Dental calculus Methanobrevibacter massiliense-Pyramidobacter piscolens, a millennium-old association

Abstract Background The metagenomic study of ancient dental calculus yielded two novel Methanobrevibacter species, TS-1 and TS-2. TS-2 was phylogenetically related to Methanobrevibacter massiliense ( M. massiliense ), a methanogen previously cultivated in close association with the bacterium Pyramidobacter piscolens ( P. piscolens ) in the oral cavity. Results Comparative genomics, including Average Nucleotide Identity, digital DNA-DNA hybridisation, and genome sequence-derived 16S rRNA gene sequence analysis revealed that TS-2 was confused with M. massiliense . Quantifying ancient and modern dental calculus metagenomic read counts yielded a positive correlation between Methanobrevibacter sp. YE315, the proxy-methanogen for M. massiliense in the Kraken2 database and P. piscolens (Pearson correlation test, p = 0.23, P -value = 0.026); whereas no such correlation was found with Fretibacterium fastidiosum (p = -0.04, P -value = 0.69) and Synergistetes phylum (p = -0.0072, P -value = 0.94) used as negative controls. Similar results were obtained with unclassified Methanobrevibacter , and the combined read counts of unclassified Methanobrevibacter and Methanobrevibacter sp. YE315. Conclusion Genomic comparisons provide evidence that M. massiliense is the named and cultured representative of Neolithic-old TS-2, suggesting a long-standing association between M. massiliense and P. piscolens. The nature, determinants, and evolutionary significance of this association remain unknown.