https://orcid.org/0000-0001-5739-8935
Figures
Abstract
The research of microbiome derived from mummified human remains, coprolites and paleofeces has gained significant interest over several decades, aiming to elucidate the evolution of microbial interactions and shed light on the lifestyles of past populations. In this study, we analyzed the gut microbiome of a pre-Hispanic male individual referred to as the Zimapán man, dated to 936 BP, discovered on the border between Mesoamerica and Aridoamerica. Employing high-throughput 16S rRNA gene sequencing on both the paleofeces and mummified intestinal tissue, we conducted a comprehensive characterization of the Zimapán man’s gut microbiome. The bacterial community was described, and a weighted UniFrac-based principal coordinates analysis (PCoA) was performed. The multivariate analysis incorporated microbiome samples from diverse environments, such as soils, compost, and both contemporary and ancient human gut microbiota. The analysis revealed bacterial groups associated with the human microbiome, including families such as Peptostreptococcaceae, Clostridiaceae, Enterobacteriaceae, and Enterococcaceae. Notably, a high abundance of the Clostridiaceae group was found, similar to those discovered in the intestinal tissue of mummies from other geographic regions. Additionally, unique groups such as Romboutsia hominis, exclusively isolated from human intestines and not previously reported in ancient human microbiomes, were identified. Furthermore, our analysis demonstrated that the bacterial composition did not resemble soil and compost environments. This initial characterization successfully achieved the goal of identifying bacterial groups of the gut microbiome in the Zimapán samples. Consequently, this study contributes to the ongoing expansion of knowledge regarding ancient microbiomes across diverse temporal, historical, geographical, and environmental contexts.
Citation: Rosas-Plaza S, Mainou L, Delgado G, Morales R, Aguilar-Romero A, Escalante AE, et al. (2025) Microbiome characterization of a pre-Hispanic man from Zimapán, Mexico: Insights into ancient gut microbial communities. PLoS One 20(10): e0331137. https://doi.org/10.1371/journal.pone.0331137
Editor: Dario Piombino-Mascali, Vilnius University: Vilniaus Universitetas, LITHUANIA
Received: September 18, 2024; Accepted: August 11, 2025; Published: October 8, 2025
Copyright: © 2025 Rosas-Plaza 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.
Data Availability: All raw amplicon sequencing data generated in this study, including 16S rRNA gene sequences from both the V3 and V3-V4 regions, have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1306640. The dataset is publicly available at https://www.ncbi.nlm.nih.gov/sra/PRJNA1306640.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
In recent years, there has been significant progress in the study of ancient samples through the analysis of DNA and other molecules, with the aim of exploring the evolutionary history of living organisms [1]. This approach encompasses the examination of remains from extinct organisms and ancient human populations, such as bones, hair, or mummified tissue, as well as archaeological artifacts, including various ancient tools. These materials can provide invaluable insights into understanding the history of our ancestors [2–3]. The integration of information from archaeological, anthropological, and molecular studies can significantly contribute to our understanding of human evolution and shed light on the context in which ancient populations lived, including the lifestyles of individuals [4,5]. This multidisciplinary approach enables a more comprehensive and detailed reconstruction of the evolutionary history of human populations, thereby contributing to a holistic understanding of our species.
Among the different types of ancient samples, coprolites, paleofeces, and in some very rare cases, mummified tissue, are considered significant sources of historical information [6]. Through these types of samples, valuable insights into the diet, diseases, and potential communities of commensal microorganisms present in the intestines of ancient populations (ancient human microbiome) can be obtained [6]. This is particularly important as it allows for a deeper understanding of the interaction between humans and the microbiome in natural environments that have not undergone significant modifications, unlike the substantial changes experienced in the globalized era [7]. While the studies of modern human microbiome have been conducted on populations maintaining lifestyles such as hunting and gathering, it is undeniable that they have been directly or indirectly affected by globalization and industrialization processes [7].
In particular, the study of the human microbiome has become a focal point of interest in the past decade, recognizing its correlation with the health status of human populations [8]. Research has demonstrated that the microbiome plays a crucial role in various vital functions, including the absorption of vitamins and protection against pathogens, among others [9]. Additionally, large-scale studies have shown that processes of urbanization have led to changes in the human gut microbiome [10]. In this context, analyzing changes across different scales of time and space becomes a key element in understanding alterations in the microbiome throughout the history of human civilization. Thus, efforts have been undertaken to explore the composition and potential functions of the ancient human microbiome from various populations living in different eras and civilizations, thereby providing a broader perspective on the human microbiome throughout history [11–16].
Through studies on the ancient human microbiome, it has been discovered that changes in the modern diet, exposure to toxic contaminants, and the continuous use of antibiotics have likely resulted in positive selection for bacterial taxa involved in specific metabolic activities within the human microbiome [17]. Similarly, a decrease in bacterial groups in contemporary human populations, such as Treponema, has been detected. Treponema is mainly reported in populations with traditional contemporary lifestyles (hunter-gatherers and agriculturalists) and in the ancient human intestinal microbiome [14,16]. On the other hand, the loss of functional genes in the contemporary human intestinal microbiome associated with the degradation of certain polysaccharides, such as chitin (a component of arthropod exoskeletons and fungal cell walls), has been suggested. This is likely due to the reduced consumption of insects in modern diets [16]. Taken together, these findings suggest adaptive changes in the human microbiome over time due to modifications in diet and the environment [17].
Despite the information generated from ancient microbiome studies, it is still necessary to continue collecting data that sheds light on changes in microbial communities and the functional profiles of the ancient human microbiome. In this sense, understanding both specific changes (at the population or regional level) and broader patterns (at the species level) throughout history is crucial in order to understand the relationship between humans and their microbiome, as well as the health status of past human populations [18].
In this context, this study aims to contribute to the expansion of knowledge about the ancient human microbiome through the characterization of bacterial communities present in paleofeces and mummified tissue samples from a pre-Hispanic individual found in El Saucillo, Zimapán, Hidalgo. The samples were provided by La Coordinación Nacional de Conservación del Patrimonio Cultural-INAH [19,20]. The individual was a young adult male, aged between 21 and 35 years at the time of death, discovered in a rock shelter in El Saucillo, within a mortuary bundle lying on a cultural organic layer composed mainly of grass and xerophytic vegetation [21]. The young adult likely belonged to the Otopame culture [22], one of the oldest cultures in Mesoamerica. While previous studies on mummies and human remains have been conducted, the challenge of finding such samples hinders a complete reconstruction of the ancient microbiome and its correlation with the lifestyle of the inhabitants. The first analysis of the ancient microbiome in mummies was carried out on an individual from the ancient Inca culture in Peru [15], followed by the description of the intestinal tissue of a male found in the glaciers of Germany, known as the Tyrolean Iceman dated to the Neolithic era (5350–5100 BP) [11].
For the analysis of the ancient microbiome, DNA extraction and subsequent sequencing of the 16S rRNA gene from the paleofeces and intestinal tissue were performed using the Illumina MiSeq platform. This initial characterization successfully addressed the challenge of identifying some bacterial groups that may have been part of the Zimapán man’s gut microbiome. All these findings contribute to the continuous expansion of knowledge regarding the ancient microbiome across various time frames, encompassing diverse historical, geographical, and environmental contexts.
Methods
Paleofeces and mummified tissue samples
In 2014, residents of El Saucillo, Zimapán, Hidalgo, reported the discovery of a mortuary bundle to the Instituto Nacional de Antropología e Historia (INAH) [19]. The mortuary bundle was located in a rock shelter where both environmental and microenvironmental conditions favored the exceptional preservation of bone tissue (The state of conservation is described in detail in S1 File). Additionally, remnants of soft tissue, including skin, fragments of the intestinal wall, fecal remains, and blood vessels, were also found in the mortuary bundle (Fig. 1) [21]. Sampling of the intestinal wall and fecal remains was conducted in a sterile setting and placed into sterile Whirl Pak® bags. The samples were stored in a biological archive at the Laboratorio de Conservación, Restauración e Investigación de Patrimonio de Origen Orgánico de la Coordinación Nacional de Conservación del Patrimonio Cultural- INAH. The individual was dated using 14C to 936 years BP, placing it in the early Postclassic period of the archaeological chronology of central Mexico (900 AD – 1200 AD).
Map of Mexico highlighting in red the state of Hidalgo and the locality of Zimapán, where the individual was recovered. Brown and green shading indicate the regions of Aridoamerica (brown) and Mesoamerica (green), B) Mortuary bundle and remains of the Zimapán individual, and C) Samples of paleofeces from the Zimapán individual.
Laboratory conditions
The extraction of ancient DNA was carried out at the Laboratorio de Genómica Bacteriana, Facultad de Medicina, UNAM. The protocol followed the recommendations of Knapp et al., 2011 [23], including the establishment of permanent sterile areas. One room was exclusively designated for changing laboratory attire, while an adjacent room was used solely for sample handling and DNA extraction. Both areas were equipped with UV lamps and air-conditioning units fitted with UV light, which were activated 3 hours before and after an extraction event. All materials used were single-use and underwent a decontamination process through autoclaving and subsequent exposure to UV light before DNA extraction. Access to the ancient DNA extraction area required wearing a disposable surgical overall and plastic booties. DNA extraction and amplification were first carried out in our specialized laboratory for handling ancient material. A second replicate was conducted in a separate laboratory located approximately 10 km away. Although this second laboratory was not specifically designed according to the specifications of Knapp et al. (2011) [23], the same handling guidelines were strictly followed.
Sample preparation
Approximately 600 mg of paleofeces were weighed, and the internal content was extracted to eliminate the external layer and minimize the risk of surface contamination. The internal material was then pulverized using a disposable mortar, and the resulting powder was homogenized. Between 150 and 200 mg of this material were weighed per sample. In total, two samples (S1 and S2) were obtained for DNA extraction.
For the mummified intestinal tissue, a 250 mg sample was used. The tissue was carefully cleaned with a 3% bleach solution, allowed to dry, and then processed for DNA extraction. Two independent DNA extractions were performed from the same sample in two separate laboratories, resulting in extracts I3 and I3.2.
Ancient DNA extraction
The samples were treated with 300 µL of lysis buffer (containing 400 mM NaCl, 10 mM Tris-HCl pH 7.5, 2 mM Na₂EDTA pH 8.2, and 20% SDS) and incubated at room temperature with agitation (250 rpm) overnight. Subsequently, 4 µL of Proteinase K (20 mg/mL) was added to each tube, followed by incubation at 65 °C for three hours. After incubation, the samples were centrifuged at 4000 rpm for 10 minutes, and the resulting supernatant was transferred to a new tube. Then, 100 µL of 5 M NaCl was added to each tube, and incubation continued for 30 minutes at −70 °C. The samples were centrifuged again at 4000 rpm for 10 minutes, and the supernatant was transferred to a clean tube. Next, 150 µL of isopropanol was added, and the samples were incubated at room temperature for 12 hours. A final centrifugation at 4000 rpm for 10 minutes was performed. The resulting DNA pellet was washed twice with 70% ethanol, air-dried, and rehydrated in 100 µL of ultrapure water. Finally, to remove excess salts that may have co-precipitated with the DNA, the extract was cleaned and concentrated using the DNeasy PowerSoil Kit (Qiagen), following the manufacturer’s instructions. A negative control (C1) was included in the extraction process.
16S rRNA sequencing
After confirming the presence of DNA through 2% agarose gel electrophoresis and quantification using NanoDrop spectrophotometry, the 16S rRNA gene was sequenced. Two independent sequencing runs were performed using samples from the same individuals but derived from different DNA extraction events. These runs were carried out to validate the results across two distinct hypervariable regions of the 16S rRNA gene.
In the first run, the V3 hypervariable region (U341F/534R) was amplified and sequenced from two paleofeces samples (S1 and S2), one intestinal tissue sample (I3), and a negative control (C1). Amplification and sequencing were conducted at the Laboratorio de Secuenciación Masiva at IBT, UNAM.
In the second run, DNA was independently extracted from the same intestinal tissue (I3.2), along with a negative control (C2), and the V3–V4 region (341F/805R) was amplified and sequenced at the Integrated Microbiome Resource (IMR) in Canada. Negative controls were included in both sequencing runs to monitor potential contamination.
16S rRNA sequence processing
Sequence data processing was conducted using QIIME2 (version 2022.2) [24]. Each sequencing dataset, defined by the utilization of either v3 or v3-v4 primer pairs, was processed independently. Adapter removal was performed using the Cutadapt program [25], and sequence quality was assessed using QIIME2 native plugins (quality-filter q-score) [26]. Sequence merging, chimera removal, OTU generation, and clustering were executed using the VSEARCH pipeline [27]. Clustering was performed against the SILVA 138 database (99% reference), using a 97% similarity threshold. Taxonomic assignment was conducted with a scikit-learn classifier trained on the SILVA 138 database [28,29].
Although USEARCH has been recommended as the optimal tool for analyzing ancient microbiome 16S rRNA data [30], it is no longer supported in QIIME2. Therefore, VSEARCH was selected as a suitable alternative due to its similar clustering strategy [27]. Finally, a phylogenetic tree was constructed using FastTree [31]. All software was run with default parameters.
Environmental 16S rRNA sequences used for comparison with the zimapán man’s microbial communities
To compare the Zimapán individual’s samples with other microbiome sources, 16S rRNA gene sequences were retrieved from various environments. These included microbiomes from soils in the state of Hidalgo [32], compost [33], the modern human gut microbiome derived from fecal samples representing populations with hunter-gatherer, agricultural, agropastoral, and urban lifestyles [34–36], as well as samples from the contemporary human colon [37]. Additionally, ancient microbiome data from mummified Andean colons and coprolites from the Middle Ages in Namur (Belgium) were included [12,38]. Accession numbers of all datasets are listed in S1 Table.
Processing of these external sequences was conducted separately using QIIME2 (version 2022.2). The resulting feature tables were then merged with the Zimapán dataset into a single file for downstream analysis in R [39].
Data analysis
To analyze the relative abundance of microbial groups from the Zimapán samples and examine abundance patterns across different taxonomic levels, relative abundance tables (abundance > 0.001) were generated using the phyloseq package in R [40]. Multivariate analyses were conducted to compare microbial composition among Zimapán samples and sequences obtained from various environments, including soil, compost, modern and ancient human intestinal microbiomes. Principal Coordinates Analysis (PCoA) with unweighted UniFrac distance at the genus and family levels were performed using the phyloseq package [40]. All plots were generated with the ggplot2 library in R [41].
Furthermore, the contribution of microbiomes from various modern sources (soil, compost, and modern human microbiome) to the Zimapán samples was estimated using the SourceTracker2 program [42]. For this analysis, we integrated the previously described datasets, excluding ancient samples (Namur Middle Ages coprolites and the mummified Andean colon). In addition, we defined a “non-industrialized” category that included populations with traditional lifestyles (agropastoralists, hunter-gatherers, and rural farming communities). The analysis was performed using OTU tables at the family level, applying the program’s default parameters.
Results
DNA extraction and sample processing
Following the previously described DNA extraction protocol, DNA concentrations ranging from 200 to 400 ng/µl were successfully achieved. Notably, sample S1 exhibited the highest concentration, while S2 showed the lowest. Remarkably, the negative control yielded a DNA concentration of 6.2 ng/µl.
Regarding the number of reads obtained in each 16S rRNA gene sequencing event, the first event (primers U341F/534R) resulted in 510,740 reads for S1, 40,210 for S2, and 2,860 reads for I3. Conversely, the second sequencing event (primers 341F-805R) produced a total of 552,371 reads for sample I3.2. After sequence denoising and quality control processing, the retained sequence percentages were 99%, 99%, 99%, and 89% for samples S1, S2, I3, and I3.2, respectively.
Relative abundances of bacterial groups
To analyze the bacterial community composition within the paleofeces and mummified intestinal tissue samples from the Zimapán individual, relative abundance tables at the family and genus levels were generated (Fig 2A). Across all Zimapán samples (S1, S2, I3, and I3.2), consistent patterns were observed, with prevalent bacterial families including Peptostreptococcaceae, Clostridiaceae, Staphylococcaceae, Morganellaceae, Enterobacteriaceae, Enterococcaceae, Bacillaceae, Carnobacteriaceae, and Lachnospiraceae. In contrast, the negative control samples (C1 and C2) exhibited distinct compositional profiles compared to the ancient samples. Notably, Nocardioidaceae dominated in C1, while Xanthomonadaceae was the most abundant group in C2 (Fig 2A)
A) Stacked bar plot showing the abundance of bacterial families identified in the Zimapán samples (relative abundance > 0.001). The plot includes negative controls (C1 and C2), mummified intestinal tissue of the Zimapán individual (I3 and I3.2), and paleofeces samples from the same individual (S1 and S2), B) Multivariate analysis integrating Zimapán samples with various environmental datasets. Principal Coordinate Analysis (PCoA) at the family level was performed using unweighted UniFrac distances, including samples from soil, compost, ancient gut microbiomes (Namur coprolite, Andean mummy tissue, and Zimapán samples), and modern gut microbiomes from populations with different lifestyles (hunter-gatherers, agropastoralists, and urban populations). Circles indicate ancient samples, while triangles indicate modern samples, and C) SourceTracker analysis of the Zimapán samples. The analysis included samples from soils, compost, gut microbiomes of urban populations (colon and feces), and gut microbiomes from non-industrialized populations (agropastoralists, hunter-gatherers, and rural farming populations).
Moreover, stacked bar plots at the genus level showed patterns similar to those observed at the family level (Supp. Fig 1A). The most abundant genera included Jeotgalicoccus, Clostridium sensu stricto, Atopostipes, Enterococcus, Escherichia−Shigella, and Providencia. Interestingly, sample I3.2 (mummified intestine) exhibited higher abundances of additional genera such as Romboutsia and Terrisporobacter. In contrast, the negative controls showed bacterial compositions distinct from those of the Zimapán samples (Supp. Fig 1A).
Multivariate analysis integrating microbiomes from different environments
In the multivariate analysis, we integrated 16S rRNA gene sequences from soil, compost, the human gut microbiome (feces and colon), and ancient microbiomes (mummified colon and coprolites), along with the samples from the Zimapán individual (Fig 2B). The objective was to visualize differences and similarities between the microbial communities identified in this study and those from other environments.
Results from the PCoA using unweighted UniFrac distance at the family level revealed a strong similarity between the Zimapán samples (S1, S2, I3, and I3.2) and colon samples from the Pre-Columbian Andean mummies (Fig 2B). Notably, the Zimapán samples clustered between soil and human intestinal microbiome samples. In particular, the intestinal tissue sample I3.2 was the closest to those from the modern human colon microbiome. In contrast, the Namur Middle Ages coprolite clustered more closely with samples from the modern human gut microbiome. Importantly, the PCoA at the genus level showed similar patterns (Supp. Fig 1B).
Sourcetracker analysis
The SourceTracker analysis was performed using 16S rRNA gene sequences from soils, compost, and the contemporary human gut microbiome (colon and feces) from populations with different lifestyles, along with the Zimapán individual’s samples (Fig 2C; S1 Table). The objective was to estimate the proportion of microbial contributions from these different environments to the microbiome of the Zimapán samples. In this analysis, more than 90% of the microbial composition in the Zimapán samples could not be assigned to any of the included source environments. Among the identifiable sources, the highest contribution corresponded to the human gut microbiome from colon and fecal samples, followed by the compost microbiome (Fig 2C).
Discussion
In this study, we analyzed the microbial composition of paleofeces and intestinal tissue from an individual dating back to the Postclassic period, situated in Zimapán, Hidalgo. This region lies on the border between Mesoamerica and Aridoamerica. Mesoamerica played a crucial role in domestication and the rise of major civilizations and cities. In contrast, the Aridoamerica region is known for its sparse human settlements, predominantly inhabited by seasonal seminomadic people who practiced hunting and gathering [43,44].
Through DNA extraction and subsequent 16S rRNA gene sequencing of paleofeces and intestinal tissue from the Zimapán man, various bacterial groups were identified. Although the most abundant group in the samples was a halotolerant microbe (Jeotgalicoccus), typically associated with marine soils and sediments [45], other identified groups are potentially related to the human gut microbiome. Among the most abundant taxa, we identified bacteria from the families Peptostreptococcaceae, Clostridiaceae, Enterobacteriaceae, Enterococcaceae, Morganellaceae, and Lachnospiraceae (Fig 2A).
The Peptostreptococcaceae family includes fermentative bacteria considered commensals of the human gut, contributing to intestinal homeostasis [46]. Notably, Romboutsia hominis (Peptostreptococcaceae family) was detected in one of the analyzed samples (I3.2) (Supp. Fig 1A). This species is the only known member of the Romboutsia genus that have been recently isolated from the human gut [47]. Although little is known about its functional roles, R. hominis has been associated with carbohydrate utilization and the biosynthesis of vitamins and other cofactors [47].
A high abundance of bacteria from the Clostridium genus was found in the Zimapán samples, consistent with previous reports from colon samples of pre-Columbian Andean mummies [48]. Clostridium is known to colonize the human intestine, performing a wide range of metabolic functions. Various Clostridium species are capable of utilizing nutrients (such as complex polysaccharides) that the human host cannot digest, producing short-chain fatty acids (SCFAs) [49]. SCFAs play a fundamental role in intestinal homeostasis [49].
A study conducted in 2021 by Wibowo et al [16] on paleofeces and coprolites revealed a broad range of metabolic functions in the Clostridiaceae family, including the degradation of carbohydrate-active enzymes (CAZymes). Particularly, chitin CAZymes were identified, possibly associated with insect consumption in ancient populations. In the Zimapán samples, a high abundance of the Clostridium genus was identified, suggesting a potential role in insect degradation. This is particularly relevant given that the Zimapán man was recovered in the state of Hidalgo, Mexico, a region with a long-standing cultural tradition of entomophagy (insect consumption) [50].
Lachnospiraceae, alongside Clostridiaceae, exhibited high abundance in the Zimapán samples. Lachnospiraceae is recognized as a core member of the human gut microbiome and is associated with the production of short-chain fatty acids (SCFAs) [51]. Within this family, certain species contribute to the breakdown of cellulose and hemicellulose, polysaccharides found in plant cell walls that humans are unable to digest. The activity of these polysaccharide-degrading bacteria enhances the bioavailability of otherwise inaccessible dietary components for the host [51]. The prevalence of such taxa suggests that the Zimapán individual may have sustained a diverse, plant-based diet, likely including agaves, yuccas, mesquites, and prickly pears, among other species characteristic of xerophilic environments [52].
Other groups from the Enterobacteriaceae and Enterococcaceae families were also identified. These groups are common members of the gastrointestinal microbiota in humans and other vertebrates. However, under conditions of intestinal dysbiosis, these groups can transition into pathobionts [53,54].
Beyond that, the multivariate analysis revealed a close similarity between the Zimapán samples and the pre-Columbian Andean mummies. Although the Zimapán samples clustered near the modern human gut microbiome samples, they didn’t share the same space in the PCoA. That could suggest a slight deviation from the human gut microbiota composition. This arrangement in the multivariate analysis is likely attributed to the presence of non-intestinal bacterial taxa in the samples from the Zimapán individual, potentially introduced through the gradual accumulation of environmental bacteria over time. Despite meticulous efforts to remove the outer layer of the paleofeces and clean the surface of the mummified intestinal tissue, soil and sediment associated groups such as Jeotgalicoccus, Sphingomonadaceae, Vagococcaceae were detected. All these factors could have influenced the microbial composition, subsequently impacting the distribution of the Zimapán samples in the multivariate analysis, resulting in a slight separation of the samples representing the human gut microbiota. These findings align with those observed in the SourceTracker2 analysis. Among all the environments included in the analysis, the human gut microbiome accounted for the largest proportion of the microbial community in the Zimapán samples. However, it is noteworthy that a substantial fraction of the microbial composition could not be assigned to any known source.
Finally, it is important to highlight that, despite detecting representative groups of the human gut microbiome such as Bacteroidaceae and Bifidobacteriaceae, were found in very low abundance, while others, including Prevotellaceae and Ruminococcaceae, were not identified in the Zimapán samples. These groups are typically abundant in the modern human microbiome and have also been reported in previous ancient gut microbiome studies [14,16]. These results suggest that alternative approaches, such as metagenomics, may be necessary to confirm the presence of these bacteria and verify their persistence in ancient samples. It has been proposed that metagenomic approaches are more appropriate when working with highly fragmented DNA, as is characteristic of ancient samples [55]. Indeed, several studies have shown that ancient DNA often consists of fragments approximately 40 base pairs in length [56], which can limit the effectiveness of 16S rRNA gene-based methods. Moreover, metagenomics could provide a more accurate view of ancient DNA damage patterns in the bacterial taxa detected in the Zimapán samples [56]. Nevertheless, conducting an initial characterization using 16S rRNA sequencing was a necessary first step to identify potential members of the ancient gut microbiome. This approach sets the foundation for deeper investigations, including taxonomic and functional characterization and the reconstruction of ancient bacterial genomes, as has been successfully achieved in other ancient microbiome studies [13,16,17].
Conclusions
The integration of anthropological, archaeological, and microbiome data serves as a powerful tool for unraveling human evolution and its intricate relationship with both biological and social environments. In this study, we present the first characterization of the gut microbiome of an individual from Zimapán, Hidalgo, using high-throughput sequencing of the 16S rRNA gene from paleofeces and mummified tissue. This approach enabled the identification of diverse bacterial groups, including taxa recognized as potential members of the human intestinal microbiome, such as Romboutsia hominis, a species exclusively isolated from the human gut and, until now, unreported in the ancient human gut microbiome. This finding raises the possibility that the Zimapán paleofeces could preserve authentic ancient gut bacteria. Conversely, the absence of other representative and typically abundant human-associated taxa underscores the need for complementary strategies to further explore the Zimapán samples. These efforts could also include functional analyses of the gut microbiome of this individual. Despite these limitations, the primary objective of identifying gut microbiome components in a seasonal seminomadic hunter-gatherer individual was achieved, thereby contributing to the ongoing expansion of knowledge about the ancient human microbiome across diverse temporal and cultural contexts.
Supporting information
S1 Fig. Bacterial composition of the Zimapán samples at the genus level.
A) Stacked bar plot showing the abundance of bacterial genera identified in the Zimapán samples (relative abundance > 0.001). The plot includes negative controls (C1 and C2), mummified intestinal tissue of the Zimapán individual (I3 and I3.2), and paleofeces samples from the same individual (S1 and S2) and, B) Multivariate analysis integrating Zimapán samples with different environmental datasets. Principal Coordinate Analysis (PCoA) at the genus level was performed using unweighted UniFrac distances, including samples from soil, compost, ancient gut microbiomes (Namur coprolite, Andean mummy tissue, and Zimapán samples), and modern gut microbiomes from populations with different lifestyles (hunter-gatherers, agropastoralists, and urban populations). Circles indicate ancient samples, while triangles indicate modern samples.
https://doi.org/10.1371/journal.pone.0331137.s001
(TIF)
S1 Table. List of accession numbers for all sequences included in the analyses.
The table includes sequences downloaded from public databases for comparison with Zimapán samples.
https://doi.org/10.1371/journal.pone.0331137.s002
(CSV)
S1 File. Detailed description of the preservation state of the mortuary bundle from El Saucillo, Zimapán.
https://doi.org/10.1371/journal.pone.0331137.s003
(DOCX)
Acknowledgments
This paper is part of the requirements for obtaining a Doctoral degree at the Posgrado en Ciencias Biológicas, UNAM of SR-P. SR-P acknowledges the scholarship supported by Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCYT) (CVU: 776199). The authors also acknowledge Rodrigo García Herrera for his support in HPC computing and LANCIS-Instituto de Ecología, UNAM for computing resources. We would like to express our gratitude to the Instituto de Antropología e Historia (INAH) and the Coordinación Nacional de Conservación del Patrimonio Cultural-INAH for their support in facilitating this research. Additionally, we thank José Luis Mendez Sanchez for his participation in setting up DNA extraction techniques.
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