Stable isotope analysis has become an essential tool in investigations of ancient migration and paleodietary reconstruction. Because the biogeochemistry of bone collagen and apatite is well known,... Show moreStable isotope analysis has become an essential tool in investigations of ancient migration and paleodietary reconstruction. Because the biogeochemistry of bone collagen and apatite is well known, current methods rely almost exclusively on analyses of bones and teeth; however, dental calculus represents a potentially additional biological source of isotopic data from ancient skeletons. Dental calculus is a mineralized bacterial biofilm that forms on the surfaces of teeth. Sampling dental calculus does not damage the dentition and thus can be used in cases where it is not possible to perform destructive analyses of conventional mineralized tissues. Like bone and dentine, dental calculus contains both inorganic and organic components, allowing measurement of C, N, O, H, and Sr isotopes. Additionally, dental calculus forms as serial, non-remodeling laminar accretions on the tooth surface, opening up the possibility of analyzing discrete time points during the lifetime of an individual. However, as a microbial biofilm and not a human tissue, the biochemistry of dental calculus is complex, containing multiple calcium phosphate mineral phases, organic and inorganic food remains, hundreds of human and bacterial proteins, and diverse biomolecules from thousands of endogenous bacterial taxa. Isotopic investigation of dental calculus is still in its infancy, and many questions remain regarding its formation and processes of diagenesis. This chapter (1) reviews the unique advantages presented by dental calculus as a novel source of biological isotopic data, (2) critically evaluates published isotopic studies of dental calculus, and (3) explores the current challenges of dental calculus stable isotope analysis through a case study of an Ancient Puebloan Basketmaker II population from the American Southwest. Show less
Fagernäs Z.; Salazar-García, D.C.; Haber Uriarte, M.; Avilés Fernández, A.; Henry A.G.; Lomba Maurandi, J.; ... ; Warinner, C. 2022
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... Show moreThe 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. Show less
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,... Show moreNeanderthals 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. Show less
ObjectivesDental 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... Show moreObjectivesDental 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 methodsTotal 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. ResultsTotal 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. DiscussionWhile 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. Show less
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... Show moreDental 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. Show less
Power, R.C.; Salazar-García, D.C.; Rubini, M.; Darlas, A.; Havarti, K.; Walker, M.; ... ; Henry, A.G. 2018
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... Show moreThe 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. Show less
Power, R.C.; Salazar-García, D.C.; Rubini, M.; Darlas, A.; Havarti, K.; Walker, M.; ... ; Henry, A.G. 2018
