Ancient DNA Genetics: 102,000‑Year Oral Microbiome Record

Ancient Mouths, Lost Worlds: Oral Microbiomes Across 102,000 Years

The study presents SMILE (Systematic Microbiome Intelligence for Lost Ecosystems), a comprehensive database examining oral microbiomes across approximately 102,000 years of human and Neanderthal history. This research uses ancient dental calculus – the mineralized tartar that accumulates on teeth – to trace microscopic life from Ice Age caves to medieval graveyards, offering remarkable insights into the evolution of oral health and microbial communities.

From Neanderthal Caves to Medieval Cemeteries

The database encompasses 1,414 archaeological specimens spanning from Pesturina Cave in Serbia (102,000 years before present) through medieval European churchyards. Among the earliest samples are Neanderthal remains from De Nadale Cave in northern Italy (70,000 years old) and Banyoles, Spain (66,000 years). Each location offers a rare glimpse into the oral microbial world of extinct hominins living in Pleistocene landscapes.

The sequence continues through Palaeolithic and Mesolithic hunter-gatherers, Neolithic farmers, Bronze and Iron Age communities, and finally extensive medieval samples from churchyards across Europe. These later specimens often accompany burials containing rosary beads, knives, belt fittings, buckles, and religious badges, turning quiet graveyards into microbiological archives in which each burial preserves a snapshot of everyday oral bacteria.

Dental Calculus: Nature's Time Capsule

Dental calculus is one of archaeology's most democratic archives – while not everyone possessed gold or weapons, virtually everyone accumulated plaque on their teeth. When dental plaque hardens and mineralizes, it traps food particles, pollen, bacteria, fungi, and viral DNA. Once sealed within this rocky matrix, these materials can remain protected for millennia, creating natural time capsules that preserve traces otherwise lost to decomposition.

Archaeologists collect these yellowish-white nodules using sterile tools, revealing rough crusts clinging to ancient enamel. From hunter-gatherers to medieval townsfolk, calculus often preserves the only surviving evidence of oral soft tissues – gums, tongue, saliva – and their invisible microbial inhabitants. The SMILE database turns these seemingly ordinary deposits into a timeline of oral cavity evolution.

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Notable Individuals and Famous Archaeological Sites

Several specimens represent familiar figures in palaeogenetics and archaeology. The database carefully tracks individuals appearing in multiple studies, ensuring accurate identification without double-counting. Notable entries include Neanderthals from El Sidrón Cave in northern Spain and Spy Cave in Belgium, whose bones have already been studied for diet and health. Their preserved plaque now adds another layer to those life histories.

The database also extends beyond humans, including thirteen non-human specimens from bears, gorillas, and reindeer. These samples illuminate oral communities across species that shared Ice Age environments. One gorilla calculus sample contained such unusual bacteria that researchers checked it again against the original publications before inclusion, a useful reminder of the database's careful verification standards.

Archaeological Context and Daily Life Connections

One of SMILE's strengths is the way it connects microscopic bacterial evidence with tangible archaeological settings. In Neolithic and Bronze Age cemeteries, calculus samples come from individuals buried with ceramic vessels, flint blades, bronze daggers, beads, and animal offerings. The same teeth that once processed early domesticated cereals now preserve bacterial patterns linked to diet change and emerging disease.

At Iron Age and early historic sites, individuals buried with iron weapons, jewellery, and imported goods provide material for comparison with simpler graves, allowing researchers to test whether elite diet, trade contacts, and urban living altered oral health and microbial ecosystems. In medieval towns, crowded conditions, refined grains, sugars, and regular fasting practices left distinctive traces in dental calculus, capturing not just decay and gum disease but also microbes that flourished in dense, often unsanitary environments.

Global Distribution and Microbial Diversity

By recording geographical coordinates for more than one thousand samples, the database maps ancient mouths from high northern latitudes of about 79° north to southern sites near 34° south, spanning a broad range of longitudes as well. This geographical spread turns the evidence into something close to a global map of ancient oral life.

Behind that broad distribution, SMILE contains 16,180 microbiome records covering 2,150 distinct organism types, including bacteria, archaea, fungi, and viruses. For each individual, the database records microbial presence and abundance exactly as reported in the original studies, whether as relative abundance percentages, direct read counts, or simple presence/absence data. That standardization makes it possible to compare medieval towns with Neanderthal caves, or Neolithic farms with modern oral communities.

Archaeological Sites as Microbial Archives

The research encourages us to see familiar archaeological sites in a new way: as archives of microscopic history. Pesturina Cave becomes more than a place of scattered stone tools and bones. Through calculus analysis, it also preserves traces of Neanderthal gum disease, dietary fibre processing, and bacteria involved in breaking down meat and plants. Medieval cemeteries with orderly grave rows likewise become records of changing oral health as cities expanded, diets shifted, and populations mixed.

Each burial, identified in excavation notebooks by trench and context numbers, acquires a parallel identity in SMILE through sample coordinates, dates, and microbial inventories. That dual framework allows researchers to investigate diet change, disease transmission, migration and oral health, the effects of social status on microbial communities, and environmental influences on the mouth's ecosystem.

Authentication and Ancient DNA Verification

Critical to SMILE's reliability is the rigorous authentication of ancient DNA. The database records authentication metrics for 212 samples with detailed damage-pattern analysis. Genuine ancient DNA shows characteristic age-related degradation, especially increased C-to-T and G-to-A transitions at fragment ends, where cytosine converts to uracil over time.

These authentication measures include damage percentages, host DNA proportions, and sequencing methodology. Together, they help researchers select the most reliable samples and reduce the risk that detected microbes reflect modern contamination introduced during excavation or laboratory work rather than genuine ancient oral communities.

Technological Innovation and AI Integration

The creation of the database also relied on innovative artificial intelligence methods to extract information from scattered archaeological publications. A five-step AI-assisted workflow analyzed PDFs and supplementary files from 45 studies, systematically converting tables, figures, and text into structured records. This approach allowed the project to incorporate publications in multiple languages, including Japanese, French, and Russian, greatly extending its reach beyond English-language research alone.

The system worked through document segmentation, table and figure identification, column mapping to archaeological and genetic categories, systematic record creation, and quality validation with unit normalization. Some data still had to be extracted manually, especially when locked inside complex visualizations, but the AI-assisted process nevertheless made it possible to compile more than 16,000 microbiome records that would otherwise have required months of manual work.

Research Applications and Future Directions

SMILE opens the door to research spanning evolutionary biology, medical history, and archaeology. It can be used to track the evolution of antibiotic resistance genes, trace pathogen emergence and spread, analyze the effects of dietary transitions on oral health, examine the impact of urbanization on microbial communities, and investigate climatic influences on ancient populations. Its timespan, from early Neanderthals to the late medieval period, provides an unusually long view of oral microbiome history.

By bringing together calculus samples from Neanderthal caves, hunter-gatherer shelters, Neolithic long barrows, and medieval churchyards, the project shows just how much history survives in ordinary dental deposits. SMILE turns these encrustations into a curated, searchable archive, allowing past mouths to speak again through their microbial signatures and revealing intimate details of daily life, health, and diet across more than 100 millennia.

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Original source article

https://www.researchsquare.com/article/rs-9586742/v1

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