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Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains1,3-6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information. E.C. and F.W. are supported by VILLUM FONDEN (17649) and by the European Commission through a Marie Skłodowska-Curie (MSCA) Individual Fellowship (795569). T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), NIHM grant U01 MH106874, Howard Hughes International Early Career grant, Obra Social ‘La Caixa’ and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C., J.C., J.V.O., D.S. and P.G. are supported by the Marie Skłodowska-Curie European Training Network (ETN) TEMPERA, a project funded by the European Union’s EU Framework Program for Research and Innovation Horizon 2020 under grant agreement no. 722606. M.J.C. and M.M. are supported by the Danish National Research Foundation award PROTEIOS (DNRF128). Work at the Novo Nordisk Foundation Center for Protein Research is funded in part by a donation from the Novo Nordisk Foundation (NNF14CC0001). Research at Chuifeng Cave is made possible by support from the National Natural Science Foundation of China (41572023) and by a grant from the Bagui Scholar of Guangxi. M.K. was supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship (KU 3467/1-1) and the Postdoctoral Junior Leader Fellowship Programme from ‘la Caixa’ Banking Foundation (LCF/BQ/PR19/11700002). M.E.A. is supported by the Independent Research Fund Denmark (7027-00147B). We thank E. Willerslev for critical reading of the manuscript, scientific support and guidance.

Ancient protein analysis is a rapidly developing field of research. Proteins ranging in age from the Quaternary to Jurassic are being used to answer questions about phylogeny, evolution, and extinction. However, these analyses are sometimes contentious, and focus primarily on large vertebrates in sedimentary fossilisation environments; there are few studies of protein preservation in fossils in amber. Here we show exceptionally slow racemisation rates during thermal degradation experiments of resin enclosed feathers, relative to previous thermal degradation experiments of ostrich eggshell, coral skeleton, and limpet shell. We also recover amino acids from two specimens of fossil feathers in amber. The amino acid compositions are broadly similar to those of degraded feathers, but concentrations are very low, suggesting that much of the original protein has been degraded and lost. High levels of racemisation in more apolar, slowly racemising amino acids suggest that some of the amino acids were ancient and therefore original. Our findings indicate that the unique fossilisation environment inside amber shows potential for the recovery of ancient amino acids and proteins.

We used palaeoproteomics and peptide mass fingerprinting to obtain secure species identifications of key specimens of early domesticated fauna from South Africa, dating to ca. 2000 BP. It can be difficult to distinguish fragmentary remains of early domesticates (sheep) from similar-sized local wild bovids (grey duiker, grey rhebok, springbok—southern Africa lacks wild sheep) based on morphology alone. Our analysis revealed a Zooarchaeology by Mass Spectrometry (ZooMS) marker (m/z 1532) present in wild bovids and we demonstrate through LC–MS/MS that it is capable of discriminating between wild bovids and caprine domesticates. We confirm that the Spoegrivier specimen dated to 2105 ± 65 BP is indeed a sheep. This is the earliest directly dated evidence of domesticated animals in southern Africa. As well as the traditional method of analysing bone fragments, we show the utility of minimally destructive sampling methods such as PVC eraser and polishing films for successful ZooMS identification. We also show that collagen extracted more than 25 years ago for the purpose of radiocarbon dating can yield successful ZooMS identification. Our study demonstrates the importance of developing appropriate regional frameworks of comparison for future research using ZooMS as a method of biomolecular species identification.

The dodo Raphus cucullatus Linnaeus, 1758, an extinct and flightless, giant pigeon endemic to Mauritius, hasfascinated people since its discovery, yet has remained surprisingly poorly known. Until the mid-19th century, almost all thatwas known about the dodo was based on illustrations and written accounts by 17th century mariners, often of questionableaccuracy. Furthermore, only a few fragmentary remains of dodos collected prior to the bird’s extinction exist. Ourunderstanding of the dodo’s anatomy was substantially enhanced by the discovery in 1865 of subfossil bones in a marsh calledthe Mare aux Songes, situated in southeastern Mauritius. However, no contextual information was recorded during earlyexcavation efforts, and the majority of excavated material comprised larger dodo bones, almost all of which wereunassociated. Here we present a modern interdisciplinary analysis of the Mare aux Songes, a 4200-year-old multitaxicvertebrate concentration Lagerst€atte. Our analysis of the deposits at this site provides the first detailed overview of theecosystem inhabited by the dodo. The interplay of climatic and geological conditions led to the exceptional preservation ofthe animal and associated plant remains at the Mare aux Songes and provides a window into the past ecosystem of Mauritius.This interdisciplinary research approach provides an ecological framework for the dodo, complementing insights on itsanatomy derived from the only associated dodo skeletons known, both of which were collected by Etienne Thirioux and arethe primary subject of this memoir.

The domestic dog has inhabited the anthropogenic niche for at least 15 000 years, but despite their impact on human strategies, the lives of dogs and their interactions with humans have only recently become a subject of interest to archaeologists. In the Arctic, dogs rely exclusively on humans for food during the winter, and while stable isotope analyses have revealed dietary similarities at some sites, deciphering the details of provisioning strategies have been challenging. In this study, we apply zooarchaeology by mass spectrometry (ZooMS) and liquid chromatography tandem mass spectrometry to dog palaeofaeces to investigate protein preservation in this highly degradable material and obtain information about the diet of domestic dogs at the Nunalleq site, Alaska. We identify a suite of digestive and metabolic proteins from the host species, demonstrating the utility of this material as a novel and viable substrate for the recovery of gastrointestinal proteomes. The recovered proteins revealed that the Nunalleq dogs consumed a range of Pacific salmon species (coho, chum, chinook and sockeye) and that the consumed tissues derived from muscle and bone tissues as well as roe and guts. Overall, the study demonstrated the viability of permafrost-preserved palaeofaeces as a unique source of host and dietary proteomes. 1. Introduction 2. Material and methods (a) Samples (b) Palaeoproteomics (c) Zooarchaeology by mass spectrometry 3. Results and discussion (a) Metaproteomics (b) Host proteins (c) Dietary proteins (d) Challenges and future directions 4. Conclusion

Unidad de excelencia María de Maeztu CEX2019-000940-M The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

We report a simple histological study on skin biopsies from young domestic sheep following each step in transformation from skin to parchment production. During the recreation of historical parchment manufacture, histological analyses were conducted; before and after lime treatment, hair removal, and stretching. Sections were fixed and stained using a variety of histological stains to identify the presence of different molecular classes and the fibrous proteins, collagen and elastin. The results reveal surprisingly few histological changes in most steps in the production process. However, very visible changes in the supramolecular ordering of skin macromolecules (elastin, collagen) occur during the final stage of parchment production when stretched on the frame. Collagen fibres and hair follicles were all strongly re-oriented in the direction of strain. Surprisingly despite the thinness of the lambskin and the exhaustive treatment in lime, not all fats were saponified and even in the final product Oil Red O stained fat bodies were detectable on the hair side of the skin. We believe this study will help compensate for the lack of sources on microscopic changes in parchment during the recreation of its historical manufacture.

In this paper we describe a dry non-invasive extraction method to detect palaeoproteomic evidence from stained manuscripts. The manuscript analysed in this study is a medieval parchment birth girdle (Wellcome Collection Western MS. 632) made in England and thought to be used by pregnant women while giving birth. Using a dry non-invasive sampling method we were able to extract both human and non-human peptides from the stains, including evidence for the use of honey, cereals, ovicaprine milk and legumes. In addition, a large number of human peptides were detected on the birth roll, many of which are found in cervico-vaginal fluid. This suggests that the birth roll was actively used during childbirth. This study is the first to extract and analyse non-collagenous peptides from a parchment document using a dry non-invasive sampling method and demonstrates the potential of this type of analysis for stained manuscripts, providing direct biomolecular evidence for active use.

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 under standing of protein survival far back in time and under different environmental conditions.

The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA., Birch pitch is thought to have been used in prehistoric times as hafting material or antiseptic and tooth imprints suggest that it was chewed. Here, the authors report a 5,700 year-old piece of chewed birch pitch from Denmark from which they successfully recovered a complete ancient human genome and oral microbiome DNA.

Jetsam ambergris, found on beaches worldwide, has always been assumed to originate as a natural product of sperm whales (Physeteroidea). However, only indirect evidence has ever been produced for this, such as the presence of whale prey remains in ambergris. Here, we extracted and analysed DNA sequences from jetsam ambergris from beaches in New Zealand and Sri Lanka, and sequences from ambergris of a sperm whale beached in The Netherlands. The lipid-rich composition of ambergris facilitated high preservation-quality of endogenous DNA, upon which we performed shotgun Illumina sequencing. Alignment of mitochondrial and nuclear genome sequences with open-access reference data for multiple whale species confirms that all three jetsam samples derived originally from sperm whales ( Physeter macrocephalus ). Shotgun sequencing here also provides implications for metagenomic insights into ambergris-preserved DNA. These results demonstrate significant implications for elucidating the origins of jetsam ambergris as a prized natural product, and also for the understanding of sperm whale metabolism and diet, and the ecological mechanisms underlying these coproliths.

Paleoproteomics and the study of ancient proteins has become an important consideration in bioarchaeological research as we seek to understand the relationship between the physical skeleton and its underlying biochemistry. Osteocalcin is an abundant, non-collagenous protein that is accessible archaeologically due to its affinity for hydroxyapatite in bone. Manufactured by the osteoblast cells during normal bone metabolism, osteocalcin is inherently linked to the process of bone remodelling. Clinical studies have consistently shown the impact of biological (i.e., sex and age) and pathological (i.e., disease) factors on osteocalcin concentrations within the body; however, this connection has yet to be fully explored bioarchaeologically. For this study, 46 individuals from the Danish Sct. Albert’s cemetery (CE 1250–1536) were analyzed with cortical bone samples harvested from the femur. Each sample was demineralized and quantified using established paleoproteomic methods (ELISA) and tested for diagenesis using FTIR-ATR. No significant differences were found in the osteocalcin concentrations between adult males and females. However, males had a wider range of osteocalcin variability perhaps explained in part by their increased prevalence of pathological conditions characterized by bone remodelling. Similarly, non-adults had a wide range of osteocalcin variability but there were no significant differences when compared to the adult mean. No correlations were found between osteocalcin concentrations and diagenesis suggesting that the protein can survive across a range of temporal periods and variable burial environments. While no clear pattern emerged between osteocalcin concentrations and biological and/or pathological factors, this study is significant in that it further explores the use of paleoproteomics in bioarchaeological contexts and how we may unite our macroscopic and biochemical assessments of human skeletal remains.

Pacific salmon (Oncorhynchus spp.) are ecological and cultural keystone species along the Northwest Coast of North America and are ubiquitous in archaeological sites of the region. The inability to morphologically identify salmonid post-cranial remains to species, however, can limit our understanding of the ecological and cultural role different taxa played in the seasonal subsistence practices of Indigenous groups in the past. Here, we present a rapid, cost-effective ZooMS method to distinguish salmonid species based on collagen peptide mass-fingerprinting. Using modern reference material and an assemblage of 28 DNA-identified salmonid bones from the pre-contact Yup'ik site of Nunalleq, Western Alaska, we apply high-resolution mass spectrometry (LC-MS/MS) to identify a series of potential collagen peptide markers to distinguish Pacific salmon. We then confirm these peptide markers with a blind ZooMS analysis (MALDI-TOF-MS) of the archaeological remains. We successfully distinguish five species of anadromous salmon with this ZooMS approach, including one specimen that could not be identified through ancient DNA analysis. Our biomolecular identification of chum (43%), sockeye (21%), chinook (18%), coho (11%) and pink (7%), confirm the exploitation of all five available species of salmonid at Nunalleq.

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics.SignificanceMaximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.

Avian eggshell survives well in alkaline and neutral soils, but its potential as an archaeological resource remains largely unexplored, mainly due to difficulties in its identification. Here we exploit the release of novel bird genomes and, for the first time on eggshell, use MALDI-ToF (matrix-assisted laser desorption ionisation-time of flight) mass spectrometry in combination with peptide sequencing by LC-MS/MS. The eggshell proteome is revealed as unexpectedly complex, with 5755 proteins identified for a reference collection comprising 23 bird species. We determined 782 m/z markers useful for eggshell identification, 583 of which could be assigned to known eggshell peptide sequences. These were used to identify eggshell fragments recovered from a medieval site at Freeschool Lane, Leicester. We discuss the specificity of the peptide markers and highlight the importance of assessing the level of taxonomic identification achievable for archaeological interpretation.

There has long been debate over the origins of dairy consumption within European populations. Whilst it was previously assumed that lactase persistence (LP) was under positive selection following the advent of agriculture, recent genetic studies of prehistoric human remains have revealed LP may have only emerged in Europe in the last 4000 years. These findings stand in contrast to organic residue analysis of Neolithic pottery indicating the utilisation of dairy products, and zooarchaeological mortality profiles consistent with dairying herds at Neolithic sites. The recent discovery of the milk protein β-lactoglobulin (BLG) within human dental calculus presents a new method via which to explore dairy product consumption in the archaeological past. Here, we apply shotgun proteomic analysis to dental calculus samples from three British Neolithic sites, revealing the earliest identification of BLG in human dental calculus to date. The presence of BLG peptides in individuals who are unlikely to possess LP provides new insight into dairying in the British Neolithic, suggesting the potential processing of milk by Neolithic populations to reduce the lactose content of dairy products.

Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology.

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

Cattle were domesticated ∼10,000 years ago, but analysis of modern breeds has not elucidated their origins. Verdugo et al. performed genome-wide analysis of 67 ancient Near Eastern Bos taurus DNA samples. Several populations of ancient aurochs were progenitors of domestic cows. These genetic lineages mixed ∼4000 years ago in a region around the Indus Valley. Interestingly, mitochondrial analysis indicated that genetic material likely derived from arid-adapted Bos indicus (zebu) bulls was introduced by introgression.Science, this issue p. 173Genome-wide analysis of 67 ancient Near Eastern cattle, Bos taurus, remains reveals regional variation that has since been obscured by admixture in modern populations. Comparisons of genomes of early domestic cattle to their aurochs progenitors identify diverse origins with separate introgressions of wild stock. A later region-wide Bronze Age shift indicates rapid and widespread introgression of zebu, Bos indicus, from the Indus Valley. This process was likely stimulated at the onset of the current geological age, ~4.2 thousand years ago, by a widespread multicentury drought. In contrast to genome-wide admixture, mitochondrial DNA stasis supports that this introgression was male-driven, suggesting that selection of arid-adapted zebu bulls enhanced herd survival. This human-mediated migration of zebu-derived genetics has continued through millennia, altering tropical herding on each continent.

We present a complete ancient human genome and oral microbiome sequenced from a piece of resinous "chewing gum" recovered from a Stone Age site on the island of Lolland, Denmark, and directly dated to 5,858-5,661 cal. BP (GrM-13305; 5,007+/-11). We sequenced the genome to an average depth-of-coverage of 2.3x and find that the individual who chewed the resin was female and genetically more closely related to western hunter-gatherers from mainland Europe, than hunter-gatherers from central Scandinavia. We use imputed genotypes to predict physical characteristics and find that she had dark skin and hair, and blue eyes. Lastly, we also recovered microbial DNA that is characteristic of an oral microbiome and faunal reads that likely associate with diet. The results highlight the potential for this type of sample material as a new source of ancient human and microbial DNA.

Bacteria play an important role in the degradation of bone material. However, much remains to be learnt about the structure of their communities in degrading bone, and how the depositional environment influences their diversity throughout the exposure period. We genetically profiled the bacterial community in an experimental series of pig bone fragments (femur and humeri) deposited at different well-defined environments in Denmark. The bacterial community in the bone fragments and surrounding depositional environment were studied over one year, and correlated with the bioerosion damage patterns observed microscopically in the bones. We observed that the bacterial communities within the bones were heavily influenced by the local microbial community, and that the general bone microbial diversity increases with time after exposure. We found the presence of several known collagenase producing bacterial groups, and also observed increases in the relative abundance of several of these in bones with tunneling. We anticipate that future analyses using shotgun metagenomics on this and similar datasets will be able to provide insights into mechanisms of microbiome driven bone degradation. © 2020 Eriksen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Following the report of Mycobacterium tuberculosis proteins found in archaeological bone by Boros-Major et al. (2011), we attempted to identify M. tuberculosis proteins in mummified lung tissues from which ancient DNA success had already been reported. Using a filter-aided sample preparation protocol modified for ancient samples we applied shotgun proteomics to seven samples of mummified lung, chest and pleura tissues. However, we only identified four peptides with unique matches to the M. tuberculosis complex, none of which were unique to M. tuberculosis, although we did identify a range of human proteins and non-mycobacterial bacterial proteins. In light of these results, we question the validity of the peptide mass fingerprint (PMF) approach presented by Boros-Major et al. (2011), especially because the PMF spectrum presented in Boros-Major et al. (2011) has similarities to that of human collagen, the dominant protein in the tissue under investigation. We explore the challenges of using proteomic approaches to detect M. tuberculosis, and propose that, given the contentious outcomes that have plagued ancient protein research in the past, the susceptibility of ancient material to modern contamination, and the degradation inherent in archaeological samples, caution is needed in the acquisition, analysis and reporting of proteomic data from such material.

Contamination is a potential problem in the study of ancient proteins, either from prior handling of the sample, laboratory consumables, or cross-sample carryover from mass spectrometers. Recently, deamidation of glutamine has been proposed as a measure for assessing the degradation of ancient proteins. Here, we present deamiDATE 1.0, a method for the authentication of ancient proteins using measure of site-specific deamidation rates. We test this approach on shotgun proteomic data derived from bone collagen from modern, archaeological and extinct taxa. We further demonstrate how this method may be used to differentiate between modern contaminants and authentic ancient proteins using a case study from Neolithic dental calculus.

We present the analysis of an osseous finger ring from a predominantly early Neolithic context in Denmark. To characterize the artefact and identify the raw material used for its manufacture, we performed micro-computed tomography scanning, zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting, as well as protein sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). We conclude that the ring was made from long bone or antler due to the presence of osteons (Haversian canals). Subsequent ZooMS analysis of collagen I and II indicated that it was made fromAlces alcesorCervus elaphusmaterial. We then used LC-MS/MS analysis to refine our species identification, confirming that the ring was made fromCervus elaphus, and to examine the rest of the proteome. This study demonstrates the potential of ancient proteomics for species identification of prehistoric artefacts made from osseous material.

How humans got their goatsLittle is known regarding the location and mode of the early domestication of animals such as goats for husbandry. To investigate the history of the goat, Dalyet al.sequenced mitochondrial and nuclear sequences from ancient specimens ranging from hundreds to thousands of years in age. Multiple wild populations contributed to the origin of modern goats during the Neolithic. Over time, one mitochondrial type spread and became dominant worldwide. However, at the whole-genome level, modern goat populations are a mix of goats from different sources and provide evidence for a multilocus process of domestication in the Near East. Furthermore, the patterns described support the idea of multiple dispersal routes out of the Fertile Crescent region by domesticated animals and their human counterparts.Science, this issue p.85

Technological innovations such as next generation sequencing and DNA hybridisation enrichment have resulted in multi-fold increases in both the quantity of ancient DNA sequence data and the time depth for DNA retrieval. To date, over 30 ancient genomes have been sequenced, moving from 0.7× coverage (mammoth) in 2008 to more than 50× coverage (Neanderthal) in 2014. Studies of rapid evolutionary changes, such as the evolution and spread of pathogens and the genetic responses of hosts, or the genetics of domestication and climatic adaptation, are developing swiftly and the importance of palaeogenomics for investigating evolutionary processes during the last million years is likely to increase considerably. However, these new datasets require new methods of data processing and analysis, as well as conceptual changes in interpreting the results. In this review we highlight important areas of future technical and conceptual progress and discuss research topics in the rapidly growing field of palaeogenomics.

The domestication and transmission of cereals is one of the most fundamental components of early farming, but direct evidence of their use in early culinary practices and economies has remained frustratingly elusive. Using analysis of a well-preserved Early Bronze Age wooden container from Switzerland, we propose novel criteria for the identification of cereal residues. Using gas chromatography mass spectrometry (GC-MS), we identified compounds typically associated with plant products, including a series of phenolic lipids (alkylresorcinols) found only at appreciable concentration in wheat and rye bran. The value of these lipids as cereal grain biomarkers were independently corroborated by the presence of macrobotanical remains embedded in the deposit, and wheat and rye endosperm peptides extracted from residue. These findings demonstrate the utility of a lipid-based biomarker for wheat and rye bran and offer a methodological template for future investigations of wider range of archaeological contexts. Alkylresorcinols provide a new tool for residue analysis which can help explore the spread and exploitation of cereal grains, a fundamental component of the advent and spread of farming.

The persistence of DNA over archaeological and paleontological timescales in diverse environments has led to a revolutionary body of paleogenomic research, yet the dynamics of DNA degradation are still poorly understood. We analyzed 185 paleogenomic datasets and compared DNA survival with environmental variables and sample ages. We find cytosine deamination follows a conventional thermal age model, but we find no correlation between DNA fragmentation and sample age over the timespans analyzed, even when controlling for environmental variables. We propose a model for ancient DNA decay wherein fragmentation rapidly reaches a threshold, then subsequently slows. The observed loss of DNA over time may be due to a bulk diffusion process in many cases, highlighting the importance of tissues and environments creating effectively closed systems for DNA preservation. This model of DNA degradation is largely based on mammal bone samples due to published genomic dataset availability. Continued refinement to the model to reflect diverse biological systems and tissue types will further improve our understanding of ancient DNA breakdown dynamics.

Peptide mass fingerprinting of bone collagen (ZooMS) has previously been proposed as a method to calculate the extent of the non-enzymatic degradation of glutamine into glutamic acid (deamidation). Temporal and spatial variation of glutamine deamidation at a single site, however, has not been investigated. Here we apply ZooMS screening of Châtelperronian and Early Holocene bone specimens from Quinçay, France, to explore temporal and spatial variation in glutamine deamidation. Our results indicate that chronological resolution is low, while spatial variation is high. Nevertheless, our analysis allows the identification of bone specimens that have undergone diagenetic histories remarkably different (either in length or in type) from spatially related bone specimens. Therefore, ZooMS ammonium-bicarbonate screening is capable of testing bone assemblage homogeneity, which could guide subsequent analysis and interpretation.

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 C-14 years before present (BP) (13,000 to 12,600 calendar years BP)(1,2). 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(3). 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(2). An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum(4). 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 C-14 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 14.4x and show that the gene flow from the Siberian Upper Palaeolithic Mal'ta population(5) 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.

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Eggshell is a potentially common archaeological resource, but it tends to be ignored. The recent development of ZooMS (zooarchaeology by mass spectrometry) as a rapid and robust system for taxonomic identification of preserved eggshell fragments has facilitated new insights into patterns of egg use in the past. This paper presents a case study of egg use at two sites in Anglo-Scandinavian York (Hungate and Coppergate). The results described below suggest that the relative prevalence of goose eggshell may become a useful indicator of status, consistent with other characteristics of the two sites, and also demonstrate an apparent lack of exploitation of eggs of wild birds in York during the Anglo-Scandinavian period. These results highlight the interpretative potential of eggshell, which can now begin to be more fully explored. Copyright © 2013 John Wiley & Sons, Ltd.

Woollen textiles recovered from archaeological excavations are usually fragile, discoloured, mineralised, or highly biodeteriorated fragmentary remains. The nature and extent of preservation is highly dependent on the site of burial and factors such as soil composition, pH, temperature, oxygen content, and contact with a wood coffin or metals. Understanding the particular biodegradation in archaeological sites is important for biomolecular studies of textiles, and to assist in the conservation of these finds. Wool fabrics dyed and buried for up to 8 yr in bog-type soils in Denmark (Lejre) and Norway (Rormyra), and in marine sediments in Sweden (Marstrand) were evaluated by proteomics analysis. Wool degradation was found to occur through a range of differing mechanisms, mainly due to the complex nature of wool itself with its many families of proteins (keratin and keratin-associated proteins) and structures. Microbial activity was a large contributory factor to the physical deterioration of the wool fabrics at Lejre and Marstrand, and might result in faster loss of keratin-associated proteins over keratins. Additional hydrolysis took place at Marstrand, influenced by the environmental conditions of the sediment, and in particular the alkaline pH, contributing to the degradation of keratins. However, cross-linking was associated with the long-term preservation of the fabrics at Rormyra, where pH, temperature, and vegetative composition of the bog prevented microbial activity, and sphagnum moss might preserve wool by binding with keratins.

We report the characterisation of meat and bone meal (MBM) standards (Set B-EFPRA) derived from cattle, sheep, pig and chicken, each rendered at four different temperatures (133, 137, 141 and 145 °C). The standards, prepared for an EU programme STRATFEED (to develop new methodologies for the detection and quantification of illegal addition of mammalian tissues in feeding stuffs), have been widely circulated and used to assess a range of methods for identification of the species composition of MBM. The overall state of mineral alteration and protein preservation as a function of temperature was monitored using small angle X-ray diffraction (SAXS), amino acid composition and racemization analyses. Progressive increases in protein damage and mineral alteration in chicken and cattle standards was observed. In the case of sheep and pig, there was greater damage to the proteins and alteration of the minerals at the lowest treatment temperature (133 °C), suggesting that the thermal treatments must have been compromised in some way. This problem has probably impacted upon the numerous studies which tested methods against these heat treatments. We use protein mass spectrometric methods to explore if thermostable proteins could be used to identify rendered MBM. In more thermally altered samples, so-called 'thermostable' proteins such as osteocalcin which has been proposed as a ideal target to speciate MBM were no longer detectable, but the structural protein type I collagen could be used to differentiate all four species, even in the most thermally altered samples.

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C). DOI: http://dx.doi.org/10.7554/eLife.17092.001, eLife digest The pattern of chemical reactions that break down the molecules that make our bodies is still fairly mysterious. Archaeologists and geologists hope that dead organisms (or artefacts made from them) might not decay entirely, leaving behind clues to their lives. We know that some molecules are more resistant than others; for example, fats are tough and survive for a long time while DNA is degraded very rapidly. Proteins, which are made of chains of smaller molecules called amino acids, are usually sturdier than DNA. Since the amino acid sequence of a protein reflects the DNA sequence that encodes it, proteins in fossils can help researchers to reconstruct how extinct organisms are related in cases where DNA cannot be retrieved. Time, temperature, burial environment and the chemistry of the fossil all influence how quickly a protein decays. However, it is not clear what mechanisms slow down decay so that full protein sequences can be preserved and identified after millions of years. As a result, it is difficult to know where to look for these ancient sequences. In the womb of ostriches, several proteins are responsible for assembling the minerals that make up the ostrich eggshell. These proteins become trapped tightly within the mineral crystals themselves. In this situation, proteins can potentially be preserved over geological time. Demarchi et al. have now studied 3.8 million-year-old eggshells found close to the equator and, despite the extent to which the samples have degraded, discovered fully preserved protein sequences. Using a computer simulation method called molecular dynamics, Demarchi et al. calculated that the protein sequences that are able to survive the longest are stabilized by strong binding to the surface of the mineral crystals. The authenticity of these sequences was tested thoroughly using a combination of several approaches that Demarchi et al. recommend using as a standard for ancient protein studies. Overall, it appears that biominerals are an excellent source of ancient protein sequences because mineral binding ensures survival. A systematic survey of fossil biominerals from different environments is now needed to assess whether these biomolecules have the potential to act as barcodes for interpreting the evolution of organisms. DOI: http://dx.doi.org/10.7554/eLife.17092.002

The purported migrations that have formed the peoples of Britain have been the focus of generations of scholarly controversy. However, this has not benefited from direct analyses of ancient genomes. Here we report nine ancient genomes (∼1 ×) of individuals from northern Britain: seven from a Roman era York cemetery, bookended by earlier Iron-Age and later Anglo-Saxon burials. Six of the Roman genomes show affinity with modern British Celtic populations, particularly Welsh, but significantly diverge from populations from Yorkshire and other eastern English samples. They also show similarity with the earlier Iron-Age genome, suggesting population continuity, but differ from the later Anglo-Saxon genome. This pattern concords with profound impact of migrations in the Anglo-Saxon period. Strikingly, one Roman skeleton shows a clear signal of exogenous origin, with affinities pointing towards the Middle East, confirming the cosmopolitan character of the Empire, even at its northernmost fringes., Here, Martiniano et al. examine the genetic structure of northern Britain in the late BC/early AD using ancient genome sequencing of 9 individuals. They uncover evidence of far-reaching Roman and later Anglo-Saxon migrations within a background of Britons similar to the earlier Iron Age.

The growth of proteomics-based methods in archaeology prompted an investigation of the survival of non-collagenous proteins, specifically immunoglobulin G (IgG), in archaeological human bone and dentine. Over a decade ago reports were published on extracted, immunoreactive archaeological IgG, and the variable yields of IgG molecules detected by Western blots of 1D and 2D SDS-PAGE gels. If IgG can indeed be recovered from archaeological skeletal material, it offers remarkable opportunities for exploring the history of disease - for example in applying functional anti-malarial IgGs to study past patterns of malaria. More recently, the field has seen a move away from immunological approaches and towards the use of shotgun proteomics via mass spectrometry. Using previously published techniques, this study attempted to extract and characterize archaeological IgG proteins. In only one extraction method were immunoglobulin derived peptides identified, and these displayed extensive evidence of degradation. The failure to extract immunoglobulins by all but one method, along with observed patterns of protein degradation, suggests that IgG may be an unsuitable target for detecting disease-associated antigens. This research highlights the importance of revisiting previously ‘successful’ biomolecular methodologies using emerging technologies.

Shamanic belief systems represent the first form of religious practice visible within the global archaeological record. Here we report on the earliest known evidence of shamanic costume: modified red deer crania headdresses from the Early Holocene site of Star Carr (c. 11 kya). More than 90% of the examples from prehistoric Europe come from this one site, establishing it as a place of outstanding shamanistic/cosmological significance. Our work, involving a programme of experimental replication, analysis of macroscopic traces, organic residue analysis and 3D image acquisition, metrology and visualisation, represents the first attempt to understand the manufacturing processes used to create these artefacts. The results produced were unexpected-rather than being carefully crafted objects, elements of their production can only be described as expedient.

International audience; One contribution of 16 to a theme issue 'From DNA barcodes to biomes'. Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species' distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets. This article is part of the themed issue 'From DNA barcodes to biomes'.

The last few years have seen an enormous proliferation of ancient biomolecules research, especially in the field of ancient DNA. Ancient DNA studies have been transformed by the advent of next generation sequencing, with the first Pleistocene sample being analysed in 2005, and several complete and draft genomes that have been compiled from ancient DNA to date. At the same time, although less conspicuous, research on ancient proteins has also seen advances, with the time limit for research on ancient biomolecules now extending to over 1 million years. Here we review which effects these developments have on research in Quaternary science. We identify several lines of research that have the potential to profit substantially from these recent developments in ancient biomolecules research. First, the identification of taxa can be made using ancient biomolecules, and in the case of ancient DNA, specimens can even be assigned to specific populations within a species. Second, increasingly large DNA data sets from Pleistocene animals allow the elucidation of ever more precise pictures of the population dynamic processes whereby organisms respond to climate and environmental change. With the accompanying better understanding of process in the Quaternary, past ecologies can also more realistically be interpreted from proxy data sets. The dominant message from this research so far is that the Quaternary saw a great deal more dynamism in populations than had been forecast by conventional palaeoecology. This suggests that reconstructions of past environmental conditions need to be done with caution. Third, ancient DNA can also now be obtained directly from sediments to elucidate the presence of both plant and animal species in an area even in the absence of identifiable fossils, be it macro- or micro-fossils. Finally, the analysis of proteins enables the identification of bone remains to genus and sometimes species level far beyond the survival time of DNA, at least in temperate regions, illustrating that precise data is now forthcoming from seemingly unlikely sources. Together, these approaches allow the study of environmental dynamics throughout a substantial part, and perhaps even the entire Quaternary (the last 2.6 million years).

In this work, two sets of experiments were carried out to assess the potential of soil proteomics for archaeological site interpretation. First, we examined the effects of various protein isolation reagents and soil constituents on peptide mass fingerprinting (PMF) of soil-like materials spiked with bovine serum albumin (BSA). In a subsequent case study, we assessed the relative age of soils from an ancient clay floor of a Roman farmhouse using amino acid racemization and then applied MALDI-TOF-MS-MS to detect and identify biomarkers for human occupation. The results from the first experiments indicate that BSA isolation efficiencies are hampered by the presence of clays and reactive organic matter (glucose), whereas these effects are, to some extent, reversed by the presence of humic acids. We also show that specific regions of the BSA molecule (the out most α-helical regions of domain 2 and 3) are more susceptible to isolation than other regions and this suggest that soil proteins can be only partly isolated. Soil-protein interactions were also found to inhibit tryptic cleavage of BSA, resulting in an enhanced specificity of BSA peptides. Our results further stress the importance of multiple or sequential protein isolation protocols with a focus on citrate and hydrofluoric acid. Results from the case study indicate that the ancient floor samples contain degraded and most likely aged proteinaceous matter, probably of keratinaceous origin. © 2012 Elsevier Ltd.

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

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.

Identifying animals to species from relict proteins is a powerful new archaeological tool. Here the authors apply the method to answer questions relating to the Salish of west coast North America. Did they weave their blankets out of dog hair? The proteomic analysis shows that they did, interweaving it with goat, and that the woolly dog was increasingly superseded by sheep in the later nineteenth century.

It is generally a challenge to interpret incomplete and degraded skeletal assemblages found during archaeological excavations. Several events after the death of animals and humans, before and during burial, will have influenced the situation as seen upon excavation. This postmortem sequence of events can be called the taphonomic history of bones. Taphonomic signatures as detected using histology can provide additional data on deposition/burial and the evolution of the burial environment. This article reports on the results of a histological characterisation of diagenetic alterations in a bone assemblage from the Roman period burial and settlement site of Castricum, located in the province of Noord-Holland in the Netherlands. The aim is to assess the relationship between bone histology and known taphonomic events. Both transmitted light microscopy and scanning electron microscopy were used to investigate histological changes. In 1995, the excavation at Castricum uncovered the skeletal material of humans and a variety of animal species that had received different burial treatments. The humans, as well as some dogs and cattle, were buried as complete inhumations. The skeletal remains of horses provided evidence of surface exposure before deposition. In addition, both environmental changes (e.g. variations in groundwater levels) and disturbances by later human activity are known to have occurred since the internment of the bones. Together with the animal refuse bones from the site, this assemblage provides an excellent opportunity to investigate the relationship between early taphonomy, burial conditions and histological appearance. The complex taphonomic history of the Castricum skeletal material was found to be reflected in several characteristic alterations of the bone microstructure such as the extent of bioerosion, cracking, staining and inclusions of framboidal pyrite crystals. This allowed for the reconstruction of a postmortem sequence of events. Copyright © 2011 John Wiley & Sons, Ltd.

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.

Near-complete collagen (I) sequences are proposed for elephantid and mammutid taxa, based upon available African elephant genomic data and supported with LC–MALDI-MS/MS and LC–ESI-MS/MS analyses of collagen digests from proboscidean bone. Collagen sequence coverage was investigated from several specimens of two extinct mammoths (Mammuthus trogontherii and Mammuthus primigenius), the extinct American mastodon (Mammut americanum), the extinct straight-tusked elephant (Elephas (Palaeoloxodon) antiquus) and extant Asian (Elephas maximus) and African (Loxodonta africana) elephants and compared between the two ionization techniques used. Two suspected mammoth fossils from the British Middle Pleistocene (Cromerian) deposits of the West Runton Forest Bed were analysed to investigate the potential use of peptide mass spectrometry for fossil identification. Despite the age of the fossils, sufficient peptides were obtained to identify these as elephantid, and sufficient sequence variation to discriminate elephantid and mammutid collagen (I). In-depth LC–MS analyses further failed to identify a peptide that could be used to reliably distinguish between the three genera of elephantids (Elephas, Loxodonta and Mammuthus), an observation consistent with predicted amino acid substitution rates between these species.

Over the past 30 years, ancient DNA studies have grown from a scientific curiosity into a powerful research tool with applications in molecular evolution, archaeology, paleontology, conservation genetics, and forensics. The analysis of ancient proteins has lagged behind that of ancient DNA, but recent developments in high-throughput, high-resolution mass spectrometry (MS) have the potential to provide the accuracy and robustness required for confident and reliable sequencing of ancient proteins. This methodology may allow biomolecular recovery to be extended further back in time, because DNA chains fall apart 10 times faster ( 1 ) than proteins ( 2 ). Furthermore, mass spectrometry enables investigation of protein expression specific for different tissues, developmental phases, or biological processes, including disease states.

During the late Pleistocene, a large number of species colonized North America from Eurasia through the Bering Land Bridge. At least four different genera within the tribe Ovibovini are known from this time, the holarctically distributed Ovibo s moschatus (the extant musk ox) and Praeovibos , and the nearctically distributed Bootherium and Euceratherium . Questions about the taxonomic relationships of the four North American musk oxen arose soon after their description and continue to be unresolved at the present. In this study we present the first DNA and protein analyses, of the four mentioned genera. Phylogenetic analysis at the mitochondrial level indicates that Praeovibos falls within the diversity of modern Ovibos , which might indicate that Praeovibos is simply an earlier morphotype of the extant species O. moschatus . In contrast Euceratherium and Bootherium are clearly distinct genera, in agreement with the findings of previous morphological studies.