Late Quaternary dynamics of Arctic biota from ancient environmental genomics.

During the last glacial-interglacial cycle, Arctic biotas experienced substantial climatic changes, yet the nature, extent and rate of their responses are not fully understood1-8. Here we report a large-scale environmental DNA metagenomic study of ancient plant and mammal communities, analysing 535 permafrost and lake sediment samples from across the Arctic spanning the past 50,000 years. Furthermore, we present 1,541 contemporary plant genome assemblies that were generated as reference sequences. Our study provides several insights into the long-term dynamics of the Arctic biota at the circumpolar and regional scales. Our key findings include: (1) a relatively homogeneous steppe-tundra flora dominated the Arctic during the Last Glacial Maximum, followed by regional divergence of vegetation during the Holocene epoch; (2) certain grazing animals consistently co-occurred in space and time; (3) humans appear to have been a minor factor in driving animal distributions; (4) higher effective precipitation, as well as an increase in the proportion of wetland plants, show negative effects on animal diversity; (5) the persistence of the steppe-tundra vegetation in northern Siberia enabled the late survival of several now-extinct megafauna species, including the woolly mammoth until 3.9 ± 0.2 thousand years ago (ka) and the woolly rhinoceros until 9.8 ± 0.2 ka; and (6) phylogenetic analysis of mammoth environmental DNA reveals a previously unsampled mitochondrial lineage. Our findings highlight the power of ancient environmental metagenomics analyses to advance understanding of population histories and long-term ecological dynamics.

Late Pleistocene shrub expansion preceded megafauna turnover and extinctions in eastern Beringia.

The collapse of the steppe-tundra biome (mammoth steppe) at the end of the Pleistocene is used as an important example of top-down ecosystem cascades, where human hunting of keystone species led to profound changes in vegetation across high latitudes in the Northern Hemisphere. Alternatively, it is argued that this biome transformation occurred through a bottom-up process, where climate-driven expansion of shrub tundra (Betula, Salix spp.) replaced the steppe-tundra vegetation that grazing megafauna taxa relied on. In eastern Beringia, these differing hypotheses remain largely untested, in part because the precise timing and spatial pattern of Late Pleistocene shrub expansion remains poorly resolved. This uncertainty is caused by chronological ambiguity in many lake sediment records, which typically rely on radiocarbon (14C) dates from bulk sediment or aquatic macrofossils-materials that are known to overestimate the age of sediment layers. Here, we reexamine Late Pleistocene pollen records for which 14C dating of terrestrial macrofossils is available and augment these data with 14C dates from arctic ground-squirrel middens and plant macrofossils. Comparing these paleovegetation data with a database of published 14C dates from megafauna remains, we find the postglacial expansion of shrub tundra preceded the regional extinctions of horse (Equus spp.) and mammoth (Mammuthus primigenius) and began during a period when the frequency of 14C dates indicates large grazers were abundant. These results are not consistent with a model of top-down ecosystem cascades and support the hypothesis that climate-driven habitat loss preceded and contributed to turnover in mammal communities.

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.

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.

Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse.

The rich fossil record of equids has made them a model for evolutionary processes. Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560-780 thousand years before present (kyr BP). Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr BP), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski's horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0-4.5 million years before present (Myr BP), twice the conventionally accepted time to the most recent common ancestor of the genus Equus. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski's and domestic horse populations diverged 38-72 kyr BP, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski's horse investigated. This supports the contention that Przewalski's horses represent the last surviving wild horse population. We find similar levels of genetic variation among Przewalski's and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski's horse. Such regions could correspond to loci selected early during domestication.

Bison phylogeography constrains dispersal and viability of the Ice Free Corridor in western Canada.

The Ice Free Corridor has been invoked as a route for Pleistocene human and animal dispersals between eastern Beringia and more southerly areas of North America. Despite the significance of the corridor, there are limited data for when and how this corridor was used. Hypothetical uses of the corridor include: the first expansion of humans from Beringia into the Americas, northward postglacial expansions of fluted point technologies into Beringia, and continued use of the corridor as a contact route between the north and south. Here, we use radiocarbon dates and ancient mitochondrial DNA from late Pleistocene bison fossils to determine the chronology for when the corridor was open and viable for biotic dispersals. The corridor was closed after ∼23,000 until 13,400 calendar years ago (cal y BP), after which we find the first evidence, to our knowledge, that bison used this route to disperse from the south, and by 13,000 y from the north. Our chronology supports a habitable and traversable corridor by at least 13,000 cal y BP, just before the first appearance of Clovis technology in interior North America, and indicates that the corridor would not have been available for significantly earlier southward human dispersal. Following the opening of the corridor, multiple dispersals of human groups between Beringia and interior North America may have continued throughout the latest Pleistocene and early Holocene. Our results highlight the utility of phylogeographic analyses to test hypotheses about paleoecological history and the viability of dispersal routes over time.

Timing and causes of mid-Holocene mammoth extinction on St. Paul Island, Alaska.

Relict woolly mammoth (Mammuthus primigenius) populations survived on several small Beringian islands for thousands of years after mainland populations went extinct. Here we present multiproxy paleoenvironmental records to investigate the timing, causes, and consequences of mammoth disappearance from St. Paul Island, Alaska. Five independent indicators of extinction show that mammoths survived on St. Paul until 5,600 ± 100 y ago. Vegetation composition remained stable during the extinction window, and there is no evidence of human presence on the island before 1787 CE, suggesting that these factors were not extinction drivers. Instead, the extinction coincided with declining freshwater resources and drier climates between 7,850 and 5,600 y ago, as inferred from sedimentary magnetic susceptibility, oxygen isotopes, and diatom and cladoceran assemblages in a sediment core from a freshwater lake on the island, and stable nitrogen isotopes from mammoth remains. Contrary to other extinction models for the St. Paul mammoth population, this evidence indicates that this mammoth population died out because of the synergistic effects of shrinking island area and freshwater scarcity caused by rising sea levels and regional climate change. Degradation of water quality by intensified mammoth activity around the lake likely exacerbated the situation. The St. Paul mammoth demise is now one of the best-dated prehistoric extinctions, highlighting freshwater limitation as an overlooked extinction driver and underscoring the vulnerability of small island populations to environmental change, even in the absence of human influence.

Testate amoeba records indicate regional 20th-century lowering of water tables in ombrotrophic peatlands in central-northern Alberta, Canada.

Testate amoebae are abundant in the surface layers of northern peatlands. Analysis of their fossilized shell (test) assemblages allows for reconstructions of local water-table depths (WTD). We have reconstructed WTD dynamics for five peat cores from peatlands ranging in distance from the Athabasca bituminous sands (ABS) region in western Canada. Amoeba assemblages were combined with plant macrofossil records, acid-insoluble ash (AIA) fluxes and instrumental climate data to identify drivers for environmental change. Two functional traits of testate amoebae, mixotrophy and the tendency to integrate xenogenic mineral matter in test construction, were quantified to infer possible effects of AIA flux on testate amoeba presence. Age-depth models showed the cores each covered at least the last ~315 years, with some spanning the last millennium. Testate amoeba assemblages were likely affected by permafrost development in two of the peatlands, yet the most important shift in assemblages was detected after 1960 CE. This shift represents a significant apparent lowering of water tables in four out of five cores, with a mean drop of ~15 cm. Over the last 50 years, assemblages shifted towards more xerophilous taxa, a trend which was best explained by increasing Sphagnum s. Acutifolia and, to a lesser extent, mean summer temperature. This trend was most evident in the two cores from the sites located farthest away from the ABS region. AIA flux variations did not show a clear effect on mineral-agglutinating taxa, nor on S. s. Acutifolia presence. We therefore suggest the drying trend was forced by the establishment of S. s. Acutifolia, driven by enhanced productivity following regional warming. Such recent apparent drying of peatlands, which may only be reconstructed by appropriate indicators combined with high chronological control, may affect vulnerability to future burning and promote emissions of CO2 .

Peat Bogs Document Decades of Declining Atmospheric Contamination by Trace Metals in the Athabasca Bituminous Sands Region.

Peat cores were collected from five bogs in the vicinity of open pit mines and upgraders of the Athabasca Bituminous Sands, the largest reservoir of bitumen in the world. Frozen cores were sectioned into 1 cm slices, and trace metals determined in the ultraclean SWAMP lab using ICP-QMS. The uppermost sections of the cores were age-dated with 210Pb using ultralow background gamma spectrometry, and selected plant macrofossils dated using 14C. At each site, trace metal concentrations as well as enrichment factors (calculated relative to the corresponding element/Th ratio of the Upper Continental Crust) reveal maximum values 10 to 40 cm below the surface which shows that the zenith of atmospheric contamination occurred in the past. The age-depth relationships show that atmospheric contamination by trace metals (Ag, Cd, Sb, Tl, but also V, Ni, and Mo which are enriched in bitumen) has been declining in northern Alberta for decades. In fact, the greatest contemporary enrichments of Ag, Cd, Sb, and Tl (in the top layers of the peat cores) are found at the control site (Utikuma) which is 264 km SW, suggesting that long-range atmospheric transport from other sources must be duly considered in any source assessment.

Antibiotic resistance is ancient.

The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to ß-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.

Species-specific responses of Late Quaternary megafauna to climate and humans.

Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary period remain contentious. Here we use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, emphasizing the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.

Airborne Petcoke Dust is a Major Source of Polycyclic Aromatic Hydrocarbons in the Athabasca Oil Sands Region.

Oil sands mining has been linked to increasing atmospheric deposition of polycyclic aromatic hydrocarbons (PAHs) in the Athabasca oil sands region (AOSR), but known sources cannot explain the quantity of PAHs in environmental samples. PAHs were measured in living Sphagnum moss (24 sites, n = 68), in sectioned peat cores (4 sites, n = 161), and snow (7 sites, n = 19) from ombrotrophic bogs in the AOSR. Prospective source samples were also analyzed, including petroleum coke (petcoke, from both delayed and fluid coking), fine tailings, oil sands ore, and naturally exposed bitumen. Average PAH concentrations in near-field moss (199 ng/g, n = 11) were significantly higher (p = 0.035) than in far-field moss (118 ng/g, n = 13), and increasing temporal trends were detected in three peat cores collected closest to industrial activity. A chemical mass-balance model estimated that delayed petcoke was the major source of PAHs to living moss, and among three peat core the contribution to PAHs from delayed petcoke increased over time, accounting for 45-95% of PAHs in contemporary layers. Petcoke was also estimated to be a major source of vanadium, nickel, and molybdenum. Scanning electron microscopy with energy-dispersive X-ray spectroscopy confirmed large petcoke particles (>10 µm) in snow at near-field sites. Petcoke dust has not previously been considered in environmental impact assessments of oil sands upgrading, and improved dust control from growing stockpiles may mitigate future risks.

True single-molecule DNA sequencing of a pleistocene horse bone.

Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates, indicating the presence of 3'-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches.

Improving the performance of true single molecule sequencing for ancient DNA.

BACKGROUND: Second-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase. RESULTS: We found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted. CONCLUSIONS: We propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted.

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.