Irina Marie Velsko

DNA library construction methods can affect the recovery of ancient DNA, thus influencing downstream analyses. While single‐stranded library preparation (ssLib) has been shown to outperform double‐stranded (dsLib) for highly degraded vertebrate host DNA, especially for samples older than 40,000 years, few studies have examined how library protocols shape ancient microbial community reconstruction. Here, we compare the sequencing output of paired ssLib and dsLib dental calculus libraries generated from 12 Neanderthals and two Chalcolithic humans, prepared using implementations of the Gansauge et al. and Meyer and Kircher protocols, respectively, and sequenced with identical Illumina chemistry. We compared read length and GC%, read duplication and taxonomic profiles across normalization strategies to assess protocol‐specific biases. Double‐stranded libraries retained a significantly higher proportion of sequenced reads throughout data processing (dsLib 72.1%, ssLib 37.9%), a higher proportion of oral reads (dsLib 9.78%, ssLib 6.75%), significantly longer median oral DNA read lengths (dsLib 57.5 bp, ssLib 50.5 bp) and more GC‐rich fragments (dsLib 60.5% GC, ssLib 52.5% GC). In contrast, ssLibs exhibited slightly higher Shannon diversity and a greater proportion of unique reads. Despite these differences, species richness and overall community composition was not significantly different between protocols, with individual and preservation status explaining the most variance. Stratifying reads by length (< 50 bp vs. ≥ 50 bp) resulted in different classification rates but only had minor effects on diversity estimates. Together, these results demonstrate that dsLib and ssLib protocols impose distinct trade‐offs and library choice should be guided by study‐specific goals.

Objectives: Metagenomic investigations of ancient dental calculus provide insights into oral health, disease, and diet. Here, we analyze the dental calculus metagenome of King Richard III of England (1452–1485). Materials and Methods: Dental calculus DNA was extracted from three teeth of King Richard III and shotgun sequenced to a depth of nearly 400 million reads. The metagenomic data were taxonomically profiled and compared to new and previously published dental calculus metagenomes from England, Ireland, the Netherlands, and Germany spanning the Neolithic to the present. Sequencing data were de novo assembled, and metagenome‐assembled genomes assigned to the genus Tannerella were investigated for phylogenetic relatedness and virulence. Putative dietary DNA was assessed for authenticity. Results: The dental calculus of King Richard III was well‐preserved and yielded an exceptionally high quantity of DNA. Oral microbiome species diversity fell within the range previously observed among other northern European populations, suggesting that a royal lifestyle and a rich diet did not substantially impact his oral microbiota. The reconstructed Tannerella genomes contained many virulence factors found today among oral Tannerella species. No putative dietary DNA could be authenticated. Discussion: The dental calculus of King Richard III produced one of the richest ancient oral metagenomes published to date, yet the species diversity was indistinguishable from that of commoners living in northern Europe over the last 7000 years. Insufficient plant and animal DNA were recovered to investigate diet, suggesting that dental calculus may not be a sufficient source of dietary DNA even when exceptionally well‐preserved.

De novo assembly of ancient and modern bacterial metagenomes can shed light on evolution and ecology of bacterial species that are challenging to culture. Tannerella and Porphyromonas are bacterial genera linked to periodontal disease, and understanding their evolution may reveal insights into their role in oral disease development. We performed pangenomic and phylogenetic analyses on a global set of isolates and metagenome-assembled genomes of the genera Tannerella (n = 238) and Porphyromonas (n = 976), including 66 genomes from ancient dental calculus samples (up to 14,800 years old), and modern oral samples from present-day living populations. We identify a novel species of oral Tannerella in modern and ancient humans, which we call Ca. Tannerella abscondita, that is related to and often mistaken for Tannerella forsythia but differs in its virulence repertoire. We reveal distinct niche tropism in Tannerella species and Porphyromonas pasteri, but not Porphyromonas gingivalis. There is limited phylogeographic structuring, and virulence genes are homogeneously distributed across continents and oral niches. Saliva-derived strains of T. forsythia and P. gingivalis from Oceania and T. serpentiformis and P. pasteri from Asia show enrichment of pseudogenes related to ecological niche transitions. A phylogenetic analysis of the P. gingivalis major fimbrial protein gene fimA reveals the genes cluster by genotypes, and that no ancient genes are found in genotypes I and Ib. Using de novo assembly for bacterial pangenomics improves the representation of oral genera found in reference databases and enhances our ability to study the evolutionary history of these taxa.

De novo assembly of ancient and modern bacterial metagenomes can shed light on evolution and ecology of bacterial species that are challenging to culture. Tannerella and Porphyromonas are bacterial genera linked to periodontal disease, and understanding their evolution may reveal insights into their role in oral disease development. We performed pangenomic and phylogenetic analyses on a global set of isolates and metagenome-assembled genomes of the genera Tannerella (n=238) and Porphyromonas (n=976), including 66 genomes from ancient dental calculus samples (up to 14,800 years old), and modern oral samples from present-day living populations. We identify a novel species of oral Tannerella in modern and ancient humans, which we call Ca. Tannerella abscondita , that is related to and often mistaken for Tannerella forsythia but differs in its virulence repertoire. We reveal distinct niche tropism in Tannerella species and Porphyromonas pasteri , but not Porphyromonas gingivalis . There is limited phylogeographic structuring, and virulence genes are homogeneously distributed across continents and oral niches. Saliva-derived strains of T. forsythia and P. gingivalis from Oceania and T. serpentiformis and P. pasteri from Asia show enrichment of pseudogenes related to ecological niche transitions. A phylogenetic analysis of the P. gingivalis major fimbrial protein gene fimA reveals the genes cluster by genotypes, and that no ancient genes are found in genotypes I and Ib. Using de novo assembly for bacterial pangenomics improves the representation of oral genera found in reference databases and enhances our ability to study the evolutionary history of these taxa.

Metagenomic investigation of archaeological dental calculus has provided insights into the changing oral health, disease, and diet of past human societies, but little is known about the oral microbiota of exceptionally high-status individuals, whose diet and lifestyle sharply differed from the general population. Here we analyze the dental calculus metagenome of King Richard III of England (1452-1485) and compare it to new and previously published dental calculus metagenomes from predominantly non-elite contexts in England, Ireland, the Netherlands, and Germany spanning the Neolithic to the present. Deep sequencing and de novo assembly enabled the investigation of metagenome-assembled genomes (MAGs) from periopathogens within the genus Tannerella . DNA preservation within the dental calculus of King Richard III was found to be exceptional, yielding an extraordinarily well-preserved oral microbiota. Oral microbiome species diversity fell within the range previously observed among other northern European populations over the past 7,000 years, suggesting that a royal lifestyle and a rich diet did not substantially shift his oral microbiome composition. Reconstructed Tannerella genomes contained many virulence factors found today among periopathogens, including Tannerella forsythia . Insufficient plant and animal DNA was recovered to investigate diet, suggesting that dental calculus may not be a sufficient source of dietary DNA for dietary reconstruction, even when well-preserved. The dental calculus of King Richard III has produced one of the richest and best preserved ancient oral metagenomes studied to date and contributes to understanding the ecology and evolution of the human oral microbiome.

The genus Streptococcus is highly diverse and a core member of the primate oral microbiome. Streptococcus species are grouped into at least eight phylogenetically-supported clades, five of which are found almost exclusively in the oral cavity. We explored the dominant Streptococcus phylogenetic clades in samples from multiple oral sites and from ancient and modern-day humans and non-human primates and found that clade dominance is conserved across human oral sites, with most Streptococcus reads assigned to species falling in the Sanguinis or Mitis clades. However, minor differences in the presence and abundance of individual species within each clade differentiated human lifestyles, with loss of S. sinensis appearing to correlate with toothbrushing. Of the non-human primates, only baboons show clade abundance patterns similar to humans, suggesting that a habitat and diet similar to that of early humans may favor the growth of Sanguinis and Mitis clade species.

The Pacific islands and Island Southeast Asia have experienced multiple waves of human migrations, providing a case study for exploring the potential of ancient microbiomes to study human migration. We perform a metagenomic study of archaeological dental calculus from 102 individuals, originating from 10 Pacific islands and 1 island in Island Southeast Asia spanning ~3000 years. Oral microbiome DNA preservation in calculus is far higher than that of human DNA in archaeological bone, and comparable to that of calculus from temperate regions. Oral microbial community composition is minimally driven by time period and geography in Pacific and Island Southeast Asia calculus, but is found to be distinctive compared to calculus from Europe, Africa, and Asia. Phylogenies of individual bacterial species in Pacific and Island Southeast Asia calculus reflect geography. Archaeological dental calculus shows good preservation in tropical regions and the potential to yield information about past human migrations, complementing studies of the human genome.

The genus Streptococcus is highly diverse and a core member of the primate oral microbiome. Streptococcus species are grouped into at least eight phylogenetically-supported clades, five of which are found almost exclusively in the oral cavity. We explored the dominant Streptococcus phylogenetic clades in samples from multiple oral sites and from ancient and modern-day humans and non-human primates and found that clade dominance is conserved across human oral sites, with most species falling in the Sanguinis or Mitis clades. However, minor differences in the presence and abundance of individual species within each clade differentiated human lifestyles, with loss of S. sinensis appearing to correlate with toothbrushing. Of the non-human primates, only baboons show clade abundance patterns similar to humans, suggesting that a habitat and diet similar to that of early humans may favor the growth of Sanguinis and Mitis clade species.

The Pacific islands have experienced multiple waves of human migrations, providing a case study for exploring the potential of using the microbiome to study human migration. We performed a metagenomic study of archaeological dental calculus from 103 ancient individuals, originating from 12 Pacific islands and spanning a time range of ∼3000 years. Oral microbiome DNA preservation in calculus is far higher than that of human DNA in archaeological bone from the Pacific, and comparable to that seen in calculus from temperate regions. Variation in the microbial community composition was minimally driven by time period and geography within the Pacific, while comparison with samples from Europe, Africa, and Asia reveal the microbial communities of Pacific calculus samples to be distinctive. Phylogenies of individual bacterial species in Pacific calculus reflect geography. Archaeological dental calculus shows potential to yield information about past human migrations, complementing studies of the human genome.

A bstract: Dental calculus is increasingly used by researchers to study dietary patterns in past populations. The benefits of using dental calculus for this purpose have been clearly demonstrated in previous studies, with dental calculus harbouring a wealth of microremains and biomarkers for health and diet within its mineral matrix. Previous studies have demonstrated some of the limitations and biases of how methods of processing may overlook, or even remove, some of the important information contained within the mineralised matrix. However, there are many factors that are impossible to account for in vivo and in archaeological material, such as exact dietary intake, and individual factors such as pH and enzyme activity, leaving some limitations that may not be addressed through these types of studies and will require a different approach. We present a protocol for creating a calcifying oral biofilm model that can be used to explore the biases and limitations of dental calculus as a medium for paleodietary reconstructions. We report the microbial and mineral composition of our model in an effort to validate the model calculus as an appropriate proxy to natural dental calculus. The microbial profile and species diversity of our model was determined using metagenomic classification with the nf-core/eager pipeline and Kraken2, and compared to various reference samples from oral sites, including saliva, plaque, and dental calculus. We then assessed whether our model calculus mineralises in a manner similar to natural dental calculus using Fourier transform infrared (FTIR) spectroscopy. The metagenomic classification showed a microbial profile predominantly made up of (facultative) anaerobes, with a community structure that was somewhat distinct from other oral reference samples. The core genera of the model consisted of oral species, but clustered separately from oral reference samples, with a higher abundance of anaerobes. Mineral and organic components of our model mimic that of the modern and archaeological reference calculus that was used as a comparison. There was an overall increase in the inorganic component relative to organic over the course of the experiment, with carbonated hydroxyapatite as the principal compound, consistent with natural human-derived calculus. We conclude that oral biofilm models, such as the one presented in this study, have great potential to validate current methods used in the analysis of archaeological dental calculus, and should be used to complement, rather than replace current in vivo studies.