Genetic insights into the social organization of Neanderthals.

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave.

Denisova Cave, a Pleistocene site in the Altai Mountains of Russian Siberia, has yielded significant fossil and lithic evidence for the Pleistocene in Northern Asia. Abundant animal and human bones have been discovered at the site, however, these tend to be highly fragmented, necessitating new approaches to identifying important hominin and faunal fossils. Here we report the results for 8253 bone fragments using ZooMS. Through the integration of this new ZooMS-based data with the previously published macroscopically-identified fauna we aim to create a holistic picture of the zooarchaeological record of the site. We identify trends associated with climate variability throughout the Middle and Upper Pleistocene as well as patterns explaining the process of bone fragmentation. Where morphological analysis of bones from the site have identified a high proportion of carnivore bones (30.2%), we find that these account for only 7.6% of the ZooMS assemblage, with large mammals between 3 and 5 more abundant overall. Our analysis suggests a cyclical pattern in fragmentation of bones which sees initial fragmentation by hominins using percussive tools and secondary carnivore action, such as gnawing and digestion, likely furthering the initial human-induced fragmentation.

Dating the last Middle Palaeolithic of the Crimean Peninsula: New hydroxyproline AMS dates from the site of Kabazi II.

Radiocarbon dating of bone and charcoal from sites dating to the Middle and Upper Paleolithic is challenging due to low residual levels of radiocarbon. This means that small amounts of contaminating carbon can wield a great influence over accuracy unless they are fully removed. The site of Kabazi II in the Crimea is important because radiocarbon dates previously obtained from bones in archaeological horizons that date to the Western Crimean Mousterian (WCM) are surprisingly young. We redated the same samples using a single compound dating method that focuses on extracting and dating the amino acid hydroxyproline. We show that single amino acid dates produce significantly older determinations than those that use bulk collagen pretreatment procedures. Our results suggest that instead of dating to 35,000-40,000 cal BP, the bones actually date to >50,000 cal BP. This implies that the WCM at this site is much older than previously thought. In light of these current findings, we considered the dates of other key Crimean sites and concluded that in the absence of reliable pretreatment methods, it would be wise to consider many of them minimum ages. We conclude that there is little robust evidence to suggest Neanderthals were present in the Crimea after 40,000 cal BP.

Compound-specific radiocarbon dating and mitochondrial DNA analysis of the Pleistocene hominin from Salkhit Mongolia.

A skullcap found in the Salkhit Valley in northeast Mongolia is, to our knowledge, the only Pleistocene hominin fossil found in the country. It was initially described as an individual with possible archaic affinities, but its ancestry has been debated since the discovery. Here, we determine the age of the Salkhit skull by compound-specific radiocarbon dating of hydroxyproline to 34,950-33,900 Cal. BP (at 95% probability), placing the Salkhit individual in the Early Upper Paleolithic period. We reconstruct the complete mitochondrial genome (mtDNA) of the specimen. It falls within a group of modern human mtDNAs (haplogroup N) that is widespread in Eurasia today. The results now place the specimen into its proper chronometric and biological context and allow us to begin integrating it with other evidence for the human occupation of this region during the Paleolithic, as well as wider Pleistocene sequences across Eurasia.

Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave.

Denisova Cave in the Siberian Altai (Russia) is a key site for understanding the complex relationships between hominin groups that inhabited Eurasia in the Middle and Late Pleistocene epoch. DNA sequenced from human remains found at this site has revealed the presence of a hitherto unknown hominin group, the Denisovans1,2, and high-coverage genomes from both Neanderthal and Denisovan fossils provide evidence for admixture between these two populations3. Determining the age of these fossils is important if we are to understand the nature of hominin interaction, and aspects of their cultural and subsistence adaptations. Here we present 50 radiocarbon determinations from the late Middle and Upper Palaeolithic layers of the site. We also report three direct dates for hominin fragments and obtain a mitochondrial DNA sequence for one of them. We apply a Bayesian age modelling approach that combines chronometric (radiocarbon, uranium series and optical ages), stratigraphic and genetic data to calculate probabilistically the age of the human fossils at the site. Our modelled estimate for the age of the oldest Denisovan fossil suggests that this group was present at the site as early as 195,000 years ago (at 95.4% probability). All Neanderthal fossils-as well as Denisova 11, the daughter of a Neanderthal and a Denisovan4-date to between 80,000 and 140,000 years ago. The youngest Denisovan dates to 52,000-76,000 years ago. Direct radiocarbon dating of Upper Palaeolithic tooth pendants and bone points yielded the earliest evidence for the production of these artefacts in northern Eurasia, between 43,000 and 49,000 calibrated years before present (taken as AD 1950). On the basis of current archaeological evidence, it may be assumed that these artefacts are associated with the Denisovan population. It is not currently possible to determine whether anatomically modern humans were involved in their production, as modern-human fossil and genetic evidence of such antiquity has not yet been identified in the Altai region.

Evolution and extinction of the giant rhinoceros Elasmotherium sibiricum sheds light on late Quaternary megafaunal extinctions.

Understanding extinction events requires an unbiased record of the chronology and ecology of victims and survivors. The rhinoceros Elasmotherium sibiricum, known as the 'Siberian unicorn', was believed to have gone extinct around 200,000 years ago-well before the late Quaternary megafaunal extinction event. However, no absolute dating, genetic analysis or quantitative ecological assessment of this species has been undertaken. Here, we show, by accelerator mass spectrometry radiocarbon dating of 23 individuals, including cross-validation by compound-specific analysis, that E. sibiricum survived in Eastern Europe and Central Asia until at least 39,000 years ago, corroborating a wave of megafaunal turnover before the Last Glacial Maximum in Eurasia, in addition to the better-known late-glacial event. Stable isotope data indicate a dry steppe niche for E. sibiricum and, together with morphology, a highly specialized diet that probably contributed to its extinction. We further demonstrate, with DNA sequencing data, a very deep phylogenetic split between the subfamilies Elasmotheriinae and Rhinocerotinae that includes all the living rhinoceroses, settling a debate based on fossil evidence and confirming that the two lineages had diverged by the Eocene. As the last surviving member of the Elasmotheriinae, the demise of the 'Siberian unicorn' marked the extinction of this subfamily.

Early human dispersals within the Americas.

Studies of the peopling of the Americas have focused on the timing and number of initial migrations. Less attention has been paid to the subsequent spread of people within the Americas. We sequenced 15 ancient human genomes spanning from Alaska to Patagonia; six are ≥10,000 years old (up to ~18× coverage). All are most closely related to Native Americans, including those from an Ancient Beringian individual and two morphologically distinct "Paleoamericans." We found evidence of rapid dispersal and early diversification that included previously unknown groups as people moved south. This resulted in multiple independent, geographically uneven migrations, including one that provides clues of a Late Pleistocene Australasian genetic signal, as well as a later Mesoamerican-related expansion. These led to complex and dynamic population histories from North to South America.

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.

New protocol for compound-specific radiocarbon analysis of archaeological bones.

RATIONALE: For radiocarbon results to be accurate, samples must be free of contaminating carbon. Sample pre-treatment using a high-performance liquid chromatography (HPLC) approach has been developed at the Oxford Radiocarbon Accelerator Unit (ORAU) as an alternative to conventional methods for dating heavily contaminated bones. This approach isolates hydroxyproline from bone collagen, enabling a purified bone-specific fraction to then be radiocarbon dated by accelerator mass spectrometry (AMS). METHODS: Using semi-preparative chromatography and non-carbon-based eluents, this technique enables the separation of underivatised amino acids liberated by hydrolysis of extracted bone collagen. A particular focus has been the isolation of hydroxyproline for single-compound AMS dating since this amino acid is one of the main contributors to the total amount of carbon in mammalian collagen. Our previous approach, involving a carbon-free aqueous mobile phase, required a two-step separation using two different chromatographic columns. RESULTS: This paper reports significant improvements that have been recently made to the method to enable faster semi-preparative separation of hydroxyproline from bone collagen, making the method more suitable for routine radiocarbon dating of contaminated and/or poorly preserved bone samples by AMS. All steps of the procedure, from the collagen extraction to the correction of the AMS data, are described. CONCLUSIONS: The modifications to the hardware and to the method itself have reduced significantly the time required for the preparation of each sample. This makes it easier for other radiocarbon facilities to implement and use this approach as a routine method for preparing contaminated bone samples.

Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition.

Previous dating of the Vi-207 and Vi-208 Neanderthal remains from Vindija Cave (Croatia) led to the suggestion that Neanderthals survived there as recently as 28,000-29,000 B.P. Subsequent dating yielded older dates, interpreted as ages of at least ∼32,500 B.P. We have redated these same specimens using an approach based on the extraction of the amino acid hydroxyproline, using preparative high-performance liquid chromatography (Prep-HPLC). This method is more efficient in eliminating modern contamination in the bone collagen. The revised dates are older than 40,000 B.P., suggesting the Vindija Neanderthals did not live more recently than others across Europe, and probably predate the arrival of anatomically modern humans in Eastern Europe. We applied zooarchaeology by mass spectrometry (ZooMS) to find additional hominin remains. We identified one bone that is Neanderthal, based on its mitochondrial DNA, and dated it directly to 46,200 ± 1,500 B.P. We also attempted to date six early Upper Paleolithic bone points from stratigraphic units G1, Fd/d+G1 and Fd/d, Fd. One bone artifact gave a date of 29,500 ± 400 B.P., while the remainder yielded no collagen. We additionally dated animal bone samples from units G1 and G1-G3 These dates suggest a co-occurrence of early Upper Paleolithic osseous artifacts, particularly split-based points, alongside the remains of Neanderthals is a result of postdepositional mixing, rather than an association between the two groups, although more work is required to show this definitively.

Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling.

The Middle to Upper Paleolithic transition is often linked with a bio-cultural shift involving the dispersal of modern humans outside of Africa, the concomitant replacement of Neanderthals across Eurasia, and the emergence of new technological traditions. The Zagros Mountains region assumes importance in discussions concerning this period as its geographic location is central to all pertinent hominin migration areas, pointing to both east and west. As such, establishing a reliable chronology in the Zagros Mountains is crucial to our understanding of these biological and cultural developments. Political circumstance, coupled with the poor preservation of organic material, has meant that a clear chronological definition of the Middle to Upper Paleolithic transition for the Zagros Mountains region has not yet been achieved. To improve this situation, we have obtained new archaeological samples for AMS radiocarbon dating from three sites: Kobeh Cave, Kaldar Cave, and Ghar-e Boof (Iran). In addition, we have statistically modelled previously published radiocarbon determinations for Yafteh Cave (Iran) and Shanidar Cave (Iraqi Kurdistan), to improve their chronological resolution and enable us to compare the results with the new dataset. Bayesian modelling results suggest that the onset of the Upper Paleolithic in the Zagros Mountains dates to 45,000-40,250 cal BP (68.2% probability). Further chronometric data are required to improve the precision of this age range.

Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis.

DNA sequencing has revolutionised our understanding of archaic humans during the Middle and Upper Palaeolithic. Unfortunately, while many Palaeolithic sites contain large numbers of bones, the majority of these lack the diagnostic features necessary for traditional morphological identification. As a result the recovery of Pleistocene-age human remains is extremely rare. To circumvent this problem we have applied a method of collagen fingerprinting to more than 2000 fragmented bones from the site of Denisova Cave, Russia, in order to facilitate the discovery of human remains. As a result of our analysis a single hominin bone (Denisova 11) was identified, supported through in-depth peptide sequencing analysis, and found to carry mitochondrial DNA of the Neandertal type. Subsequent radiocarbon dating revealed the bone to be >50,000 years old. Here we demonstrate the huge potential collagen fingerprinting has for identifying hominin remains in highly fragmentary archaeological assemblages, improving the resources available for wider studies into human evolution.

Sonoelectrochemical degradation of phenol in aqueous solutions.

The sonoelectrochemical degradation of phenol in aqueous solutions with stainless steel electrodes and high-frequency ultrasound (850kHz) was investigated. A 60% synergetic effect was obtained in the combined reaction system. High concentration of electrolyte (sodium sulfate) and a high electrical voltage are favorable conditions for the degradation of phenol. A nearly complete degradation of phenol was achieved with 4.26g/L Na(2)SO(4) and 30V electrical voltages at 25°C in 1h. The degradation of phenol follows pseudo-first order kinetics. Considering costs and application, the energy efficiency of the reaction system with different reaction conditions was evaluated.

Quantification of folate in fruits and vegetables: A fluorescence-based homogeneous assay.

A high-throughput, homogeneous, fluorescence polarization, and fluorescence intensity assay has been developed for the measurement of folate in fruits and vegetables. This assay is based on the competitive displacement of the fluorescent folate ligands Alexa Fluor (Alexa) 594-folate and Alexa 660-folate from bovine milk folate-binding protein by folates in fruit and vegetable extracts. These fluorescent ligands are employed because their excitation and emission maxima are in regions of the spectrum with minimal autofluorescence in many extracts. Folate-binding protein and Alexa-folate were typically used at concentrations of 0.5 microg/ml and 5nM, respectively, in 20-microl volumes in 384-well microplates. The assay is complete within 100 min. The folate estimate is unaffected by the heterogeneity of polyglutamyl residues that complicates the liquid chromatography-mass spectrometry (LC-MS)-based methods of quantification. In this assay, folic acid had an apparent affinity 2.5-fold greater than 5-methyltetrahydrofolate (5MTHF); therefore, it cannot be used to quantify folate when both natural and synthetic folate are present. 5MTHF-equivalent values were measured in broccoli (240 microg/100g), strawberry (113 microg/100g), white grape (32 microg/100g), orange (44 microg/100g), tomato (12 microg/100g), raspberry (31 microg/100g), banana (29 microg/g), and kiwifruit (36 microg/100g). These data are similar to published values. However, the assay will not detect 5-formyltetrahydrofolate which is a significant constituent of the total folate in lettuce, spinach, carrot, and peppers.

Characterization and quantification of anthocyanins in red kiwifruit ( Actinidia spp.).

Red-fleshed fruit occur in a small number of distantly related taxa in different sections of the genus Actinidia (kiwifruit). We describe and identify the anthocyanin profile of fruit of several Actinidia species. Differences in the relative amounts of cyanidin- and delphinidin-based anthocyanins determine whether the fruit appear red or purple. Cyanidin derivatives have been found in all Actinidia species that contain anthocyanins, whereas delphinidin derivatives are limited to two taxa: A. melanandra and A. arguta var. purpurea . The fruit of these not only contain a wider range of anthocyanins, but they also have greater concentrations. Anthocyanins of most Actinidia species are usually conjugated with either xylosyl-galactose or galactose, whereas A. deliciosa anthocyanins are conjugated with glucose and galactose.

Isolation and structural identification of the anthocyanin components of red kiwifruit.

The anthocyanins responsible for the red color of red kiwifruit were extracted in acidified ethanol and isolated by solid phase extraction (SPE) followed by preparative HPLC. Five anthocyanins were obtained and subsequently identified as delphinidin 3-[2-(xylosyl)galactoside], delphinidin 3-galactoside, cyanidin 3-[2-(xylosyl)galactoside], cyanidin 3-galactoside, and cyanidin 3-glucoside by a combination of LC-MS/MS, GC-MS, and 2D NMR. Delphinidin 3-[2-(xylosyl)galactoside] and delphinidin 3-galactoside have not previously been reported in the genus Actinidia.