Viral Metagenomics and Anthropology in the Americas

Viruses are the most abundant and diverse biological entities on earth. As humans, we could not exist without them. For billions of years they have been the unseen mediators of microbial and multicellular life. They are in the air, water, dirt and within every living thing on the planet. Although they are not technically alive since all viruses need a host cell to replicate, they outnumber our own cells an estimated 100 to 1 and therefore cannot be undervalued in their importance to human health and biology. Viruses are in close contact with humans from the moment we are born and as anthropologists we have commonly examined the cultural, social and political implications and responses of human viral infections. Yet there is so much untapped research potential when it comes to viruses. There are essentially five subject areas to discuss when addressing viruses from an anthropological perspective (Figure A1). First is that of host identity. Viruses are an integral part of who we are. This human-virus connection extends back hundreds of thousands of years, as these infectious agents are deeply ingrained within our genomes. In the first chapter I integrate literature of microbiology, virology and many others to posit that the traditional visual metaphor of the tree of life is inadequate and we would be better suited to describe each branch as its own treehouse built upon microbial and viral foundations. A second subject area is that of novelty. In other words, what the discoveries of novel viruses and understanding of understudied viral ecosystems can contribute to our definition of the human superorganism. In the second chapter I address novelty and future research by examining all of the current viral taxonomic information and their associations within the field of anthropology. Next generation sequencing of organisms capable of zoonotic transmission could identify viruses that may eventually be the next great health risk to humans. The third subject area that intersects viruses and anthropology is host history. In the past, viruses have been used to study complex human migratory patterns but this technique has yet to be fully realized within a viral metagenomic dataset. In chapter three, I use gut viral information from four populations in Oklahoma (Cheyenne and Arapaho) and Peru (Matses, Chincha, Tambo de Mora) in order to address the question of whether gut viral proteins are geographically structured. I hypothesize that DNA viruses are unique enough to the host that their sequences are closely tied to the geographic origin of the host. A fourth subject to be addressed is that of human health. Viruses obviously have a tremendous influence on human cells and microbial cells, but there is still much to be uncovered. In chapter four I use gut information from Oklahoma (Cheyenne and Arapaho, Norman) and Peru (Matses) to examine a recently discovered abundant DNA bacteriophage known as crAssphage. I biologically characterize the phage within the three populations by discerning its method of replication, potential microbial hosts, and its association with gut metabolites. I hypothesize that the abundance of crAssphage sequences found within the Cheyenne and Arapaho is not a result of their geography, but a host health association due to a decreased gastrointestinal health state. The final subject that links anthropology and viruses is the future, which is referred to throughout the document and summarized in the conclusion section. This dissertation provides a thorough literature review, novel data analysis and potential future directions, all of which are intent to stress the unexplored connections between viruses and the field of anthropology

The Impact of Delivery Mode on the Human Microbiome: A Global Perspective

The mode of childbirth in humans (vaginal or cesarean section) is considered to be one of the most influential factors in the development of gut microbiota of an infant. A microbiome is defined as a collection of microorganisms that are concentrated together in a single location or body site. The genetic material of all microbes, including bacteria, viruses, and fungi, comprises this complex ecosystem. The gut microbiota regulate digestion and strengthen the immune system in humans, helping to maintain homeostasis and are at the early stages of life, dictated by the method of delivery. Vaginal versus cesarean deliveries have increased amounts of microbial diversity, which is considered part of a ‘healthy’ microbiome. Literature has documented associations between cesarean delivery and the risk of developing illnesses such as ADHD, autism spectrum disorders, obesity, type 1 diabetes, and asthma. However, most research in this field has been dominated by studies originating from metropolitan areas within the United States, thus not giving a complete picture of the extent to which birthing methods impact human microbiomes on a global scale. This literature review discusses how these different delivery methods have affected the gut microbiome of infants in regions around the world (including countries in North America, Europe, Australia, Asia, and Central America)

Metagenomic analysis of dental calculus in ancient Egyptian baboons

Dental calculus, or mineralized plaque, represents a record of ancient biomolecules and food residues. Recently, ancient metagenomics made it possible to unlock the wealth of microbial and dietary information of dental calculus to reconstruct oral microbiomes and lifestyle of humans from the past. Although most studies have so far focused on ancient humans, dental calculus is known to form in a wide range of animals, potentially informing on how human-animal interactions changed the animals' oral ecology. Here, we characterise the oral microbiome of six ancient Egyptian baboons held in captivity during the late Pharaonic era (9th-6th centuries BC) and of two historical baboons from a zoo via shotgun metagenomics. We demonstrate that these captive baboons possessed a distinctive oral microbiome when compared to ancient and modern humans, Neanderthals and a wild chimpanzee. These results may reflect the omnivorous dietary behaviour of baboons, even though health, food provisioning and other factors associated with human management, may have changed the baboons' oral microbiome. We anticipate our study to be a starting point for more extensive studies on ancient animal oral microbiomes to examine the extent to which domestication and human management in the past affected the diet, health and lifestyle of target animals

Oral Microbiome Diversity in Chimpanzees from Gombe National Park

Historic calcified dental plaque (dental calculus) can provide a unique perspective into the health status of past human populations but currently no studies have focused on the oral microbial ecosystem of other primates, including our closest relatives, within the hominids. Here we use ancient DNA extraction methods, shotgun library preparation, and next generation Illumina sequencing to examine oral microbiota from 19 dental calculus samples recovered from wild chimpanzees (Pan troglodytes schweinfurthii) who died in Gombe National Park, Tanzania. The resulting sequences were trimmed for quality, analyzed using MALT, MEGAN, and alignment scripts, and integrated with previously published dental calculus microbiome data. We report significant differences in oral microbiome phyla between chimpanzees and anatomically modern humans (AMH), with chimpanzees possessing a greater abundance of Bacteroidetes and Fusobacteria, and AMH showing higher Firmicutes and Proteobacteria. Our results suggest that by using an enterotype clustering method, results cluster largely based on host species. These clusters are driven by Porphyromonas and Fusobacterium genera in chimpanzees and Haemophilus and Streptococcus in AMH. Additionally, we compare a nearly complete Porphyromonas gingivalis genome to previously published genomes recovered from human gingiva to gain perspective on evolutionary relationships across host species. Finally, using shotgun sequence data we assessed indicators of diet from DNA in calculus and suggest exercising caution when making assertions related to host lifestyle. These results showcase core differences between host species and stress the importance of continued sequencing of nonhuman primate microbiomes in order to fully understand the complexity of their oral ecologies

Biogeographic Study of Human Gut-Associated CrAssphage Suggests Impacts From Industrialization and Recent Expansion

CrAssphage (cross-assembly phage) is a bacteriophage that was first discovered in human gut metagenomic data. CrAssphage belongs to a diverse family of crAss-like bacteriophages thought to infect gut commensal bacteria belonging to Bacteroides species. However, not much is known about the biogeography of crAssphage and whether certain strains are associated with specific human populations. In this study, we screened publicly available human gut metagenomic data from 3,341 samples for the presence of crAssphage sensu stricto (NC_024711.1). We found that crAssphage prevalence is low in traditional, hunter-gatherer populations, such as the Hadza from Tanzania and Matses from Peru, as compared to industrialized, urban populations. Statistical comparisons showed no association of crAssphage prevalence with variables such as age, sex, body mass index, and health status of individuals. Phylogenetic analyses show that crAssphage strains reconstructed from the same individual over multiple time-points, cluster together. CrAssphage strains from individuals from the same study population do not always cluster together. Some evidence of clustering is seen at the level of broadly defined geographic regions, however, the relative positions of these clusters within the crAssphage phylogeny are not well-supported. We hypothesize that this lack of strong biogeographic structuring is suggestive of an expansion event within crAssphage. Using a Bayesian dating approach, we estimate that this expansion has occurred fairly recently. Overall, we determine that crAssphage presence is associated with an industrialized lifestyle and the absence of strong biogeographic structuring within global crAssphage strains is likely due to a recent population expansion within this bacteriophage.This study was supported by a grant from the National Institutes of Health (https://www.nih.gov/), NIH R01 GM089886, awarded to C.M.L., C.W., and K.S. Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

Metagenomic Analysis of Dental Calculus in Ancient Egyptian Baboons

Dental calculus, or mineralized plaque, represents a record of ancient biomolecules and food residues. Recently, ancient metagenomics made it possible to unlock the wealth of microbial and dietary information of dental calculus to reconstruct oral microbiomes and lifestyle of humans from the past. Although most studies have so far focused on ancient humans, dental calculus is known to form in a wide range of animals, potentially informing on how human-animal interactions changed the animals’ oral ecology. Here, we characterise the oral microbiome of six ancient Egyptian baboons held in captivity during the late Pharaonic era (9th–6th centuries BC) and of two historical baboons from a zoo via shotgun metagenomics. We demonstrate that these captive baboons possessed a distinctive oral microbiome when compared to ancient and modern humans, Neanderthals and a wild chimpanzee. These results may reflect the omnivorous dietary behaviour of baboons, even though health, food provisioning and other factors associated with human management, may have changed the baboons’ oral microbiome. We anticipate our study to be a starting point for more extensive studies on ancient animal oral microbiomes to examine the extent to which domestication and human management in the past affected the diet, health and lifestyle of target animals

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Objectives The tropics harbor a large part of the world\u27s biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semi‐tropical sites in Tanzania, Mexico, and Puerto Rico (N = 12). Materials and Methods All samples were extracted twice, built into double‐stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step. Results No significant difference was found in overall ancient DNA yields or post‐mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality‐filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments. Discussion Both methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future

Origins of an Unmarked Georgia Cemetery Using Ancient DNA Analysis

Determining the origins of those buried within undocumented cemeteries is of incredible importance to historical archaeologists and in many cases, the nearby communities. In the case of Avondale Burial Place, a cemetery in Bibb County, Georgia, in use from 1820 to 1950, all written documentation of those interred within it has been lost. Osteological and archaeological evidence alone could not describe, with confidence, the ancestral origins of the 101 individuals buried there. In the present study, we utilize ancient DNA extraction methods to investigate the origins of Avondale Burial Place through the use of well-preserved skeletal fragments from 20 individuals buried in the cemetery. Through examination of hypervariable region I in the mitochondrial genome (HVR1, mtDNA), we determine haplotypes for all 20 of these individuals. A total of 18 of the 20 individuals belong to the L or U haplogroups, suggesting that Avondale Burial Place was most likely used primarily as a resting place for African Americans. After the surrounding Bibb County community expressed interest in investigating potential ancestral relationships to those within the cemetery, a total of eight potential descendants provided saliva in order to obtain mtDNA HVR1 information. This phase of the study revealed that two different individuals from Avondale Burial Place matched two individuals with oral history ties to the cemetery. Using the online tool EMPOP, we calculated the likelihood of these exact matches occurring by chance alone

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. Inmost 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