Evidence of a millennia-old association between a methanogenic archaeon and a bacterium in dental calculus: a re-analysis of ancient and modern metagenomic datasets.

Granehäll et al. (2021) identified TS-2 as an unknown Methanobrevibacter lineage abundant in ancient dental calculus, less prevalent in modern samples, and not linked to any cultivated representative. We aimed (i) to determine whether TS-2 corresponds to the cultivated oral archaeon Methanobrevibacter massiliense using genome-based species delineation, and (ii) to assess the antiquity of the association between M. massiliense and Pyramidobacter piscolens in ancient and modern dental calculus. This fully in silico study combined comparative genomics with re-analysis of 97 ancient and modern dental calculus metagenomes. Species-level relationships were assessed using average nucleotide identity, digital DNA–DNA hybridization, and 16S rRNA phylogeny. Metagenomic associations were examined using Kraken2-based taxonomic profiling, with Methanobrevibacter sp. YE315 as a proxy because M. massiliense was absent from the classifier database, and direct competitive read mapping to M. massiliense . Associations with P. piscolens were evaluated using Spearman correlation and negative binomial regression. Comparative genomics supported TS-2 and M. massiliense as the same species-level taxon, with >95% average nucleotide identity and >90% digital DNA–DNA hybridization. In metagenomic analyses, the YE315 proxy was positively associated with P. piscolens in Kraken2 Spearman analysis (ρ = 0.3506, q = 0.0026), and mapped M. massiliense reproduced this pattern (ρ = 0.2939, q = 0.0153). Negative binomial models showed concordant but weaker support, whereas the signal for M. oralis was less consistent. These results identify M. massiliense as the cultivated representative of TS-2 and support an ancient, recurrent association between M. massiliense and P. piscolens in dental calculus. • TS-2 corresponds to the cultivated archaeon Methanobrevibacter massiliense. • M. massiliense was linked to Pyramidobacter piscolens in dental calculus. • This association was detected in both ancient and modern metagenomic datasets. • Kraken2 proxy analysis and direct mapping yielded concordant association signals. • Oral methanogen turnover may reflect long-term ecological and dietary shifts.