High conservation of the dental plaque microbiome across populations with differing subsistence strategies and levels of market integration.

Industrialization-including urbanization, participation in the global food chain and consumption of heavily processed foods-is thought to drive substantial shifts in the human microbiome. While diet strongly influences stool microbiome composition, the influence of diet on the oral microbiome is largely speculative. Multiple ecologically distinct surfaces in the mouth, each harbouring a unique microbial community, pose a challenge to assessing changes in the oral microbiome in the context of industrialization, as the results depend on the oral site under study. Here, we investigated whether microbial communities of dental plaque, the dense biofilm on non-shedding tooth surfaces, are distinctly different across populations with dissimilar subsistence strategies and degree of industrialized market integration. Using a metagenomic approach, we compared the dental plaque microbiomes of Baka foragers and Nzime subsistence agriculturalists in Cameroon (n = 46) with the dental plaque and calculus microbiomes of highly industrialized populations in North America and Europe (n = 38). We found that differences in microbial taxonomic composition between populations were minimal, with high conservation of abundant microbial taxa and no significant differences in microbial diversity related to dietary practices. Instead, we find that the major source of variation in dental plaque microbial species composition is related to tooth location and oxygen availability, which may be influenced by toothbrushing or other dental hygiene measures. Our results support that dental plaque, in contrast to the stool microbiome, maintains an inherent stability against ecological perturbations in the oral environment.

The cost of chewing: The energetics and evolutionary significance of mastication in humans.

Any change in the energetic cost of mammalian mastication will affect the net energy gain from foods. Although the energetic efficiency of masticatory effort is fundamental in understanding the evolution of the human masticatory system, nothing is known currently about the associated metabolic costs of chewing different items. Here, using respirometry and electromyography of the masseter muscle, we demonstrate that chewing by human subjects represents a measurable energy sink. Chewing a tasteless odorless gum elevates metabolic rate by 10 to 15% above basal levels. Energy expenditure increases with gum stiffness and is paid for by greater muscle recruitment. For modern humans, it is likely that mastication represents a small part of the daily energy budget. However, for our ancestors, before the onset of cooking and sophisticated food processing methods, the costs must have been relatively high, adding a previously unexplored energetic dimension to the interpretation of hominin dentofacial fossils.

Synchrotron radiation-based phase-contrast microtomography of human dental calculus allows nondestructive analysis of inclusions: implications for archeological samples.

Purpose: Dental calculus forms on teeth during the life of an individual and its investigation can yield information about diet, health status, and environmental pollution. Currently, the analytical techniques used to visualize the internal structure of human dental calculus and entrapped inclusions are limited and require destructive sampling, which cannot always be justified. Approach: We used propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SR- µ CT ) to non-destructively examine the internal organization of dental calculus, including its microstructure and entrapped inclusions, on both modern and archeological samples. Results: The virtual histological exploration of the samples shows that PPC-SR- µ CT is a powerful approach to visualize the internal organization of dental calculus. We identified several important features, including previously undetected negative imprints of enamel and dentine growth markers (perikymata and periradicular bands, respectively), the non-contiguous structure of calculus layers with multiple voids, and entrapped plant remains. Conclusions: PPC-SR- µ CT is an effective technique to explore dental calculus structural organization, and is especially powerful for enabling the identification of inclusions. The non-destructive nature of synchrotron tomography helps protect samples for future research. However, the irregular layers and frequent voids reveal a high heterogeneity and variability within calculus, with implications for research focusing on inclusions.

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.

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.

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.

Multi-contrast anatomical subcortical structures parcellation.

The human subcortex is comprised of more than 450 individual nuclei which lie deep in the brain. Due to their small size and close proximity, up until now only 7% have been depicted in standard MRI atlases. Thus, the human subcortex can largely be considered as terra incognita. Here, we present a new open-source parcellation algorithm to automatically map the subcortex. The new algorithm has been tested on 17 prominent subcortical structures based on a large quantitative MRI dataset at 7 Tesla. It has been carefully validated against expert human raters and previous methods, and can easily be extended to other subcortical structures and applied to any quantitative MRI dataset. In sum, we hope this novel parcellation algorithm will facilitate functional and structural neuroimaging research into small subcortical nuclei and help to chart terra incognita.

Phytoliths can cause tooth wear.

Comparative laboratory sliding wear tests on extracted human molar teeth in artificial saliva with third-body particulates demonstrate that phytoliths can be as effective as silica grit in the abrasion of enamel. A pin-on-disc wear testing configuration is employed, with an extracted molar cusp as a pin on a hard disc antagonist, under loading conditions representative of normal chewing forces. Concentrations and sizes of phytoliths in the wear test media match those of silica particles. Cusp geometries and ensuing abrasion volumes are measured by digital profilometry. The wear data are considered in relation to a debate by evolutionary biologists concerning the relative capacities of intrinsic mineral bodies within plant tissue and exogenous grit in the atmosphere to act as agents of tooth wear in various animal species.

Seasonal and habitat effects on the nutritional properties of savanna vegetation: Potential implications for early hominin dietary ecology.

The African savannas that many early hominins occupied likely experienced stark seasonality and contained mosaic habitats (i.e., combinations of woodlands, wetlands, grasslands, etc.). Most would agree that the bulk of dietary calories obtained by taxa such as Australopithecus and Paranthropus came from the consumption of vegetation growing across these landscapes. It is also likely that many early hominins were selective feeders that consumed particular plants/plant parts (e.g., leaves, fruit, storage organs) depending on the habitat and season within which they were foraging. Thus, improving our understanding of how the nutritional properties of potential hominin plant foods growing in modern African savanna ecosystems respond to season and vary by habitat will improve our ability to model early hominin dietary behavior. Here, we present nutritional analyses (crude protein and acid detergent fiber) of plants growing in eastern and southern African savanna habitats across both wet and dry seasons. We find that many assumptions about savanna vegetation are warranted. For instance, plants growing in our woodland habitats have higher average protein/fiber ratios than those growing in our wetland and grassland transects. However, we find that the effects of season and habitat are complex, an example being the unexpectedly higher protein levels we observe in the grasses and sedges growing in our Amboseli wetlands during the dry season. Also, we find significant differences between the vegetation growing in our eastern and southern African field sites, particularly among plants using the C4 photosynthetic pathway. This may have implications for the differences we see between the stable carbon isotope compositions and dental microwear patterns of eastern and southern African Paranthropus species, despite their shared, highly derived craniodental anatomy.

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.

Grass leaves as potential hominin dietary resources.

Discussions about early hominin diets have generally excluded grass leaves as a staple food resource, despite their ubiquity in most early hominin habitats. In particular, stable carbon isotope studies have shown a prevalent C4 component in the diets of most taxa, and grass leaves are the single most abundant C4 resource in African savannas. Grass leaves are typically portrayed as having little nutritional value (e.g., low in protein and high in fiber) for hominins lacking specialized digestive systems. It has also been argued that they present mechanical challenges (i.e., high toughness) for hominins with bunodont dentition. Here, we compare the nutritional and mechanical properties of grass leaves with the plants growing alongside them in African savanna habitats. We also compare grass leaves to the leaves consumed by other hominoids and demonstrate that many, though by no means all, compare favorably with the nutritional and mechanical properties of known primate foods. Our data reveal that grass leaves exhibit tremendous variation and suggest that future reconstructions of hominin dietary ecology take a more nuanced approach when considering grass leaves as a potential hominin dietary resource.

Fecal metabolome of the Hadza hunter-gatherers: a host-microbiome integrative view.

The recent characterization of the gut microbiome of traditional rural and foraging societies allowed us to appreciate the essential co-adaptive role of the microbiome in complementing our physiology, opening up significant questions on how the microbiota changes that have occurred in industrialized urban populations may have altered the microbiota-host co-metabolic network, contributing to the growing list of Western diseases. Here, we applied a targeted metabolomics approach to profile the fecal metabolome of the Hadza of Tanzania, one of the world's few remaining foraging populations, and compared them to the profiles of urban living Italians, as representative of people in the post-industrialized West. Data analysis shows that during the rainy season, when the diet is primarily plant-based, Hadza are characterized by a distinctive enrichment in hexoses, glycerophospholipids, sphingolipids, and acylcarnitines, while deplete in the most common natural amino acids and derivatives. Complementary to the documented unique metagenomic features of their gut microbiome, our findings on the Hadza metabolome lend support to the notion of an alternate microbiome configuration befitting of a nomadic forager lifestyle, which helps maintain metabolic homeostasis through an overall scarcity of inflammatory factors, which are instead highly represented in the Italian metabolome.

Enterocyte-Associated Microbiome of the Hadza Hunter-Gatherers.

By means of a recently developed non-invasive ex vivo minimal model based on the interaction of the human enterocyte-like HT29 cell line and fecal slurries, we explored the enterocyte-associated microbiome of 21 Hadza hunter-gatherers and nine urban living Italians. Though reductionist, this model allows inferring the microbiota structural and functional arrangement as it interacts with enterocytes. Microbial suspensions obtained from Hadza or Italian stools were first evaluated for structural integrity by high resolution-scanning electron microscopy and co-incubated with HT29 cell monolayers. The enterocyte adherent microbiota fraction was then characterized by 16S rRNA gene sequencing and predictive functional profiling using PICRUSt. Compared to Italians, the Hadza enterocyte-associated microbiome was characterized by a greater amount of adhesive microorganisms with pathogenic potential, such as Proteobacteria, Erysipelotrichaceae, Enterococcus, Clostridium and Sarcina. These compositional characteristics were reflected in a functional enrichment in membrane transport, signal transduction, signaling molecules and interaction. Our results depict a new interesting mutualistic configuration of the enterocyte-associated microbiome in Hadza, stressing the importance of microbe-host interaction at the mucosal surface along the course of human evolution.

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.

Assessing digestibility of Hadza tubers using a dynamic in-vitro model.

OBJECTIVES: Bioaccessibility is a useful measure for assessing the biological value of a particular nutrient from food, especially foods such as tubers. The wild tubers exploited by Hadza foragers in Tanzania are of interest because they are nontoxic, consumed raw or briefly roasted, and entail substantial physical barriers to consumers. In this study, we attempted to elucidate the biological value of Hadza tubers by measuring the absorption of glucose through in-vitro digestion. METHODS: We quantified digestibility using data from 24 experimental trials on four species of Hadza tuber using a dynamic in-vitro model that replicates digestion in the stomach and small intestine. Analysis of glucose in the input meal and output dialysate revealed the accessible glucose fraction. We also conducted assays for protein, vitamin, and mineral content on whole tubers and meal fractions. RESULTS: Bioaccessibility of glucose varies depending on tuber species. Holding effects of chewing constant, brief roasting had negligible effects, but high intraspecific variation precludes interpretive power. Overall, Hadza tubers are very resistant to digestion, with between one- and two-thirds of glucose absorbed on average. Glucose absorption negatively correlated with glucose concentration of the tubers. CONCLUSIONS: Roasting may provide other benefits such as ease of peeling and chewing to extract edible parenchymatous tissue. A powerful factor in glucose acquisition is tuber quality, placing emphasis on the skill of the forager. Other nutrient assays yielded unexpectedly high values for protein, iron, and iodine, making tubers potentially valuable resources beyond caloric content.

Earliest evidence of dental caries manipulation in the Late Upper Palaeolithic.

Prehistoric dental treatments were extremely rare, and the few documented cases are known from the Neolithic, when the adoption of early farming culture caused an increase of carious lesions. Here we report the earliest evidence of dental caries intervention on a Late Upper Palaeolithic modern human specimen (Villabruna) from a burial in Northern Italy. Using Scanning Electron Microscopy we show the presence of striations deriving from the manipulation of a large occlusal carious cavity of the lower right third molar. The striations have a "V"-shaped transverse section and several parallel micro-scratches at their base, as typically displayed by cutmarks on teeth. Based on in vitro experimental replication and a complete functional reconstruction of the Villabruna dental arches, we confirm that the identified striations and the associated extensive enamel chipping on the mesial wall of the cavity were produced ante-mortem by pointed flint tools during scratching and levering activities. The Villabruna specimen is therefore the oldest known evidence of dental caries intervention, suggesting at least some knowledge of disease treatment well before the Neolithic. This study suggests that primitive forms of carious treatment in human evolution entail an adaptation of the well-known toothpicking for levering and scratching rather than drilling practices.

Metagenome Sequencing of the Hadza Hunter-Gatherer Gut Microbiota.

Through human microbiome sequencing, we can better understand how host evolutionary and ontogenetic history is reflected in the microbial function. However, there has been no information on the gut metagenome configuration in hunter-gatherer populations, posing a gap in our knowledge of gut microbiota (GM)-host mutualism arising from a lifestyle that describes over 90% of human evolutionary history. Here, we present the first metagenomic analysis of GM from Hadza hunter-gatherers of Tanzania, showing a unique enrichment in metabolic pathways that aligns with the dietary and environmental factors characteristic of their foraging lifestyle. We found that the Hadza GM is adapted for broad-spectrum carbohydrate metabolism, reflecting the complex polysaccharides in their diet. Furthermore, the Hadza GM is equipped for branched-chain amino acid degradation and aromatic amino acid biosynthesis. Resistome functionality demonstrates the existence of antibiotic resistance genes in a population with little antibiotic exposure, indicating the ubiquitous presence of environmentally derived resistances. Our results demonstrate how the functional specificity of the GM correlates with certain environment and lifestyle factors and how complexity from the exogenous environment can be balanced by endogenous homeostasis. The Hadza gut metagenome structure allows us to appreciate the co-adaptive functional role of the GM in complementing the human physiology, providing a better understanding of the versatility of human life and subsistence.

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.