Environmental DNA (eDNA) is genetic material extracted from the environment from the likes of water, soil, sediment, sand, snow and ice where there is no sign of biological source material. There is also an emerging field of eDNA analysis from air for use in forensic investigations1.
As far back as 1987, T. Barkay of the USA Environmental Protection Agency and A. Orgam of University of Tennessee wrote a paper on the extraction and purification of microbial DNA from sediment and referred to environmental DNA2. However, it is the application of next generation sequencing (NGS) and targeted NGS in this field that has been a game changer.
A team of scientists from the Universities of Cincinnati, Minnesota, Calgary and Florida, along with colleagues from the Universidad Autónoma de Campeche, Universidad Nacional Autónoma de México and the Universidad de San Carlos de Guatemala, have been investigating the vegetation growing at the artificial reservoirs of Tikal, a major city of the ancient Maya World in the Yucatan Peninsula in Guatemala. They also studied the psychoactive and ceremonial plants used in Maya rituals from 2000-year-old deposits at Yaxnohcah in South Mexico near Guatemala.
Understanding ancient agricultural practices at Tikal, Guatemala
Research paper: Forests, fields, and the edge of sustainability at the ancient Maya city of Tikal
The land use practices of the great Maya city of Tikal sustained a ‘low density urban population for many centuries’3, which can be partly attributed to intensive agriculture. They created artificial reservoirs to supply drinking water and irrigation, constructed terraces, developed arboriculture, planted household gardens and utilised short fallow swidden3. Scientists studying this city have wondered how the leaders and farmers managed to provide food, fuel and other sustenance for the occupants of this city, which was advanced for its time, 400 BC-900 AD4.
The city’s relationship with the Neotropical Forest
Despite over a century of study of the city the relationship between the Maya and the surrounding forest was difficult to interpret and understand. In order to further understand this relationship, the team used ‘a powerful new technology, environmental DNA analysis, that enabled us to characterise the site core vegetation growing in association with the artificial reservoirs that provided the city’s water supply’5. 
To do this, eDNA was extracted from 30 sediment samples collected in the reservoirs and waterways. These sediment samples contained trace quantities of DNA from plant material that fell into the reservoirs, sank and was buried, thus preserving it. To boost the ability to detect plant DNA within the samples a genetic probe set was developed to target genes from chloroplasts, which are more abundant than nuclear genes, increasing the likelihood of recovering segments of DNA that can be used to identify species they came from.
Taking a probe-based approach
The researchers in conjunction with the team at Rapid Genomics, now part of LGC Biosearch Technologies, used the large database of publicly available DNA sequencing data to develop a custom Capture-Seq probe panel tailored around Mesoamerica plants. This panel consisted of thousands of probes designed to target and enrich for chloroplast and nuclear genes that would allow for identification of plants to genus, family or order. These probes were hybridised to the eDNA samples, enriching for these targets over the non-plant background DNA in the sample. These were then sequenced, and the resulting data were assembled into gene fragments (contigs) and compared against the reference database using a series of BLAST (Basic Logical Alignment Search Tool) searches to find matches between the sample DNA and known plant species. This effort resulted in the identification of species such as wild onion, hog plum, bay leaf, wild cherry, breadnut and fig.
Capture-Seq is a targeted probe hybridisation technology to selectively recover regions of interest throughout the genome of any species with up to 99% data return and high levels of reproducibility.
Results of the Tikal project
The scientists concluded that the research strongly supported the hypothesis that Maya maintained native forest plants on the banks of the reservoirs. They also found no clear evidence of cultigens, as many of the species identified, for example from the Cucurbitaceae family, are wild species native to the neotropics. Maize was also not clearly identified. The scientific team concluded that ‘the Maya did not plant cultigens around these two reservoirs but allowed or encouraged native forest species to grow on the banks’5.They further concluded that, ‘perhaps the most important result from this study was that we were able to extract DNA from ancient Mesoamerican reservoir sediments, sequence the extracts and identify species-specific sequences. This achievement represents a technological leap forward for the field of archaeology’5.
Discovering plants used for ceremonial purposes in Yaxnohcah, Mexico
Research paper: Psychoactive and other ceremonial plants from a 2,000-year-old Maya ritual deposit at Yaxnohcah, Mexico - PMC (nih.gov)
Archaeobotany is the study of ancient plant remains and is a growing sector of archaeology as scientists strive to further understand the relationship between ancient civilisations and their connection to plant life. The Mayan culture has been connected to rituals and ceremonies from ethnographic sources such as murals, stone carvings and Mayan books made of tree bark. Most research has focussed on pollen grains, starch analysis and phytolith analysis, all of which have shortcomings.
Picture: Mayan Sacrifice Altar Q's, at Copán Ruins
An eDNA probe-based approach to discover the plants used in Mayan ceremonials
Following their success with eDNA analysis at Tikal, Dr Lentz and his team decided to use eDNA analysis on deposits found in the Maya city Yaxnohcah. The deposits were ‘discovered beneath an early plaza floor of a civic ceremonial platform upon which was constructed a ballcourt at the ancient Maya city’6.
With the success delivered by Capture-Seq on the Tika project, the team collaborated with Rapid Genomics to create probes for 104 plant species across 54 genera, spanning 33 families of plants.
The sequencing data from the samples recovered 105,577 assembled gene fragments ranging in size from 208-3692 bp in length (median 263 bp)6. Many of these fragments were from background, non-plant DNA within the samples (i.e. fungi and bacteria) but 15 were identifiable as being from vascular plants and were able to be placed at the family or order level. Four sequences were unique enough for identification to genus or species level representing Ipomoea corymbose (morning glory family), Capsicum sp (chili pepper), Hampea trilobata and Oxandra lanceolata (lancewood)6. These four plants have special properties which make them likely to be used in ancient rituals and for medicinal purposes, including one with psychoactive properties.
Results of the Yaxnohcah project
The research team concluded that ‘with the ceremonial plants found at Yaxnohcah, a greater understanding of the ritual and other sacred practices of ancient cultures can now come into clearer focus with the assistance of eDNA evidence, a methodology whose promise for archaeology is only beginning to be explored’6.
Further reading
References
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Goray M, Taylor D, Bibbo E, Fantinato C, Fonneløp AE, Gill P, et al. Emerging use of air eDNA and its application to forensic investigations – A review. Electrophoresis. 2024; 45: 916–932. https://doi.org/10.1002/elps.202300228
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Andrew Ogram, Gary S. Sayler, Tamar Barkay, The extraction and purification of microbial DNA from sediments, Journal of Microbiological Methods, Volume 7, Issues 2–3, 1987, Pages 57-66, ISSN 0167-7012, https://doi.org/10.1016/0167-7012(87)90025-X.
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Lentz D et al. Forests, fields, and the edge of sustainability at the ancient Maya city of Tikal, Proceeding of the National Academy of Sciences, Volume 111, Number 52, 2014. https://doi.org/10.1073/pnas.1408631111
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Dunning P, Lentz D et al. Tikal land water and forest: An introduction Tikal: Paleoecology of an Ancient Maya City, Chater 1, page 1 2015. Tikal: Paleoecology of an Ancient Maya City - Google Books
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Lentz DL, Hamilton TL, Dunning NP, Tepe EJ, Scarborough VL, Meyers SA, Grazioso L, Weiss AA. Environmental DNA reveals arboreal cityscapes at the Ancient Maya Center of Tikal. Sci Rep. 2021 Jun 16;11(1):12725. doi: 10.1038/s41598-021-91620-6. PMID: 34135357; PMCID: PMC8209062
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Lentz DL, Hamilton TL, Meyers SA, Dunning NP, Reese-Taylor K, Hernández AA, Walker DS, Tepe EJ, Esquivel AF, Weiss AA. Psychoactive and other ceremonial plants from a 2,000-year-old Maya ritual deposit at Yaxnohcah, Mexico. PLoS One. 2024 Apr 26;19(4):e0301497.
doi: 10.1371/journal.pone.0301497. PMID: 38669253; PMCID: PMC11051596.
