A glimpse of the paleome in endolithic microbial communities.

BACKGROUND: The terrestrial subsurface is home to a significant proportion of the Earth's microbial biomass. Our understanding about terrestrial subsurface microbiomes is almost exclusively derived from groundwater and porous sediments mainly by using 16S rRNA gene surveys. To obtain more insights about biomass of consolidated rocks and the metabolic status of endolithic microbiomes, we investigated interbedded limestone and mudstone from the vadose zone, fractured aquifers, and deep aquitards. RESULTS: By adapting methods from microbial archaeology and paleogenomics, we could recover sufficient DNA for downstream metagenomic analysis from seven rock specimens independent of porosity, lithology, and depth. Based on the extracted DNA, we estimated between 2.81 and 4.25 × 105 cells × g-1 rock. Analyzing DNA damage patterns revealed paleome signatures (genetic records of past microbial communities) for three rock specimens, all obtained from the vadose zone. DNA obtained from deep aquitards isolated from surface input was not affected by DNA decay indicating that water saturation and not flow is controlling subsurface microbial survival. Decoding the taxonomy and functional potential of paleome communities revealed increased abundances for sequences affiliated with chemolithoautotrophs and taxa such as Cand. Rokubacteria. We also found a broader metabolic potential in terms of aromatic hydrocarbon breakdown, suggesting a preferred utilization of sedimentary organic matter in the past. CONCLUSIONS: Our study suggests that limestones function as archives for genetic records of past microbial communities including those sensitive to environmental stress at modern times, due to their specific conditions facilitating long-term DNA preservation. Video Abstract.

High conservation of the dental plaque microbiome across populations with differing subsistence strategies and levels of market integration.

Industrialization-including urbanization, participation in the global food chain and consumption of heavily processed foods-is thought to drive substantial shifts in the human microbiome. While diet strongly influences stool microbiome composition, the influence of diet on the oral microbiome is largely speculative. Multiple ecologically distinct surfaces in the mouth, each harbouring a unique microbial community, pose a challenge to assessing changes in the oral microbiome in the context of industrialization, as the results depend on the oral site under study. Here, we investigated whether microbial communities of dental plaque, the dense biofilm on non-shedding tooth surfaces, are distinctly different across populations with dissimilar subsistence strategies and degree of industrialized market integration. Using a metagenomic approach, we compared the dental plaque microbiomes of Baka foragers and Nzime subsistence agriculturalists in Cameroon (n = 46) with the dental plaque and calculus microbiomes of highly industrialized populations in North America and Europe (n = 38). We found that differences in microbial taxonomic composition between populations were minimal, with high conservation of abundant microbial taxa and no significant differences in microbial diversity related to dietary practices. Instead, we find that the major source of variation in dental plaque microbial species composition is related to tooth location and oxygen availability, which may be influenced by toothbrushing or other dental hygiene measures. Our results support that dental plaque, in contrast to the stool microbiome, maintains an inherent stability against ecological perturbations in the oral environment.

Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic.

Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name "paleofurans." This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.

Genetic population structure of the Xiongnu Empire at imperial and local scales.

The Xiongnu established the first nomadic imperial power, controlling the Eastern Eurasian steppe from ca. 200 BCE to 100 CE. Recent archaeogenetic studies identified extreme levels of genetic diversity across the empire, corroborating historical records of the Xiongnu Empire being multiethnic. However, it has remained unknown how this diversity was structured at the local community level or by sociopolitical status. To address this, we investigated aristocratic and local elite cemeteries at the western frontier of the empire. Analyzing genome-wide data from 18 individuals, we show that genetic diversity within these communities was comparable to the empire as a whole, and that high diversity was also observed within extended families. Genetic heterogeneity was highest among the lowest-status individuals, implying diverse origins, while higher-status individuals harbored less genetic diversity, suggesting that elite status and power was concentrated within specific subsets of the broader Xiongnu population.

Ecology, Not Host Phylogeny, Shapes the Oral Microbiome in Closely Related Species.

Host-associated microbiomes are essential for a multitude of biological processes. Placed at the contact zone between external and internal environments, the little-studied oral microbiome has important roles in host physiology and health. Here, we investigate the roles of host evolutionary relationships and ecology in shaping the oral microbiome in three closely related gorilla subspecies (mountain, Grauer's, and western lowland gorillas) using shotgun metagenomics of 46 museum-preserved dental calculus samples. We find that the oral microbiomes of mountain gorillas are functionally and taxonomically distinct from the other two subspecies, despite close evolutionary relationships and geographic proximity with Grauer's gorillas. Grauer's gorillas show intermediate bacterial taxonomic and functional, and dietary profiles. Altitudinal differences in gorilla subspecies ranges appear to explain these patterns, suggesting a close connection between dental calculus microbiomes and the environment, likely mediated through diet. This is further supported by the presence of gorilla subspecies-specific phyllosphere/rhizosphere taxa in the oral microbiome. Mountain gorillas show a high abundance of nitrate-reducing oral taxa, which may promote adaptation to a high-altitude lifestyle by modulating blood pressure. Our results suggest that ecology, rather than evolutionary relationships and geographic distribution, shape the oral microbiome in these closely related species.

Remembering St. Louis individual-structural violence and acute bacterial infections in a historical anatomical collection.

Incomplete documentary evidence, variable biomolecular preservation, and limited skeletal responses have hindered assessment of acute infections in the past. This study was initially developed to explore the diagnostic potential of dental calculus to identify infectious diseases, however, the breadth and depth of information gained from a particular individual, St. Louis Individual (St.LI), enabled an individualized assessment and demanded broader disciplinary introspection of ethical research conduct. Here, we document the embodiment of structural violence in a 23-year-old Black and/or African American male, who died of lobar pneumonia in 1930s St. Louis, Missouri. St.LI exhibits evidence of systemic poor health, including chronic oral infections and a probable tuberculosis infection. Metagenomic sequencing of dental calculus recovered three pre-antibiotic era pathogen genomes, which likely contributed to the lobar pneumonia cause of death (CoD): Klebsiella pneumoniae (13.8X); Acinetobacter nosocomialis (28.4X); and Acinetobacter junii (30.1X). Ante- and perimortem evidence of St.LI's lived experiences chronicle the poverty, systemic racism, and race-based structural violence experienced by marginalized communities in St. Louis, which contributed to St.LI's poor health, CoD, anatomization, and inclusion in the Robert J. Terry Anatomical Collection. These same embodied inequalities continue to manifest as health disparities affecting many contemporary communities in the United States.

HAYSTAC: A Bayesian framework for robust and rapid species identification in high-throughput sequencing data.

Identification of specific species in metagenomic samples is critical for several key applications, yet many tools available require large computational power and are often prone to false positive identifications. Here we describe High-AccuracY and Scalable Taxonomic Assignment of MetagenomiC data (HAYSTAC), which can estimate the probability that a specific taxon is present in a metagenome. HAYSTAC provides a user-friendly tool to construct databases, based on publicly available genomes, that are used for competitive read mapping. It then uses a novel Bayesian framework to infer the abundance and statistical support for each species identification and provide per-read species classification. Unlike other methods, HAYSTAC is specifically designed to efficiently handle both ancient and modern DNA data, as well as incomplete reference databases, making it possible to run highly accurate hypothesis-driven analyses (i.e., assessing the presence of a specific species) on variably sized reference databases while dramatically improving processing speeds. We tested the performance and accuracy of HAYSTAC using simulated Illumina libraries, both with and without ancient DNA damage, and compared the results to other currently available methods (i.e., Kraken2/Bracken, KrakenUniq, MALT/HOPS, and Sigma). HAYSTAC identified fewer false positives than both Kraken2/Bracken, KrakenUniq and MALT in all simulations, and fewer than Sigma in simulations of ancient data. It uses less memory than Kraken2/Bracken, KrakenUniq as well as MALT both during database construction and sample analysis. Lastly, we used HAYSTAC to search for specific pathogens in two published ancient metagenomic datasets, demonstrating how it can be applied to empirical datasets. HAYSTAC is available from https://github.com/antonisdim/HAYSTAC.

Paleoproteomics.

Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human-animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored "dark" proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

A primer for ZooMS applications in archaeology.

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

Emergence and intensification of dairying in the Caucasus and Eurasian steppes.

Archaeological and archaeogenetic evidence points to the Pontic-Caspian steppe zone between the Caucasus and the Black Sea as the crucible from which the earliest steppe pastoralist societies arose and spread, ultimately influencing populations from Europe to Inner Asia. However, little is known about their economic foundations and the factors that may have contributed to their extensive mobility. Here, we investigate dietary proteins within the dental calculus proteomes of 45 individuals spanning the Neolithic to Greco-Roman periods in the Pontic-Caspian Steppe and neighbouring South Caucasus, Oka-Volga-Don and East Urals regions. We find that sheep dairying accompanies the earliest forms of Eneolithic pastoralism in the North Caucasus. During the fourth millennium BC, Maykop and early Yamnaya populations also focused dairying exclusively on sheep while reserving cattle for traction and other purposes. We observe a breakdown in livestock specialization and an economic diversification of dairy herds coinciding with aridification during the subsequent late Yamnaya and North Caucasus Culture phases, followed by severe climate deterioration during the Catacomb and Lola periods. The need for additional pastures to support these herds may have driven the heightened mobility of the Middle and Late Bronze Age periods. Following a hiatus of more than 500 years, the North Caucasian steppe was repopulated by Early Iron Age societies with a broad mobile dairy economy, including a new focus on horse milking.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

Ancient genomes from the Himalayas illuminate the genetic history of Tibetans and their Tibeto-Burman speaking neighbors.

Present-day Tibetans have adapted both genetically and culturally to the high altitude environment of the Tibetan Plateau, but fundamental questions about their origins remain unanswered. Recent archaeological and genetic research suggests the presence of an early population on the Plateau within the past 40 thousand years, followed by the arrival of subsequent groups within the past 10 thousand years. Here, we obtain new genome-wide data for 33 ancient individuals from high elevation sites on the southern fringe of the Tibetan Plateau in Nepal, who we show are most closely related to present-day Tibetans. They derive most of their ancestry from groups related to Late Neolithic populations at the northeastern edge of the Tibetan Plateau but also harbor a minor genetic component from a distinct and deep Paleolithic Eurasian ancestry. In contrast to their Tibetan neighbors, present-day non-Tibetan Tibeto-Burman speakers living at mid-elevations along the southern and eastern margins of the Plateau form a genetic cline that reflects a distinct genetic history. Finally, a comparison between ancient and present-day highlanders confirms ongoing positive selection of high altitude adaptive alleles.

Understanding the microbial biogeography of ancient human dentitions to guide study design and interpretation.

The oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species in dental plaque, but it is not known how well these patterns are reflected in archaeological dental calculus. In most archaeological studies, a single sample of dental calculus is studied per individual and is assumed to represent the entire oral cavity. However, it is not known if this sampling strategy introduces biases into studies of the ancient oral microbiome. Here, we present the results of a shotgun metagenomic study of a dense sampling of dental calculus from four Chalcolithic individuals from the southeast Iberian peninsula (ca. 4500-5000 BP). Interindividual differences in microbial composition are found to be much larger than intraindividual differences, indicating that a single sample can indeed represent an individual in most cases. However, there are minor spatial patterns in species distribution within the oral cavity that should be taken into account when designing a study or interpreting results. Finally, we show that plant DNA identified in the samples is likely of postmortem origin, demonstrating the importance of including environmental controls or additional lines of biomolecular evidence in dietary interpretations.

Ancient dental calculus preserves signatures of biofilm succession and interindividual variation independent of dental pathology.

Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here, we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed interindividual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

The genomic origins of the Bronze Age Tarim Basin mummies.

The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000-2800 BC from the Dzungarian Basin and 13 individuals dating to around 2100-1700 BC from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early-Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria-Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert.

PyDamage: automated ancient damage identification and estimation for contigs in ancient DNA de novo assembly.

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

Shifts in gut and vaginal microbiomes are associated with cancer recurrence time in women with ovarian cancer.

Many studies investigating the human microbiome-cancer interface have focused on the gut microbiome and gastrointestinal cancers. Outside of human papillomavirus driving cervical cancer, little is known about the relationship between the vaginal microbiome and other gynecological cancers, such as ovarian cancer. In this retrospective study, we investigated the relationship between ovarian cancer, platinum-free interval (PFI) length, and vaginal and gut microbiomes. We observed that Lactobacillus-dominated vaginal communities were less common in women with ovarian cancer, as compared to existing datasets of similarly aged women without cancer. Primary platinum-resistance (PPR) disease is strongly associated with survivability under one year, and we found over one-third of patients with PPR (PFI < 6 months, n = 17) to have a vaginal microbiome dominated by Escherichia (>20% relative abundance), while only one platinum super-sensitive (PFI > 24 months, n = 23) patient had an Escherichia-dominated microbiome. Additionally, L. iners was associated with little, or no, gross residual disease, while other Lactobacillus species were dominant in women with >1 cm gross residual disease. In the gut microbiome, we found patients with PPR disease to have lower phylogenetic diversity than platinum-sensitive patients. The trends we observe in women with ovarian cancer and PPR disease, such as the absence of Lactobacillus and presence of Escherichia in the vaginal microbiome as well as low gut microbiome phylogenetic diversity have all been linked to other diseases and/or pro-inflammatory states, including bacterial vaginosis and autoimmune disorders. Future prospective studies are necessary to explore the translational potential and underlying mechanisms driving these associations.

Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.