Microbial archaeology is flourishing in the era of high-throughput sequencing, revealing the agents behind devastating historical plagues, identifying the cryptic movements of pathogens in prehistory, and reconstructing the ancestral microbiota of humans. Here, we introduce the fundamental concepts and theoretical framework of the discipline, then discuss applied methodologies for pathogen identification and microbiome characterization from archaeological samples. We give special attention to the process of identifying, validating, and authenticating ancient microbes using high-throughput DNA sequencing data. Finally, we outline standards and precautions to guide future research in the field.

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Significance: The microbiome plays key roles in human health, but little is known about its evolution. We investigate the evolutionary history of the African hominid oral microbiome by analyzing dental biofilms of humans and Neanderthals spanning the past 100,000 years and comparing them with those of chimpanzees, gorillas, and howler monkeys. We identify 10 core bacterial genera that have been maintained within the human lineage and play key biofilm structural roles. However, many remain understudied and unnamed. We find major taxonomic and functional differences between the oral microbiomes of Homo and chimpanzees but a high degree of similarity between Neanderthals and modern humans, including an apparent Homo -specific acquisition of starch digestion capability in oral streptococci, suggesting microbial coadaptation with host diet.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007–0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003–70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Documenting the natural diversity of eukaryotic organisms in the nonhuman primate (NHP) gut is important for understanding the evolution of the mammalian gut microbiome, its role in digestion, health and disease, and the consequences of anthropogenic change on primate biology and conservation. Despite the ecological significance of gut-associated eukaryotes, little is known about the factors that influence their assembly and diversity in mammals. In this study, we used an 18S rRNA gene fragment metabarcoding approach to assess the eukaryotic assemblage of 62 individuals representing 16 NHP species. We find that cercopithecoids, and especially the cercopithecines, have substantially higher alpha diversity than other NHP groups. Gut-associated protists and nematodes are widespread among NHPs, consistent with their ancient association with NHP hosts. However, we do not find a consistent signal of phylosymbiosis or host-species specificity. Rather, gut eukaryotes are only weakly structured by primate phylogeny with minimal signal from diet, in contrast to previous reports of NHP gut bacteria. The results of this study indicate that gut-associated eukaryotes offer different information than gut-associated bacteria and add to our understanding of the structure of the gut microbiome.

Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism primarily treated through a phenylalanine-restrictive diet that is frequently supplemented with an amino acid formula to maintain proper nutrition. Little is known of the effects of these dietary interventions on the gut microbiome of PKU patients, particularly in adults. In this study, we sequenced the V4 region of the 16S rRNA gene from stool samples collected from adults with PKU (n = 11) and non-PKU controls (n = 21). Gut bacterial communities were characterized through measurements of diversity and taxa abundance. Additionally, metabolic imputation was performed based on detected bacteria. Gut community diversity was lower in PKU individuals, though this effect was only statistically suggestive. A total of 65 genera across 5 phyla were statistically differentially abundant between PKU and control samples (p < 0.001). Additionally, we identified six metabolic pathways that differed between groups (p < 0.05), with four enriched in PKU samples and two in controls. While the child PKU gut microbiome has been previously investigated, this is the first study to explore the gut microbiome of adult PKU patients. We find that microbial diversity in PKU children differs from PKU adults and highlights the need for further studies to understand the effects of dietary restrictions.