Archaeological Dental Calculus Research Articles

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.

Tracking the transition to agriculture in Southern Europe through ancient DNA analysis of dental calculus

Archaeological dental calculus, or mineralized plaque, is a key tool to track the evolution of oral microbiota across time in response to processes that impacted our culture and biology, such as the rise of farming during the Neolithic. However, the extent to which the human oral flora changed from prehistory until present has remained elusive due to the scarcity of data on the microbiomes of prehistoric humans. Here, we present our reconstruction of oral microbiomes via shotgun metagenomics of dental calculus in 44 ancient foragers and farmers from two regions playing a pivotal role in the spread of farming across Europe-the Balkans and the Italian Peninsula. We show that the introduction of farming in Southern Europe did not alter significantly the oral microbiomes of local forager groups, and it was in particular associated with a higher abundance of the species Olsenella sp. oral taxon 807. The human oral environment in prehistory was dominated by a microbial species, Anaerolineaceae bacterium oral taxon 439, that diversified geographically. A Near Eastern lineage of this bacterial commensal dispersed with Neolithic farmers and replaced the variant present in the local foragers. Our findings also illustrate that major taxonomic shifts in human oral microbiome composition occurred after the Neolithic and that the functional profile of modern humans evolved in recent times to develop peculiar mechanisms of antibiotic resistance that were previously absent.

Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics.

Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins β-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.

Effects of chemical pre-treatments on modified starch granules: Recommendations for dental calculus decalcification for ancient starch research

Starch granules and other plant microremains embedded in dental calculus provide information about the diet and culinary practices of past individuals. While most researchers use either hydrochloric acid (HCl) or ethylenediaminetetraacetic acid (EDTA) to demineralise samples and release embedded microremains, protocols vary significantly in regard to chemical concentration and duration. Furthermore, there are conflicting reports concerning the suitability of these chemicals for archaeological starch analyses, particularly when granules are modified (damaged) such as from food processing or enzymatic digestion. This study tests 0.5 M HCl and 0.5 M EDTA on modified modern wheat, chickpea and potato starch with direct observation of individual starch granules over 168 h, the maximum duration reported so far for full decalcification of archaeological calculus samples. While there were no observable changes to gross morphological characteristics of any granules, metric analysis indicates slight changes to average granule size in milled potato and wheat starch in all solutions. Furthermore, interactions between the chemical reagent and ions released from the calculus matrix have the potential to result in the diminished recovery of archaeological starch from HCl extractions. We recommend that of these two chemical decalcification options, EDTA is more suitable for processing archaeological dental calculus for starch microremains as it chelates ions released during decalcification of the calculus matrix that might otherwise adversely affect archaeological starch granules.

Do I have something in my teeth? The trouble with genetic analyses of diet from archaeological dental calculus

Dental calculus and other preserved microbiome substrates are an attractive target for dietary reconstruction in past populations through a variety of physical, chemical, and molecular means. Recently, studies have attempted to reconstruct diet from archaeological dental calculus using archaeogenetic techniques. While dental calculus may provide a relatively stable environment for DNA preservation, the detection of plants and animals possibly consumed by an individual through DNA analysis is primarily hindered by microbial richness and incomplete reference databases. Moreover, high genomic similarity within eukaryotic groups - such as mammals - can obfuscate precise taxonomic identification. In the current study we demonstrate the challenges associated with accurate taxonomic identification and authentication of dietary taxa in ancient DNA data using both synthetic and ancient dental calculus datasets. We highlight common errors and sources of contamination across ancient DNA datasets, provide recommendations for dietary DNA validation, and call for caution in the interpretation of diet from dental calculus and other archaeological microbiome substrates.

Multi-omic detection of Mycobacterium leprae in archaeological human dental calculus.

Mineralized dental plaque (calculus) has proven to be an excellent source of ancient biomolecules. Here we present a Mycobacterium leprae genome (6.6-fold), the causative agent of leprosy, recovered via shotgun sequencing of sixteenth-century human dental calculus from an individual from Trondheim, Norway. When phylogenetically placed, this genome falls in branch 3I among the diversity of other contemporary ancient strains from Northern Europe. Moreover, ancient mycobacterial peptides were retrieved via mass spectrometry-based proteomics, further validating the presence of the pathogen. Mycobacterium leprae can readily be detected in the oral cavity and associated mucosal membranes, which likely contributed to it being incorporated into this individual's dental calculus. This individual showed some possible, but not definitive, evidence of skeletal lesions associated with early-stage leprosy. This study is the first known example of successful multi-omics retrieval of M. leprae from archaeological dental calculus. Furthermore, we offer new insights into dental calculus as an alternative sample source to bones or teeth for detecting and molecularly characterizing M. leprae in individuals from the archaeological record.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.

A unified protocol for simultaneous extraction of DNA and proteins from archaeological dental calculus

Archaeological materials are a finite resource, and efforts should be made to minimize destructive analyses. This can be achieved by using protocols combining extraction of several types of biomolecules or microparticles, which decreases the material needed for analyses while maximizing the information yield. Archaeological dental calculus is a source of several different types of biomolecules, as well as microfossils, and can tell us about the human host, microbiome, diet, and even occupational activities. Here, we present a unified protocol allowing for simultaneous extraction of DNA and proteins from a single sample of archaeological dental calculus. We evaluate the protocol on dental calculus from six individuals from a range of time periods and estimated preservation states, and compare it against previously published DNA-only and protein-only protocols. We find that most aspects of downstream analyses are unaltered by the unified protocol, although minor shifts in the recovered proteome can be detected, such as a slight loss of hydrophilic proteins. Total protein recovery depends on both the amount of starting material and choice of extraction protocol, whereas total DNA recovery is significantly reduced using the unified protocol (mean 43%). Nevertheless, total DNA recovery from dental calculus is generally very high, and we found no differences in DNA fragment characteristics or taxonomic profile between the protocols. In conclusion, the unified protocol allows for simultaneous extraction of two complementary lines of biomolecular evidence from archaeological dental calculus without compromising downstream results, thereby minimizing the need for destructive analysis of this finite resource.

Cleaning protocol of archaeological dental calculus: A methodological proposal for vegetable microremains analysis.

One of the major drawbacks involves the contamination produced during the sampling of dental calculus samples due to their manipulation with non-sterilized latex gloves containing maize starch. These gloves have been commonly used for the sampling, cleaning and conservation of archaeological materials. The objective of the present work is to propose a protocol for the cleaning of dental calculus samples that have been contaminated, allowing the removal of such material from the calculus surface. The application of this protocol guarantees the exogenous elements elimination without causing the loss of the archaeological material, since as it is a nondestructive and/or invasive cleaning method, it remains contained within the dental calculus which protects and guarantees its preservation. Fifty-seven fragments of dental calculus were recovered from several archaeological sites in the province of Córdoba, Argentina, dated by AMS between 4058 and 387 14C years BP. A sample comprising 22 fragments larger than 2 mm, which represent 38.59% of the total, were subjected to four cleaning stages through distilled and centrifugal water. As a control test, 10 fragments of human dental tartar were taken, known to have not been previously handled with powdered latex gloves. The results show significant differences between the four cleaning stages, representing an important reduction of modern starch grains adhered to the surface of archaeological samples of human dental calculus.

Proteomic evidence of dietary sources in ancient dental calculus.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein β-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

The dental calculus metabolome in modern and historic samples

IntroductionDental calculus is a mineralized microbial dental plaque biofilm that forms throughout life by precipitation of salivary calcium salts. Successive cycles of dental plaque growth and calcification make it an unusually well-preserved, long-term record of host-microbial interaction in the archaeological record. Recent studies have confirmed the survival of authentic ancient DNA and proteins within historic and prehistoric dental calculus, making it a promising substrate for investigating oral microbiome evolution via direct measurement and comparison of modern and ancient specimens.ObjectiveWe present the first comprehensive characterization of the human dental calculus metabolome using a multi-platform approach.MethodsUltra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) quantified 285 metabolites in modern and historic (200 years old) dental calculus, including metabolites of drug and dietary origin. A subset of historic samples was additionally analyzed by high-resolution gas chromatography–MS (GC–MS) and UPLC–MS/MS for further characterization of metabolites and lipids. Metabolite profiles of modern and historic calculus were compared to identify patterns of persistence and loss.ResultsDipeptides, free amino acids, free nucleotides, and carbohydrates substantially decrease in abundance and ubiquity in archaeological samples, with some exceptions. Lipids generally persist, and saturated and mono-unsaturated medium and long chain fatty acids appear to be well-preserved, while metabolic derivatives related to oxidation and chemical degradation are found at higher levels in archaeological dental calculus than fresh samples.ConclusionsThe results of this study indicate that certain metabolite classes have higher potential for recovery over long time scales and may serve as appropriate targets for oral microbiome evolutionary studies.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus.

ABSTRACTObjectivesArchaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis.Materials and MethodsExtracted DNA from six individuals at the 700‐year‐old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in‐solution capture techniques, followed by Illumina high‐throughput sequencing.ResultsFull mitogenomes (7–34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth.DiscussionDental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220–228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

Plant microremains in dental calculus as a record of plant consumption: A test with Twe forager-horticulturalists

Starch granules and phytoliths trapped in dental calculus preserve a record of plant consumption. Analysis of these microscopic plant remains has increased in popularity in recent years, providing information on diet that complements dental microwear and stable isotope studies. However, it is unclear how accurately these microremains reflect plant consumption. This study examines how well starch granules and phytoliths in dental calculus from a living population (the Twe) with a well-documented diet capture the range and intensity of plant consumption. We find that plant microremains are a poor predictor of plant consumption on an individual level, but may provide a good signal of plant consumption across a population, as well as evidence for plant processing in the mouth. This is the first study to test how well plant microremains in dental calculus reflect plant consumption in a population with a known diet. Results from this project have implications for interpreting plant microremain data from archaeological dental calculus samples.

Ancient DNA analysis of dental calculus

Dental calculus (calcified tartar or plaque) is today widespread on modern human teeth around the world. A combination of soft starchy foods, changing acidity of the oral environment, genetic pre-disposition, and the absence of dental hygiene all lead to the build-up of microorganisms and food debris on the tooth crown, which eventually calcifies through a complex process of mineralisation. Millions of oral microbes are trapped and preserved within this mineralised matrix, including pathogens associated with the oral cavity and airways, masticated food debris, and other types of extraneous particles that enter the mouth. As a result, archaeologists and anthropologists are increasingly using ancient human dental calculus to explore broad aspects of past human diet and health. Most recently, high-throughput DNA sequencing of ancient dental calculus has provided valuable insights into the evolution of the oral microbiome and shed new light on the impacts of some of the major biocultural transitions on human health throughout history and prehistory. Here, we provide a brief historical overview of archaeological dental calculus research, and discuss the current approaches to ancient DNA sampling and sequencing. Novel applications of ancient DNA from dental calculus are discussed, highlighting the considerable scope of this new research field for evolutionary biology and modern medicine.

Corrigendum to de la Fuente et al. (2013)

ArchaeometryVolume 56, Issue 6 p. 1105-1105 CorrigendumFree Access Corrigendum to de la Fuente et al. (2013) This article corrects the following: DNA FROM HUMAN ANCIENT BACTERIA: A NOVEL SOURCE OF GENETIC EVIDENCE FROM ARCHAEOLOGICAL DENTAL CALCULUS C. DE LA FUENTE, S. FLORES, M. MORAGA, Volume 55Issue 4Archaeometry pages: 767-778 First Published online: August 2, 2012 First published: 20 November 2014 https://doi.org/10.1111/arcm.12150AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat At the time of submission of the manuscript the authors forgot to include in the Acknowledgments section, the research grant FONDECYT 1110461. This project contributed to the completion of this study and should have been included in the acknowledgments. The corrected version of the last paragraph of the acknowledgments should read: This research was supported by grants FONDECYT 11060442, FONDECYT 1100643, FONDECYT 1110461 and Programa de Investigación Asociativa Conicyt, Anillo ACT-096 (www.cienciaymemoria.cl). Reference de la Fuente, C., Flores, S., and Moraga, M., 2013, DNA from human ancient bacteria: a novel source of genetic evidence from archaeological dental calculus, Archaeometry, 55, 766– 778. Wiley Online LibraryCASWeb of Science®Google Scholar Volume56, Issue6December 2014Pages 1105-1105 ReferencesRelatedInformation

DNA FROM HUMAN ANCIENT BACTERIA: A NOVEL SOURCE OF GENETIC EVIDENCE FROM ARCHAEOLOGICAL DENTAL CALCULUS

We report a molecular methodology to obtain and analyse ancient bacterial DNA from archaeological dental calculus. Recent and archaeological DNA samples, as old as 4000 bp, were successfully extracted and amplified with species-specific PCR primers. We propose this approach in order to: detect the presence of specific bacterial species infecting past human populations; compare the composition of ancient oral microbiomes among human populations; and analyse the genetic variability and covariation of bacteria and human host populations. Genomic analysis of bacteria from dental calculus is a promising source of evidence for palaeopathological and micro-evolutionary studies, focused either on micro-organisms or their human hosts.