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Utilising multiple lines of evidence for palaeoenvironmental reconstruction improves our understanding of the past landscapes in which human populations interacted with other species. Illuminating such processes is key for a nuanced understanding of fundamental transitions in human history, such as the shift from hunting and gathering to farming, which move beyond simple deterministic interpretations of climate-driven innovation. Avifaunal remains can provide detailed indications of complex multi-species interactions at the local scale. They allow us to infer relationships between human and non-human animals, but also to reconstruct their niche, because many bird species are sensitive to specific ecological conditions and will often relocate and change their breeding patterns. In this paper, we illustrate how novel evidence that waterfowl reproduced at Levantine wetlands, which we obtained through biomolecular archaeology, as well as modern ornithological data, can reveal conditions of wetlands that are conducive for breeding waterfowl. By understanding the interplay between wetland cover cycles and waterfowl ecology, we argue that human modifications to the environment would have promoted wetland productivity inviting waterfowl to remain year-round. Within this landscape of "mutual ecologies", the feedback resulting from the agency of all species is involved in the construction of the human niche.

The controversy over the taxonomic identity of the eggs exploited by Australia’s first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless “mihirung” Genyornis and a large megapode Progura, had proven impossible using morphological and geochemical methods. Ancient DNA sequencing remains inconclusive because of the age and burial temperature of the eggshell. In contrast, ancient protein sequences recovered from the eggshell enabled estimation of the evolutionary affinity between the egg and a range of extant taxa. The eggs are those of a Galloanseres (a group that includes extinct Dromornithidae, as well as extant landfowl and waterfowl), Genyornis, and not of the megapode (Megapodiidae, crown Galliformes). The realization that ancient biomolecules are preserved in “fossil” samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia’s indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller etnbsp;al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.

SignificanceThe controversy over the taxonomic identity of the eggs exploited by Australia's first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless "mihirung"

Mollusc shells represent excellent systems for the preservation and retrieval of genuine biomolecules from archaeological or palaeontological samples. As a consequence, the post-mortem breakdown of intracrystalline mollusc shell proteins has been extensively investigated, particularly with regard to its potential use as a "molecular clock" for geochronological applications. But despite seventy years of ancient protein research, the fundamental aspects of diagenesis-induced changes to protein structures and sequences remain elusive. In this study we investigate the degradation of intracrystalline proteins by performing artificial degradation experiments on the shell of the thorny oyster, Spondylus gaederopus, which is particularly important for archaeological research. We used immunochemistry and tandem mass tag (TMT) quantitative proteomics, a novel analytical approach that allowed us to simultaneously track patterns of structural loss and of peptide bond hydrolysis. Powdered and bleached shell samples were heated in water at four different temperatures (80, 95, 110, 140 °C) for different time durations. The structural loss of carbohydrate and protein groups was investigated by immunochemical techniques (ELLA and ELISA) and peptide bond hydrolysis was studied by tracking the changes in protein/peptide relative abundances over time using TMT quantitative proteomics. We find that heating does not induce instant organic matrix decay, but first facilitates the uncoiling of cross-linked structures, thus improving matrix detection. We calculated apparent activation energies of structural loss: Ea (carbohydrate groups) = 104.7 kJ/mol, Ea (protein epitopes) = 104.4 kJ/mol, which suggests that secondary matrix structure degradation may proceed simultaneously with protein hydrolysis. While prolonged heating at 110 °C (10 days) results in complete loss of the structural signal, surviving peptide sequences were still observed. Eight hydrolysis-prone peptide bonds were identified in the top scoring shell sequence, the uncharacterised protein LOC117318053 from Pecten maximus. Interestingly, these were not the expected "weak" bonds based on published theoretical stabilities calculated for peptides in solution. This further confirms that intracrystalline protein degradation patterns are complex and that the overall microchemical environment plays an active role in protein stability. Our TMT approach represents a major stepping stone towards developing a model for studying protein diagenesis in biomineralised systems.

© 2019 Elsevier Ltd Bird eggs can become part of the archaeological record either accidentally or as a result of human activities but, in both instances, they can reveal important aspects of the environment at the site, the ways in which people chose to exploit it, and even the existence of subtle ecological balances between humans and other animals. This is the case for El Mirόn, one of the most important cave sites in Cantabrian Spain, with occupation levels spanning around 40,000 years, from the late Middle Palaeolithic to the Bronze Age. This mountainous area in Cantabria was an ideal environment for hunting medium-sized game and, as such, supported both human and non-human predators, including birds of prey. Here we use a combination of peptide mass fingerprinting (by MALDI-MS) and protein sequencing (by LC-MS/MS) in order to taxonomically identify ninety-five fragments of eggshells recovered from nineteen archaeological layers. We firmly identify these as diurnal birds of prey (Accipitridae) and suggest that the species might have been bearded vulture, based on previous taphonomic studies that highlighted its presence at the cave. The implication is that both species of diurnal predators, humans and birds, inhabited the cave and used the surrounding environment during different periods of the year.

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.

10 pages; International audience; Molluscs were one of the most widely-used natural resources in the past, and their shells are abundant among archaeological findings. However, our knowledge of the variety of shells that were circulating in prehistoric times (and thus their socio-economic and cultural value) is scarce due to the difficulty of achieving taxonomic determination of fragmented and/or worked remains. This study aims to obtain molecular barcodes based on peptide mass fingerprints (PMFs) of intracrystalline proteins, in order to obtain shell identification. Palaeoproteomic applications on shells are challenging, due to low concentration of molluscan proteins and an incomplete understanding of their sequences. We explore different approaches for protein extraction from small-size samples (

Rates of peptide bond hydrolysis and other diagenetic reactions are not favourable for Mesozoic protein survival. Proteins hydrolyse into peptide fragments and free amino acids that, in open systems such as bone, can leach from the specimen and be further degraded. However, closed systems are more likely to retain degradation products derived from endogenous proteins. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, high-performance liquid chromatography reveals that the intra-crystalline fraction of Late Cretaceous (estimated ~80 Ma) titanosaur sauropod eggshell is enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and those that racemise are fully racemic, despite being some of the slowest racemising amino acids. These results are consistent with degradation trends deduced from modern, thermally matured, sub-fossil, and ~3.8 Ma avian eggshell, as well as ~30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, along with similar concentrations of free versus total hydrolysable amino acids, likely suggests complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, suggestive of kerogen formation deriving from eggshell lipids. Raman spectroscopy yields bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation even if such reactions might be slowed in exceptional circumstances. The evidence for complete hydrolysis and degradation of most amino acids in the eggshell raises concern about the validity of reported polypeptide sequences from open-system non-avian dinosaur bone and other Mesozoic fossils.

The scientific analysis of mummified individuals can reveal important details on the way people lived and died in the past. Palaeoproteomic approaches are theoretically suitable for obtaining information on the extent of tissue preservation, on the use of protein-based substances for embalming and/or restoration, as well as for characterising the microbiota from both the individual and the environment. However, these analyses usually require the destruction of a sample of tissue, a practice which is (obviously) discouraged by most museums. Unfortunately, this means that in-depth studies, for example by taking multiple samples from each individual, are seldom feasible. Here we show that a non-invasive sampling technique, based on mixed-bed chromatographic media embedded on ethylene vinyl acetate membranes (EVA), which had previously been used exclusively on historical material, was successful in extracting ancient proteins from the surface of Egyptian mummies. We tested the method on a decontextualised fragment of skin and assessed the endogeneity of its metaproteome by comparison with a procedural blank. Furthermore, we retrieved and authenticated sequences of human collagen and keratin, as well as potential bacterial/fungal biodeteriogens, from the mummy of a young woman (Supp. 16747 of the Museum of Anthropology and Ethnography of the University of Turin) who lived and died during the Old Kingdom of Egypt.

17 pages; International audience; Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised.We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe’s cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers.Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences.This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids – fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Marine faunal remains from Grotta d’Oriente (Favignana Island, NW Sicily) offer invaluable snapshots of human-coastal environment interaction in the central Mediterranean from the Late Pleistocene to the Middle Holocene. The long-term shellfish and fish records reflect human exploitation of coastal environments undergoing considerable reorganizations during the postglacial sea level rise and the progressive isolation of Favignana from mainland Sicily. We detected an intensification of marine resource exploitation between ∼9.6 ka and ∼7.8 ka BP, which corresponds with the isolation of Favignana Island and, later on, with the introduction of early agro-pastoral economy in this region. We suggest that a higher investment in marine resource exploitation by late foragers and early farmers in NW Sicily was also supported by an increase in marine productivity in the south Tyrrhenian Sea in the Middle Holocene.

Palaeoproteomics is an emerging neologism used to describe the application of mass spectrometry-based approaches to the study of ancient proteomes. As with palaeogenomics (the study of ancient DNA), it intersects evolutionary biology, archaeology and anthropology, with applications ranging from the phylogenetic reconstruction of extinct species to the investigation of past human diets and ancient diseases. However, there is no explicit consensus at present regarding standards for data reporting, data validation measures or the use of suitable contamination controls in ancient protein studies. Additionally, in contrast to the ancient DNA community, no consolidated guidelines have been proposed by which researchers, reviewers and editors can evaluate palaeoproteomics data, in part due to the novelty of the field. Here we present a series of precautions and standards for ancient protein research that can be implemented at each stage of analysis, from sample selection to data interpretation. These guidelines are not intended to impose a narrow or rigid list of authentication criteria, but rather to support good practices in the field and to ensure the generation of robust, reproducible results. As the field grows and methodologies change, so too will best practices. It is therefore essential that researchers continue to provide necessary details on how data were generated and authenticated so that the results can be independently and effectively evaluated. We hope that these proposed standards of practice will help to provide a firm foundation for the establishment of palaeoproteomics as a viable and powerful tool for archaeologists, anthropologists and evolutionary biologists.

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.

Microscopic analyses can be used to determine whether fragments of eggshell come from hatched, incubated or non-incubated eggs. This information is essential for their interpretation since the developmental state of eggs often permits archaeologists to draw conclusions about the function of these finds at a site. However, what has often been neglected in previous studies is the fact that not only incubation but also taphonomy may affect the microstructure of shells. This preliminary study aims to demonstrate that taphonomic processes can in fact imitate site specific dissolution features that are commonly interpreted as traces of incubation. One likely cause of this could be bacteria or other microorganisms. The paper further introduces an approach by which a distinction between taphonomic and embryonic dissolution may be possible. The successful application of this technique on seemingly incubated eggs from a late Roman burial of Ober-Olm (Germany) indicates that these shells were altered only by taphonomy and not by embryonic development as initially assumed. It is finally emphasized that the preliminary data of this investigation need to be validated in future research.

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including ‘palaeoshellomics’, the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of ‘double-buttons’. This craft is found throughout Europe between 4200–3800 BCE, highlighting the ornament-makers’ profound knowledge of the biogeosphere and the existence of cross-cultural traditions., eLife digest Just like people do today, prehistoric humans liked to adorn themselves with beautiful objects. Shells, from creatures like clams and snails, were used to decorate clothing or worn as jewelry at least as far back as 100,000 years ago. Later people used shells as the raw materials to make beads or bracelets. Learning where the shells came from may help scientists understand why prehistoric people chose certain shells and not others. It may also offer clues about how they used natural resources and the cultural significance of these objects. But identifying the shells is difficult because they lose many of their original distinctive features when worked into ornaments. New tools that use DNA or proteins to identify the raw materials used to craft ancient artifacts have emerged that may help. So far, scientists have mostly used these genomic and proteomic tools to identify the source of materials made from animal hide, ivory or bone – where collagen is the most abundant protein molecule. Yet it is more challenging to extract and characterize proteins or genetic material from mollusc shells. This is partly because the amount of proteins in shells is at least 300 times lower than in bone, and also because the makeup of proteins in shells is not as well-known as in collagen. Sakalauskaite et al. have now overcome these issues by combining the analytical tools used to study the proteins and mineral content of modern shells with those of ancient protein research. They then used this approach, which they named palaeoshellomics, to extract proteins from seven “double-buttons” – pearl-like ornaments crafted by prehistoric people in Europe. The double-buttons were made between 4200 and 3800 BC and found at archeological sites in Denmark, Germany and Romania. Comparing the extracted proteins to those from various mollusc shells showed that the double-buttons were made from freshwater mussels belonging to a group known as the Unionoida. The discovery helps settle a decade-long debate in archeology about the origin of the shells used to make double-buttons in prehistoric Europe. Ancient people often crafted ornaments from marine shells, because they were exotic and considered more prestigious. But the results on the double-buttons suggest instead that mother-of-pearl from fresh water shells was valued and used by groups throughout Europe, even those living in coastal areas. The palaeoshellomics technique used by Sakalauskaite et al. may now help identify the origins of shells from archeological and palaeontological sites.

The level of glutamine (Gln) deamidation in bone collagen provides information on the diagenetic history of bone but, in order to accurately assess the extent of Gln deamidation, it is important to minimise the conditions that may induce deamidation during the sample preparation. Here we report the results of a preliminary investigation of the variability in glutamine deamidation levels in an archaeological bone due to: a) sampling location within a bone; b) localised diagenesis; and c) sample preparation methods. We then investigate the effects of pre-treatment on three bone samples: one modern, one Medieval and one Pleistocene. The treatment of bone with acidic solutions was found to both induce deamidation and break down the collagen fibril structure. This is particularly evident in the Pleistocene material (∼80,000 years BP) considered in this study. We show that ethylenediaminetetraacetic acid (EDTA), when used as an alternative to hydrochloric acid (HCl) demineralisation, induces minimal levels of deamidation and maintains the collagen fibril structure. Areas of bone exhibiting localised degradation are shown to be correlated with an increase in the levels of Gln deamidation. This indicates that the extent of Gln deamidation could provide a marker for diagenesis but that sampling is important, and that, whenever possible, subsamples should be taken from areas of the bone that are visually representative of the bone as a whole. Although validation of our observations will require analysis of a larger sample set, deamidation measurements could be a valuable screening tool to evaluate the suitability of bone for further destructive collagen analyses such as isotopic or DNA analysis, as well as assessing the overall preservation of bone material at a site. The measure of bone preservation may be useful to help conservators identify bones that may require special long-term storage conditions.

Intra-crystalline protein diagenesis (IcPD), a recent development of amino acid racemization dating (AAR), is now established as a reliable geochronological tool for the Quaternary. However, extending the method to new biominerals requires extensive testing in order to provide evidence for the closed-system behaviour of the intra-crystalline proteins and to assess the temporal span that can be covered.Here we present results from high-temperature experiments on the IcPD of the bivalve Pecten, demonstrating that a fraction of proteins can be isolated from a bleach-resistant mineral matrix, which effectively operates as a closed system under conditions of accelerated diagenesis in the laboratory. Analyses of Pecten from the well-dated terrace system of the Gulf of Corinth (Greece) provided a pilot test for the integrity of the intra-crystalline fraction in subfossil shells. The small sample sizes in this preliminary study preclude a full assessment of the aminostratigraphic power of Pecten IcPD, but a concordance is observed between the extent of IcPD and sites dating from between MIS 5 and MIS 11.We conclude that Pecten is a potentially good substrate for IcPD dating in the Mediterranean, and that the temporal limit of the technique in this area lies beyond MIS 11. © 2015 Published by Elsevier B.V.

A recommendation of: Delphine Vettese, Trajanka Stavrova, Antony Borel, Juan Marin, Marie-Helene Moncel, Marta Arzarello, Camille Daujeard A way to break bones? The weight of intuitiveness 10.31219/osf.io/rebwt

In the North Sea basin the marine bivalve Macoma balthica first appears within the Early Pleistocene ‘Weybourne Crag’, which forms an important biostratigraphical datum. Here we review the fossil assemblages from sites of this age, prompted by new discoveries from Sidestrand, Norfolk, UK. The molluscan assemblages from this horizon are dominated by intertidal species with some colder/deeper water taxa and a few temperate non-marine species. A high boreal/low arctic marine environment with reduced salinities is indicated. An extensive assemblage of small mammals dominated by voles includes two species (Mimomys hordijki and Ungaromys dehmi) previously unknown from the British Pleistocene. The assemblage can be assigned to Tesakov’s Mammal Biozone MNR1 (=MN17, Middle Villafranchian), which according to current estimates corresponds to a date of ∼2.2-2.1 Ma (MIS 84-79). It matches another assemblage from -61 m to -65 m in the Zuurland-2 borehole in The Netherlands, and is similar to that from the Dutch Tiglian type site at Tegelen, although this has more temperate elements. A late Tiglian age is consistent with the co-occurrence of the marine bivalves Macoma balthica, Mya arenaria and the freshwater gastropod Viviparus glacialis in the Zuurland-2 borehole and in a North Sea borehole (BGS 52-02-472). A Macoma balthica – Mya arenaria Concurrent Range Zone is defined for this assemblage, which can be traced across the North Sea basin. Amino acid dating provides strong independent support for these correlations and indicates that the Baventian cold stage post-dates the Bramertonian (Norwich Crag). It also confirms that Early Pleistocene molluscan assemblages with M. balthica are younger than those without it. The correlation of this marine marker horizon with Mammal Biozone MNR1 provides a secure link between continental and marine sequences during the Early Pleistocene. It also provides a basis for dating events in the pre-glacial fluvial drainage history and linking it to the East European mammal zonation.

11 pages; International audience; The exceptional eggshell assemblage from Çatalhöyük was studied using an integrated approach combining morphology (by optical and scanning electron microscopy) and palaeoproteomics (by mass spectrometry). We provide taxonomic classification for 90 fragments, of which only 11 remain undetermined. The striking predominance of Anseriformes (probably including greylag geese, as well as ducks and swans) in all types of deposits examined, particularly middens and burial fills, suggests that these eggs were exploited as food and, at the same time, had a special significance for the inhabitants of the site. We detected the presence of crane eggshell in contexts associated with both the world of the living and the world of the dead (consistent with the well-known importance of this bird at Çatalhöyük), as well as the possible but infrequent occurrence of bustards and herons. Overall, we suggest that eggshell analysis, together with osteological data and the archaeological context, can provide the basis for a nuanced understanding of the relationship between humans and birds in the past.

In the version of this Perspective originally published, in the third paragraph of the section 'Selection and sampling', the sentence beginning 'Pyrolysis-gas chromatography' should have also referred to high-performance liquid chromatography; the sentence has now been amended to read 'Pyrolysis-gas chromatography/mass spectrometry (py-GC/MS) and high-performance liquid chromatography (HPLC) can be used to detect the presence of amino acids62 in any putative proteinaceous sample.'

This paper discusses some of the approaches and results from two multi-disciplinary projects. The first is the AHRC-funded ‘Cultural and Scientific Perceptions of Human-Chicken Interactions’ Project. This is investigating the history of the exploitation of chickens in Europe. The second is the Leverhulme Trust-funded ‘Rural Settlement of Roman Britain’ Project, which has collated evidence from excavation reports from thousands of sites. This paper updates the evidence for the exploitation of chickens in Roman Britain, showing that there were significant variations in the abundance of chicken bones found on different types of settlement. There was also a modest increase in their abundance during the Roman period suggesting chickens became slightly more frequent contributors to the diet, albeit still only a rare commodity. However, they continued to be frequently represented in graves, shrines and other ritual deposits. The paper also discusses evidence of egg production and avian osteopetrosis, demonstrating that when traditional zooarchaeological research is integrated with scientific analyses, a deeper understanding of past human diet can be acquired.

Eggshells are unusual finds in the Iron Age of eastern Europe (500 BC–1200 AD) deserving extra attention in terms of analysis as well as interpretation. This paper discusses two rare eggshell finds, discovered in female burials at the conversion period (12th–13th century AD) cemetery at Kukruse, NE Estonia. Our multianalytical study combining FT-IR, SEM(-EDS), microscopy and ZooMS provides an overview of methods applicable for identifying egg species, their predepositional history and curation. Based on the analytical results and the comparative analysis of the content and context of these two burials, we argue that different aims and connotations lay behind depositing eggs as burial goods, allowing well-supported interpretations of both pagan and Christian religious worldviews simultaneously.

Modern human dispersal into Europe is thought to have occurred with the start of the Upper Paleolithic around 50,000-40,000 y ago. The Levantine corridor hypothesis suggests that modern humans from Africa spread into Europe via the Levant. Ksâr 'Akil (Lebanon), with its deeply stratified Initial (IUP) and Early (EUP) Upper Paleolithic sequence containing modern human remains, has played an important part in the debate. The latest chronology for the site, based on AMS radiocarbon dates of shell ornaments, suggests that the appearance of the Levantine IUP is later than the start of the first Upper Paleolithic in Europe, thus questioning the Levantine corridor hypothesis. Here we report a series of AMS radiocarbon dates on the marine gastropod Phorcus turbinatus associated with modern human remains and IUP and EUP stone tools from Ksâr 'Akil. Our results, supported by an evaluation of individual sample integrity, place the EUP layer containing the skeleton known as "Egbert" between 43,200 and 42,900 cal B.P. and the IUP-associated modern human maxilla known as "Ethelruda" before ∼45,900 cal B.P. This chronology is in line with those of other Levantine IUP and EUP sites and demonstrates that the presence of modern humans associated with Upper Paleolithic toolkits in the Levant predates all modern human fossils from Europe. The age of the IUP-associated Ethelruda fossil is significant for the spread of modern humans carrying the IUP into Europe and suggests a rapid initial colonization of Europe by our species.

Recognition of palaeoclimatic instability in the Greenland ice cores has spurred researchers to identify corresponding evidence in other terrestrial records from the last glacial stage. Such evidence is critical for establishing how much environmental stress precipitated Neanderthal and Late Pleistocene megafaunal extinctions, although a need for improved chronology has been consistently highlighted. In formerly glaciated and periglaciated areas of northern Europe, palaeoenvironmental sequences are frequently discontinuous. These often yield high-resolution proxy-based quantitative palaeotemperature estimates but can be hard to date, due to difficulties in removing contamination from biological samples at the limits of the radiocarbon technique (c.30-50kya). Here we demonstrate, for the first time using samples with independent age control, that different radiocarbon pretreatments can generate different age data and that gentler, less effective treatments applied to avoid sample loss may not yield reliable age-estimates. We advocate alternative harsher pretreatment using a strong acid-base-acid protocol. This provides an acceptable balance between contamination removal and excessive sample loss and generates more accurate ages, significantly enhancing our ability to detect and understand the impacts of palaeoclimatic instability in the terrestrial record of the last glacial., Natural Environment Research Council (NERC) NRCF grant 1848.1014

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.

The facile racemization of protein-bound amino acids plays an important role in the aging and pathologies of living tissues, and it can be exploited for protein geochronological studies in subfossil biominerals. However, the in-chain degradation pathways of amino acids are complex and difficult to elucidate. Serine has proven to be particularly elusive, and its ability to racemize as a peptide-bound residue (like asparagine and aspartic acid) has not been demonstrated. This study investigates the patterns of degradation of a model peptide (WNSVWAW) at elevated temperatures, quantifying the extent of racemization and peptide bond hydrolysis using reverse-phase high-performance liquid chromatography (RP-HPLC) and tracking the presence of degradation products by MALDI-MS. We provide direct evidence that, under these experimental conditions, both serine and asparagine are able to undergo racemization as internally bound residues, which shows their potential for initiating protein breakdown and provides an explanation for the presence of d-enantiomers in living mammalian tissues.

This study successfully isolates a fraction of intra-crystalline proteins from shells of the marine gastropod Patella vulgata and assesses the suitability of these proteins for IcPD (Intra-crystalline Protein Diagenesis) geochronology. We discuss the mineralogical composition of this gastropod, investigated for the first time by X-ray diffraction mapping, and use the results to inform our sampling strategy. The potential of the calcitic rim and of a bulk sample (containing both apex and rim) of the shell to act as stable repositories for the intra-crystalline proteins during diagenesis is examined. The composition and the diagenetic behaviour of the intra-crystalline proteins isolated from different locations within the shell are compared, highlighting the necessity of targeting consistent sampling positions.We induced artificial diagenesis of both intra-crystalline and whole-shell proteins by conducting high-temperature experiments in hydrous environment; this allowed us to quantify the loss of amino acids by leaching and therefore evaluate the open- or closed-system behaviour of the different fractions of proteins. The results obtained provide further confirmation that patterns of diagenesis vary according to the protein sequence, structure, and location within or outside the intra-crystalline fraction. As Patella is frequently found in the fossil record, both in archaeological and geological contexts, the application of IcPD geochronology to this biomineral opens up the possibility to obtain reliable age information from a range of sites in different areas of the world.

Examples of wetland deposits can be found across the globe and are known for preserving organic archaeological and environmental remains that are vitally important to our understanding of past human–environment interactions. The Mesolithic site of Star Carr (Yorkshire, United Kingdom) represents one of the most influential archives of human response to the changing climate at the end of the last glacial in Northern Europe. A hallmark of the site since its discovery in 1948 has been the exceptional preservation of its organic remains. Disturbingly, recent excavations have suggested that the geochemistry of the site is no longer conducive to such remarkable survival of organic archaeological and environmental materials. Microcosm (laboratory-based) burial experiments have been undertaken, alongside analysis of artifacts excavated from the site, to assess the effect of these geochemical changes on the remaining archaeological material. By applying a suite of macroscopic and molecular analyses, we demonstrate that the geochemical changes at Star Carr are contributing to the inexorable and rapid loss of valuable archaeological and paleoenvironmental information. Our findings have global implications for other wetland sites, particularly archaeological sites preserved in situ.