Dental calculus as a proxy for animal microbiomes.

The field of dental calculus research has exploded in recent years, predominantly due to the multitude of studies related to human genomes and oral pathogens. Despite having a subset of these studies devoted to non-human primates, little progress has been made in the distribution of oral pathogens across domestic and wild animal populations. This overlooked avenue of research is particularly important at present when many animal populations with the potentiality for zoonotic transmission continue to reside in close proximity to human groups due to reasons such as deforestation and climatic impacts on resource availability. Here, we analyze all previously available published oral microbiome data recovered from the skeletal remains of animals, all of which belong to the Mammalia class. Our genus level results emphasize the tremendous diversity of oral ecologies across mammals in spite of the clustering based primarily on host species. We also discuss the caveats and flaws in analyzing ancient animal oral microbiomes at the species level of classification. Lastly, we assess the benefits, challenges, and gaps in the current knowledge of dental calculus research within animals and postulate the future of the field as a whole.

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.

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments.

OBJECTIVES: The tropics harbor a large part of the world's biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semi-tropical sites in Tanzania, Mexico, and Puerto Rico (N = 12). MATERIALS AND METHODS: All samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step. RESULTS: No significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments. DISCUSSION: Both methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.

Oral microbiome diversity among Cheyenne and Arapaho individuals from Oklahoma.

OBJECTIVES: There is a major ascertainment bias in microbiome research, with individuals of predominately European ancestry living within metropolitan areas dominating most studies. Here we present a study of the salivary microbiome within a North American Indian community. This research is the culmination of four years of collaboration and community engagement with Cheyenne & Arapaho (C&A) tribal members from western Oklahoma. MATERIALS AND METHODS: Using 16S rRNA gene amplification and next-generation sequencing, we generated microbial taxonomic inventories for 37 individuals representing five towns within the C&A tribes. For comparison, we performed the same laboratory techniques on saliva samples from 20 non-native individuals (NNI) from Norman, Oklahoma. RESULTS: The C&A participants differ from the NNI in having reduced within-individual species richness and higher between-individual variation. Unsupervised clustering analyses reveal that three ecological groupings best fit the data, and while C&A individuals include assignments to all three groups, the NNI individuals are assigned to only one group. One of the ecological groups found exclusively among C&A participants was characterized by high abundance of the oral bacterial genus Prevotella. DISCUSSION: The C&A and NNI participants from Oklahoma have notable differences in their microbiome diversity, with a wider range of variation observed among the C&A individuals, including a higher frequency of bacteria implicated in systemic disorders. Overall, this study highlights the importance of engagement with indigenous communities, and the need for an improved understanding of human microbiome diversity among underrepresented groups and those individuals living outside of metropolitan areas.

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

OBJECTIVES: Archaeological 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 METHODS: Extracted 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. RESULTS: Full 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. DISCUSSION: Dental 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.

Poor Oral Health Is Associated With Inflammation, Aortic Valve Calcification, and Brain Volume Among Forager-Farmers.

Poor oral health is associated with cardiovascular disease and dementia. Potential pathways include sepsis from oral bacteria, systemic inflammation, and nutritional deficiencies. However, in post-industrialized populations, links between oral health and chronic disease may be confounded because the lower socioeconomic exposome (poor diet, pollution, and low physical activity) often entails insufficient dental care. We assessed tooth loss, caries, and damaged teeth, in relation to cardiovascular and brain aging among the Tsimane, a subsistence population living a relatively traditional forager-horticulturalist lifestyle with poor dental health, but minimal cardiovascular disease and dementia. Dental health was assessed by a physician in 739 participants aged 40-92 years with cardiac and brain health measured by chest computed tomography (CT; n = 728) and brain CT (n = 605). A subset of 356 individuals aged 60+ were also assessed for dementia and mild cognitive impairment (n = 33 impaired). Tooth loss was highly prevalent, with 2.2 teeth lost per decade and a 2-fold greater loss in women. The number of teeth with exposed pulp was associated with higher inflammation, as measured by cytokine levels and white blood cell counts, and lower body mass index. Coronary artery calcium and thoracic aortic calcium were not associated with tooth loss or damaged teeth. However, aortic valve calcification and brain tissue loss were higher in those who had more teeth with exposed pulp. Overall, these results suggest that dental health is associated with indicators of chronic diseases in the absence of typical confounds, even in a population with low cardiovascular and dementia risk factors.

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.

Urine as a high-quality source of host genomic DNA from wild populations.

The ability to generate genomic data from wild animal populations has the potential to give unprecedented insight into the population history and dynamics of species in their natural habitats. However, for many species, it is impossible legally, ethically or logistically to obtain tissue samples of quality sufficient for genomic analyses. In this study we evaluate the success of multiple sources of genetic material (faeces, urine, dentin and dental calculus) and several capture methods (shotgun, whole-genome, exome) in generating genome-scale data in wild eastern chimpanzees (Pan troglodytes schweinfurthii) from Gombe National Park, Tanzania. We found that urine harbours significantly more host DNA than other sources, leading to broader and deeper coverage across the genome. Urine also exhibited a lower rate of allelic dropout. We found exome sequencing to be far more successful than both shotgun sequencing and whole-genome capture at generating usable data from low-quality samples such as faeces and dental calculus. These results highlight urine as a promising and untapped source of DNA that can be noninvasively collected from wild populations of many species.

Oral microbiome diversity in chimpanzees from Gombe National Park.

Historic calcified dental plaque (dental calculus) can provide a unique perspective into the health status of past human populations but currently no studies have focused on the oral microbial ecosystem of other primates, including our closest relatives, within the hominids. Here we use ancient DNA extraction methods, shotgun library preparation, and next generation Illumina sequencing to examine oral microbiota from 19 dental calculus samples recovered from wild chimpanzees (Pan troglodytes schweinfurthii) who died in Gombe National Park, Tanzania. The resulting sequences were trimmed for quality, analyzed using MALT, MEGAN, and alignment scripts, and integrated with previously published dental calculus microbiome data. We report significant differences in oral microbiome phyla between chimpanzees and anatomically modern humans (AMH), with chimpanzees possessing a greater abundance of Bacteroidetes and Fusobacteria, and AMH showing higher Firmicutes and Proteobacteria. Our results suggest that by using an enterotype clustering method, results cluster largely based on host species. These clusters are driven by Porphyromonas and Fusobacterium genera in chimpanzees and Haemophilus and Streptococcus in AMH. Additionally, we compare a nearly complete Porphyromonas gingivalis genome to previously published genomes recovered from human gingiva to gain perspective on evolutionary relationships across host species. Finally, using shotgun sequence data we assessed indicators of diet from DNA in calculus and suggest exercising caution when making assertions related to host lifestyle. These results showcase core differences between host species and stress the importance of continued sequencing of nonhuman primate microbiomes in order to fully understand the complexity of their oral ecologies.

Metagenomic analysis of dental calculus in ancient Egyptian baboons.

Dental calculus, or mineralized plaque, represents a record of ancient biomolecules and food residues. Recently, ancient metagenomics made it possible to unlock the wealth of microbial and dietary information of dental calculus to reconstruct oral microbiomes and lifestyle of humans from the past. Although most studies have so far focused on ancient humans, dental calculus is known to form in a wide range of animals, potentially informing on how human-animal interactions changed the animals' oral ecology. Here, we characterise the oral microbiome of six ancient Egyptian baboons held in captivity during the late Pharaonic era (9th-6th centuries BC) and of two historical baboons from a zoo via shotgun metagenomics. We demonstrate that these captive baboons possessed a distinctive oral microbiome when compared to ancient and modern humans, Neanderthals and a wild chimpanzee. These results may reflect the omnivorous dietary behaviour of baboons, even though health, food provisioning and other factors associated with human management, may have changed the baboons' oral microbiome. We anticipate our study to be a starting point for more extensive studies on ancient animal oral microbiomes to examine the extent to which domestication and human management in the past affected the diet, health and lifestyle of target animals.

"The dead shall be raised": Multidisciplinary analysis of human skeletons reveals complexity in 19th century immigrant socioeconomic history and identity in New Haven, Connecticut.

In July 2011, renovations to Yale-New Haven Hospital inadvertently exposed the cemetery of Christ Church, New Haven, Connecticut's first Catholic cemetery. While this cemetery was active between 1833 and 1851, both the church and its cemetery disappeared from public records, making the discovery serendipitous. Four relatively well-preserved adult skeletons were recovered with few artifacts. All four individuals show indicators of manual labor, health and disease stressors, and dental health issues. Two show indicators of trauma, with the possibility of judicial hanging in one individual. Musculoskeletal markings are consistent with physical stress, and two individuals have arthritic indicators of repetitive movement/specialized activities. Radiographic analyses show osteopenia, healed trauma, and other pathologies in several individuals. Dental calculus analysis did not identify any tuberculosis indicators, despite osteological markers. Isotopic analyses of teeth indicate that all four were likely recent immigrants to the Northeastern United States. Nuclear and mitochondrial DNA were recovered from three individuals, and these analyses identified ancestry, hair/eye color, and relatedness. Genetic and isotopic results upended our initial ancestry assessment based on burial context alone. These individuals provide biocultural evidence of New Haven's Industrial Revolution and the plasticity of ethnic and religious identity in the immigrant experience. Their recovery and the multifaceted analyses described here illuminate a previously undescribed part of the city's rich history. The collective expertise of biological, geochemical, archaeological, and historical researchers interprets socioeconomic and cultural identity better than any one could alone. Our combined efforts changed our initial assumptions of a poor urban Catholic cemetery's membership, and provide a template for future discoveries and analyses.

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.

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.