Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions

Abstract

The importance of commensal microbes for human health is increasingly recognized, yet the impacts of evolutionary changes in human diet and culture on commensal microbiota remain almost unknown. Two of the greatest dietary shifts in human evolution involved the adoption of carbohydrate-rich Neolithic (farming) diets (beginning ∼10,000 years before the present) and the more recent advent of industrially processed flour and sugar (in ∼1850). Here, we show that calcified dental plaque (dental calculus) on ancient teeth preserves a detailed genetic record throughout this period. Data from 34 early European skeletons indicate that the transition from hunter-gatherer to farming shifted the oral microbial community to a disease-associated configuration. The composition of oral microbiota remained unexpectedly constant between Neolithic and medieval times, after which (the now ubiquitous) cariogenic bacteria became dominant, apparently during the Industrial Revolution. Modern oral microbiotic ecosystems are markedly less diverse than historic populations, which might be contributing to chronic oral (and other) disease in postindustrial lifestyles.

Figure 1. Phylum level microbial composition of the ancient dental calculus deposits

This is similar to modern oral samples, and distinct from non-template controls (EBC), ancient human teeth and environmental samples. The phylum frequencies for the V3 region are presented for the ancient calculus samples (LBK; Linear Pottery Culture, BB; Bell Beaker), modern oral samples, which included pyrosequenced (calculus, plaque and saliva) and cloned (plaque^,,) data, non-template controls (or extraction blanks), ancient human teeth and environmental samples (freshwater, sediments and soils^-) (Supplementary Table 1). Phylum frequencies from the HOMD were generated from partial and full-length sequences of the 16S rRNA gene. The phyla with a frequency < 1% include: ABY1_OD1, AD3, Armatimonadetes, BRC1, CCM11b, Chlamydiae, Chlorobi, Cyanobacteria, Elusimicrobia, Euryarchaeota, Fibrobacteres, GAL15, Gemmatimonadetes, GN02, GN04, GOUTA4, KSB1, Lentisphaerae, NC10, Nitrospirae, NKB19, OP11, OP3, OP9, PAUC34f, Planctomycetes, SBR1093, SC3, SC4, SM2F11, SPAM, Spirochaetes, SR1, Tenericutes, Thermi, TM6, Verrucomicrobia, WPS-2, WS3 and ZB2.

Figure 2. Principal components plot of β-diversity

PCoA reveals a close phylogenetic relationship between ancient dental calculus and modern oral samples, both of which are distinct from the non-template controls and environmental samples. β-diversity was calculated for all samples (Supplementary Note) using the UniFrac metric for the V3 region, and PCoA was applied to the unweighted, UniFrac distances. The plot of the first and second components (PC1 and PC2) (a) and the first and third components (PC1 and PC3) (b) of the PCoA clustered the ancient dental calculus samples with the modern oral pyrosequenced data (calculus, plaque and saliva), which were separated from the environmental samples and extraction blanks. A restricted PCoA plot of PC1 and PC2 (c) and PC1 and PC3 (d) that only includes ancient and modern oral pyrosequencing samples separated the hunter-gatherer (Mesolithic) individuals from modern, Medieval, and Neolithic samples.

Figure 3. Changes in the diversity and composition of oral microbiota

(a) For the V3 region sequences, we estimated the phylogenetic diversity (Supplementary Note) of the archaeological dental calculus samples (n = 34) and compared them to modern calculus (n = 6) and plaque (n = 13). We estimated phylogenetic diversity from only classified, Gram-positive bacterial sequences to minimize the influence of taphonomic bias (see Supplementary Note). Diversity was calculated at a depth of 34 sequences and bootstrapped to assess the robustness of the pattern. Error bars represent bootstrapped frequencies generated by sampling 255 replicates without replacement. (b) Specific primers were used to amplify sequences unique to the oral pathogens S. mutans (light) and P. gingivalis (dark).

Figure 4. Discriminant analysis of β-diversity

Discriminant analysis was applied to the principal coordinates generated from the unweighted UniFrac distances calculated from the V3 region sequences. Each individual is represented by a circle and coloured according to archaeological grouping (HG; Hunter-Gatherer, LBK; Linear Pottery Culture, BB; Bell Beaker, LN/BA; Late Neolithic/Bronze Age, StHW; St. Helen-on-the-Walls). The majority of phylogenetic variation (91.2%) was described by the first discriminant function, showing that individuals from the same archaeological groups cluster according to microbial composition