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Abstract

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (~8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.

Citation: Tito RY, Knights D, Metcalf J, Obregon-Tito AJ, Cleeland L, et al. (2012) Insights from Characterizing Extinct Human Gut Microbiomes. PLoS ONE 7(12): e51146. doi:10.1371/journal.pone.0051146

Editor: Michael Hofreiter, University of York, United Kingdom

Received: June 24, 2012; Accepted: November 1, 2012; Published: December 12, 2012

Copyright: © 2012 Tito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Support for this research is from the National Institutes of Health (grants R01 HG005172-01 and R01 GM089886-01A1) and the National Science Foundation (NSF#0845314). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The human distal gut is a complex bacterial bioreactor housing a 100 times the number of genes than its human host genome [1] and functions as a vital adaptive “organ” [2]. The genomics of microbial ecologies (microbiomes) has gained great attention recently, in part, because the Human Microbiome Project (HMP) a U.S. National Institutes of Health Initiative [3]. One primary objective of the HMP is to determine whether there are core aspects of microbiomes shared by healthy humans. One consideration is that core aspects of microbiomes observed in modern cosmopolitan populations today may underrepresent core aspects of human microbiomes that had existed historically, or prehistorically.

The modern cosmopolitan transformation, such as the advent of processed foods, antibiotics and other systemic drugs, and various sanitation technologies, has impacted our interaction with microbes. This transformation has reduced the spread of aggressive infectious diseases, which are chiefly problematic for the densely populated populations. Unfortunately, these interventions are far from targeted strikes, and a wide range of potentially beneficial microbes are caught in the crossfire [4]. Analogous to James Neel's hypothesis regarding syndromes of impaired genetic homeostasis [5], our modern lifestyle may have impacted ancestral mutualistic relationships between humans and microbes. The result is a potential increased risk for autoimmune diseases among other health related conditions [4], [6], [7], [8], [9], [10].

Understanding the evolution of human-microbe ecosystems greatly benefits from a baseline reflecting an ancestral state of the human microbiome. The study of our closest living cousins, the other great apes, provides one path to reconstruct ancestral microbiomes. But the human-chimp common ancestor was over 6.5 million years ago, providing ample time for extensive evolution in the human line. Alternatively, the study of modern people living a more traditional and isolated lifestyle provides a valuable perspective on the ancestral state of human microbiomes, but arguably, there are no traditional communities unaffected by modern globalization and even if we make exceptions for those communities deep within South American jungles, these communities provide a very restricted view of the potential variation in ancestral microbiomes recovered from other environments.

Retrieving human microbiome information from samples left behind by our distant ancestors would provide an ideal approach to understanding the coevolution of humans and microbes. Fecal material is the typical sample proxy for characterizing distal gut microbiomes. Therefore, ancient fecal samples (coprolites) have the potential to reveal the ancestral state of the human gut microbiome [11], are common within some archaeological sites representing sedentary lifestyles, as well as for some hunter-gatherer sites where coprolites have been retrieved from cave deposits and from mummies. Ideally, coprolites provide a view of how humans and microbes coevolved in response to different environments over time, including responses to both natural and cultural change. Previous molecular analyses of coprolites have been used to retrieve dietary information [12], [13]. However, the potential for retrieving ancient microbiome data is confounded by continuing microbial activity, environmental contamination, degradation and other post-depositional processes.

We provide a systematic examination of coprolite microbial communities from three different archaeological sites, each exposed to different environmental conditions. We assess the challenges of ancient gut microbiome research attributed to post-depositional processes including molecular degradation and contamination, and we retrieve ancient microbiome profiles consistent with the primate gut. The results suggest that there are aspects of ancestral human microbiomes that are atypical of modern cosmopolitan populations, and they reveal novel avenues to explore the prehistoric human condition.

Results

We analyzed the microbial composition of two coprolite samples from each of the three archaeological sites: Hinds Cave (~8000 years B.P.) in southwestern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.) shown in Figure 1 (see Figure S1 for an overview of methods). These three sites provide a broad range of environmental conditions. Hinds Cave is a rock shelter with extensive and repeated human occupation for thousands of years. The Hinds Cave coprolites (BE04 and BE21) were morphologically intact and part of abundant geological lenses of coprolites found throughout the site [14]. In contrast, the Caserones coprolites (CA10 and CA18) were retrieved directly from the intestines of a mummy and had no exposure to soil. The coprolites from Rio Zape (ZA04 and ZA23) were recovered from the La Cueva de los Chiquitos Muertos [15], a deep, dry cave. The Rio Zape coprolites were originally deposited in a midden composed of sand and refuse in the cave. The midden was used for seven child burials, which were made at, or around, the same time. The midden with the burials, refuse, and coprolites were sealed under an adobe layer that prevented disturbance. The intact preservation of material under the adobe layer included food offerings of agave, beans, corn, cucurbits and piñón.

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Figure 1. The geographic distribution and bacterial diversity of the included samples.

These data resulted from comparison of the 16S rRNA V3. Taxon distribution and cluster dendrogram were limited to phyla with a frequency of 5% or more.

doi:10.1371/journal.pone.0051146.g001

Results for negative controls are included. EX02 refers to a negative control used during DNA extraction in which the coprolite sample was replaced by water. EX03 refers to a negative control used during amplification in which the DNA extract was replaced by water. Results for these controls required a greatly extended qPCR reaction than that used for the ancient DNA reactions (see Methods).

Figure 1 provides the geographic and phyla distribution for the ancient samples, in comparison to the phyla inferred from modern primates, while table S1 provides QIIME 1.3.0 [16] taxonomic assignments in detail. The coprolites from Rio Zape have phyletic representation that is consistent with that observed in humans and primates, while the coprolites from Caserones have very low diversity with respect to phyla. A higher phyletic diversity for Rio Zape samples compared to Caserones samples is observed in a species-level rarefaction analysis (Figure S2). Analysis of the Hinds Cave BE04 sample showed phyla typical of the gut. Sample BE21 harbored phyla observed in pooled negative controls, raising an initial concern about contamination, which was later resolved by additional analyses.

Venn-Euler diagrams (Figure 2) provide a general pattern where coprolites from the same site tend to cluster. Specifically, the microbes present in the Rio Zape samples clustered together and represent constituents in the primate gut. In contrast, although the Caserones and Hinds Cave data were clustered among Rio Zape, they did not contained microbial similarities with primate gut as observed in Rio Zape. These Venn-Euler diagrams therefore reveal that the coprolites from the same site shared a more similar coprolite microbiome than those from different sites and that the coprolites varied greatly in their degree of similarity to the modern and primate gut. A Principal Coordinates Analysis (Figure S3) is consistent with Venn-Euler analysis; both approaches depict resemblances between Rio Zape and primate gut microbiome.

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