In our analysis of modern dental calculus, a cluster consisting of four Japanese samples was observed at lower PC1 values (Fig.
In contrast, results from ancient dental calculus studies have reported greater microbial diversity across different regions of Europe and Asia18.
These observations suggest that Methanobrevibacter species exhibit distinct prevalence patterns in ancient dental calculus.
3a, consistent with previous findings from ancient dental calculus in the Netherlands60, suggesting that these taxa may represent common features of ancient oral microbiomes across different geographic regions.
This admixture and subsequent cultural and demographic continuity ultimately gave rise to the modern Japanese population through later historical periods, including the Edo period.
In our analysis of modern dental calculus, a cluster consisting of four Japanese samples was observed at lower PC1 values (Fig. 1). Although this pattern might initially appear to reflect geographic variation, further examination of the taxonomic composition suggests an alternative explanation. The cluster was characterized by higher abundances of members of the “Red Complex,” including T. denticola, P. gingivalis, and T. forsythia, which are well known to be associated with periodontal disease54. In contrast, S. sanguinis, a species commonly associated with oral health57, was more abundant in samples outside this cluster. Clinical assessments revealed that the mean probing pocket depth (PPD) of the modern Japanese individuals was 5.2 ± 2.2 mm, indicating that all participants suffered from moderate to severe periodontitis. This clinical status is closely associated with the taxonomic clustering observed in Fig. 1. Specifically, the high abundance of “Red Complex” taxa, such as T. denticola, in these samples aligns with the high PPD values. In the absence of direct clinical assessment for the ancient individuals, dental calculus was collected from specimens in which the relationship between calculus deposits and the underlying alveolar bone could be evaluated. Visual inspection revealed varying degrees of alveolar bone resorption consistent with chronic periodontal attachment loss. Importantly, these observations are consistent with the distribution of ancient samples in Fig. 2, where some of them are positioned between the two clusters observed in modern individuals along PC2, and therefore do not contradict the interpretation that these clusters reflect differences in oral health status. Taken together, these observations suggest that the clustering pattern may reflect differences in oral hygiene or periodontal status rather than geographic variation.
More broadly, these findings indicate that, particularly in modern populations from industrialized countries, widespread lifestyle westernization may have contributed to reduced inter-individual diversity within the oral microbiome. In contrast, results from ancient dental calculus studies have reported greater microbial diversity across different regions of Europe and Asia18.
Consistent with a previous 16 S rRNA-based study20, our shotgun metagenomic analysis revealed clear compositional differences between modern and ancient Japanese oral microbiomes (Fig. 2). Notably, M. oralis was frequently detected in ancient samples, consistent with findings from the UK58, and its relative abundance varied substantially across individuals [Fig. S11], in agreement with a previous report59. In contrast, M. oralis was not detected in modern Japanese samples in this study, possibly due to the limited sample size. These observations suggest that Methanobrevibacter species exhibit distinct prevalence patterns in ancient dental calculus. Beyond taxonomic composition, functional differences were also evident between modern and ancient samples (Fig. 4). Previous studies of paleofeces have highlighted genes related to the starch utilization system (SUS) as distinguishing features between ancient and modern populations14. Motivated by these observations, we considered whether similar differences might be detectable in our dental calculus data and found that the relative abundance of SUS genes was significantly different between ancient and modern samples (Welch’s t-test, t(16) = −5.8, p < 0.001). This result suggests that the transition to starch-rich diets in modern populations may have influenced the structure of the oral microbiome.
Furthermore, analysis of ancient individuals revealed subtle regional differences in microbial composition between Mainland Japan and Okinawa (Fig. 3a), potentially reflecting lifestyle differences between the two regions. Notably, M. oralis and S. sanguinis were identified in the biplot of Fig. 3a, consistent with previous findings from ancient dental calculus in the Netherlands60, suggesting that these taxa may represent common features of ancient oral microbiomes across different geographic regions. By contrast, no clear differences were observed by social status within Mainland Japan (Fig. 3b) or between urban and rural areas (Fig. S8). Our results also showed that altering thresholds for read count and species richness affected both PCoA and PERMANOVA results, highlighting how environmental contamination, DNA fragmentation, and deamination complicate accurate reconstruction of the ancient oral microbiome. Notably, the use of the HOMD—a reference database constructed primarily from Western populations—may have led to underestimation of microbial diversity in ancient Japanese samples. Future studies incorporating more comprehensive oral microbiome data from East Asian populations may improve the accuracy of ancient oral microbiome reconstruction by enabling the identification of truly ancient and oral-specific taxa, based on DNA damage signatures and their presence in modern Japanese datasets.
The Japanese archipelago was originally inhabited by hunter-gatherer Jomon people, who were later admixed with agricultural migrants from the Asian continent around the 10th century BCE, marking the beginning of the Yayoi period and introducing wet-rice farming to Japan61. This admixture and subsequent cultural and demographic continuity ultimately gave rise to the modern Japanese population through later historical periods, including the Edo period. With respect to temporal variation, our analysis confirmed the previously reported phylogenetic divergence of Anaerolineaceae sp. oral taxon 439 between the Edo and Jomon periods22, using newly sequenced samples from multiple archaeological sites (Fig. 5a). In addition, several other species—including S. sinensis and E. minutum—formed Jomon-specific clades (Fig. 5b, Fig. S9), suggesting phylogenetic turnover associated with the migration, admixture, and cultural interactions that began in the Yayoi period and continued thereafter, leading to the transition from hunting and gathering to agriculture and the resulting transformation of dietary habits. Further analysis of dental calculus from other periods (e.g., the Yayoi period) may provide additional insights into the relationship between bacterial evolution and human demographic history in ancient Japan. In particular, the successful phylogenetic analysis of S. sinensis, a species reported to be abundant in ancient dental calculus but rare in modern industrialized populations59, suggests that ancient dental calculus may preserve evolutionary diversity of oral bacterial taxa that are now diminishing in modern human populations.
We also focused on M. oralis, a periodontal-associated species62 frequently detected in ancient dental calculus, and performed phylogenetic analyses using both reference-based and assembly-based approaches. Both methods consistently revealed two major clades (Fig. 6a, Fig. S10), one of which diverged from the M. oralis reference sequence. This finding suggests that diversification occurred not only at the species level within Methanobrevibacter15 but also within M. oralis itself in ancient Japan [Fig. S12]. Estimated divergence times for the two clades indicate that their split occurred several centuries before or after the beginning of the Common Era, raising the possibility that this diversification emerged domestically. Notably, all 11 samples from Hitotsubashi High School (Tokyo) belonged to a single clade, suggesting potential geographic structuring in clade distribution. We also observed a statistically significant association between clade membership and host sex; however, this pattern should be interpreted with caution given the substantial proportion of individuals with unknown sex. In one father–daughter pair, the father’s M. oralis strain belonged to one clade, whereas the daughter’s strain belonged to the other. This observation may reflect sex-associated patterns in clade distribution and could, for example, be consistent with processes such as sex-specific adaptation or transmission dynamics.
Comparative genomic analysis of the two clades revealed that clade A, which includes the reference strain, possessed a greater number of clade-specific genes (Table 3). Among these, the presence of the SpoVT/AbrB-like domain, known to regulate the transcription of genes involved in antibiotic resistance and biofilm formation63,64, suggests possible adaptation to antibiotic-influenced oral environments in modern individuals However, due to the limited detection of M. oralis sequences in modern Japanese samples, it remains unclear whether this phylogenetic diversity persists in present-day populations. Broader analyses of the modern Japanese oral microbiome will be necessary to clarify the origin, functional roles, and health implications of M. oralis clades. Previous studies have suggested that M. oralis interacts with diverse oral bacteria through metabolic processes such as hydrogen consumption during methanogenesis, and has also been implicated in extra-oral, systemic conditions21. If M. oralis can be more consistently detected in modern Japanese populations, it may be possible to investigate whether such microbial interactions and potential health associations have changed over time.
All samples previously reported to exhibit traces of tooth blackening (ohaguro)22 were assigned to clade B, suggesting a potential link between this Edo period custom—predominantly practiced by adult women—and the observed sex-specific clade distribution. Since ohaguro involved the application of iron-rich substances55, we investigated clade-specific variants in iron-associated metabolic genes. Notably, substitutions unique to clade B were identified in mvhA and mvhB, which encode methyl viologen hydrogenase—a ferredoxin-type enzyme involved in methanogenesis that requires iron at its active site56. Comparison with reference sequences from related taxa indicated that two of the mvhB substitutions were exclusive to clade B (Table 4). One possible hypothesis is that these variants may have conferred an energetic advantage under iron-rich oral conditions associated with ohaguro. Alternatively, if the substitutions were metabolically disadvantageous, the iron-rich environment may have enabled the persistence of otherwise suboptimal strains. As our study did not systematically record ohaguro traces on a per-sample basis, future research incorporating standardized and objective assessment of tooth blackening will be essential to further investigate the relationship between this cultural practice and the oral microbiome. In addition, detailed chemical analyses of residual compounds may help to evaluate whether antimicrobial substances, such as flavonoids65, which may be present in plant-derived materials used in ohaguro, influenced oral hygiene and microbial community composition.
Consistent with previous studies20,22, our results confirmed distinct differences in oral microbiome composition between ancient and modern Japanese individuals, as well as phylogenetic divergence between the Jomon and Edo periods. We also identified regional variation in ancient microbiome composition and clade-level diversity within the periodontal-associated species M. oralis. Continued progress in oral microbiome research will likely yield valuable insights into the dynamic relationships between microbial evolution, cultural practices, and human demographic history in Japan.
