A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa, Spain.

The characterization of Neandertals' diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ66Zn value observed in the Neandertal's tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ15N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ66Zn and highest δ15N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Exotic foods reveal contact between South Asia and the Near East during the second millennium BCE.

Although the key role of long-distance trade in the transformation of cuisines worldwide has been well-documented since at least the Roman era, the prehistory of the Eurasian food trade is less visible. In order to shed light on the transformation of Eastern Mediterranean cuisines during the Bronze Age and Early Iron Age, we analyzed microremains and proteins preserved in the dental calculus of individuals who lived during the second millennium BCE in the Southern Levant. Our results provide clear evidence for the consumption of expected staple foods, such as cereals (Triticeae), sesame (Sesamum), and dates (Phoenix). We additionally report evidence for the consumption of soybean (Glycine), probable banana (Musa), and turmeric (Curcuma), which pushes back the earliest evidence of these foods in the Mediterranean by centuries (turmeric) or even millennia (soybean). We find that, from the early second millennium onwards, at least some people in the Eastern Mediterranean had access to food from distant locations, including South Asia, and such goods were likely consumed as oils, dried fruits, and spices. These insights force us to rethink the complexity and intensity of Indo-Mediterranean trade during the Bronze Age as well as the degree of globalization in early Eastern Mediterranean cuisine.

The evolution and changing ecology of the African hominid oral microbiome.

The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine-platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

Dietary evidence from Central Asian Neanderthals: A combined isotope and plant microremains approach at Chagyrskaya Cave (Altai, Russia).

Neanderthals are known primarily from their habitation of Western Eurasia, but they also populated large expanses of Northern Asia for thousands of years. Owing to a sparse archaeological record, relatively little is known about these eastern Neanderthal populations. Unlike in their western range, there are limited zooarchaeological and paleobotanical studies that inform us about the nature of their subsistence. Here, we perform a combined analysis of carbon and nitrogen stable isotopes on bone collagen and microbotanical remains in dental calculus to reconstruct the diet of eastern Neanderthals at Chagyrskaya Cave in the Altai Mountains of Southern Siberia, Russia. Stable isotopes identify one individual as possessing a high trophic level due to the hunting of large- and medium-sized ungulates, while the analysis of dental calculus also indicates the presence of plants in the diet of this individual and others from the site. These findings indicate eastern Neanderthals may have had broadly similar subsistence patterns to those elsewhere in their range.

New insights on Neolithic food and mobility patterns in Mediterranean coastal populations.

OBJECTIVES: The aims of this research are to explore the diet, mobility, social organization, and environmental exploitation patterns of early Mediterranean farmers, particularly the role of marine and plant resources in these foodways. In addition, this work strives to document possible gendered patterns of behavior linked to the neolithization of this ecologically rich area. To achieve this, a set of multiproxy analyses (isotopic analyses, dental calculus, microremains analysis, ancient DNA) were performed on an exceptional deposit (n = 61) of human remains from the Les Bréguières site (France), dating to the transition of the sixth to the fifth millennium BCE. MATERIALS AND METHODS: The samples used in this study were excavated from the Les Bréguières site (Mougins, Alpes-Maritimes, France), located along the southeastern Mediterranean coastline of France. Stable isotope analyses (C, N) on bone collagen (17 coxal bones, 35 craniofacial elements) were performed as a means to infer protein intake during tissue development. Sulfur isotope ratios were used as indicators of geographical and environmental points of origin. The study of ancient dental calculus helped document the consumption of plants. Strontium isotope analysis on tooth enamel (n = 56) was conducted to infer human provenance and territorial mobility. Finally, ancient DNA analysis was performed to study maternal versus paternal diversity within this Neolithic group (n = 30). RESULTS: Stable isotope ratios for human bones range from -20.3 to -18.1‰ for C, from 8.9 to 11.1‰ for N and from 6.4 to 15‰ for S. Domestic animal data range from -22.0 to -20.2‰ for C, from 4.1 to 6.9‰ for N, and from 10.2 to 12.5‰ for S. Human enamel 87 Sr/86 Sr range from 0.7081 to 0.7102, slightly wider than the animal range (between 0.7087 and 0.7096). Starch and phytolith microremains were recovered as well as other types of remains (e.g., hairs, diatoms, fungal spores). Starch grains include Triticeae type and phytolith includes dicotyledons and monocot types as panicoid grasses. Mitochondrial DNA characterized eight different maternal lineages: H1, H3, HV (5.26%), J (10.53%), J1, K, T (5.2%), and U5 (10.53%) but no sample yielded reproducible Y chromosome SNPs, preventing paternal lineage characterization. DISCUSSION: Carbon and nitrogen stable isotope ratios indicate a consumption of protein by humans mainly focused on terrestrial animals and possible exploitation of marine resources for one male and one undetermined adult. Sulfur stable isotope ratios allowed distinguishing groups with different geographical origins, including two females possibly more exposed to the sea spray effect. While strontium isotope data do not indicate different origins for the individuals, mitochondrial lineage diversity from petrous bone DNA suggests the burial includes genetically differentiated groups or a group practicing patrilocality. Moreover, the diversity of plant microremains recorded in dental calculus provide the first evidence that the groups of Les Bréguières consumed a wide breadth of plant foods (as cereals and wild taxa) that required access to diverse environments. This transdisciplinary research paves the way for new perspectives and highlights the relevance for novel research of contexts (whether recently discovered or in museum collections) excavated near shorelines, due to the richness of the biodiversity and the wide range of edible resources available.

The efficacy of whole human genome capture on ancient dental calculus and dentin.

OBJECTIVES: Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. MATERIALS AND METHODS: Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in-solution hybridization capture and high-throughput sequencing. RESULTS: Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four-fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on-target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum-level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off-target sequences in human genome-enriched calculus libraries may still be useful for oral microbiome reconstruction. DISCUSSION: While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.

Dental calculus indicates widespread plant use within the stable Neanderthal dietary niche.

The ecology of Neanderthals is a pressing question in the study of hominin evolution. Diet appears to have played a prominent role in their adaptation to Eurasia. Based on isotope and zooarchaeological studies, Neanderthal diet has been reconstructed as heavily meat-based and generally similar across different environments. This image persists, despite recent studies suggesting more plant use and more variation. However, we have only a fragmentary picture of their dietary ecology, and how it may have varied among habitats, because we lack broad and environmentally representative information about their use of plants and other foods. To address the problem, we examined the plant microremains in Neanderthal dental calculus from five archaeological sites representing a variety of environments from the northern Balkans, and the western, central and eastern Mediterranean. The recovered microremains revealed the consumption of a variety of non-animal foods, including starchy plants. Using a modeling approach, we explored the relationships among microremains and environment, while controlling for chronology. In the process, we compared the effectiveness of various diversity metrics and their shortcomings for studying microbotanical remains, which are often morphologically redundant for identification. We developed Minimum Botanical Units as a new way of estimating how many plant types or parts are present in a microbotanical sample. In contrast to some previous work, we found no evidence that plant use is confined to the southern-most areas of Neanderthal distribution. Although interpreting the ecogeographic variation is limited by the incomplete preservation of dietary microremains, it is clear that plant exploitation was a widespread and deeply rooted Neanderthal subsistence strategy, even if they were predominately game hunters. Given the limited dietary variation across Neanderthal range in time and space in both plant and animal food exploitation, we argue that vegetal consumption was a feature of a generally static dietary niche.

To meat or not to meat? New perspectives on Neanderthal ecology.

Neanderthals have been commonly depicted as top predators who met their nutritional needs by focusing entirely on meat. This information mostly derives from faunal assemblage analyses and stable isotope studies: methods that tend to underestimate plant consumption and overestimate the intake of animal proteins. Several studies in fact demonstrate that there is a physiological limit to the amount of animal proteins that can be consumed: exceeding these values causes protein toxicity that can be particularly dangerous to pregnant women and newborns. Consequently, to avoid food poisoning from meat-based diets, Neanderthals must have incorporated alternative food sources in their daily diets, including plant materials as well.

Small-scale mobility fostering the interaction networks of Patagonian (Argentina) hunter-gatherers during the Late Holocene: Perspectives from strontium isotopes and exotic items.

During the Late Holocene, hunter-gatherer interaction networks significantly grew in intensity and extension across Patagonia. Although this growth is evidenced by the increased flow of exotic items across the region, the mechanisms behind these strengthening social networks remain unclear. Since evidence suggests that some individuals might have performed long-distance trips, this article aims to address the potential relationship between these individuals and the flows of exotic items in North Patagonia. We analyzed 54 enamel teeth for strontium isotopes and reconstructed their probable mobility using mixed-effect models and isotope-based geographic assignments. We inferred population and individual mobility trends and compared them against the flow of exotic items built from a standardized compilation. Our results indicate that most individuals have isotopic composition compatible with residence within their burial and surrounding areas. However, a few individuals show isotopic composition incompatible with their burial areas, which suggests axes -from the burial location to the most likely isotope integration area- of extraordinary mobility. At the same time, the flows of exotic items overlap with these axes around the eastern sector of the study area suggesting that this location could have been a central point of convergence for people and items. We argue that small-scale socially driven mobility could have played a relevant role as a general mechanism of interaction that fostered and materialized Patagonian interaction networks during the Late Holocene.

Understanding the microbial biogeography of ancient human dentitions to guide study design and interpretation.

The oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species in dental plaque, but it is not known how well these patterns are reflected in archaeological dental calculus. In most archaeological studies, a single sample of dental calculus is studied per individual and is assumed to represent the entire oral cavity. However, it is not known if this sampling strategy introduces biases into studies of the ancient oral microbiome. Here, we present the results of a shotgun metagenomic study of a dense sampling of dental calculus from four Chalcolithic individuals from the southeast Iberian peninsula (ca. 4500-5000 BP). Interindividual differences in microbial composition are found to be much larger than intraindividual differences, indicating that a single sample can indeed represent an individual in most cases. However, there are minor spatial patterns in species distribution within the oral cavity that should be taken into account when designing a study or interpreting results. Finally, we show that plant DNA identified in the samples is likely of postmortem origin, demonstrating the importance of including environmental controls or additional lines of biomolecular evidence in dietary interpretations.

Ancient dental calculus preserves signatures of biofilm succession and interindividual variation independent of dental pathology.

Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here, we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed interindividual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

Archaeometric evidence for the earliest exploitation of lignite from the bronze age Eastern Mediterranean.

This paper presents the earliest evidence for the exploitation of lignite (brown coal) in Europe and sheds new light on the use of combustion fuel sources in the 2nd millennium BCE Eastern Mediterranean. We applied Thermal Desorption/Pyrolysis-Gas Chromatography-Mass Spectrometry and Polarizing Microscopy to the dental calculus of 67 individuals and we identified clear evidence for combustion markers embedded within this calculus. In contrast to the scant evidence for combustion markers within the calculus samples from Egypt, all other individuals show the inhalation of smoke from fires burning wood identified as Pinaceae, in addition to hardwood, such as oak and olive, and/or dung. Importantly, individuals from the Palatial Period at the Mycenaean citadel of Tiryns and the Cretan harbour site of Chania also show the inhalation of fire-smoke from lignite, consistent with the chemical signature of sources in the northwestern Peloponnese and Western Crete respectively. This first evidence for lignite exploitation was likely connected to and at the same time enabled Late Bronze Age Aegean metal and pottery production, significantly by both male and female individuals.

Microwear and isotopic analyses on cave bear remains from Toll Cave reveal both short-term and long-term dietary habits.

Dietary habits of the extinct Ursus spelaeus have always been a controversial topic in paleontological studies. In this work, we investigate carbon and nitrogen values in the bone collagen and dental microwear of U. spelaeus specimens recovered in Level 4 from Toll Cave (Moià, Catalonia, NE Iberian Peninsula). These remains have been dated to > 49,000 14C BP. The ability of both proxies to provide data on the diet of U. spelaeus at different times in the life-history (isotopes: average diet of life; microwear: last days/weeks before death), allows us to generate high-resolution and complementary data. Our results show lower values (δ13C & δ15N) in cave bears than in strict herbivores (i.e. Cervus elaphus) recovered from the same level of Toll Cave. On the other hand, 12 lower molars (m1) were analysed through low-magnification microwear technique. The cave bears from Toll Cave show a microwear pattern like that of extant bears with omnivorous and carnivorous diets. These data are discussed in the framework of all available data in Europe and add new information about the plasticity of the dietary habits of this species at the southern latitudes of Europe during Late Pleistocene periods.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007-0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003-70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Dental calculus evidence of Taï Forest Chimpanzee plant consumption and life history transitions.

Dental calculus (calcified dental plaque) is a source of multiple types of data on life history. Recent research has targeted the plant microremains preserved in this mineralised deposit as a source of dietary and health information for recent and past populations. However, it is unclear to what extent we can interpret behaviour from microremains. Few studies to date have directly compared the microremain record from dental calculus to dietary records, and none with long-term observation dietary records, thus limiting how we can interpret diet, food acquisition and behaviour. Here we present a high-resolution analysis of calculus microremains from wild chimpanzees (Pan troglodytes verus) of Taï National Park, Côte d'Ivoire. We test microremain assemblages against more than two decades of field behavioural observations to establish the ability of calculus to capture the composition of diet. Our results show that some microremain classes accumulate as long-lived dietary markers. Phytolith abundance in calculus can reflect the proportions of plants in the diet, yet this pattern is not true for starches. We also report microremains can record information about other dietary behaviours, such as the age of weaning and learned food processing techniques like nut-cracking.

The Oldest Case of Decapitation in the New World (Lapa do Santo, East-Central Brazil).

We present here evidence for an early Holocene case of decapitation in the New World (Burial 26), found in the rock shelter of Lapa do Santo in 2007. Lapa do Santo is an archaeological site located in the Lagoa Santa karst in east-central Brazil with evidence of human occupation dating as far back as 11.7-12.7 cal kyBP (95.4% interval). An ultra-filtered AMS age determination on a fragment of the sphenoid provided an age range of 9.1-9.4 cal kyBP (95.4% interval) for Burial 26. The interment was composed of an articulated cranium, mandible and first six cervical vertebrae. Cut marks with a v-shaped profile were observed in the mandible and sixth cervical vertebra. The right hand was amputated and laid over the left side of the face with distal phalanges pointing to the chin and the left hand was amputated and laid over the right side of the face with distal phalanges pointing to the forehead. Strontium analysis comparing Burial 26's isotopic signature to other specimens from Lapa do Santo suggests this was a local member of the group. Therefore, we suggest a ritualized decapitation instead of trophy-taking, testifying for the sophistication of mortuary rituals among hunter-gatherers in the Americas during the early Archaic period. In the apparent absence of wealth goods or elaborated architecture, Lapa do Santo's inhabitants seemed to use the human body to express their cosmological principles regarding death.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

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

Pathogens and host immunity in the ancient human oral cavity.

Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first, to our knowledge, high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human-associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past.