Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

The ancestry and affiliations of Kennewick Man.

Kennewick Man, referred to as the Ancient One by Native Americans, is a male human skeleton discovered in Washington state (USA) in 1996 and initially radiocarbon dated to 8,340-9,200 calibrated years before present (BP). His population affinities have been the subject of scientific debate and legal controversy. Based on an initial study of cranial morphology it was asserted that Kennewick Man was neither Native American nor closely related to the claimant Plateau tribes of the Pacific Northwest, who claimed ancestral relationship and requested repatriation under the Native American Graves Protection and Repatriation Act (NAGPRA). The morphological analysis was important to judicial decisions that Kennewick Man was not Native American and that therefore NAGPRA did not apply. Instead of repatriation, additional studies of the remains were permitted. Subsequent craniometric analysis affirmed Kennewick Man to be more closely related to circumpacific groups such as the Ainu and Polynesians than he is to modern Native Americans. In order to resolve Kennewick Man's ancestry and affiliations, we have sequenced his genome to ∼1× coverage and compared it to worldwide genomic data including for the Ainu and Polynesians. We find that Kennewick Man is closer to modern Native Americans than to any other population worldwide. Among the Native American groups for whom genome-wide data are available for comparison, several seem to be descended from a population closely related to that of Kennewick Man, including the Confederated Tribes of the Colville Reservation (Colville), one of the five tribes claiming Kennewick Man. We revisit the cranial analyses and find that, as opposed to genome-wide comparisons, it is not possible on that basis to affiliate Kennewick Man to specific contemporary groups. We therefore conclude based on genetic comparisons that Kennewick Man shows continuity with Native North Americans over at least the last eight millennia.

Reassessing the chronology of the archaeological site of Anzick.

Found in 1968, the archaeological site of Anzick, Montana, contains the only known Clovis burial. Here, the partial remains of a male infant, Anzick-1, were found in association with a Clovis assemblage of over 100 lithic and osseous artifacts-all red-stained with ochre. The incomplete, unstained cranium of an unassociated, geologically younger individual, Anzick-2, was also recovered. Previous chronometric work has shown an age difference between Anzick-1 and the Clovis assemblage (represented by dates from two antler rod samples). This discrepancy has led to much speculation, with some discounting Anzick-1 as Clovis. To resolve this issue, we present the results of a comprehensive radiocarbon dating program that utilized different pretreatment methods on osseous material from the site. Through this comparative approach, we obtained a robust chronometric dataset that suggests that Anzick-1 is temporally coeval with the dated antler rods. This implies that the individual is indeed temporally associated with the Clovis assemblage.

Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans.

The origins of the First Americans remain contentious. Although Native Americans seem to be genetically most closely related to east Asians, there is no consensus with regard to which specific Old World populations they are closest to. Here we sequence the draft genome of an approximately 24,000-year-old individual (MA-1), from Mal'ta in south-central Siberia, to an average depth of 1×. To our knowledge this is the oldest anatomically modern human genome reported to date. The MA-1 mitochondrial genome belongs to haplogroup U, which has also been found at high frequency among Upper Palaeolithic and Mesolithic European hunter-gatherers, and the Y chromosome of MA-1 is basal to modern-day western Eurasians and near the root of most Native American lineages. Similarly, we find autosomal evidence that MA-1 is basal to modern-day western Eurasians and genetically closely related to modern-day Native Americans, with no close affinity to east Asians. This suggests that populations related to contemporary western Eurasians had a more north-easterly distribution 24,000 years ago than commonly thought. Furthermore, we estimate that 14 to 38% of Native American ancestry may originate through gene flow from this ancient population. This is likely to have occurred after the divergence of Native American ancestors from east Asian ancestors, but before the diversification of Native American populations in the New World. Gene flow from the MA-1 lineage into Native American ancestors could explain why several crania from the First Americans have been reported as bearing morphological characteristics that do not resemble those of east Asians. Sequencing of another south-central Siberian, Afontova Gora-2 dating to approximately 17,000 years ago, revealed similar autosomal genetic signatures as MA-1, suggesting that the region was continuously occupied by humans throughout the Last Glacial Maximum. Our findings reveal that western Eurasian genetic signatures in modern-day Native Americans derive not only from post-Columbian admixture, as commonly thought, but also from a mixed ancestry of the First Americans.

The genome of a Late Pleistocene human from a Clovis burial site in western Montana.

Clovis, with its distinctive biface, blade and osseous technologies, is the oldest widespread archaeological complex defined in North America, dating from 11,100 to 10,700 (14)C years before present (bp) (13,000 to 12,600 calendar years bp). Nearly 50 years of archaeological research point to the Clovis complex as having developed south of the North American ice sheets from an ancestral technology. However, both the origins and the genetic legacy of the people who manufactured Clovis tools remain under debate. It is generally believed that these people ultimately derived from Asia and were directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western Montana. The human bones date to 10,705 ± 35 (14)C years bp (approximately 12,707-12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal'ta population into Native American ancestors is also shared by the Anzick-1 individual and thus happened before 12,600 years bp. We also show that the Anzick-1 individual is more closely related to all indigenous American populations than to any other group. Our data are compatible with the hypothesis that Anzick-1 belonged to a population directly ancestral to many contemporary Native Americans. Finally, we find evidence of a deep divergence in Native American populations that predates the Anzick-1 individual.

Late Pleistocene horse and camel hunting at the southern margin of the ice-free corridor: reassessing the age of Wally's Beach, Canada.

The only certain evidence for prehistoric human hunting of horse and camel in North America occurs at the Wally's Beach site, Canada. Here, the butchered remains of seven horses and one camel are associated with 29 nondiagnostic lithic artifacts. Twenty-seven new radiocarbon ages on the bones of these animals revise the age of these kill and butchering localities to 13,300 calibrated y B.P. The tight chronological clustering of the eight kill localities at Wally's Beach indicates these animals were killed over a short period. Human hunting of horse and camel in Canada, coupled with mammoth, mastodon, sloth, and gomphothere hunting documented at other sites from 14,800-12,700 calibrated y B.P., show that 6 of the 36 genera of megafauna that went extinct by approximately 12,700 calibrated y B.P. were hunted by humans. This study shows the importance of accurate geochronology, without which significant discoveries will go unrecognized and the empirical data used to build models explaining the peopling of the Americas and Pleistocene extinctions will be in error.

Genome-wide ancestry of 17th-century enslaved Africans from the Caribbean.

Between 1500 and 1850, more than 12 million enslaved Africans were transported to the New World. The vast majority were shipped from West and West-Central Africa, but their precise origins are largely unknown. We used genome-wide ancient DNA analyses to investigate the genetic origins of three enslaved Africans whose remains were recovered on the Caribbean island of Saint Martin. We trace their origins to distinct subcontinental source populations within Africa, including Bantu-speaking groups from northern Cameroon and non-Bantu speakers living in present-day Nigeria and Ghana. To our knowledge, these findings provide the first direct evidence for the ethnic origins of enslaved Africans, at a time for which historical records are scarce, and demonstrate that genomic data provide another type of record that can shed new light on long-standing historical questions.

Bayesian chronological analyses consistent with synchronous age of 12,835-12,735 Cal B.P. for Younger Dryas boundary on four continents.

The Younger Dryas impact hypothesis posits that a cosmic impact across much of the Northern Hemisphere deposited the Younger Dryas boundary (YDB) layer, containing peak abundances in a variable assemblage of proxies, including magnetic and glassy impact-related spherules, high-temperature minerals and melt glass, nanodiamonds, carbon spherules, aciniform carbon, platinum, and osmium. Bayesian chronological modeling was applied to 354 dates from 23 stratigraphic sections in 12 countries on four continents to establish a modeled YDB age range for this event of 12,835-12,735 Cal B.P. at 95% probability. This range overlaps that of a peak in extraterrestrial platinum in the Greenland Ice Sheet and of the earliest age of the Younger Dryas climate episode in six proxy records, suggesting a causal connection between the YDB impact event and the Younger Dryas. Two statistical tests indicate that both modeled and unmodeled ages in the 30 records are consistent with synchronous deposition of the YDB layer within the limits of dating uncertainty (∼ 100 y). The widespread distribution of the YDB layer suggests that it may serve as a datum layer.

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.

Millennial-scale isotope records from a wide-ranging predator show evidence of recent human impact to oceanic food webs.

Human exploitation of marine ecosystems is more recent in oceanic than near shore regions, yet our understanding of human impacts on oceanic food webs is comparatively poor. Few records of species that live beyond the continental shelves date back more than 60 y, and the sheer size of oceanic regions makes their food webs difficult to study, even in modern times. Here, we use stable carbon and nitrogen isotopes to study the foraging history of a generalist, oceanic predator, the Hawaiian petrel (Pterodroma sandwichensis), which ranges broadly in the Pacific from the equator to near the Aleutian Islands. Our isotope records from modern and ancient, radiocarbon-dated bones provide evidence of over 3,000 y of dietary stasis followed by a decline of ca. 1.8‰ in δ(15)N over the past 100 y. Fishery-induced trophic decline is the most likely explanation for this sudden shift, which occurs in genetically distinct populations with disparate foraging locations. Our isotope records also show that coincident with the apparent decline in trophic level, foraging segregation among petrel populations decreased markedly. Because variation in the diet of generalist predators can reflect changing availability of their prey, a foraging shift in wide-ranging Hawaiian petrel populations suggests a relatively rapid change in the composition of oceanic food webs in the Northeast Pacific. Understanding and mitigating widespread shifts in prey availability may be a critical step in the conservation of endangered marine predators such as the Hawaiian petrel.

Ancient DNA reveals genetic stability despite demographic decline: 3,000 years of population history in the endemic Hawaiian petrel.

In the Hawaiian Islands, human colonization, which began approximately 1,200 to 800 years ago, marks the beginning of a period in which nearly 75% of the endemic avifauna became extinct and the population size and range of many additional species declined. It remains unclear why some species persisted whereas others did not. The endemic Hawaiian petrel (Pterodroma sandwichensis) has escaped extinction, but colonies on two islands have been extirpated and populations on remaining islands have contracted. We obtained mitochondrial DNA sequences from 100 subfossil bones, 28 museum specimens, and 289 modern samples to investigate patterns of gene flow and temporal changes in the genetic diversity of this endangered species over the last 3,000 years, as Polynesians and then Europeans colonized the Hawaiian Islands. Genetic differentiation was found to be high between both modern and ancient petrel populations. However, gene flow was substantial between the extirpated colonies on Oahu and Molokai and modern birds from the island of Lanai. No significant reductions in genetic diversity occurred over this period, despite fears in the mid-1900s that this species may have been extinct. Simulations show that even a decline to a stable effective population size of 100 individuals would result in the loss of only 5% of the expected heterozygosity. Simulations also show that high levels of genetic diversity may be retained due to the long generation time of this species. Such decoupling between population size and genetic diversity in long-lived species can have important conservation implications. It appears that a pattern of dispersal from declining colonies, in addition to long generation time, may have allowed the Hawaiian petrel to escape a severe genetic bottleneck, and the associated extinction vortex, and persist despite a large population decline after human colonization.

Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins.

We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.

Shock-synthesized hexagonal diamonds in Younger Dryas boundary sediments.

The long-standing controversy regarding the late Pleistocene megafaunal extinctions in North America has been invigorated by a hypothesis implicating a cosmic impact at the Allerød-Younger Dryas boundary or YDB (approximately 12,900 +/- 100 cal BP or 10,900 +/- 100 (14)C years). Abrupt ecosystem disruption caused by this event may have triggered the megafaunal extinctions, along with reductions in other animal populations, including humans. The hypothesis remains controversial due to absence of shocked minerals, tektites, and impact craters. Here, we report the presence of shock-synthesized hexagonal nanodiamonds (lonsdaleite) in YDB sediments dating to approximately 12,950 +/- 50 cal BP at Arlington Canyon, Santa Rosa Island, California. Lonsdaleite is known on Earth only in meteorites and impact craters, and its presence strongly supports a cosmic impact event, further strengthened by its co-occurrence with other nanometer-sized diamond polymorphs (n-diamonds and cubics). These shock-synthesized diamonds are also associated with proxies indicating major biomass burning (charcoal, carbon spherules, and soot). This biomass burning at the Younger Dryas (YD) onset is regional in extent, based on evidence from adjacent Santa Barbara Basin and coeval with broader continent-wide biomass burning. Biomass burning also coincides with abrupt sediment mass wasting and ecological disruption and the last known occurrence of pygmy mammoths (Mammuthus exilis) on the Channel Islands, correlating with broader animal extinctions throughout North America. The only previously known co-occurrence of nanodiamonds, soot, and extinction is the Cretaceous-Tertiary (K/T) impact layer. These data are consistent with abrupt ecosystem change and megafaunal extinction possibly triggered by a cosmic impact over North America at approximately 12,900 +/- 100 cal BP.

The genome and diet of a 35,000-year-old Canis lupus specimen from the Paleolithic painted cave, Chauvet-Pont d'Arc, France.

The Chauvet-Pont-d'Arc Cave (Ardèche, France) contains some of the oldest Paleolithic paintings recorded to date, as well as thousands of bones of the extinct cave bear, and some remains and footprints of other animals. As part of the interdisciplinary research project devoted to this reference cave site, we analyzed a coprolite collected within the deep cave. AMS radiocarbon dating of bone fragments from the coprolite yielded an age of 30,450 ± 550 RC yr. BP (AAR-19656; 36,150-34,000 cal BP), similar to ages assigned to Paleolithic artwork and cave bear remains from the same cave sector. Using high-throughput shotgun DNA sequencing, we demonstrated a high abundance of canid DNA and lesser amounts of DNA from the extinct cave bear. We interpret the sample as feces from a canid that had consumed cave bear tissue. The high amount of canid DNA allowed us to reconstruct a complete canid mitochondrial genome sequence (average coverage: 83×) belonging to a deeply divergent clade of extinct mitochondrial wolf lineages that are most closely related to coeval (~35 ka) Belgian wolves. Analysis of the nuclear genome yielded a similar coverage for the X chromosome (2.4×) and the autosomes (range: 2.3-3.2×), indicating that the Chauvet canid was a female. Comparing the relationship of the nuclear genome of this specimen with that of a variety of canids, we found it more closely related to gray wolves' genomes than to other wild canid or dog genomes, especially wolf genomes from Europe and the Middle East. We conclude that the coprolite is feces from an animal within an extinct wolf lineage. The consumption of cave bear by this wolf likely explains its intrusion into the dark cave sectors and sheds new light on the paleoecology of a major cave site.

Revisiting proboscidean phylogeny and evolution through total evidence and palaeogenetic analyses including Notiomastodon ancient DNA.

The extinct Gomphotheriidae is the only proboscidean family that colonized South America. The phylogenetic position of the endemic taxa has been through several revisions using morphological comparisons. Morphological studies are enhanced by paleogenetic analyses, a powerful tool to resolve phylogenetic relationships; however, ancient DNA (aDNA) preservation decreases in warmer regions. Despite the poor preservation conditions for aDNA in humid, sub-tropical climates, we recovered ∼3,000 bp of mtDNA of Notiomastodon platensis from the Arroyo del Vizcaíno site, Uruguay. Our calibrated phylogeny places Notiomastodon as a sister taxon to Elephantidae, with a divergence time of ∼13.5 Ma. Additionally, a total evidence analysis combining morphological and paleogenetic data shows that the three most diverse clades within Proboscidea diverged during the early Miocene, coinciding with the formation of a land passage between Africa and Eurasia. Our results are a further step toward aDNA analyses on Pleistocene samples from subtropical regions and provide a framework for proboscidean evolution.

The age of Clovis-13,050 to 12,750 cal yr B.P.

Thirty-two radiocarbon ages on bone, charcoal, and carbonized plant remains from 10 Clovis sites range from 11,110 ± 40 to 10,820 ± 10 14C years before the present (yr B.P.). These radiocarbon ages provide a maximum calibrated (cal) age range for Clovis of ~13,050 to ~12,750 cal yr B.P. This radiocarbon record suggests that Clovis first appeared at the end of the Allerød and is one of at least three contemporary archaeological complexes in the Western Hemisphere during the terminal Pleistocene. Stemmed projectile points in western North America are coeval and even older than Clovis, and the Fishtail point complex is well established in the southern cone of South America by ~12,900 cal yr B.P. Clovis disappeared ~12,750 cal yr B.P. at the beginning of the Younger Dryas, coincident with the extinction of the remaining North American megafauna (Proboscideans) and the appearance of multiple North American regional archaeological complexes.

Rapid range shifts and megafaunal extinctions associated with late Pleistocene climate change.

Large-scale changes in global climate at the end of the Pleistocene significantly impacted ecosystems across North America. However, the pace and scale of biotic turnover in response to both the Younger Dryas cold period and subsequent Holocene rapid warming have been challenging to assess because of the scarcity of well dated fossil and pollen records that covers this period. Here we present an ancient DNA record from Hall's Cave, Texas, that documents 100 vertebrate and 45 plant taxa from bulk fossils and sediment. We show that local plant and animal diversity dropped markedly during Younger Dryas cooling, but while plant diversity recovered in the early Holocene, animal diversity did not. Instead, five extant and nine extinct large bodied animals disappeared from the region at the end of the Pleistocene. Our findings suggest that climate change affected the local ecosystem in Texas over the Pleistocene-Holocene boundary, but climate change on its own may not explain the disappearance of the megafauna at the end of the Pleistocene.

Campo Laborde: A Late Pleistocene giant ground sloth kill and butchering site in the Pampas.

The extinction of Pleistocene megafauna and the role played by humans have been subjects of constant debate in American archeology. Previous evidence from the Pampas region of Argentina suggested that this environment might have provided a refugium for the Holocene survival of several megamammals. However, recent excavations and more advanced accelerator mass spectrometry radiocarbon dating at Campo Laborde site in the Argentinian Pampas challenge the Holocene survival of Pleistocene megamammals and provide original and high-quality information documenting direct human impact on the Pleistocene fauna. The new data offer definitive evidence for hunting and butchering of Megatherium americanum (giant ground sloth) at 12,600 cal years BP and dispute previous interpretations that Pleistocene megamammals survived into the Holocene in the Pampas.

Pre-Clovis occupation 14,550 years ago at the Page-Ladson site, Florida, and the peopling of the Americas.

Stone tools and mastodon bones occur in an undisturbed geological context at the Page-Ladson site, Florida. Seventy-one radiocarbon ages show that ~14,550 calendar years ago (cal yr B.P.), people butchered or scavenged a mastodon next to a pond in a bedrock sinkhole within the Aucilla River. This occupation surface was buried by ~4 m of sediment during the late Pleistocene marine transgression, which also left the site submerged. Sporormiella and other proxy evidence from the sediments indicate that hunter-gatherers along the Gulf Coastal Plain coexisted with and utilized megafauna for ~2000 years before these animals became extinct at ~12,600 cal yr B.P. Page-Ladson expands our understanding of the earliest colonizers of the Americas and human-megafauna interaction before extinction.