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Cycle 9 (2020 Deadline)

Biodiversity correlates of sustainable value chain expansion in the Brazilian Amazon: developing combined environmental DNA (eDNA) and camera trapping protocols to assess vertebrate diversity in managed Brazil nut forests

PI: Ludmilla Aguiar (, University of Brasilia/Foundation for Scientific and Technological Enterprises, with co-PI Pedro Galetti, Federal University of São Carlos
U.S. Partner: Kirsten Silvius, Virginia Polytechnic Institute and State University
Project Dates: May 2021 - April 2024

Project Overview:
9-236 Setting up traps
A project team member sets an invertebrate trap during sample collection training. Photo courtesy of Dr. Aguiar.
DNA metabarcoding of environmental DNA (eDNA)—simultaneously identifying multiple species using short sections of DNA from soil or water samples— holds great promise for biodiversity research, but these tools have not been fully refined for assessing animal community structure in general, considering species abundance as well as presence-absence, and terrestrial vertebrate communities in particular. eDNA work is more advanced for aquatic than terrestrial systems, facilitated by DNA’s more rapid dispersal in water. This project will deploy camera trapping and sign and visual encounter surveys—traditional tools whose accuracy and precision for describing animal abundance and richness are well understood—simultaneously with eDNA sampling from soil and water and invertebrate DNA (iDNA) and sampling from insect blood-meals to calibrate the DNA-based methods through comparison with the traditional methods. Such calibration efforts have begun in the temperate zone, but this project will be the first to use multiple methods in a high biodiversity tropical area and use them to test hypotheses about both individual species abundance and species composition.

Through the simultaneous deployment of these methods, the project will describe the species composition of mammal communities associated with Brazil nut (Berhtolletia excelsa) rich forests and address the still-open question of whether Brazil nut harvest and management practices affect vertebrate communities and species abundance. The project will use metabarcoding for broad species detection and community-wide surveys, as well as quantitative PCR (qPCR) to test hypotheses for a subset of mammal species whose abundance is expected to be impacted by harvesting. Regardless of the success of the eDNA methods, the traditional sampling methods will allow the project team to address the question of biodiversity consequences of harvest and make recommendations for improved natural resource management in Brazil nut extraction areas.

The publicly available barcode database compiled by the project will be made available to the academic, public (biodiversity management and protection agencies), and private (consulting companies) sectors for research, monitoring, enforcement, and environmental impact assessments, respectively. Graduate students and postdoctoral associates at the two participating Brazilian universities will be trained in the use of qPCR techniques with broad applicability to biomedical/human health fields, as well as ecological contexts. Importantly, this project will enable new Brazil-U.S. research collaborations through exchanges among the USDA National Genomics Center and the Aguiar and Galetti labs at the University of Brasilia (UNB) and the Federal University of São Carlos (UFSCar).

Summary of Recent Activities: 

In April 2022, the eDNA and iDNA team, led by co-PI Pedro Galetti, carried out a pilot experiment in a natural environment. Postdocs C. Gestich and B. Saranholi, accompanied by project member J. Fragoso, traveled to Terra Ronca State Park (PETeR, Goiás, Brazil), a protected area of the Cerrado biome, to investigate the mammal community through eDNA and iDNA samples with metabarcoding sequencing and to reproduce the ongoing experiment with Panthera onca using an RT-PCR approach. During six days of sampling, they collected 17 one-liter water samples and 12 bulk samples of mosquitoes and flies from insect traps, totaling 24 insect trap-days across the PETeR. The water samples were processed in the laboratory by extracting DNA from filters, amplifying two mini-barcodes (12SrRNA and 16SrRNA genes), and amplifying the specific region of ATP6 gene for P. onca to determine its presence in each sampling point by RT-PCR. The amplified minibarcodings were then delivered to be sequenced in an external lab on the Illumina platform. The row sequences were processed with bioinformatic tools, with the results showing the presence of 33 mammal species occurring in the PETeR, including P. onca. The results obtained in the pilot of PETeR will be presented in the XI Brazilian Congress of Mammalogists (CBMZ 2022, scheduled for October 17-21, 2022.

Between April 19 and May 2, 2022, USFS and UnB researchers K. Silvius and J. Fragoso accompanied an expedition organized by project partner Fundação Vitória Amazônica (FVA) up the Unini River to visit Brazil nut-producing communities in Jaú National Park and Unini River Extractive Reserve. During the expeditions, the USFS and UnB researchers presented the PEER project at eight community meetings, each in a different community of traditional ribeirinho families living on both banks of the Unini River. After each meeting, the researchers held conversations with individuals identified as among the most active Brazil nut harvesters in each community. Through these conversations, potential study sites (forest areas with high densities of Brazil nut trees, known as castanhais, where transects and cameras can be placed) were identified. Residents of all communities expressed interest in seeing the project implemented in their territory. Because individual families hold recognized rights to specific Brazil nut-harvesting areas, research in any Brazil nut stand must be negotiated and agreed to with the family that uses and manages it.

Between September 9 and 30, three researchers (C. Gestich and B. Saranholi from UFSCar and D. Sana from UNB) carried out the first field expedition for data collection along the Unini River. In this expedition, they visited the Lago das Pedras, Terra Nova and Patauá communities, to present the project again and advance the involvement of local people in the research. They selected four castanhais for the first eDNA and iDNA sampling; at each site they walked 3-km linear transects in which they placed four fly traps during three consecutive days for iDNA analysis, totaling 48 insect trap-days. They also collected 16 water samples (1.2 liters each) along the transects and from the river edge near to transect entrances, for eDNA analysis. All four selected sites have not been used for nut harvesting for a few years. At each site, family members with harvest rights for that area accompanied the team in the transect sampling. Although camera traps have not yet been installed or linear transect sightings initiated, in this expedition the researchers evaluated the local conditions for these methods, talked with local people about their interest in working directly on such data collection, and planned a training course with local residents to prepare them to work as parabiologists in the next stages of the project.

Dr. Galetti’s team have maintained the collaboration with the researcher Gonzalo Barquero, director-president of the Tropical Sustainability Institute (TSI), which has a long-term program surveying the mammal community in Terra Ronca State Park. Barquero assisted the team during the field trip to the PETeR, including local logistical support, and shared TSI results of mammal monitoring, in order to compare the results being obtained with iDNA and eDNA approaches. Additionally, during the reported period, the PEER researchers started a collaboration with the non-governmental organization Fundação Vitória Amazônica (FVA), a long -term USFS and USAID partner organization with strong presence in the two protected areas along with Unini River (Jaú National Park and Unini Extractive Reserve). The leadership of the Association representing residents of the two protected areas, AMORU, also expressed strong support for the project, understanding it as a potential contribution to AMORUs agenda to implement tourism and research activities by the communities that make up the association.

After field data collection, during their stay in Novo Airão and Manaus, the researchers met with the government agencies ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade) and SEMA (Secretaria Estadual de Meio Ambiente - State Department of Environment) to update the authorization for studying in the conservation units where the communities live, the Resex Unini and National Park of Jaú, administered by ICMBio, and RDS Amanã (Reserva de Desenvolvimento Sustentável - Sustainable Development Reserve) by SEMA. The furthest community from Rio Unini is within the territory of RDS Amanã, and as the PEER team intends to include this community in their study, they requested authorization to include RDS Amanã. Also in Novo Airão, researchers met with a representative of the COOMARU cooperative that manages a Brazil nut processing plant in the reserve and sells the processed nuts. During this meeting researchers further clarified protocols for engaging with families and contracting logistical support in the study area.

In late 2022 and into 2023, plans call for completing laboratory-based analysis of water samples and insect samples from the PETeR and initiating analysis of fly and water samples from the Unini field site. From information about the communities and nut forests collected during the first field expedition, the researchers will select the other potential areas to establish the transect and continue the data collection, including camera traps and sighting surveying. UnB and USFS researchers plan on visiting Novo Airão and the Unini area in November 2022 to participate in the annual management council meetings for the two protected areas; identify additional families and communities to work with; and advance with the field component related to camera trapping and animal observations along transects. A longer expedition with the full research team is planned for the second trimester of 2023, after the Brazil-nut harvesting season is complete, with a return visit to the site in the third to fourth trimesters. Planned exchanges with the USFS National Genomic Center will also take place in 2023. The project timeline was seriously impacted both by COVID-related disruptions and seasonal weather conditions. Therefore, a no-cost extension has been issued to allow the project to continue through April 2024.


Carvalho, C., Oliveira, M., Rodriguez-Castro, K., Saranholi, B., & Galetti Jr, P. 2021. Efficiency of eDNA and iDNA in assessing vertebrate diversity and its abundance. Molecular Ecology Resources. DOI:10.1111/1755- 0998.13543

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