Neolithic culinary traditions revealed by cereal, milk and meat lipids in pottery from Scottish crannogs.

Cereal cultivation in Britain dates back to ca. 4000 BCE, probably introduced by migrant farmers from continental Europe. Widespread evidence for livestock appears in the archaeozoological record, also reflected by ubiquitous dairy lipids in pottery organic residues. However, despite archaeobotanical evidence for domesticated plants (such as cereals), organic residue evidence has been near-absent. Our approach, targeting low-abundance cereal-specific markers, has now revealed evidence for cereals (indicating wheat) in Neolithic pottery from Scottish 'crannogs', dating to ca. 3600 - 3300 BCE. Their association with dairy products suggests cereals may have been regularly prepared together as a milk-based gruel. We also observed a strong association between the occurrence of dairy products and smaller-mouthed vessels. Here, we demonstrate that cereal-specific markers can survive in cooking pots for millennia, revealing the consumption of specific cereals (wheat) that are virtually absent from the archaeobotanical record for this region and illuminating culinary traditions among early farming communities.

Dairying, diseases and the evolution of lactase persistence in Europe.

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 years1. Although the selection of LP and the consumption of prehistoric milk must be linked, considerable uncertainty remains concerning their spatiotemporal configuration and specific interactions2,3. 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 Biobank4,5 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.

Interpreting ancient food practices: stable isotope and molecular analyses of visible and absorbed residues from a year-long cooking experiment.

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 C3 and C4 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.

Mechanisms of lipid preservation in archaeological clay ceramics revealed by mass spectrometry imaging.

Traces of lipids, absorbed and preserved for millennia within the inorganic matrix of ceramic vessels, act as molecular fossils and provide manifold information about past people's subsistence, diet, and rituals. It is widely assumed that lipids become preserved after adsorption into nano- to micrometer-sized pores, but to this day the distribution of these lipids in the ceramics was virtually unknown, which severely limits our understanding about the process of lipid preservation. Here we use secondary ion mass spectrometry (SIMS) imaging for direct in situ analysis of lipids absorbed in 700- to 2,000-y-old archaeological pottery. After sectioning from larger sherds, wall cross-sections of smaller fragments were used for SIMS analysis. Lipids were found in relatively large zones of 5- to 400-µm diameter, which does not support the notion of absorption only into individual nanometer-scale pores but indicates that more macroscopic structures in the ceramics are involved in lipid preservation as well. Furthermore, lipids were found concentrated on calcium carbonate inclusions in the ceramics, which suggests that precipitation of fatty acids as calcium salts is an important aspect of lipid preservation in archaeological samples. This has important implications for analytical methods based on extraction of lipids from archaeological ceramics and needs to be considered to maximize the yield and available information from each unique sample.

Chemical evidence of dairying by hunter-gatherers in highland Lesotho in the late first millennium AD.

The recovery of Early Iron Age artefacts and domestic animal remains from hunter-gatherer contexts at Likoaeng, Lesotho, has been argued to indicate contact between highland hunter-gatherers and Early Iron Age agropastoralist communities settled in lowland areas of southeastern Africa during the second half of the first millennium AD. However, disagreement between archaeozoological studies and ancient DNA means that the possibility that those hunter-gatherers kept livestock themselves remains controversial. Here we report analyses of pottery-absorbed organic residues from two hunter-gatherer sites and one agriculturalist site in highland Lesotho to reconstruct prehistoric subsistence practices. Our results demonstrate the exploitation of secondary products from domestic livestock by hunter-gatherers in Lesotho, directly dated to the seventh century AD at Likoaeng and the tenth century AD at the nearby site of Sehonghong. The data provide compelling evidence for the keeping of livestock by hunter-gatherer groups and their probable incorporation as ancillary resources into their subsistence strategies.

Digging deeper - A new data mining workflow for improved processing and interpretation of high resolution GC-Q-TOF MS data in archaeological research.

Gas chromatography-mass spectrometry profiling is the most established method for the analysis of organic residues, particularly lipids, from archaeological contexts. This technique allows the decryption of hidden chemical information associated with archaeological artefacts, such as ceramic pottery fragments. The molecular and isotopic compositions of such residues can be used to reconstruct past resource use, and hence address major questions relating to patterns of subsistence, diet and ritual practices in the past. A targeted data analysis approach, based on previous findings reported in the literature is common but greatly depends on the investigator's prior knowledge of specific compound classes and their mass spectrometric behaviour, and poses the risk of missing unknown, potentially diagnostic compounds. Organic residues from post-prehistoric archaeological samples often lead to highly complex chromatograms, which makes manual chromatogram inspection very tedious and time consuming, especially for large datasets. This poses a significant limitation regarding the scale and interpretative scopes of such projects. Therefore, we have developed a non-targeted data mining workflow to extract a higher number of known and unknown compounds from the raw data to reduce investigator's bias and to vastly accelerate overall analysis time. The workflow covers all steps from raw data handling, feature selection, and compound identification up to statistical interpretation.

Regional diversity in subsistence among early farmers in Southeast Europe revealed by archaeological organic residues.

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.

Lipid profiling and analytical discrimination of seven cereals using high temperature gas chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry.

Minor lipids in cereals (such as phytosterols and alkylresorcinols) can be important for human nutrition and/or be used as biomarkers for cereal intake. However, the analysis of cereal lipids is very challenging due to the complex lipidome comprising several hundred individual compounds present over a wide range of concentrations. Here we present a method for the profiling of cereal lipids using high temperature gas chromatography coupled to high resolution mass spectrometry (GC/Q-TOF MS). The method was used to investigate the lipid profiles of 77 samples of bread wheat, spelt, einkorn, emmer, barley, rye and oats. Distinct differences in the patterns of alkylresorcinols, free and conjugated sterols and tocopherols between the cereals could be observed. Furthermore, traces of tocomonoenols and diunsaturated and methyl-alkylresorcinols (not previously reported in cereals) could be detected. Finally, the lipid patterns in the cereals could be used to separate the cereals by Principal Component Analysis.

Widespread exploitation of the honeybee by early Neolithic farmers.

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.

Neolithic dairy farming at the extreme of agriculture in northern Europe.

The conventional 'Neolithic package' comprised animals and plants originally domesticated in the Near East. As farming spread on a generally northwest trajectory across Europe, early pastoralists would have been faced with the challenge of making farming viable in regions in which the organisms were poorly adapted to providing optimal yields or even surviving. Hence, it has long been debated whether Neolithic economies were ever established at the modern limits of agriculture. Here, we examine food residues in pottery, testing a hypothesis that Neolithic farming was practiced beyond the 60th parallel north. Our findings, based on diagnostic biomarker lipids and δ(13)C values of preserved fatty acids, reveal a transition at ca 2500 BC from the exploitation of aquatic organisms to processing of ruminant products, specifically milk, confirming farming was practiced at high latitudes. Combining this with genetic, environmental and archaeological information, we demonstrate the origins of dairying probably accompanied an incoming, genetically distinct, population successfully establishing this new subsistence 'package'.

Immediate replacement of fishing with dairying by the earliest farmers of the Northeast Atlantic archipelagos.

The appearance of farming, from its inception in the Near East around 12 000 years ago, finally reached the northwestern extremes of Europe by the fourth millennium BC or shortly thereafter. Various models have been invoked to explain the Neolithization of northern Europe; however, resolving these different scenarios has proved problematic due to poor faunal preservation and the lack of specificity achievable for commonly applied proxies. Here, we present new multi-proxy evidence, which qualitatively and quantitatively maps subsistence change in the northeast Atlantic archipelagos from the Late Mesolithic into the Neolithic and beyond. A model involving significant retention of hunter-gatherer-fisher influences was tested against one of the dominant adoptions of farming using a novel suite of lipid biomarkers, including dihydroxy fatty acids, ω-(o-alkylphenyl)alkanoic acids and stable carbon isotope signatures of individual fatty acids preserved in cooking vessels. These new findings, together with archaeozoological and human skeletal collagen bulk stable carbon isotope proxies, unequivocally confirm rejection of marine resources by early farmers coinciding with the adoption of intensive dairy farming. This pattern of Neolithization contrasts markedly to that occurring contemporaneously in the Baltic, suggesting that geographically distinct ecological and cultural influences dictated the evolution of subsistence practices at this critical phase of European prehistory.