of now-extinct mammalian taxa. However, a number of enigmatic Quaternary species have never been characterized with molecular data, often because available fossils are rare or are found in environments that are not optimal for DNA preservation. Here, we analyze paleogenomic data extracted from bones attributed to the late Pleistocene western camel, Camelops cf. hesternus, a species that was distributed across central and western North America until its extinction approximately 13,000 years ago. Despite a modal sequence length of only around 35 base pairs, we reconstructed high-coverage complete mitochondrial genomes and low-coverage partial nuclear genomes for each specimen. We find that Camelops is sister to African and Asian bactrian and dromedary camels, to the exclusion of South American camelids (llamas, guanacos, alpacas, and vicuñas). These results contradict previous morphology-based phylogenetic models for Camelops, which suggest instead a closer relationship between Camelops and the South American camelids. The molecular data imply a Late Miocene divergence of the Camelops clade from lineages that separately gave rise to the extant camels of Eurasia. Our results demonstrate the increasing capacity of modern paleogenomic methods to resolve evolutionary relationships among distantly related lineages.

Interordinal gene capture, the phylogenetic position of Steller's sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia.

The recently extinct (ca. 1768) Steller's sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller's sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

The delayed rise of present-day mammals.

Did the end-Cretaceous mass extinction event, by eliminating non-avian dinosaurs and most of the existing fauna, trigger the evolutionary radiation of present-day mammals? Here we construct, date and analyse a species-level phylogeny of nearly all extant Mammalia to bring a new perspective to this question. Our analyses of how extant lineages accumulated through time show that net per-lineage diversification rates barely changed across the Cretaceous/Tertiary boundary. Instead, these rates spiked significantly with the origins of the currently recognized placental superorders and orders approximately 93 million years ago, before falling and remaining low until accelerating again throughout the Eocene and Oligocene epochs. Our results show that the phylogenetic 'fuses' leading to the explosion of extant placental orders are not only very much longer than suspected previously, but also challenge the hypothesis that the end-Cretaceous mass extinction event had a major, direct influence on the diversification of today's mammals.

Ancient DNA analyses exclude humans as the driving force behind late Pleistocene musk ox (Ovibos moschatus) population dynamics.

The causes of the late Pleistocene megafaunal extinctions are poorly understood. Different lines of evidence point to climate change, the arrival of humans, or a combination of these events as the trigger. Although many species went extinct, others, such as caribou and bison, survived to the present. The musk ox has an intermediate story: relatively abundant during the Pleistocene, it is now restricted to Greenland and the Arctic Archipelago. In this study, we use ancient DNA sequences, temporally unbiased summary statistics, and Bayesian analytical techniques to infer musk ox population dynamics throughout the late Pleistocene and Holocene. Our results reveal that musk ox genetic diversity was much higher during the Pleistocene than at present, and has undergone several expansions and contractions over the past 60,000 years. Northeast Siberia was of key importance, as it was the geographic origin of all samples studied and held a large diverse population until local extinction at approximately 45,000 radiocarbon years before present ((14)C YBP). Subsequently, musk ox genetic diversity reincreased at ca. 30,000 (14)C YBP, recontracted at ca. 18,000 (14)C YBP, and finally recovered in the middle Holocene. The arrival of humans into relevant areas of the musk ox range did not affect their mitochondrial diversity, and both musk ox and humans expanded into Greenland concomitantly. Thus, their population dynamics are better explained by a nonanthropogenic cause (for example, environmental change), a hypothesis supported by historic observations on the sensitivity of the species to both climatic warming and fluctuations.

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.

Ancient DNA reveals late survival of mammoth and horse in interior Alaska.

Causes of late Quaternary extinctions of large mammals ("megafauna") continue to be debated, especially for continental losses, because spatial and temporal patterns of extinction are poorly known. Accurate latest appearance dates (LADs) for such taxa are critical for interpreting the process of extinction. The extinction of woolly mammoth and horse in northwestern North America is currently placed at 15,000-13,000 calendar years before present (yr BP), based on LADs from dating surveys of macrofossils (bones and teeth). Advantages of using macrofossils to estimate when a species became extinct are offset, however, by the improbability of finding and dating the remains of the last-surviving members of populations that were restricted in numbers or confined to refugia. Here we report an alternative approach to detect 'ghost ranges' of dwindling populations, based on recovery of ancient DNA from perennially frozen and securely dated sediments (sedaDNA). In such contexts, sedaDNA can reveal the molecular presence of species that appear absent in the macrofossil record. We show that woolly mammoth and horse persisted in interior Alaska until at least 10,500 yr BP, several thousands of years later than indicated from macrofossil surveys. These results contradict claims that Holocene survival of mammoths in Beringia was restricted to ecologically isolated high-latitude islands. More importantly, our finding that mammoth and horse overlapped with humans for several millennia in the region where people initially entered the Americas challenges theories that megafaunal extinction occurred within centuries of human arrival or were due to an extraterrestrial impact in the late Pleistocene.

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.

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.

Out of America: ancient DNA evidence for a new world origin of late quaternary woolly mammoths.

Although the iconic mammoth of the Late Pleistocene, the woolly mammoth (Mammuthus primigenius), has traditionally been regarded as the end point of a single anagenetically evolving lineage, recent paleontological and molecular studies have shown that successive allopatric speciation events must have occurred within Pleistocene Mammuthus in Asia, with subsequent expansion and hybridization between nominal taxa [1, 2]. However, the role of North American mammoth populations in these events has not been adequately explored from an ancient-DNA standpoint. To undertake this task, we analyzed mtDNA from a large data set consisting of mammoth samples from across Holarctica (n = 160) and representing most of radiocarbon time. Our evidence shows that, during the terminal Pleistocene, haplotypes originating in and characteristic of New World populations replaced or succeeded those endemic to Asia and western Beringia. Also, during the Last Glacial Maximum, mammoth populations do not appear to have suffered an overall decline in diversity, despite differing responses on either side of the Bering land bridge. In summary, the "Out-of-America" hypothesis holds that the dispersal of North American woolly mammoths into other parts of Holarctica created major phylogeographic structuring within Mammuthus primigenius populations, shaping the last phase of their evolutionary history before their demise.

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.

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.