Journal Pone 0292077

PLOS ONE
RESEARCH ARTICLE
Edible flora in pre-Columbian Caribbean

coprolites: Expected and unexpected data
Jelissa Reynoso-Garcı́a ID1¤a¤b*, Tasha M. Santiago-Rodriguez2, Yvonne Narganes-
Storde3, Raul J. Cano4, Gary A. Toranzos1*
1 Environmental Microbiology Laboratory, Biology Department, University of Puerto Rico, San Juan, Puerto
Rico, 2 Diversigen, Inc., New Brighton, Minnesota, United States of America, 3 Center for Archaeological
Research, University of Puerto Rico, San Juan, Puerto Rico, 4 Biological Sciences Department, California
Polytechnic State University, San Luis Obispo, California, United States of America
a1111111111 ¤a Current address: School of Medicine, MIND Institute, Department of Psychiatry and Behavioral Sciences,
a1111111111 University of California – Davis, Sacramento, California, United States of America
a1111111111 ¤b Current address: Department of Anatomy, Physiology and Cell Biology, University of California – Davis,
a1111111111 Davis, California, United States of America
* gary.toranzos@upr.edu (GAT); jelissa.reynoso@upr.edu (JRG)
a1111111111
Abstract
OPEN ACCESS Coprolites, or mummified feces, are valuable sources of information on ancient cultures as
Citation: Reynoso-Garcı́a J, Santiago-Rodriguez they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated
TM, Narganes-Storde Y, Cano RJ, Toranzos GA from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vie-
(2023) Edible flora in pre-Columbian Caribbean ques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and life-
coprolites: Expected and unexpected data. PLoS
styles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested
ONE 18(10): e0292077. https://doi.org/10.1371/
journal.pone.0292077 during food consumption, to further support dietary habits. Our findings show that pre-
Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet
Editor: John P. Hart, New York State Museum,
UNITED STATES potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.),
papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton
Received: May 6, 2023
(Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phyto-
Accepted: September 12, 2023
pathogenic fungi and plant interactions confirmed the potential consumption of these plants
Published: October 11, 2023 as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize
Copyright: © 2023 Reynoso-Garcı́a et al. This is an and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucino-
open access article distributed under the terms of genic plants likely played an important role in ancient human subsistence and societal cus-
the Creative Commons Attribution License, which
toms. We compared our results with coprolites found in Mexico and the United States, as
permits unrestricted use, distribution, and
reproduction in any medium, provided the original well as present-day faeces from Mexico, Peru, and the United States. The results suggest
author and source are credited. that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers,
Data Availability Statement: Sequence data are while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-
available in the National Center for Biotechnology Columbian cultures and larger scale farmers and United States individuals. Our study high-
Information (NCBI) Sequence Read Archive under lights differences in dietary patterns related to human lifestyles and provides insight into the
the BioProject PRJNA961747 (accession numbers
SAMN34367738, SAMN34367739).
flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal
specimens demonstrate the importance of ancient DNA studies to better understand pre-
Funding: (JRG) This study was partially supported
by the National Institute of Health (NIH) Research
Columbian populations.
Initiative for Scientific Enhancement (RISE)
Program (Grant No. 5R25GM061151) https://brtc.
uprrp.edu/rise/. The funders had no role in study
design, data collection and analysis, decision to
PLOS ONE | https://doi.org/10.1371/journal.pone.0292077 October 11, 2023 1 / 21

PLOS ONE The edible flora and associated phythopathogens in pre-Columbian coprolites
publish, or preparation of the manuscript. This Introduction

study was also supported by an Institutional
Development Award (IDeA) INBRE Grant Number The Huecoid and the Saladoid, two pre-Columbian indigenous cultures, migrated from differ-
P20GM103475 from the National Institute of ent regions of the Americas in independent migratory waves to settle in the Caribbean Islands
General Medical Science (NIGMS), a component of [1–3]. Since its discovery, it has been hypothesized that each culture was distinct based on
the National Institutes of Health (NIH), and the major differences in the pottery, lapidary and faunal cultural assemblage [3], and that both co-
Bioinformatics Research Core of the INBRE. Its
habited the site of Sorcé, Vieques, Puerto Rico for more than 1,000 years [4]. A counter
contents are solely the responsibility of the authors
and do not necessarily represent the official view of hypothesis was presented in which the Huecoid materials belonged to the Saladoid tradition
NIGMS or NIH. The funders had no role in study [5]. While the Saladoid culture is believed to have migrated from the Orinoco River Valley of
design, data collection and analysis, decision to Venezuela [6], and inhabited the island of Vieques around the sixth century B.C. [7], the Hue-
publish, or preparation of the manuscript. coid culture is believed to have originated on the eastern slopes of the Andean mountains of
Competing interests: Tasha Santiago is a current present-day Bolivia and Peru [8], and to have arrived to Puerto Rico around the third century
Diversigen employee, a microbiome services B.C. In addition to differences in pottery and faunal materials, as well as migratory patterns, a
company. This does not alter our adherence to considerable amount of lapidary objects consisting of semiprecious stone ornaments, which
PLOS ONE policies on sharing data and materials.
include jadeite condors distinguished the Huecoid culture and supports the proposed Andean
origin of this culture [9, 10]. In contrast, the Saladoid culture was characterized by polychro-
mic (white and orange over red) painted pottery tradition [6, 11], distinct faunal assemblage
and significant shell ornament industry.
Highly developed phytocultural practices that connected these pre-Columbian cultures in
the Caribbean to South America resulted in complex social systems [12]. Early European
chroniclers [13, 14], and more recently starch remains stored on plant-processing artifacts (as
well as human dental calculus) have shown a complex food system in the Caribbean [15, 16].
During the early ceramic age, the ancient South American and Caribbean Amerindians har-
vested a variety of plants including maize (Zea mays), sweet potato (Ipomoea batatas), com-
mon bean (Phaseolus vulgaris), manioc (Manihot esculenta), marunguey (Zamia spp.),
cocoyam (Xanthosoma sp.), and peanut (Arachis hypogaea) [15–17]. Minor dietary compo-
nents included achira (Cannaceae) and arrowroot (Maranta arundinacea) [18], while chili
peppers (Capsicum spp.) were used as a condiment. Many of these plants continued being
used during the Late Ceramic Age, although indigenous people included a larger repertoire of
plants (including fruits) [17, 19] and the importance of Cannaceae and Marantaceae increased.
While paleoethnobotanical data are corroborating the information given by the Spanish
chroniclers, significant gaps in knowledge of the pre-Columbian diet, and regional and tempo-
ral differences in dietary habits remain. The chroniclers described mainly the culture, flora
and fauna of Hispaniola, holding the notion that all the Caribbean islands were alike. Thus, the
information about Puerto Rico and other islands of the Caribbean is limited. In addition, the
Huecoid and Saladoid are early undescribed cultures since the chroniclers only described later
cultures. Information on consumed plants contributing to the diet of pre-Columbian and pres-
ent-day ethnic groups with contrasting geocultural regions and temporal scales is needed to
better understand diet as an important part of present culture and identity.
Mummified feces, or coprolites, recovered from archaeological sites have provided a wealth
of valuable information about pre-Columbian diets and the past environment in which theses
cultures lived [20]. For instance, micro- (e.g., pollen), and macroscopic remains (e.g., bones,
seeds, and fibers) recovered from coprolites have provided dietary and lifestyle information
[21, 22]. Similarly, the presence of pollen from famine foods in coprolites may suggest that the
individual may have lived in arid environments [20, 23]. In addition to micro- and macro-
scopic remains, DNA analysis has revealed the diet of extinct sloths [24, 25], dogs [26, 27],
moas [28, 29], and mummies [30, 31]. Moreover, the reconstruction of ancient human diets
has been possible in part by studies of the gut microbiome [32, 33], virome [34], parasitome
[35], and mycobiome [36] found in coprolites. Notably, ancient microbial communities

detected in coprolites can also reflect the evolution of human lifestyles through time [23, 36–
38]. Other DNA sequences, such as those originating from plants, may provide a refined taxo-
nomic classification of edible plants, which, in turn, may aid in the reconstruction of ancient
dietary habits and lifestyles.
Our study presents data on plant DNA extracted from coprolites (mummified feces) recov-
ered from Vieques, Puerto Rico, which are approximately 1500 years old. Our goal was to
reconstruct the diet and surrounding flora of the pre-Columbian Huecoid and Saladoid cul-
tures using this plant DNA data. To better understand how diet varied across different geocul-
tural regions and historical periods, we also analyzed coprolites from other cultures, including
the Loma San Gabriel culture in La Cueva de los Muertos Chiquitos (Rio Zape, Mexico), the
Ancestral Puebloans in the Arid West Cave (Arizona, USA), and the Boomerang Shelter
(Utah, USA). Additionally, we included data obtained from present-day fecal samples from
various indigenous groups, such as the Matses (hunter-gatherers from the Peruvian Amazon),
traditional Tunapuco (agriculturalists from the Peruvian Andean highlands), and Mazahua
(farmers from Mexico), as well as urban-industrial individuals from the United States. To fur-
ther support our analysis of dietary habits, we investigated DNA sequences from phytopatho-
genic fungi, which can be ingested along with the corresponding plant species. Overall, our
study aimed to use plant and fungal DNA extracted from coprolites to shed light on the
ancient dietary habits of pre-Columbian cultures of Puerto Rico, and to identify any differ-
ences in diet across different geocultural and historical periods.
Materials and methods

Archaeological samples and site
Coprolites from the Huecoid and Saladoid cultures from La Hueca archeological site in Sorcé,
Vieques (18º 05’ 56” Latitude North and 65º 29’ 34” Longitude West), a semi-arid island
located about 13 km southeast of the main island of Puerto Rico were used. Archaeologists
Luis Chanlatte and Yvonne Narganes conducted the excavations on private land with the own-
er’s approval and followed all relevant regulations. In total, ten coprolites from the two pre-
Columbian cultures were used: six of the coprolites corresponded to the Huecoid culture and
four of the coprolites corresponded to the Saladoid culture. Detailed information about the
samples is presented in S1 Table. The collection is deposited at the Center for Archaeological
Research at the University of Puerto Rico, San Juan, Puerto Rico. Specimen registration num-
bers have been previously described [35]. Coprolite age was estimated using radiocarbon dat-
ing from associated archeological material (charcoal and shells) [8]. All samples were carbon-
dated at Teledyne Isotopes (Westwood, NJ) and BETA Analytic, Inc. (Miami, FL) using a stan-
dard protocol. Radiocarbon dating estimates for the Huecoid coprolite samples ranged from
245 to 600 A.D., whereas the Saladoid coprolites ranged from 230 to 395 A.D. [34]. Coprolites
have yielded well-preserved gut microbiome DNA [34, 35] as well as human and plant DNA.
DNA extraction and contamination prevention

DNA was extracted from all coprolite samples as previously described [34]. Briefly, ten copro-
lites from the Huecoid (n = 6) and Saladoid (n = 4) cultures were processed in a class II biolog-
ical safety cabinet exclusively dedicated to ancient DNA following strict procedures: protective
clothes, disinfection of surfaces, sterilization of instruments, and ultraviolet radiation. The
class II biosafety cabinet was cleaned with 70% ethanol and exposed to ultraviolet radiation for
30 minutes before and after use. To avoid modern exogenous contamination, DNA extraction
was performed using only the inner core of each coprolite after the removal of the exterior por-
tion using sterile and flamed scalpels. Total DNA was extracted using the PowerSoil DNA

extraction kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s
instructions. The inner core of the coprolites was pulverized using a sterile mortar and pestle
and hydrated overnight in sterile C1 solution at 4 ˚C. Because of low concentrations of DNA,
samples were then pooled into one composite sample per culture using standard glycogen pre-
cipitation protocols (Thermo Scientific).
Metagenomic library construction and shotgun sequencing

Whole-genome amplification from small quantities of DNA was performed using a REPLI-g
Midi kit (Qiagen). Amplified DNA was purified using the PowerClean DNAClean-Up Kit
(MO BIO Laboratories) and sample concentrations were calculated using the Qubit1 dsDNA
HS Assay Kit (Life Technologies). Library preparation was completed using the Nextera DNA
Sample Preparation kit (Illumina) according to the manufacturer’s recommendations. Librar-
ies concentrations were evaluated using the Qubit1 dsDNA HS Assay Kit (Life Technologies)
and the average library size was quantified using Experion (Bio-Rad). Libraries were then
pooled in equimolar ratios and shotgun sequenced on the Illumina Miseq sequencing platform
at MR DNA Research lab (Shallowater, TX) [34].
Comparison with other coprolite and present-day fecal sequences

Publicly available sequence data were obtained from the NCBI’s Sequence Read Archive (SRA)
using the SRA Toolkit (v2.10.4). Archaeological samples constitute sequencing data from 13
coprolites, including coprolites from the Loma San Gabriel culture in La Cueva de Los Muer-
tos Chiquitos (n = 8, Rio Zape, Mexico; under BioProject ID: PRJEB31971, PRJEB33577, and
PRJEB35362) [23, 39, 40]; from the Ancestral Puebloans from the Arid West Cave (n = 3, Ari-
zona, USA; BioProject ID: PRJNA561510) [23]; and from the Ancestral Puebloans from the
Boomerang Shelter (n = 2, Utah, USA; BioProject ID: PRJNA561510) [23] (Table 1).
Present-day samples comprised published sequence data from 86 extant stools, including
feces from the Matses hunter-gatherers (n = 24, Peru, BioProject ID: PRJNA268964) [41]; the
Tunapuco farmers (n = 12, BioProject ID: PRJNA268964); the Mazahua farmers (n = 22,
Mexico, BioProject ID: PRJNA561510) [23]; and United States individuals from the Human
Microbiome Project (n = 28, USA, BioProject ID: PRJNA48479) [42] (Table 2). All samples
were computationally analyzed along with the data from our study [43].
Bioinformatics and statistical analysis

Read processing and quality control. Raw paired-end reads were trimmed and filtered
from adapters and low-quality reads (Phred score < 20) through trim-galore using default
parameters as implemented in the metaWRAP Read_qc module (v1.2.4) [44]. Contaminating
human DNA sequences were then removed from the metagenomic datasets through align-
ment of reads to the Homo sapiens reference genome (build Hg38) using the BMTagger
approach implemented in the metaWRAP Read_qc module [44]. The human origin of the
Table 1. Description of the archaeological samples analyzed in this study.

Pre-Columbian culture Archaeological site Geographical regions Coprolite C-14 data (range) Reference
Huecoid La Hueca Sorcé Vieques, Puerto Rico 1500 BP This study, [34, 35]
Saladoid La Hueca, Sorcé Vieques, Puerto Rico 1500 BP This study, [34, 35]
Loma San Gabriel La Cueva de los Muertos Chiquitos, Rio Zape Durango, Mexico 1300 BP [39, 40]
Puebloans Arid West Cave Arizona, United States 2500–1500 BP [23]
Puebloans Boomerang Shelter Utah, United States 2000–1000 BP [23]
https://doi.org/10.1371/journal.pone.0292077.t001

Table 2. Description of the published present-day samples analyzed in this study.

Present-day culture N Subsistence strategy Geographical region Reference
Matses 24 Hunter-gatherers Peru [41]
Tunapuco 12 Agriculturalists Peru [41]
Mazahua 22 Agriculturalists Mexico [23]
United States 28 Urban-industrial United States [42]
coprolites was evaluated through the detection of human-specific Bacteroides by PCR, as pre-
sented in previous studies from our laboratory [33]. Quality control improvement on sequenc-
ing reads was assessed using FastQC [45]. Pre-processed reads were considered for all
downstream analyses.
Metagenomic profiling. Taxonomic assignment of high-quality sequencing reads was
performed through Kaiju as implemented in command-line (v1.5.0) [46] using the following
parameters: −a greedy −E 0.05 for e-value filtering. Kaiju classified reads using a subset of the
NCBI BLAST non-redundant (nr) reference database (argument -nr_euk) comprising anno-
tated protein-coding genes from bacteria, archaea, viruses, and fungi (accessed on 25 May
2020). Taxon IDs from plant sequences from the NCBI nr database were also included. It has
been shown that a database comprising all domains of life is better suited for taxonomic profil-
ing of microbial eukaryotes [47].
Functional ecological guilds. Ecological functions (trophic and guilds) of fungal genera
were parsed using FUNGuild (v.1.2) (https://github.com/UMNFuN/FUNGuild) [48]. Fungal
genera that classified within the plant pathogen functional guild were considered for further
analysis.
Source tracking of microbial communities. The proportion of DNA reads from each
potential source contributing to the Huecoid and Saladoid sink coprolite samples was esti-
mated using Meta-SourceTracker (mSourceTracker) [49]. Publicly available shotgun libraries
from human feces, coprolites, and human skin were downloaded from the Sequence Read
Archive (SRA) using SRA Toolkit (v2.10.4) and the soil metagenomes were downloaded from
MG-RAST using grabseqs [50]. These reference metagenomes were selected as potential
sources and contaminants (i.e., soil and skin) for coprolite samples. The environmental source
samples included: 58 non-industrial human feces, 28 industrial feces, 13 coprolites, 16 human
skin, and 16 soil samples. All samples were processed using the metagenome classifier Kaiju
and then combined using the mSourceTracker script kaiju_table_to_OTU_table.py. The
resulting table for the Eukaryotic domain was converted to HDF5 biom format using the
biom-format python package (v.2.1.10) and then used as an input for mSourceTracker.
Plant-pathogen interaction network. We used the rglobi (global biotic interactions) R
package to extract all the interactions between the plants and phytopathogenic fungi (queried
as “Fungi”) in the dataset using the get_interactions_by_taxa function. In addition, we
retrieved plant disease data from the American Phytopathological Society website (https://
www.apsnet.org/edcenter/resources/commonnames/Pages/default.aspx). We only retained
potential phytopathogenic fungi that were identified through Kaiju in our dataset. Pathogen-
host interaction network was constructed from a taxonomic table by generating a directional
data frame of pathogen-host interactions as identified using rglobi and plant disease data. We
then imported the dataset table into Cytoscape to build a directed network. For the resulting
network, we calculated the degree of connectivity, and the eigenvector centrality using
CytoNCA, to assess the importance of each node.
Statistical analysis and visualization. Sequencing data were primarily analyzed and visu-
alized using the R statistical environment, version v4.1.3 (R Foundation for Statistical

Computing). For beta diversity, dimensional reduction of Aitchison distances was visualized
in a principal coordinates analysis (PCoA) using the phyloseq R package (v.1.38.0) [51]. Statis-
tical differences in beta diversity were tested through Permutational Multivariate Analysis of
Variance (PERMANOVA) using the adonis function in the phyloseq R package. A hierarchical
clustering dendrogram based on Bray-Curtis dissimilarity distances was constructed on plant
abundance per sample using the Ward’s clustering algorithm in the vegan R package (v. 2.5.7)
[52]. Maps and piedonut plots were generated using the R packages sf (v.1.0.7), webr (v.0.1.6),
and ggplot2 (v. 3.3.5).
Results
General patterns of plant DNA in coprolites from the Huecoid and
Saladoid
We studied ten coprolites recovered from an archaeological site in Vieques, Puerto Rico in an
attempt to reconstruct dietary habits of the pre-Columbian Huecoid and Saladoid cultures.
We analyzed DNA sequences from plants and their potential phytopathogenic fungi using
shotgun metagenomic sequencing, which may contain fewer biases in ancient microbiome
reconstruction compared to amplicon-based sequencing [53]. Following bioinformatic pro-
cessing, plant sequence reads were classified into one phylum, one class, five orders, five fami-
lies, eight genera, and nine species (Table 3). Six of the taxa have not been previously detected
in paleoethnobotanical studies and thus are putative taxonomic assignments. In addition, the
putative plant sequences identified could be a closely related plant taxon.
Starchy tubers, legumes, pseudograins, fruits, and a hallucinogenic plant

were likely part of the Huecoid and Saladoid vegetal diet and culture
Plant sequencing reads in this study revealed a variety of food plants in the Huecoid and Sala-
doid coprolites. We found a high abundance of maize (Zea mays; relative abundance = 48.4%)
followed by chili pepper (Capsicum annuum; 29.0%), sweet potato (Ipomoea batatas; 6.5%),
wild peanut (Arachis duranensis; 6.5%), domesticated peanut (Arachis hypogaea; 3.2%), cotton
(Gossypium barbadense; 3.2%) and tomato (Solanum lycopersicum; 3.2%) in the Huecoid
Table 3. Description of the identified taxa from DNA sequencing of the Huecoid and Saladoid coprolites.
Order Family Genus Species Common name Possible Origina Uses
Poales Poaceae Zea Zea mays Maize Mesoamerica Foodstuff
Brassicales Caricaceae Carica* Carica papaya Papaya Tropical America Foodstuff
Fabales Fabaceae Arachis* Arachis hypogaea Domesticated Brazilian–Paraguayan Center Foodstuff
peanut
Fabales Fabaceae Arachis* Arachis duranensis Wild peanut Brazilian–Paraguayan Center Foodstuff
Solanales Solanaceae Ipomoea Ipomoea batatas Sweet potato Central America Foodstuff
Solanales Solanaceae Capsicum Capsicum annuum Chili pepper South America, northern Peru Condiment and medicinal
Solanales Solanaceae Nicotiana* Nicotiana sylvestris Tobacco Probably Mexico, Central America Narcotic and
hallucinogenic
Solanales Solanaceae Solanum* Solanum lycopersicum Tomato Western South America Foodstuff
Malvales Malvaceae Gossypium* Gossypium Cotton Northwestern Peru and southwestern Ecuador Fiber and oil
barbadense [54]
*Putative taxonomic assignment of plant sequences in the present dataset.

a
Data obtained from [55].

coprolite sample. Notably, we observed less plant DNA sequences Saladoid coprolite sample
compared to the Huecoid coprolite sample. Specifically, plant sequence reads identified in the
Saladoid coprolite sample included chili pepper (Capsicum annuum; 63.2%), tobacco (Nicoti-
ana sylvestris; 21.1%), papaya (Carica papaya; 15.8%), and tomato (Solanum lycopersicum,
5.0%). Sequencing reads of chili peppers and tomato were shared between the Huecoid and
Saladoid coprolite samples, while seven plant taxa were only identified in one sample (Fig 1A).
Using network analysis, we examined the relationship between fungal pathogens and plant
hosts in the coprolites of the Huecoid and Saladoid cultures, considering previous archaeolog-
ical findings. Due to the limited taxa in the Saladoid coprolite sample, we merged the two ethnic
groups and created clusters in the network model. The maize node had the highest degree of
connectivity (degree = 16) and eigenvector centrality (eigenvalue = 0.39), indicating that maize
could potentially host many fungi and plays a critical role in connecting other nodes. The
tobacco node had the second highest degree of connectivity (degree = 13), followed by sweet
potato (degree = 9), and peanuts (degree = 7). However, sweet potato had a higher eigenvector
centrality (eigenvalue = 0.35) than tobacco (eigenvalue = 0.34), suggesting that sweet potato is
more influential in the network. Plants with the lowest degree of connectivity and eigenvector
centrality were Papaya (degree = 3, eigenvalue = 0.12), tomato (degree = 4, eigenvalue = 0.08),
chili pepper (degree = 2, eigenvalue = 0.08), and cotton (degree = 1, eigenvalue = 0) (Fig 1B).
Phytocultural practices of ancient cultures may be different from those of

present-day cultures
For comparative purposes, we analyzed publicly available coprolite sequence data from Rio
Zape Cave (Mexico), Boomerang Shelter (United States), and Arid West Cave (United States)
as well as present-day feces from the Matses hunter-gatherers (Peru), Tunapuco (Peru) and
Mazahuas (Mexico) agricultural communities, and industrial populations (from the United
States). We quantified Bray-Curtis dissimilarity and Aitchison distances to investigate differ-
ences in the plant beta diversity across the samples. Hierarchical cluster analysis based on
Bray-Curtis dissimilarity, and the plant relative abundance of each sample reflected two main
clusters; combining present-day feces from Mazahua and United States (Cluster 1); and copro-
lites from Ancestral Puebloans, Loma San Gabriel, Huecoid and Saladoid as well as present-
day feces from Matses and Tunapuco (Cluster 2) (Fig 2A). PCoA based on Aitchison distances
and relative abundance of plant families showed significant segregation (PERMANOVA,
R2 = 0.27 and p-value = 0.001) in the plants across samples from the Amerindian groups (Fig
2B), suggesting differences in community structure. The PCoA axis 1 (PC1) explained 22.2%
of the variance, which correlated with a gradient in human lifestyles. For instance, the pre-
Columbian cultures and present-day hunter-gatherers were located on the left, the Tunapuco
agriculturalists in the middle and the Mazahua farmers and United States individuals on the
right (Fig 2B). Thus, despite differences in geography, coprolites from Arid West, Boomerang
Shelter, Loma San Gabriel, Huecoid, and Saladoid were more similar to each other, and to
present-day feces from Matses hunter-gatherers than to present-day feces from Mazahua and
the United States (Fig 2B). However, the Tunapuco feces showed a transitional state between
the coprolites and present-day feces from hunter-gatherers, and the present-day feces from the
Mazahua farmers and individuals from the United States. The findings indicate a shift in
behaviors leading to modifications in dietary patterns (Fig 2B).
Discussion
Through the integration of molecular data, pathogen-host interaction modeling, and pub-
lished literature, we present the first attempt to reconstruct the plant-based diets of pre-

Fig 1. Piedonut diagram for plant distribution and directed network analysis of pathogen-host interactions of the pre-Columbian
Huecoid and Saladoid cultures. Panel (A) Inner pie chart represents the percentage of plants identified by culture, whereas the outer
donut shows the distribution of the plants. Panel (B) Pathogen-host interaction network constructed using the rglobi (global biotic
interactions) database. Relationships between fungal pathogens and plant hosts are represented as directed edges from source (fungi) to
target (plants). Each node represents either plant (green) or fungal (blue) taxa. Plant node size represents indegree and plant node
transparency depicts the Eigenvector centrality.
https://doi.org/10.1371/journal.pone.0292077.g001

Fig 2. Composition and structure of plants differentiate the ethnic groups according to dietary lifestyles. Panel (A) Bray-Curtis distance dendrogram
constructed on plant family abundance showing hierarchical clustering/relationships between similar samples. Panel (B) Principal component analysis of
Aitchison distances showing that plant families-diversity segregated pre-Columbian ethnic groups and hunter-gatherers from present-day large-scale farmers
and industrialized individuals, whereas the agriculturalists showed a transitional state. Each color code represents the ethnicity of each group, whereas the
circle and triangles symbols represent plant communities of each sample in ancient and present-day ethnic groups, respectively. Adonis test was performed
on Aitchison distances.
https://doi.org/10.1371/journal.pone.0292077.g002

Columbian cultures in Puerto Rico using preserved ancient plant and phytopathogen DNA
sequences. We analyzed plant sequence reads obtained from coprolites of the Huecoid and Sal-
adoid cultures and confirmed the identity of plant DNA using phytopathogenic fungi DNA
that may have impacted their horticultural ecosystem. We also compared our results with pre-
viously published coprolite sequence data from Mexico and the United States, as well as pres-
ent-day fecal samples from Mexico, Peru, and the United States, to investigate the phyto-
cultural diversity between ancient and present-day populations in various social environments.
Our study provides insights into the lifestyle and dietary habits of the Huecoid and Saladoid
cultures, which have contributed to the cultural identity of present-day Caribbeans, by identi-
fying the plants used for consumption by these pre-Columbian groups. Furthermore, our find-
ings suggest that the incorporation of native plant sequences into DNA databases is crucial to
use DNA-based approaches for reconstructing dietary habits. Although historical records of
the plants used in the Huecoid and Saladoid cultures are scarce, it is possible that closely
related plant phyla and families served as food for these ethnic groups and could be extinct,
replaced, and forgotten due to the introduction of other crops resulting from the colonization
of the Americas.
Overcoming challenges with contamination

Sample contamination with modern exogenous DNA is a major challenge in the analysis of
ancient DNA from coprolites [56, 57]. We used SourceTracker to test for contamination and
verify the authenticity of coprolite DNA, and found that unknown sources contributed the
highest number of eukaryotic sequences (S1 Fig). This may suggest either that no soil contami-
nation affected the results, or that other sources not included in the mSourceTracker analysis
may have contributed to the results in the present study. Moreover, the high proportion of
unknown sources contributing taxa is consistent with previous studies on coprolites and mum-
mies [30, 38]. Interestingly, we also found that published metagenomes of coprolites were the
main known sources contributing to the Huecoid coprolite samples, suggesting that the Hue-
coid eukaryote sequences are endogenous to the coprolite sample. In contrast, soil was the pri-
marily source contributing to the Saladoid coprolite, followed by coprolite samples. This may
suggest that the coprolite samples were possibly contaminated with soil as a result of being
exposed to the environment, or vigorously washing edible plants was potentially not practiced
by pre-Columbian cultures. Another plausible explanation may rely on soil as an important
microbial seeding source (possible geophagy) [58]. Geophagy (ingestion of soil intentionally or
unintentionally), has been used by humans to protect them from dietary chemicals and patho-
gens [59]. It has been observed in many different cultures around the world [59], and archaeo-
logical evidence suggests that geophagy dates back to Homo habilis [60, 61].
Evidence of various food items

Earlier research has revealed a wide range of plants that were processed using lithic artifacts
such as burén, stones, and shells, as evidenced by the presence of preserved starch grains [19].
This suggests a sophisticated food system, which is in contrast to historical accounts that
emphasize the indigenous cultures’ dependance on manioc [62–65]. Consistent with this early
archaeobotanical study, we identified DNA sequences from a diversity of plants, including a
starchy tuber (sweet potato), legume (peanut), solanaceous fruit (chili peppers and tomato),
caricaceous fruit (papaya), pseudograin (maize), and other crops (tobacco and cotton). Such
plants identified in the Huecoid and Saladoid coprolites analyzed suggest a variety of dietary,
medicinal, and hallucinogenic plants as part of these pre-Columbian cultures diet and culture.
Although very useful and insightful, the results and conclusions in the present study are

limited by the fact that there are relatively few available plant genomes sequenced and available
in the current databases. As the number of genome sequences increases, sequences obtained
from coprolites will probably be more defined.
Tobacco
Tobacco originates in the New World, particularly in Central and South America [66]. It has
been reported that indigenous cultures from the 16th century would consume tobacco for hal-
lucinogenic, as well as medicinal purposes as it would alleviate pain and induce sleep [66]. It
has also been hypothesized that tobacco was consumed in social activities [66]. Consumption
of tobacco comes from chroniclers dealing with pre-Columbian cultures, such as the Aztecs
[66], the Maya [67] and indigenous people from La Hispaniola [13, 14]. Traces of nicotine
were detected in a Late Mayan period flask (Campeche, Mexico) dating around 700 AD by
using chromatography and mass spectrometry, indicating the ancient use of tobacco in the
Mayan culture [67]. The Mayan flask may have been used for tobacco enema preparations,
likely for rituals and medicinal purposes [68]. However, limited records exist on tobacco con-
sumption by other pre-Columbian cultures. Nevertheless, information from specific pre-
Columbian cultures may provide insights into tobacco consumption in the Huecoid and Sala-
doid cultures. As such, there are several plausible practices that may explain the presence of
tobacco sequences in the Huecoid and Saladoid coprolite samples. First, tobacco may be
chewed, and although we could not find any references of this practice in pre-Columbian rec-
ords, there is a likelihood that tobacco could have been consumed in this manner. An alterna-
tive explanation for the presence of tobacco sequences in the coprolite samples include the use
of wood or ceramic inhalers, where pulverized tobacco (and other herbs, or mixtures) can be
placed. The inhaler is then inserted in the nostrils of the recipient and a second person would
blow into the inhaler to force the powder deep into the nostrils. Tobacco could also have been
used as an additive for food and drink [67].
Sweet potato and legumes

Sweet potato and legumes (including common beans) played an important function in the
agricultural economies of ancient Puerto Rico [18]. We identified DNA sequences likely corre-
sponding to sweet potato and legumes in the Huecoid coprolite sample. Pre-Columbian cul-
tures relied on specific food items such as sweet potato, which would be consumed with fruits,
vegetables, meat, fish [5, 17], mollusks, and crustaceans. In addition to sweet potato sequences,
we observed Fabaceae sequences (putatively assigned as peanut), suggesting that it may have
been a component of the Huecoid diet. Indeed, peanuts are native to the New World, originat-
ing in South America, and new species continue to be discovered [69]. Sweet potato and
legumes have also been found on lithic and shell artifacts, and dental calculus from Puerto
Rico and other regions of the Caribbean [18, 19, 70, 71], suggesting the consumption of these
plant items by pre-Columbian cultures. Legumes persisted in the ancient Caribbean diet of
pre-Columbian cultures from the early ceramic age to the early colonial period, whereas the
sweet potato was a key starchy crop during the pre- and post-Columbian eras [18]. The low
abundance of sweet potato sequences in the Huecoid coprolite sample may be a result of food
traces from a previous meal that were obscured by the abundant foods of the latest meal [21],
or the sporadic consumption of this item.
Maize
Maize (Zea Mays), a plant domesticated in Mesoamerica [72, 73], was introduced from the cir-
cum-Caribbean region (Central America and the northern countries of South America) to

Puerto Rico probably during the archaic age approximately 5,000 B.P [74]. Early European
chroniclers indicate that indigenous cultures cultivated maize twice a year and consumed it
tender, as well as raw, and roasted. They also included maize in certain stews, and would also
consume it ground and with water [13]. Maize could be ground or pounded and further
baked, grilled, or toasted by these pre-Columbian cultures to possibly prepare bread [15, 75].
While maize has been previously considered a restricted crop [17, 76], evidence of human iso-
tope and pre-Columbian dental calculus from Puerto Rico and the Caribbean suggest that
maize was frequently consumed [15, 77]. Such findings were further extended by Pagan and
Mickleburgh, who suggested that maize was the most ubiquitous edible crop of the insular
Caribbean [18]. Overall, these results suggest that maize had an important role in pre-Colum-
bian dietary habits. We detected a high abundance of maize in the Huecoid coprolite sample,
suggesting that maize was an important crop in this culture, likely consumed daily, which is
consistent with previous paleomicrobiological findings [32]. In addition, the analysis of starch
residues in lithic tools from two Huecoid settlements in Puerto Rico demonstrated that the
Huecoid culture maintained and used this plant [19]. The detection of certain plants, like
maize, exclusively in the Huecoid culture may further support differences in cultural back-
grounds observed in archeological records, as well as microbiome, virome and mycobiome
analyses. However, perhaps the Saladoid culture consumed maize sporadically and thus,
sequences were not detected in these particular coprolite samples. Moreover, the presence of
Ustilago spp. sequence reads, a fungal genus known to infect maize, in the coprolites not only
provided further evidence of maize consumption, but possibly point to the consumption of
huitlacoche (musuro or sara musuru in quechua) [78], a common fungal phytopathogen that
is prized as a delicacy, even by today’s cultures.
Chili peppers
Chili peppers have been used for food, medicinal and religious purposes throughout the Amer-
icas [55]. Paleo-biolinguistics along with genetic and archaeobotanical evidence have shown
that domesticated chili pepper originated in central-east Mexico approximately 6,500 years
ago [79]. Chili peppers are not frequently found in the archaeological record likely due to poor
starch or capsain resiliency over time [80]. However, starches of chili pepper have been
detected in food-processing tools of the early southern Caribbean and the late pre-Columbian
period of the northern Caribbean [18, 70], and were likely consumed as a condiment, stimu-
lant and medicine in the pre-Columbian era [13, 14]. We identified sequencing reads match-
ing chili peppers in both the Huecoid and Saladoid coprolites. Notably, it has been shown that
chili peppers and maize occurred together in food-processing tools, suggesting a preferred
food-complex [80]. Consistent with these observations, we found a high abundance of maize
and chili pepper DNA sequences in the Huecoid coprolite sample.
Unexpected findings and absence of expected sequences

Tomato. Tomato is currently divided into S. lycopersicum var. lycopersicum and S. lycoper-
sicum var. cerasiforme, and S. pimpinellifolium is considered the most closely related wild spe-
cies of tomato [81]. The roles of some of these species in the domestication of tomato remain
unclear and a matter of further investigation as some consider that S. l. cerasiforme is an ances-
tor of tomato, while others consider that S. l. cerasiforme is a combination of S. pimpinellifo-
lium and S. l. lycopersicum [81]. While it remains unclear if the domestication of tomato
occurred in the Andean region or Mesoamerica, one study suggested that pre-domestication
occurred in the Andean region, and domestication occurred in Mesoamerica [81]. The

presence of Solanum spp. (tomato) DNA sequences was somehow unexpected; however, this
food item may have been brought along with other food crops from South America.
Cotton. The presence of Gossypium spp. (cotton) sequences was unexpected since it is a
non-edible crop used for textile throughout the ages. However, a possible explanation might
be the use of the seeds as either additives or as source of oils to be used in some manner;
although cotton oils are known for their bitter flavors. Nonetheless, this finding opens up
more questions than it answers. Current cotton oils are not likely to have DNA present, but
that is because of the highly processed nature of these oils. These ethnic groups may have
found the ground seeds were a food additive to their diet in some manner. Other possible
explanation is that indigenous women ingested cotton fibers during the weaving process by
using the saliva to prepare the raw yarn. In fact, cellulose fibers of cotton have been found in
dental calculus from the Late Woodland period (900–1100 AD) of the Danbury site (Ohio),
suggesting the processing of cotton fibers for textiles and likely fishing nets using the mouth
[82]. Cotton fibers have been found to contain ample concentrations of DNA [83]. Addition-
ally, cotton processing and textile crafting was (and still is in many cultures) a female-only
activity; thus, a likely scenario is that these coprolites were deposited by female members of the
ethnic groups.
Cassava. Many archaeological narratives of the Caribbean suggest that the subsistence
strategies of the Huecoid and Saladoid cultures were primarily based on cassava/yucca/manioc
(Manihot esculenta) [62–64]. Cassava can be “sweet” or “bitter”, with the later retaining its
toxic liquid if not extracted [84, 85]. While “sweet” cassava has been selected to be non-toxic
and can be consumed either boiled or roasted, most cultivated types are “bitter” and retain the
toxicity and as such require processing to extract the toxic liquid [84, 85]. The chroniclers
widely described the “bitter” cassava in the Antilles and reported it consumption by indige-
nous people after a long preparation process that includes grating and squeezing the cassava
pulp with a sebucán to extract the toxic liquid, followed by a drying stage to produce flat tortas
[14]. The chroniclers also reported that “sweet” cassava was not observed in the Antilles but on
the mainland. The absence of cassava DNA sequences in the coprolite samples may suggest the
extensive pretreatment of Manihot spp. to remove toxins contained in the liquid. Indeed, cer-
tain methods of food preparation, are known to degrade dietary DNA [34, 86], which could
then be further degraded by enzymes and microbes during digestion [87], as well as by tapho-
nomic processes. Alternatively, the absence of some plants DNA could also explain seasonal
variation [26], or sporadic consumption of certain food items, such as cassava.
The importance of cassava has been debated over the years. One study including the analy-
sis of Huecoid lithic tools from La Hueca, Vieques, showed the recovery of ancient cassava
starches from a single tool [19]. In contrast, sweet potato, and other plants (including maize),
were identified in several lithic tools, suggesting that cassava was only part of a diverse spec-
trum of plants contributing to the diet [19]. Although archaeological narratives suggest that
cassava was introduced to Puerto Rico by the Saladoid, cassava starch grains were undetected
in twenty-four tools corresponding to this culture [88, 89]. Similarly, cassava starches were
unidentified in “burenes” (tools often associated with the cooking of cassava) from the Sala-
doid culture, where sweet potato and other plant remains (maize and beans and others) have
been frequently found.
Spatiotemporal dietary variations

Pre-Columbian cultures and present-day hunter-gatherers showed contrasting diets compared
to present-day agriculturalists and urban-industrial individuals. Plant communities among the
ethnic groups were significantly segregated based on ethnicity and lifestyles, as shown by

hierarchical clustering and PCoA. Since the pre-Columbian cultures inhabited regions similar
to those of their present-day counterparts, it is unlikely that changes in dietary habits are due
to geography. Clustering of plant communities based on lifestyles (i.e., hunter-gatherer, agri-
culturalist and urban-industrial) suggested changes in dietary habits in response to transitions
in human lifestyles. In fact, the diet of past populations is known to differ greatly from that of
modern populations depending on the environment, socioeconomic status, and available
resources [90]. During the Neolithic Era, human dietary lifestyles transitioned from game
meat and gathering of unprocessed fruits from the environment (i.e., hunter-gatherers) [91],
into one based on agriculture and animal domestication (farming). However, a westernized
diet was adopted with the industrial revolution, characterized by being high in fats and includ-
ing simple carbohydrates [92, 93].
Ancestral Puebloans (the Anasazi) were a prehistoric culture from the Colorado Plateau,
which include the States of Colorado, New Mexico, Arizona, and Utah. Macro-remains analy-
sis of coprolites from the Arid West (Arizona) and the Boomerang Shelter (Utah) have shown
that maize-derived foods (including huitlacoche) [23, 94], and prickly pear fruit (Opuntia) are
abundant components of the Ancestral Pueblo diet [23]. In contrast, coprolites from the Loma
San Gabriel culture, a prehistoric population from Rio Zape Valley in Durango, Mexico,
showed that they subsisted mainly on Agave and maize [21, 95]. Other plants that supple-
mented their diet included squash (Cucurbita spp.) and beans (Phaseolus spp.). In agreement
with previous studies, our study showed that the Huecoid and Saladoid plant diet consisted of
starchy tubers, maize, and legumes, supplemented with fruits. It is well known that food
sources can vary due to differing geographical and cultural characteristics. However, the Hue-
coid and Saladoid shared food sources with the Ancestral Pueblo culture and the Loma San
Gabriel culture, which may explain the clustering patterns. Nonetheless, this study is biased
towards domesticated taxa, whose sequences are available in databases. Thus, food sources
identified in geographically disparate pre-Columbian cultures could be showing the impor-
tance of domesticated crops in human diets after being adopted by these cultures. Notably, the
presence of certain plant sequences may also correspond to what was consumed a short period
prior to fecal deposition. Despite the fact that coprolites provide relevant information about
diet, food plant DNA in each coprolite likely reflects a few previous meals prior to defecation.
Interestingly, the present-day Matses also seems to be more similar to the Ancestral Pueblo-
ans, Loma San Gabriel, Huecoid and Saladoid pre-Columbian cultures, despite differences in
culture and temporal scales. The Matses hunter-gatherers have a diet mainly composed of
gathered tubers (Manihot spp.) and plantains (Musa spp.) [41]. On the other hand, the Tuna-
puco dietary lifestyle suggests a state of transition from hunter-gathering to agriculture. Pota-
toes (Solanum tuberosum spp.), oca (Oxalis tuberosa), and mashua (Tropaeolum tuberosum)
are part of every meal of these extant agriculturalists from the Peruvian highlands [41]. In con-
trast, the Mazahua farmers from Mexico had a greater resemblance to the United States indi-
viduals, likely due to a higher agricultural production in the Mazahua community compared
to the Tunapuco agriculturalists. The present-day Mazahua farmers base their diet on maize,
secondarily on wheat and edible mushrooms [23, 96]. Individuals from the United States
exhibit the typical western diet composed of processed foods and dairy products [42].
Although rare or limited, the Tunapuco consume dairy and processed foods [41]. In addition,
rice and bread are the main food supplementing the Tunapuco diet, while wheat contributes
the majority of the Mazahua calories after maize. Similar dietary components among the
Mazahua and United States feces, and to a lesser extent the Tunapuco, may have resulted in
the clustering patterns observed for the Mazahua and United States and the transitional state
of the Tunapuco. These results are partially supported by a recent study showing the segrega-
tion of pre-Columbian and present-day populations based on their gut mycobiome [36].

Studies have shown that dietary lifestyles strongly shape the composition of the gut micro-
biome of traditional populations, which differs from that of industrialized populations [41,
97–104].
The detection of plant DNA in coprolites may be biased towards foods that are consumed
raw or lightly cooked, as cooking and food preparation can result in the liberation of DNA
form cells. Additionally, plant materials that have been metabolized during digestion may be
difficult to identify [105, 106]. Furthermore, the degradation of DNA by nucleases during
digestion can also affect the results. Maize was commonly identified in the Huecoid coprolites
likely due to non-digestible fibers that are resistant to digestion [107]. Identifying certain plant
sequences can be challenging due to limitations in current DNA databases that primarily
include plants of commercial and economic importance. Taphonomic processes that damage
ancient DNA can also aggravate and restrict the matching of ancient DNA sequences to those
available in existing databases. It is important to note that a match or close hit between
sequences does not necessarily imply similarities across the species being compared, but rather
that the ancient DNA sequence could represent a taxon that is not represented in the database
[26].
Conclusions
We conducted DNA sequence analyses of plant sequencing reads from coprolites, which
revealed the presence of a variety of plants in the Huecoid and Saladoid cultures of the Carib-
bean, as well as in ancient and present-day America. Our study supports archaeological rec-
ords that suggest that these cultures consumed maize (Zea mays), sweet potato (Ipomoea
batatas), chili pepper (Capsicum annuum), papaya (Carica papaya), peanut (Arachis spp.),
tobacco (Nicotiana sylvestris), tomato (Solanum lycopersicum), and surprisingly, cotton (Gossy-
pium barbadense). However, it is important to note that coprolites reflect a limited range of
potential plants ingested, and the detection of plant sequences in a single culture may suggest
preferences for certain plants, occasional consumption, degradation due to specific food prep-
aration practices, or taphonomic processes.
Our analyses are limited by the current DNA sequence databases, which are focused mostly
on commercially important crops. Expansion of these databases to include plants that are not
necessarily of economic importance might allow us to better understand ancient dietary habits,
as well as those of extant remote populations. By examining plant and fungi sequences, our
data aids in the reconstruction of the dietary habits and lifestyles of the Huecoid and Saladoid
cultures, and opens the opportunity to further understand the diets and lifestyles of other pre-
Columbian groups in America.
Supporting information
S1 Table. Detailed description of the archaeological samples analyzed in this study.
(DOCX)
S1 Fig. Source proportion estimates for the Huecoid and Saladoid coprolite samples (sink)
using reference datasets of environmental samples (source). Meta-SourceTracker showed
the proportion of Eukaryote domain sequencing data that each environmental source sample
contributed to the Huecoid and Saladoid coprolite sink samples. Overall, mSourceTracker
showed that unknown sources contributed the highest proportions of Eukaryote reads in the
Huecoid (0.41%) and Saladoid (0.68%) coprolites. Besides unknown sources, mSourceTracker
estimated that a high proportion of the eukaryote reads of the Huecoid coprolite sink sample
came from well-preserved coprolite source samples (0.33%). Conversely, a high proportion of

eukaryotes exhibited soil (0.24%) and coprolite (0.07%) origin in the Saladoid coprolite sink
sample.
(PDF)
Author Contributions
Conceptualization: Jelissa Reynoso-Garcı́a, Raul J. Cano, Gary A. Toranzos.
Data curation: Jelissa Reynoso-Garcı́a, Tasha M. Santiago-Rodriguez.
Formal analysis: Jelissa Reynoso-Garcı́a.
Funding acquisition: Jelissa Reynoso-Garcı́a.
Investigation: Jelissa Reynoso-Garcı́a.
Methodology: Jelissa Reynoso-Garcı́a.
Project administration: Gary A. Toranzos.
Resources: Yvonne Narganes-Storde.
Supervision: Gary A. Toranzos.
Validation: Yvonne Narganes-Storde, Gary A. Toranzos.
Visualization: Jelissa Reynoso-Garcı́a, Tasha M. Santiago-Rodriguez.
Writing – original draft: Jelissa Reynoso-Garcı́a.
Writing – review & editing: Jelissa Reynoso-Garcı́a, Tasha M. Santiago-Rodriguez, Yvonne
Narganes-Storde, Raul J. Cano, Gary A. Toranzos.
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PLOS ONE

Edible flora in pre-Columbian Caribbean

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publish, or preparation of the manuscript. This Introduction

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Materials and methods

DNA extraction and contamination prevention

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Metagenomic library construction and shotgun sequencing

Comparison with other coprolite and present-day fecal sequences

Bioinformatics and statistical analysis

Table 1. Description of the archaeological samples analyzed in this study.

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Table 2. Description of the published present-day samples analyzed in this study.

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Starchy tubers, legumes, pseudograins, fruits, and a hallucinogenic plant

*Putative taxonomic assignment of plant sequences in the present dataset.

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Phytocultural practices of ancient cultures may be different from those of

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Overcoming challenges with contamination

Evidence of various food items

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Sweet potato and legumes

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Unexpected findings and absence of expected sequences

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Spatiotemporal dietary variations

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