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Esh-Shaheinab is a landmark in the African Neolithic. This site gave the name Shaheinab Neolithic to the Neolithic period in central Sudan, becoming its archetype. Excavated in the late 1940s by A.J. Arkell, it bears witness to the processes of domestic animal introduction from the Middle East into North and East Africa. Its excavation also uncovered the remains of an earlier Mesolithic or Early Khartoum (ca. ninth-sixth millennia BC) and a Late Neolithic occupation (ca. fourth millennium BC), providing essential insights into the Neolithic's premises and sequels. Although the influence of Esh-Shaheinab has been recognized for more than seventy years, our knowledge of its material culture has remained as it was then. In 2001, one of the present authors (EAAG) had permission to restudy the ceramic collection at the National Museum in Khartoum and subsequently export samples for laboratory analyses. Here, for the first time, we provide a multi-scale analysis of the Esh-Shaheinab ceramic material from the Early Khartoum to the Late Neolithic periods by integrating the chaîne opératoire approach into the local landscape. By combining the results of macroscopic and microscopic analyses, we performed petrographic investigations on the composition and manufacturing technology of the ceramic pastes using polarized optical microscopy (POM) and scanning electron microscopy (SEM-EDS). Organic residue analysis (ORA) was also carried out, to provide information on diet, vessel use, and subsistence practices. The results of our combined analyses showed that the inhabitants of Esh-Shaheinab developed an adaptation specific to the ecological niche they inhabited. They lived in the western valley of the Nile, which was narrower and offered different environmental conditions than the eastern bank. This resulted in partial continuity in manufacturing traditions and ceramic recipes, including more mixed wadi materials and a strong emphasis on wild meat consumption as the narrower alluvial plain restricted animal herding., Competing Interests: All necessary permits were obtained for the described study, which complied with all relevant regulations., (Copyright: © 2024 D’Ercole 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.)

During the sixth millennium BCE, the first farmers of Central Europe rapidly expanded across a varied mosaic of forested environments. Such environments would have offered important sources of mineral-rich animal feed and shelter, prompting the question: to what extent did early farmers exploit forests to raise their herds? Here, to resolve this, we have assembled multi-regional datasets, comprising bulk and compound-specific stable isotope values from zooarchaeological remains and pottery, and conducted cross-correlation analyses within a palaeo-environmental framework. Our findings reveal a diversity of pasturing strategies for cattle employed by early farmers, with a notable emphasis on intensive utilization of forests for grazing and seasonal foddering in some regions. This experimentation with forest-based animal feeds by early farmers would have enhanced animal fertility and milk yields for human consumption, concurrently contributing to the expansion of prehistoric farming settlements and the transformation of forest ecosystems. Our study emphasizes the intricate relationship that existed between early farmers and forested landscapes, shedding light on the adaptive dynamics that shaped humans, animals and environments in the past., (© 2024. The Author(s).)

Rationale: Plastic mulch film manages weed growth and moisture loss on the surface of cropping beds. The chemical components of such plastics include polymer(s), additives and non-intentionally added substances (NIASs). The unknown chemical nature and behaviours of these constituents require investigation due to their potential to add to the anthropogenic chemical burden in the agrifood system., Methods: Solvent extracts of a commercial 15% polylactic acid (PLA)/85% poly(butylene adipate-co-terephthalate) mulch film were investigated using gas chromatography-mass spectrometry (GC-MS) with electron ionisation to characterise the additive and NIAS components. The obscurity of some of the NIASs meant their identification was not readily achieved through routine MS library comparisons. As such, the identification of several polymer-derived compounds required interpretation of the MS data and re-application of the derived fragmentation patterns with reference to the wider literature. Unknowns were confirmed using commercially available compounds., Results: Unknown NIASs were identified as cyclic oligoesters comprised of the monomeric building blocks of the polymer system. Cyclic structures derived from the monomers of polybutylene adipate (PBA) and polybutylene terephthalate (PBT) fragmented through a primary pathway involving 1,5- and 1,3-H transfers at ester linkages. Characteristic ions at m/z 111, 129, 183 and 201 for PBA-derived cyclic oligoesters and m/z 104, 132, 149 and 221 for PBT-derived cyclic oligoesters were assigned in the mass spectra of unknowns. Cyclic oligoesters containing sebacate moieties were also identified, indicating the presence of polybutylene sebacate as an unexpected component of the mulch., Conclusions: Systematic analyses of the sort reported here are valuable for providing alternative approaches for the identification of plastic-related chemicals. Open publication of MS spectral data is required to build a greater understanding of the mulch film chemical components contributing to the environmental chemical load introduced to agroecosystems., (© 2024 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

The differential soil microbial assimilation of common nitrogen (N) fertilizer compounds into the soil organic N pool is revealed using novel compound-specific amino acid (AA) 15N-stable isotope probing. The incorporation of fertilizer 15N into individual AAs reflected the known biochemistry of N assimilation-e.g. 15N-labelled ammonium (15NH4+) was assimilated most quickly and to the greatest extent into glutamate. A maximum of 12.9% of applied 15NH4+, or 11.7% of 'retained' 15NH4+ (remaining in the soil) was assimilated into the total hydrolysable AA pool in the Rowden Moor soil. Incorporation was lowest in the Rowden Moor 15N-labelled nitrate (15NO3-) treatment, at 1.7% of applied 15N or 1.6% of retained 15N. Incorporation in the 15NH4+ and 15NO3- treatments in the Winterbourne Abbas soil, and the 15N-urea treatment in both soils was between 4.4% and 6.5% of applied 15N or 5.2% and 6.4% of retained 15N. This represents a key step in greater comprehension of the microbially mediated transformations of fertilizer N to organic N and contributes to a more complete picture of soil N-cycling. The approach also mechanistically links theoretical/pure culture derived biochemical expectations and bulk level fertilizer immobilization studies, bridging these different scales of understanding., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Over the last 50 years, the intense use of agricultural plastic in the form of mulch films has led to an accumulation of plastic in soil, creating a legacy of plastic in agricultural fields. Plastic often contains additives, however it is still largely unknown how these compounds affect soil properties, potentially influencing or masking effects of the plastic itself. Therefore, the aim of this study was to investigate the effects of pure plastics of varying sizes and concentrations, to improve our understanding of plastic-only interactions within soil-plant mesocosms. Maize (Zea mays L.) was grown over eight weeks following the addition of micro and macro low-density polyethylene and polypropylene at increasing concentrations (equivalent to 1, 10, 25, and 50 years mulch film use) and the effects of plastic on key soil and plant properties were measured. We found the effect of both macro and microplastic on soil and plant health is negligible in the short-term (1 to <10 years). However, ≥ 10 years of plastic application for both plastic types and sizes resulted in a clear negative effect on plant growth and microbial biomass. This study provides vital insight into the effect of both macro and microplastics on soil and plant properties., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Micro and macroplastics are emerging contaminants in agricultural settings, yet their impact on nitrogen (N) cycling and partitioning in plant-soil-microbial systems is poorly understood. In this mesocosm-scale study, spring barley (Hordeum vulgare L.) was exposed to macro or microplastic produced from low density polyethylene (LDPE) or biodegradable plastic at concentrations equivalent to 1, 10 and 20 years of plastic mulch film use. Partitioning of 15 N-labelled fertiliser into plant biomass, soil and leachate yielded a partial mass balance. Soil N partitioning was probed via compound-specific 15 N-stable isotope analyses of soil microbial protein. Concentration-dependent decreases in plant 15 N uptake occurred with increased leached nitrogen for LDPE microplastic. Assimilation into soil microbial protein was higher for biodegradable plastics, which we associate with early-stage biodegradable plastic degradation. Partitioning of 15 N into inorganic soil N pools was affected by LDPE size, with lower assimilation into the microbial protein pool. While microplastics and macroplastics altered soil N cycling, the limited impacts on plant health indicated the threshold for negative effects was not reached at agriculturally relevant concentrations. This study highlights the difference between conventional and biodegradable plastics, and emphasises that the interplay of micro and macroplastics on soil N cycling must be considered in future studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The study of painted artworks using scientific methods is fundamental for understanding the techniques used in their creation and their appropriate conservation. The ethical constraints involved in the handling of, and sampling from, these objects has steered recent developments in the field of Heritage science towards a range of new non-invasive/non-destructive spectroscopic techniques capable of providing important insights into their elemental or bulk chemical compositions. Due to the inherent complexities of heritage artefacts, however, their organic components are especially difficult to study in this way and their identification and degradation pathways are thus often best investigated using mass spectrometric (MS) techniques. The versatility, sensitivity and specificity of MS techniques are constantly increasing, with technological advances pushing the boundaries of their use in this field. The progress in the past ten years in the use of MS techniques for the analysis of paint media are described in the present review. While some historical context is included, the body of the review is structured around the five most widely used or emerging capabilities offered by MS. The first pertains to the use of spatially resolved MS to obtain chemical maps of components in cross-sections, which may yield information on both inorganic and organic materials, while the second area describes the development of novel sample preparation approaches for gas chromatography (GC)-MS to allow simultaneous analysis of a variety of components. The third focuses on thermally assisted analysis (either with direct MS or coupled with GC-MS), a powerful tool for studying macromolecules requiring zero (or minimal) sample pre-treatment. Subsequently, the use of soft ionisation techniques often combined with high-resolution MS for the study of peptides (proteomics) and other macromolecules (such as oligosaccharides and triglycerides) is outlined. The fifth area covers the advances in radiocarbon dating of painting components with accelerator MS (AMS). Lastly, future applications of other MS techniques to the study of paintings are mentioned; such as direct analysis in real time MS (DART-MS) and stable isotope ratio MS (IRMS). The latter, having proven its efficiency for the study of lipids in archaeological artefacts, is envisioned to become a valuable tool for this area, whereas DART-MS is already being utilised to study the surface composition of various museum objects. Rapid technological advances, resulting in increased sensitivity and selectivity of MS techniques, are opening up new approaches for paintings analysis, overcoming the fundamental hurdle of sample size available for destructive analysis. Importantly, while the last decade has seen proteomics applications come to the fore, this review aims to emphasise the wider potential of advanced MS techniques for the study of painting materials and their conservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The subsistence practices of Later Stone Age (LSA) foragers and herders living in Namaqualand South Africa are often difficult to differentiate based on their archaeological signatures but characterizing their dietary choices is vital to understand the economic importance of domesticates. However, ethnohistoric accounts have provided information on the cooking/boiling of marine mammal fat, mutton, plants, and milk by early herders and foragers across the Western Cape. To further investigate these reports, we use lipid residue analysis to characterize 106 potsherds from four open-air LSA sites, spanning in time from the early first millennium to the late second millennium AD. Two sites (SK2005/057A, SK2006/026) are located on the Atlantic coast whereas sites Jakkalsberg K and Jakkalsberg M are located further inland on the southern bank of the Orange River. Notably, at the coastal sites, the presence of marine biomarkers suggests the intensive and/or specialized processing of marine products in many vessels. The dominance of ruminant carcass products at inland sites and probable sheep remains confirms the importance of stockkeeping. Furthermore, and in good agreement with ethnohistoric accounts for its use, our results provide the first direct chemical evidence for the use of dairy products in LSA western South Africa., (© 2023. The Author(s).)

The Kura-Araxes (KA) cultural phenomenon (dated to the Early Bronze Age, c. 3500/3350-2500 BCE) is primarily characterised by the emergence of a homogeneous pottery style and a uniform 'material culture package' in settlements across the South Caucasus, as well as territories extending to the Ancient Near East and the Levant. It has been argued that KA societies practised pastoralism, despite a lack of direct examination of dietary and culinary practices in this region. Here, we report the first analyses of absorbed lipid residues from KA pottery to both determine the organic products produced and consumed and to reconstruct subsistence practices. Our results provide compelling evidence for a diversified diet across KA settlements in Armenia, comprising a mixed economy of meat and plant processing, aquatic fats and dairying. The preservation of diagnostic plant lipid biomarkers, notably long-chain fatty acids (C20 to C28) and n-alkanes (C23 to C33) has enabled the identification of the earliest processing of plants in pottery of the region. These findings suggest that KA settlements were agropastoral exploiting local resources. Results demonstrate the significance of applying biomolecular methods for examining dietary inferences in the South Caucasus region., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Manoukian 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.)

Direct, accurate, and precise dating of archaeological pottery vessels is now achievable using a recently developed approach based on the radiocarbon dating of purified molecular components of food residues preserved in the walls of pottery vessels. The method targets fatty acids from animal fat residues, making it uniquely suited for directly dating the inception of new food commodities in prehistoric populations. Here, we report a large-scale application of the method by directly dating the introduction of dairying into Central Europe by the Linearbandkeramik (LBK) cultural group based on dairy fat residues. The radiocarbon dates ( n = 27) from the 54th century BC from the western and eastern expansion of the LBK suggest dairy exploitation arrived with the first settlers in the respective regions and were not gradually adopted later. This is particularly significant, as contemporaneous LBK sites showed an uneven distribution of dairy exploitation. Significantly, our findings demonstrate the power of directly dating the introduction of new food commodities, hence removing taphonomic uncertainties when assessing this indirectly based on associated cultural materials or other remains.

In European and many African, Middle Eastern and southern Asian populations, lactase persistence (LP) is the most strongly selected monogenic trait to have evolved over the past 10,000 years 1 . Although the selection of LP and the consumption of prehistoric milk must be linked, considerable uncertainty remains concerning their spatiotemporal configuration and specific interactions 2,3 . Here we provide detailed distributions of milk exploitation across Europe over the past 9,000 years using around 7,000 pottery fat residues from more than 550 archaeological sites. European milk use was widespread from the Neolithic period onwards but varied spatially and temporally in intensity. Notably, LP selection varying with levels of prehistoric milk exploitation is no better at explaining LP allele frequency trajectories than uniform selection since the Neolithic period. In the UK Biobank 4,5 cohort of 500,000 contemporary Europeans, LP genotype was only weakly associated with milk consumption and did not show consistent associations with improved fitness or health indicators. This suggests that other reasons for the beneficial effects of LP should be considered for its rapid frequency increase. We propose that lactase non-persistent individuals consumed milk when it became available but, under conditions of famine and/or increased pathogen exposure, this was disadvantageous, driving LP selection in prehistoric Europe. Comparison of model likelihoods indicates that population fluctuations, settlement density and wild animal exploitation-proxies for these drivers-provide better explanations of LP selection than the extent of milk exploitation. These findings offer new perspectives on prehistoric milk exploitation and LP evolution., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Recent research has highlighted the importance of dissolved organic matter (DOM) for ecosystem function and because of this paradigm shift, it has become crucial to not only quantify its contribution to river nutrient loads but also to characterise its composition. There has been a significant research effort utilising optical methods, such as fluorescence and UV-Vis spectrophotometry, in order to start exploring DOM character. However, these methods still lack the granularity to understand the chemical composition at the molecular level, which is vital to properly understanding its functional role in freshwater ecosystems. As a direct result, there has been a shift towards including molecular-scale analyses to investigate the in-stream processing of the material. Alongside this, recent methodological advancements, particularly in mass spectrometry are opening new opportunities for probing one of the most complex environmental mixtures. However, in order to fully exploit these opportunities, it is key that the way that samples are collected, processed and stored is considered carefully such that sample integrity is maintained. There are additional challenges when collecting water samples for analysis at molecular scale, for example the ultra-low concentrations of individual compounds within DOM means that the samples are sensitive to contamination. This paper discusses current sample collection, processing and storage protocols for this C, N and P quantification and characterisation in freshwaters, and proposes a new standardised protocol suitable for both nutrient fraction quantification and molecular scale analyses, based on method development and testing undertaken in our UK Natural Environment Research Council large grant programme, characterising the nature, origins and ecological significance of Dissolved Organic Matter IN freshwater Ecosystems (DOMAINE)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The Sun sporadically produces eruptive events leading to intense fluxes of solar energetic particles (SEPs) that dramatically disrupt the near-Earth radiation environment. Such events have been directly studied for the last decades but little is known about the occurrence and magnitude of rare, extreme SEP events. Presently, a few events that produced measurable signals in cosmogenic radionuclides such as 14 C, 10 Be and 36 Cl have been found. Analyzing annual 14 C concentrations in tree-rings from Switzerland, Germany, Ireland, Russia, and the USA we discovered two spikes in atmospheric 14 C occurring in 7176 and 5259 BCE. The ~2% increases of atmospheric 14 C recorded for both events exceed all previously known 14 C peaks but after correction for the geomagnetic field, they are comparable to the largest event of this type discovered so far at 775 CE. These strong events serve as accurate time markers for the synchronization with floating tree-ring and ice core records and provide critical information on the previous occurrence of extreme solar events which may threaten modern infrastructure., (© 2022. The Author(s).)

Direct and accurate radiocarbon dating of lipid residues preserved in ceramics is a recently established method that allows direct dating of specific food products and their inception in human subsistence strategies. The method targets individual fatty acids originating from animal fats such as ruminant dairy, ruminant adipose, non-ruminant adipose and aquatic fats. Horse lipid residues found in Central Asian pottery vessels are also directly dateable using this new method. Here we present the identification of equine lipid residues preserved in two pottery assemblages from the Neolithic and Eneolithic in Kazakhstan and their direct 14 C dating. The site of Botai, previously radiocarbon-dated to the 4th millennium BC, was used as a reference to evaluate the dates obtained directly on horse lipids. The direct dating of equine products extracted from Botai potsherds are shown to be compatible with previous 14 C dates at the site. The site of Bestamak, lacking previous 14 C measurements, had been relatively dated to the Neolithic based on pottery typologies. The direct dating of equine residues made it possible to anchor the pottery assemblage of Bestamak in the 6th millennium BC confirming their Neolithic attribution. These findings demonstrate the potential for dating horse products through a compound-specific approach, while highlighting challenges in 14 C dating individual fatty acids from lipid extracts in which their abundances differ substantially., Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-022-01630-2., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2022.)

Purpose: Nitrogen (N) transfer from white clover ( Trifolium repens cv.) to ryegrass ( Lolium perenne cv.) has the potential to meet ryegrass N requirements. This study aimed to quantify N transfer in a mixed pasture and investigate the influence of the microbial community and land management on N transfer., Methods: Split root 15 N-labelling of clover quantified N transfer to ryegrass via exudation, microbial assimilation, decomposition, defoliation and soil biota. Incorporation into the microbial protein pool was determined using compound-specific 15 N-stable isotope probing approaches., Results: N transfer to ryegrass and soil microbial protein in the model system was relatively small, with one-third arising from root exudation. N transfer to ryegrass increased with no microbial competition but soil microbes also increased N transfer via shoot decomposition. Addition of mycorrhizal fungi did not alter N transfer, due to the source-sink nature of this pathway, whilst weevil grazing on roots decreased microbial N transfer. N transfer was bidirectional, and comparable on a short-term scale., Conclusions: N transfer was low in a model young pasture established from soil from a permanent grassland with long-term N fertilisation. Root exudation and decomposition were major N transfer pathways. N transfer was influenced by soil biota (weevils, mycorrhizae) and land management (e.g. grazing). Previous land management and the role of the microbial community in N transfer must be considered when determining the potential for N transfer to ryegrass., Supplementary Information: The online version contains supplementary material available at 10.1007/s11104-022-05585-0., Competing Interests: Conflicts of interestWe have no conflicts of interest nor competing interests., (© The Author(s) 2022.)

The Lake Titicaca basin was one of the major centers for cultural development in the ancient world. This lacustrine environment is unique in the high, dry Andean altiplano , and its aquatic and terrestrial resources are thought to have contributed to the florescence of complex societies in this region. Nevertheless, it remains unclear to what extent local aquatic resources, particularly fish, and the introduced crop, maize, which can be grown in regions along the lakeshores, contributed to facilitating sustained food production and population growth, which underpinned increasing social political complexity starting in the Formative Period (1400 BCE to 500 CE) and culminating with the Tiwanaku state (500 to 1100 CE). Here, we present direct dietary evidence from stable isotope analysis of human skeletal remains spanning over two millennia, together with faunal and floral reference materials, to reconstruct foodways and ecological interactions in southern Lake Titicaca over time. Bulk stable isotope analysis, coupled with compound-specific amino acid stable isotope analysis, allows better discrimination between resources consumed across aquatic and terrestrial environments. Together, this evidence demonstrates that human diets predominantly relied on C 3 plants, particularly quinoa and tubers, along with terrestrial animals, notably domestic camelids. Surprisingly, fish were not a significant source of animal protein, but a slight increase in C 4 plant consumption verifies the increasing importance of maize in the Middle Horizon. These results underscore the primary role of local terrestrial food resources in securing a nutritious diet that allowed for sustained population growth, even in the face of documented climate and political change across these periods., Competing Interests: The authors declare no competing interest.

Small ceramic vessels with spouts, from which liquid can be poured, became popular during the Late Bronze and Early Iron Ages in Central Europe ( c . 1200-600 BC). Such feeding vessels represent a functional type and are highly variable in size, shape and decoration. Found both on settlements and within graves, their association with child burials suggest they might have been used to feed babies and small children. Combined lipid and isotope analysis was performed on 24 of these feeding vessels, with seven delivering interpretable results. Feeding vessels associated with child burials tend to deliver a ruminant milk signal, whereas other vessels were used to process ruminant and non-ruminant adipose fats. Here, we highlight the potential significance of feeding vessels as indicators of changing childcare practices during times of population increase, settlement nucleation and mobility, possibly involving out-sourcing the feeding of babies and small children to persons other than the mother., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Honey and other bee products were likely a sought-after foodstuff for much of human history, with direct chemical evidence for beeswax identified in prehistoric ceramic vessels from Europe, the Near East and Mediterranean North Africa, from the 7 th millennium BC. Historical and ethnographic literature from across Africa suggests bee products, honey and larvae, had considerable importance both as a food source and in the making of honey-based drinks. Here, to investigate this, we carry out lipid residue analysis of 458 prehistoric pottery vessels from the Nok culture, Nigeria, West Africa, an area where early farmers and foragers co-existed. We report complex lipid distributions, comprising n-alkanes, n-alkanoic acids and fatty acyl wax esters, which provide direct chemical evidence of bee product exploitation and processing, likely including honey-collecting, in over one third of lipid-yielding Nok ceramic vessels. These findings highlight the probable importance of honey collecting in an early farming context, around 3500 years ago, in West Africa.

Nitrogen (N) stable isotope techniques are widely used in ecology, archaeology, and forensic science to explore trophic relationships and provenances of organisms and materials, most widely using bulk δ 15 N values of whole organisms, tissues, or other materials. However, compound-specific isotope values can provide more diagnostic isotope "fingerprints" and specific information about metabolic processes. Existing techniques for nitrogen isotope analysis allow the determination of δ 15 N values of 14 amino acids (AAs), accounting for ca. 75% of plant protein and collagen N. The majority of remaining N is from arginine, comprising 16 and 14% of collagen and plant protein N, respectively. We therefore aimed to develop a method to detect arginine and determine its δ 15 N value (δ 15 N Arg ) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), to further contribute to the understanding of the metabolic routing of this important AA. We demonstrate that arginine, as its N -acetyl isopropyl ester, is amenable to GC analysis using a 15 m midpolarity DB-35 column, eluting with baseline resolution from other AAs. The recorded δ 15 N value by GC-C-IRMS was within the error of that of the underivatized compound determined by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). The newly developed GC-C-IRMS method was applied to modern plant protein and cattle collagen, enabling their δ 15 N Arg values to be related to AA biosynthesis. Determination of archaeological cattle collagen δ 15 N Arg values confirmed the suitability of this method to provide further insights into past diets and ecosystems. Bulk collagen δ 15 N value reconstruction including δ 15 N Arg values better reflect the measured bulk values, as the isotopic ratio of 91% of collagen N can now be determined at the compound-specific level.

Rationale: Anthropogenic organic inputs to freshwaters can exert detrimental effects on aquatic ecosystems, raising growing concern for both environmental conservation and water security. Current regulation by the EU water framework directive (European Union, 2000/60/EC) relates to organic pollution by monitoring selected micropollutants; however, aquatic ecosystem responses require a comprehensive understanding of dissolved organic matter (DOM) composition. The introduction of high-resolution mass spectrometry (HRMS) is set to greatly increase our understanding of the composition of DOM of both natural and anthropogenic origin derived from diffuse and point sources., Methods: DOM was extracted from riverine and treated sewage effluent using solid-phase extraction (SPE) and analysed using dissolved organic carbon analysis, direct-infusion high-resolution mass spectrometry (DI-HRMS) and high-performance liquid chromatography (HPLC)/HRMS. The data obtained were analysed using univariate and multivariate statistics to demonstrate differences in background DOM, anthropogenic inputs and in-river mixing. Compound identifications were achieved based on MS 2 spectra searched against on-line databases., Results: DI-HRMS spectra showed the highly complex nature of all DOM SPE extracts. Classification and visualisation of extracts containing many thousands of individual compounds were achieved using principal component analysis (PCA) and hierarchical cluster analysis. Kruskal-Wallis analyses highlighted significant discriminating ions originating from the sewage treatment works for more in-depth investigation by HPLC/HRMS. The generation of MS 2 spectra in HPLC/HRMS provided the basis for identification of anthropogenic compounds including; pharmaceuticals, illicit drugs, metabolites and oligomers, although many thousands of compounds remain unidentified., Conclusions: This new approach enables comprehensive analysis of DOM in extracts without any preconceived ideas of the compounds which may be present. This approach has the potential to be used as a high throughput, qualitative, screening method to determine if the composition of point sources differs from that of the receiving water bodies, providing a new approach to the identification of hitherto unrecognised organic contribution to water bodies., (© 2019 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Chemical analyses of carbonized and absorbed organic residues from archaeological ceramic cooking vessels can provide a unique window into the culinary cultures of ancient people, resource use, and environmental effects by identifying ingredients used in ancient meals. However, it remains uncertain whether recovered organic residues represent only the final foodstuffs prepared or are the accumulation of various cooking events within the same vessel. To assess this, we cooked seven mixtures of C 3 and C 4 foodstuffs in unglazed pots once per week for one year, then changed recipes between pots for the final cooking events. We conducted bulk stable-isotope analysis and lipid residue analysis on the charred food macro-remains, carbonized thin layer organic patina residues and absorbed lipids over the course of the experiment. Our results indicate that: (1) the composition of charred macro-remains represent the final foodstuffs cooked within vessels, (2) thin-layer patina residues represent a mixture of previous cooking events with bias towards the final product(s) cooked in the pot, and (3) absorbed lipid residues are developed over a number of cooking events and are replaced slowly over time, with little evidence of the final recipe ingredients.

The application of biomolecular techniques to archaeological materials from the Balkans is providing valuable new information on the prehistory of the region. This is especially relevant for the study of the neolithisation process in SE Europe, which gradually affected the rest of the continent. Here, to answer questions regarding diet and subsistence practices in early farming societies in the central Balkans, we combine organic residue analyses of archaeological pottery, taxonomic and isotopic study of domestic animal remains and biomolecular analyses of human dental calculus. The results from the analyses of the lipid residues from pottery suggest that milk was processed in ceramic vessels. Dairy products were shown to be part of the subsistence strategies of the earliest Neolithic communities in the region but were of varying importance in different areas of the Balkan. Conversely, milk proteins were not detected within the dental calculus. The molecular and isotopic identification of meat, dairy, plants and beeswax in the pottery lipids also provided insights into the diversity of diet in these early Neolithic communities, mainly based on terrestrial resources. We also present the first compound-specific radiocarbon dates for the region, obtained directly from absorbed organic residues extracted from pottery, identified as dairy lipids., Competing Interests: The authors have declared that no competing interests exist.

The development of pastoralism transformed human diets and societies in grasslands worldwide. The long-term success of cattle herding in Africa has been sustained by dynamic food systems, consumption of a broad range of primary and secondary livestock products, and the evolution of lactase persistence (LP), which allows digestion of lactose into adulthood and enables the milk-based, high-protein, low-calorie diets characteristic of contemporary pastoralists. Despite the presence of multiple alleles associated with LP in ancient and present-day eastern African populations, the contexts for selection for LP and the long-term development of pastoralist foodways in this region remain unclear. Pastoral Neolithic ( c 5000 to 1200 BP) faunas indicate that herders relied on cattle, sheep, and goats and some hunting, but direct information on milk consumption, plant use, and broader culinary patterns is rare. Combined chemical and isotopic analysis of ceramic sherds ( n = 125) from Pastoral Neolithic archaeological contexts in Kenya and Tanzania, using compound-specific δ 13 C and Δ 13 C values of the major fatty acids, provides chemical evidence for milk, meat, and plant processing by ancient herding societies in eastern Africa. These data provide the earliest direct evidence for milk product consumption and reveal a history of reliance on animal products and other nutrients, likely extracted through soups or stews, and plant foods. They document a 5,000-y temporal framework for eastern Africa pastoralist cuisines and cultural contexts for selection for alleles distinctive of LP in eastern Africa., Competing Interests: The authors declare no competing interest.

Myo-inositol hexakisphosphate, or phytic acid, (myo-IP6) is a key organic phosphorus (P) compound in soils and manures. Determinations of myo-IP6 in soils and manure extracts are frequently performed by 31 P NMR spectroscopy. This approach is time-consuming in terms of both sample preparation and instrument time, with uncertainties existing in relation to accuracy of identification and quantification due to potentially interfering resonances from co-extracted P species. In contrast, ion chromatography (IC) in combination with high-resolution mass spectrometry (HRMS) negative ion, electrospray ionisation (ESI) has been shown to enable highly specific identifications of myo-IP6 isolated from complex mixtures. In this paper, IC and ESI-HRMS were applied to the identification and the quantification of myo-IP6 isolated from soils and manures using NaOH-EDTA extraction, and quantifications based on IC. ESI-HRMS analysis of eluate trapped from IC unequivocally confirmed identification of myo-IP6 from a soil extract. The ion suppression cell of the IC instrument provides isolates of the analyte free of ionic components that would interfere with ESI. The myo-IP6 was identified in the NMR by comparing spectra of extracts of soils with and without authentic myo-IP6 "spiked" prior to extraction. Comparison of quantification via standard addition in IC and NMR analysis gave good correlation (r = 0.955). IC with ESI-HRMS was found to be more sensitive, rapid and reliable for the identification and quantification of myo-IP6 with a limit of detection (LOD) of 0.7 mg kg -1 and limit of quantification (LOQ) of 2.1 mg kg -1 using IC versus > 10 mg kg -1 LOD using 31 P NMR., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pottery is one of the most commonly recovered artefacts from archaeological sites. Despite more than a century of relative dating based on typology and seriation 1 , accurate dating of pottery using the radiocarbon dating method has proven extremely challenging owing to the limited survival of organic temper and unreliability of visible residues 2-4 . Here we report a method to directly date archaeological pottery based on accelerator mass spectrometry analysis of 14 C in absorbed food residues using palmitic (C 16:0 ) and stearic (C 18:0 ) fatty acids purified by preparative gas chromatography 5-8 . We present accurate compound-specific radiocarbon determinations of lipids extracted from pottery vessels, which were rigorously evaluated by comparison with dendrochronological dates 9,10 and inclusion in site and regional chronologies that contained previously determined radiocarbon dates on other materials 11-15 . Notably, the compound-specific dates from each of the C 16:0 and C 18:0 fatty acids in pottery vessels provide an internal quality control of the results 6 and are entirely compatible with dates for other commonly dated materials. Accurate radiocarbon dating of pottery vessels can reveal: (1) the period of use of pottery; (2) the antiquity of organic residues, including when specific foodstuffs were exploited; (3) the chronology of sites in the absence of traditionally datable materials; and (4) direct verification of pottery typochronologies. Here we used the method to date the exploitation of dairy and carcass products in Neolithic vessels from Britain, Anatolia, central and western Europe, and Saharan Africa.

The study of childhood diet, including breastfeeding and weaning, has important implications for our understanding of infant mortality and fertility in past societies 1 . Stable isotope analyses of nitrogen from bone collagen and dentine samples of infants have provided information on the timing of weaning 2 ; however, little is known about which foods were consumed by infants in prehistory. The earliest known clay vessels that were possibly used for feeding infants appear in Neolithic Europe, and become more common throughout the Bronze and Iron Ages. However, these vessels-which include a spout through which liquid could be poured-have also been suggested to be feeding vessels for the sick or infirm 3,4 . Here we report evidence for the foods that were contained in such vessels, based on analyses of the lipid 'fingerprints' and the compound-specific δ 13 C and Δ 13 C values of the major fatty acids of residues from three small, spouted vessels that were found in Bronze and Iron Age graves of infants in Bavaria. The results suggest that the vessels were used to feed infants with milk products derived from ruminants. This evidence of the foodstuffs that were used to either feed or wean prehistoric infants confirms the importance of milk from domesticated animals for these early communities, and provides information on the infant-feeding behaviours that were practised by prehistoric human groups.

Amino acid δ 15 N values of foliage of various plant taxa, grown at the experimental farm stations of North Wyke, UK and Bad Lauchstädt, Germany were determined by GC-C-IRMS. The difference between δ 15 N values of glutamate (Glx) and phenylalanine (Phe) were found to differ significantly between woody and herbaceous plants, with mean Δ 15 N Glx-Phe (i.e. δ 15 N Phe - δ 15 N Glx ) values of -9.3 ± 1.6‰ and -5.8 ± 2.1‰, respectively. These differences in values are hypothesised to be due to the involvement of Phe in the phenylpropanoid pathway, by which lignin and other phenolic secondary metabolites are produced, leading to isotopic fractionation and enrichment of the remaining Phe pool available for protein biosynthesis. This results in the more negative Δ 15 N Glx-Phe values observed in woody plants relative to herbaceous plants, as the former are assumed to produce more lignin. To test this assumption, plant leaf tissue lignin concentrations were estimated by solid state 13 C cross-polarisation, magic-angle-spinning (CPMAS) NMR spectroscopy for a subset of plants, which showed that tree foliage has a higher concentration of lignin (12.6 wt%) than herbaceous foliage (6.3 wt%). The correlation of lignin concentration with Δ 15 N Glx-Phe values demonstrates that the difference in these values with plant type is indeed due to differential production of lignin. The ability to estimate the lignin content of plants from amino acid δ 15 N values will, to give one example, allow refinement of estimates of herbivore diet in present and past ecosystems, enabling more accurate environmental niche modelling., (Copyright © 2019. Published by Elsevier Ltd.)

Bog butters are large white or yellow waxy deposits regularly discovered within the peat bogs of Ireland and Scotland. They represent an extraordinary survival of prehistoric and later agricultural products, comprising the largest deposits of fat found anywhere in nature. Often found in wooden containers or wrapped in animal bladders, they are considered to have been buried intentionally by past farming communities. While previous analysis has determined that Irish bog butters derive from animal fat, their precise characterisation could not be achieved due to diagenetic compositional alterations during burial. Via compound-specific stable isotope analysis, we provide the first conclusive evidence of a dairy fat origin for the Irish bog butter tradition, which differs from bog butter traditions observed elsewhere. Our research also reveals a remarkably long-lived tradition of deposition and possible curation spanning at least 3500 years, from the Early Bronze Age (c. 1700 BC) to the 17 th century AD. This is conclusively established via an extensive suite of both bulk and compound-specific radiocarbon dates.

Amino sugars can be used as indices to evaluate the role of soil microorganisms in active nitrogen (N) cycling in soil. This paper details the assessment of the suitability of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the analysis of 15 N-enriched amino sugars as alditol acetate derivatives prior to application of a novel 15 N stable isotope probing (SIP) approach to amino sugars. The efficient derivatization and cleanup of alditol acetate derivatives for GC was achieved using commercially available amino sugars, including glucosamine, mannosamine, galactosamine, and muramic acid, as laboratory standards. A VF-23ms stationary phase was found to produce optimal separations of all four compounds. The structure of the alditol acetate derivatives was confirmed using gas chromatography/mass spectrometry (GC/MS). For GC-C-IRMS determinations, implementation of a two-point normalization confirmed the optimal carrier gas flow rate to be 1.7 mL min -1 . Linearity of δ 15 N value determinations up to δ 15 N t of 469 ± 3.1‰ (where δ 15 N t is the independently measured δ 15 N value) was confirmed when 30 nmol N was injected on-column, with the direction of deviation from δ 15 N t at low sample amount dependent on the 15 N abundance of the analyte. Observed between- and within-run memory effects were significant ( P < 0.007) when a highly enriched standard (469 ± 3.1‰) was run; therefore, analytical run order and variation in 15 N enrichment of analytes within the same sample must be considered. The investigated parameters have confirmed the isotopic robustness of alditol acetate derivatives of amino sugars for the GC-C-IRMS analysis of 15 N-enriched amino sugars in terms of linearity over an enrichment range (natural abundance to 469 ± 3.1‰) with on-column analyte amount over 30 nmol N.

The spread of early farming across Europe from its origins in Southwest Asia was a culturally transformative process which took place over millennia. Within regions, the pace of the transition was probably related to the particular climatic and environmental conditions encountered, as well as the nature of localized hunter-gatherer and farmer interactions. The establishment of farming in the interior of the Balkans represents the first movement of Southwest Asian livestock beyond their natural climatic range, and widespread evidence now exists for early pottery being used extensively for dairying. However, pottery lipid residues from sites in the Iron Gates region of the Danube in the northern Balkans show that here, Neolithic pottery was being used predominantly for processing aquatic resources. This stands out not only within the surrounding region but also contrasts markedly with Neolithic pottery use across wider Europe. These findings provide evidence for the strategic diversity within the wider cultural and economic practices during the Neolithic, with this exceptional environmental and cultural setting offering alternative opportunities despite the dominance of farming in the wider region.

Preparative capillary gas chromatography (pcGC) is widely used for the isolation of single compounds for radiocarbon determinations. While being effective at isolating compounds, there are still genuine concerns relating to contamination associated with the isolation procedure, such as incomplete removal of solvent used to recover isolated compounds from the traps and cross-contamination, which can lead to erroneous 14 C determinations. Herein we describe new approaches to identifying and removing these two sources of contamination. First, we replaced the common "U" trap design, which requires recovery of compounds using organic solvent, with a novel solventless trapping system (STS), consisting of a simple glass tube containing a glass wool plug, allowing condensation of a target compound in the wool and its solventless recovery by pushing the glass wool directly into a foil capsule for graphitization. With the STS trap, an average of 95.7% of the target compound was recovered, and contamination from column bleed was reduced. In addition, comparison of 14 C determinations of fatty acid methyl ester (FAME) standards determined offline to those isolated by pcGC in STS traps showed excellent reproducibility and accuracy compared to those isolated using the commercial "U" traps. Second, "coldspots" were identified in the instrument, i.e., the termini of capillaries in the preparative unit, which can be cleaned of compounds condensed from earlier runs using a heat gun. Our new procedure, incorporating these two modifications, was tested on archeological fat hoards, producing 14 C dates on isolated C 16:0 and C 18:0 fatty acids statistically consistent with the bulk dates of the archeological material.

The 8.2-thousand years B.P. event is evident in multiple proxy records across the globe, showing generally dry and cold conditions for ca. 160 years. Environmental changes around the event are mainly detected using geochemical or palynological analyses of ice cores, lacustrine, marine, and other sediments often distant from human settlements. The Late Neolithic excavated area of the archaeological site of Çatalhöyük East [Team Poznań (TP) area] was occupied for four centuries in the ninth and eighth millennia B.P., thus encompassing the 8.2-thousand years B.P. climatic event. A Bayesian analysis of 56 radiocarbon dates yielded a high-resolution chronological model comprising six building phases, with dates ranging from before 8325-8205 to 7925-7815 calibrated years (cal) B.P. Here, we correlate an onsite paleoclimate record constructed from δ 2 H values of lipid biomarkers preserved in pottery vessels recovered from these buildings with changes in architectural, archaeozoological, and consumption records from well-documented archaeological contexts. The overall sequence shows major changes in husbandry and consumption practices at ca. 8.2 thousand years B.P., synchronous with variations in the δ 2 H values of the animal fat residues. Changes in paleoclimate and archaeological records seem connected with the patterns of atmospheric precipitation during the occupation of the TP area predicted by climate modeling. Our multiproxy approach uses records derived directly from documented archaeological contexts. Through this, we provide compelling evidence for the specific impacts of the 8.2-thousand years B.P. climatic event on the economic and domestic activities of pioneer Neolithic farmers, influencing decisions relating to settlement planning and food procurement strategies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Over the past three decades, studies of ancient biomolecules-particularly ancient DNA, proteins, and lipids-have revolutionized our understanding of evolutionary history. Though initially fraught with many challenges, today the field stands on firm foundations. Researchers now successfully retrieve nucleotide and amino acid sequences, as well as lipid signatures, from progressively older samples, originating from geographic areas and depositional environments that, until recently, were regarded as hostile to long-term preservation of biomolecules. Sampling frequencies and the spatial and temporal scope of studies have also increased markedly, and with them the size and quality of the data sets generated. This progress has been made possible by continuous technical innovations in analytical methods, enhanced criteria for the selection of ancient samples, integrated experimental methods, and advanced computational approaches. Here, we discuss the history and current state of ancient biomolecule research, its applications to evolutionary inference, and future directions for this young and exciting field.

The nature and extent to which hydrological changes induced by the Asian summer monsoon affected key biogeochemical processes remain poorly defined. This study explores the relationship between peatland drying and carbon cycling on centennial timescales in central China using lipid biomarkers. The difference between peat n-alkane δ 2 H and a nearby stalagmite δ 18 O record reveals that intervals of prominent peatland drying occurred during the mid-Holocene. Synchronous with these drier conditions, leaf wax δ 13 C values show large negative excursions, with the utilization of CO 2 respired from the peatland subsurface for plant photosynthesis being a possible mechanism. Crucially, successive drying events appear to have had a cumulative impact on the susceptibility of peat carbon stores to climate change. Concurrently, bacterially derived hopane δ 13 C values suggest the occurrence of enhanced methane oxidation during the drier periods. Collectively, these observations expand our understanding of how respiration and degradation of peat are enhanced during drying events.

Current archaeological evidence indicates the transition from hunting-fishing-gathering to agriculture in Northern Europe was a gradual process. This transition was especially complex in the prehistoric North Fennoscandian landscape where the high latitude posed a challenge to both domestic animal breeding and cereal cultivation. The conditions varied, the coastal dwellers had access to rich marine resources and enjoyed a milder climate due to the Gulf Stream, while those living in the inland Boreal forest zone faced longer and colder winters and less diversity in animal and plant resources. Thus, the coastal area provided more favourable conditions for early agriculture compared to those found inland. Interestingly, a cultural differentiation between these areas is archaeologically visible from the late 2 nd millennium BC onwards. This is most clearly seen in regionally distinct pottery styles, offering unique opportunities to probe diet and subsistence through the organic residues preserved in ceramic vessels. Herein, we integrate the lipid biomarker, compound-specific stable carbon isotopes (δ 13 C), and zooarchaeological evidence to reveal culturally distinct human diets and subsistence patterns. In northern Norway, some of the coastal people adopted dairying as part of their subsistence strategy, while the inhabitants of the interior, in common with northern Finland, continued their hunter-gatherer-fisher lifestyles.

Rationale: The phosphorus storage compound in grains, phytic acid, or myo-inositol hexakisphosphate (IP6), is important for nutrition and human health, and is reportedly the most abundant organic phosphorus compound in soils. Methods for its determination have traditionally relied on complexation with iron and precipitation, acid digestion and measurement of phosphate concentration, or 31 P NMR spectroscopy. Direct determination of phytic acid (and its homologues) using mass spectrometry has, as yet, found limited application to environmental or other complex matrices. The behaviour of phytic acid in electrospray ionisation high-resolution mass spectrometry (ESI-HRMS) and its fragmentation, both in-source and via collision-induced dissociation, have not been studied so far., Methods: The negative ion mass spectrometry and tandem mass spectrometry (MS/MS) of IP6, and the lower inositol pentakisphosphate (IP5), using an ESI-Orbitrap mass spectrometer is described. The purity of the compounds was investigated using anion-exchange chromatography., Results: IP6 is highly anionic, forming multiply charged ions and sodium adduct ions, which readily undergo dissociation in the ESI source. MS/MS analysis of the phytic acid [M-2H] 2- ion and fragment ions and comparison with the full MS of the IP5 reference standard, and the MS/MS spectrum of the pentakisphosphate [M-2H] 2- ion, confirm the fragmentation pattern of inositol phosphates in ESI. Further evidence for dissociation in the ion source is shown by the effect of increasing the source voltage on the mass spectrum of phytic acid., Conclusions: The ESI-HRMS of inositol phosphates is unusual and highly characteristic. The study of the full mass spectrum of IP6 in ESI-HRMS mode indicates the detection of the compound in environmental matrices using this technique is preferable to the use of multiple reaction monitoring (MRM)., (© 2017 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.)

Since their domestication in the Mediterranean zone of Southwest Asia in the eighth millennium BC, sheep, goats, pigs and cattle have been remarkably successful in colonizing a broad variety of environments. The initial steps in this process can be traced back to the dispersal of farming groups into the interior of the Balkans in the early sixth millennium BC, who were the first to introduce Mediterranean livestock beyond its natural climatic range. Here, we combine analysis of biomolecular and isotopic compositions of lipids preserved in prehistoric pottery with faunal analyses of taxonomic composition from the earliest farming sites in southeast Europe to reconstruct this pivotal event in the early history of animal husbandry. We observe a marked divergence between the (sub)Mediterranean and temperate regions of Southeast Europe, and in particular a significant increase of dairying in the biochemical record coupled with a shift to cattle and wild fauna at most sites north of the Balkan mountain range. The findings strongly suggest that dairying was crucial for the expansion of the earliest farming system beyond its native bioclimatic zone.

Preparative capillary gas chromatography (PCGC) is the central technique used for the purification of volatile or semivolatile organic compounds for radiocarbon analysis using accelerator mass spectrometry (AMS). While thicker film columns offer efficient separations, cyclic poly(dimethylsiloxanes) (PDMS) derived from the column's stationary phase have been highlighted as a potential source of contaminant carbon in "trapped" compounds. The PDMS CH 3 groups are of "infinite" radiocarbon age due to the fossil carbon origin of the feedstock used in production. Hence, column bleed, if present at sufficiently high concentrations, would shift the radiocarbon ages of trapped compounds to older ages. Quantification of the column bleed in trapped samples, however, is extremely challenging and up to now has only been achieved through indirect 14 C determinations of chromatographic blanks, which are used for post 14 C determination "corrections". As part of wider investigations aimed at better understanding the chemical nature of contamination in compound-specific 14 C determinations, herein, we report a rigorous approach to column bleed identification and quantification. Using reference fatty acid methyl esters (FAMEs), 1 H nuclear magnetic resonance spectroscopy (NMR), employing a 700 MHz instrument equipped with a 1.7 mm microcryoprobe optimized for 1 H observation, was able to detect low submicrogram amounts of low molecular weight compounds (<500 Da). Direct quantification of PCGC "trapped" FAMEs was achieved based on the recorded 1 H NMR spectra. Gravimetrically prepared calibration mixtures of cyclic PMDSs and FAMEs showed column bleed abundance to be below 0.03% w/w of the "trapped" FAMEs, which would lead to a maximum shift in radiocarbon age of <3 years toward older values. We therefore conclude that column bleed contamination has a negligible effect on the 14 C determination of FAMEs prepared using the chromatographic method described. The 1 H NMR analysis also revealed the absence of other protonated carbon-containing components that would affect radiocarbon determinations at the precisions achievable by AMS.

The invention of thermally resistant ceramic cooking vessels around 15,000 years ago was a major advance in human diet and nutrition 1-3 , opening up new food groups and preparation techniques. Previous investigations of lipid biomarkers contained in food residues have routinely demonstrated the importance of prehistoric cooking pots for the processing of animal products across the world 4 . Remarkably, however, direct evidence for plant processing in prehistoric pottery has not been forthcoming, despite the potential to cook otherwise unpalatable or even toxic plants 2,5 . In North Africa, archaeobotanical evidence of charred and desiccated plant organs denotes that Early Holocene hunter-gatherers routinely exploited a wide range of plant resources 6 . Here, we reveal the earliest direct evidence for plant processing in pottery globally, from the sites of Takarkori and Uan Afuda in the Libyan Sahara, dated to 8200-6400 bc. Characteristic carbon number distributions and δ 13 C values for plant wax-derived n-alkanes and alkanoic acids indicate sustained and systematic processing of C 3 /C 4 grasses and aquatic plants, gathered from the savannahs and lakes in the Early to Middle Holocene green Sahara.

In the absence of any direct evidence, the relative importance of meat and dairy productions to Neolithic prehistoric Mediterranean communities has been extensively debated. Here, we combine lipid residue analysis of ceramic vessels with osteo-archaeological age-at-death analysis from 82 northern Mediterranean and Near Eastern sites dating from the seventh to fifth millennia BC to address this question. The findings show variable intensities in dairy and nondairy activities in the Mediterranean region with the slaughter profiles of domesticated ruminants mirroring the results of the organic residue analyses. The finding of milk residues in very early Neolithic pottery (seventh millennium BC) from both the east and west of the region contrasts with much lower intensities in sites of northern Greece, where pig bones are present in higher frequencies compared with other locations. In this region, the slaughter profiles of all domesticated ruminants suggest meat production predominated. Overall, it appears that milk or the by-products of milk was an important foodstuff, which may have contributed significantly to the spread of these cultural groups by providing a nourishing and sustainable product for early farming communities., Competing Interests: The authors declare no conflict of interest.

Mummification was practised in ancient Egypt for more than 3000 years, emerging from initial observations of buried bodies preserved by natural desiccation. The use of organic balms (and other funerary practices) was a later introduction necessitated by more humid burial environments, especially tombs. The dark colour of many mummies led to the assumption that petroleum bitumen (or natural asphalt) was ubiquitous in mummification; however, this has been questioned for more than 100 years. We test this by investigating 91 materials comprising balms, tissues and textiles from 39 mummies dating from ca 3200 BC to AD 395. Targeted petroleum bitumen biomarker (steranes and hopanes) analyses by gas chromatography-mass spectrometry selected ion monitoring (GC-MS SIM, m/z 217 and 191) showed no detectable bitumen use before the New Kingdom (ca 1550-1070 BC). However, bitumen was used in 50% of New Kingdom to Late Period mummies, rising to 87% of Ptolemaic/Roman Period mummies. Quantitative determinations using (14)C analyses reveal that even at peak use balms were never more than 45% w/w bitumen. Critically, the dark colour of balms can be simulated by heating/ageing mixtures of fats, resins and beeswax known to be used in balms. The application of black/dark brown balms to bodies was deliberate after the New Kingdom reflecting changing funerary beliefs and shifts in religious ideology.This article is part of the themed issue 'Quantitative mass spectrometry'., (© 2016 The Authors.)

Biodiesel Co-Product (BCP) is a complex organic material formed during the transesterification of lipids. We investigated the effect of BCP on the extracellular microbial matrix or 'extracellular polymeric substance' (EPS) in soil which is suspected to be a highly influential fraction of soil organic matter (SOM). It was hypothesised that more N would be transferred to EPS in soil given BCP compared to soil given glycerol. An arable soil was amended with BCP produced from either 1) waste vegetable oils or 2) pure oilseed rape oil, and compared with soil amended with 99% pure glycerol; all were provided with 15 N labelled KNO 3 . We compared transfer of microbially assimilated 15 N into the extracellular amino acid pool, and measured concomitant production of exopolysaccharide. Following incubation, the 15 N enrichment of total hydrolysable amino acids (THAAs) indicated that intracellular anabolic products had incorporated the labelled N primarily as glutamine and glutamate. A greater proportion of the amino acids in EPS were found to contain 15 N than those in the THAA pool, indicating that the increase in EPS was comprised of bioproducts synthesised de novo. Moreover, BCP had increased the EPS production efficiency of the soil microbial community (μg EPS per unit ATP) up to approximately double that of glycerol, and caused transfer of 21% more 15 N from soil solution into EPS-amino acids. Given the suspected value of EPS in agricultural soils, the use of BCP to stimulate exudation is an interesting tool to consider in the theme of delivering sustainable intensification.

The pressures on honeybee (Apis mellifera) populations, resulting from threats by modern pesticides, parasites, predators and diseases, have raised awareness of the economic importance and critical role this insect plays in agricultural societies across the globe. However, the association of humans with A. mellifera predates post-industrial-revolution agriculture, as evidenced by the widespread presence of ancient Egyptian bee iconography dating to the Old Kingdom (approximately 2400 BC). There are also indications of Stone Age people harvesting bee products; for example, honey hunting is interpreted from rock art in a prehistoric Holocene context and a beeswax find in a pre-agriculturalist site. However, when and where the regular association of A. mellifera with agriculturalists emerged is unknown. One of the major products of A. mellifera is beeswax, which is composed of a complex suite of lipids including n-alkanes, n-alkanoic acids and fatty acyl wax esters. The composition is highly constant as it is determined genetically through the insect's biochemistry. Thus, the chemical 'fingerprint' of beeswax provides a reliable basis for detecting this commodity in organic residues preserved at archaeological sites, which we now use to trace the exploitation by humans of A. mellifera temporally and spatially. Here we present secure identifications of beeswax in lipid residues preserved in pottery vessels of Neolithic Old World farmers. The geographical range of bee product exploitation is traced in Neolithic Europe, the Near East and North Africa, providing the palaeoecological range of honeybees during prehistory. Temporally, we demonstrate that bee products were exploited continuously, and probably extensively in some regions, at least from the seventh millennium cal BC, likely fulfilling a variety of technological and cultural functions. The close association of A. mellifera with Neolithic farming communities dates to the early onset of agriculture and may provide evidence for the beginnings of a domestication process.