# Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG)

Mattia Saccò, Abdul Elmasri, Mahima Tawal, Roman Alther, Florian Altermatt, Alejandro Martínez, Stefano Mammola, Maja Zagmajster, Pablo Saenz-Agudelo, Miwa Takahashi, et al.

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Mattia Saccò, Abdul Elmasri, Mahima Tawal, Roman Alther, Florian Altermatt, et al. . Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG). Subterranean Biology, 2025, 53, pp.31-40. 10.3897/subtbiol.53.165710 . hal-05272878

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# Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG)

Mattia Saccò¹,²,³, Abdul Elmasri⁴, Mahima Tawal¹, Roman Alther⁵,⁶,
Florian Altermatt⁵,⁶, Alejandro Martínez⁷, Stefano Mammola⁷,⁸,⁹,
Maja Zagmajster¹⁰, Pablo Saenz-Agudelo¹¹, Miwa Takahashi¹², Jake Thornhill¹³,
Aude Andouche³, Mariella Baratti¹⁴, Muhammad Bilal¹, Helena Bilandžija¹⁵,
Rossano Bolpagni², Sarah Boulamail¹⁶, Fulvio Celico¹⁷, Claire A. Chauveau¹⁸,
Efrain Chavez¹⁹, Olivier Collard¹⁸, Steven J. B. Cooper¹³,²⁰, Marjorie Couton²¹,
Francesco Cozzoli⁹,¹⁶,²², Kat Dawkins²³, Tiziana Di Lorenzo⁹,¹⁴,²⁴,²⁵,
Ximena Dubinsky²⁶, Hans Jürgen Hahn²⁷, Jean-François Flot¹⁸, Fabien Glatting²⁸,
Christian Griebler²⁹, Simone Guareschi³⁰, Tamar Guy-Haim²⁶, Shane Herbert²³,
Grant Hose³¹, William F. Humphreys³², Benjamin Hutchins³³, Sanda Iepure²⁴,³⁴,³⁵,
Tim Johns³⁶, Clemens Karwautz²⁹, Dominik Kirschner⁵, Oren Kolodny³⁷,
Marjeta Konec¹⁰,³⁸, Raoul Manenti³⁹, Tarik Meziane³, Nataša Mori⁴⁰,
Elena Pazhenkova¹⁰, Maxine P. Piggott⁴¹, Alejandra Prieto-Davó⁴², Zoé Raphalen³,
Hannah Rau²⁷, Ana Sofia Reboleira²⁵,⁴³, Hans Recknagel¹⁰, Anne Robertson⁴⁴,
Coline Royaux⁴⁵, Benjamin Schwartz³³,⁴⁶, Klaus Schwenk²⁷, Nuno Simoes¹⁹,⁴⁷,⁴⁸,
David Stanković⁴⁰, Fabio Stoch¹⁸, Joe D. Taylor⁴⁹, Mieke Van Der Heyde¹,⁵⁰,
Živa Vehovar⁴⁰,⁵¹, Valerija Zakšek¹⁰, Nicole E. White¹, Morten E. Allentoft⁴,⁵²,
Paul Nevill⁵⁰, Kathryn L. Korbel³¹*, Michelle T. Guzik¹³*

1 *Subterranean Research and Groundwater Ecology (SuRGE) Group, Trace and Environmental DNA (TrEnD) Lab, School of Molecular and Life Sciences, Curtin University, Perth, WA, Australia* 2 *Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy* 3 *Laboratoire de Biologie des Organismes et des Écosystèmes Aquatiques-BOREA, Muséum National d'Histoire naturelle, SU, CNRS, IRD, UA, Paris, France* 4 *Trace and Environmental DNA (TrEnD) Lab, School of Molecular and Life Sciences, Curtin University, Perth, WA, Australia* 5 *Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Aquatic Ecology, Dübendorf, Switzerland* 6 *Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zürich, Switzerland* 7 *Molecular Ecology Group (MEG), Water Research Institute (IRSA), National Research Council (CNR), Pallanza, Italy* 8 *Finnish Museum of Natural History, University of Helsinki, Helsinki, Finland* 9 *NBFC, National Biodiversity Future Center, Palermo, Italy* 10 *Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia* 11 *Cawthron Institute, Nelson, New Zealand* 12 *Commonwealth Scientific and Industrial Research Organisation, National Collections and Marine Infrastructure, Crawley, Western Australia, Australia* 13 *The Environment Institute and School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia*

<footnote>* Shared last author.</footnote>
14 Research Institute on Terrestrial Ecosystems, IRET CNR, Sesto Fiorentino (FI), Italy 15 Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia 16 Research Institute on Terrestrial Ecosystems (IRET) - National Research Council of Italy (CNR) URT Lecce, Palazzina A - Complesso Ecotekne, Lecce (LE), Italy 17 HydrogeoCentre, Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy 18 Department of Organismal Biology, Université Libre de Bruxelles (ULB), Belgium 19 Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán, Mexico 20 South Australian Museum, Adelaide, South Australia, Australia 21 Laboratoire d'Ecologie des Hydrosystèmes Naturels et Anthropisés, LEHNA UMR, CNRS, ENTPE, Université Claude Bernard Lyon 1, Villeurbanne, France 22 Department of Biological and Environmental Sciences and Technologies, Palazzina B - Complesso Ecotekne, Lecce (LE), Italy 23 eDNA Frontiers, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia 24 Emil Racovită Institute of Speleology, Cluj-Napoca, Romania 25 Centre for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, and Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal 26 National Institute of Oceanography, Israel Oceanographic and Limnological Research, Haifa, Israel 27 iES Landau, Institute for Environmental Sciences, University of Kaiserslautern-Landau (RPTU), Landau, Germany 28 Institut of Applied Geoscience (AGW), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany 29 Department of Functional and Evolutionary Ecology, University of Vienna, Vienna, Austria 30 Biodiversity and Conservation Area, Rey Juan Carlos University (URJC), Móstoles, Madrid, Spain 31 School of Natural Sciences, Macquarie University, Sydney, New South Wales, Australia 32 School of Biological Sciences, University of Western Australia, Crawley, Western Australia, Australia 33 Edwards Aquifer Research and Data Center, Texas State University, San Marcos, TX, USA 34 Department of Taxonomy and Ecology, University Babes-Bolyai, Cluj-Napoca, Romania 35 Centre for Systems Biology, Biodiversity and Bioresources (3B), Babeş-Bolyai University, Cluj-Napoca, Romania 36 Environment Agency, Bristol, UK 37 Department of Ecology, Evolution, and Behavior, The Silberman Institute for Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 38 DivjaLabs Ltd, Ljubljana, Slovenia 39 Department of Environmental Sciences and Policy, Università degli Studi di Milano, Milano, Italy 40 Department of Organisms and Ecosystems Research, National Institute of Biology, Ljubljana, Slovenia 41 Research Institute for the Environment and Livelihoods, Faculty of Science and Technology, Charles Darwin University, Darwin, Northern Territory, Australia 42 Unidad de Química-Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Sisal, Yucatán, Mexico 43 National Museum of Natural History and Science, University of Lisbon, Lisboa, Portugal 44 School of Life & Health Sciences, Roehampton University, London, UK 45 UMR8067 Biologie des Organismes et Ecosystèmes Aquatiques (BOREA, MNHN-CNRS SU-IRD-UCN-UA), Sorbonne Université, Station Marine de Concarneau, Concarneau, France 46 Department of Biology, Texas State University, San Marcos, TX, USA 47 Laboratorio Nacional de Resiliencia Costera, Consejo Nacional de Ciencia y Tecnología, Ciudad de Mexico, Mexico 48 International Chair for Coastal and Marine Studies, Harte Research Institute for Gulf of Mexico Studies, Texas A&M University, Corpus Christi, TX, USA 49 UK Centre for Ecology & Hydrology, Benson Ln, Crowmarsh Gifford, Wallingford, UK 50 Minesite Biodiversity Monitoring with eDNA Research Group (MBioMe), Trace and Environmental DNA (TrEnD) Laboratory, School of Molecular and Life Sciences, Curtin University, Bentley, Perth, WA, Australia 51 Jožef Stefan International Postgraduate School, Ljubljana, Slovenia 52 Section for GeoGenetics, Globe Institute, University of Copenhagen, Copenhagen, Denmark

Corresponding author: Mattia Saccò (mattia.sacco@curtin.edu.au)
**Citation:** Saccò M, Elmasri A, Tawal M, Alther R, Altermatt F, Martínez A, Mammola S, Zagmajster M, Saenz-Agudelo P, Takahashi M, Thornhill J, Andouche A, Baratti M, Bilal M, Bilandžija H, Bolpagni R, Boulamail S, Celico F, Chauveau CA, Chavez E, Collard O, Cooper SJB, Couton M, Cozzoli F, Dawkins K, Di Lorenzo T, Dubinsky X, Hahn HJ, Flot J-F, Glatting F, Griebler C, Guareschi S, Guy-Haim T, Herbert S, Hose G, Humphreys WF, Hutchins B, Iepure S, Johns T, Karwautz C, Kirschner D, Kolodny O, Konec M, Manenti R, Meziane T, Mori N, Pazhenkova E, Piggott MP, Prieto-Davó A, Raphalen Z, Rau H, Reboleira AS, Recknagel H, Robertson A, Royaux C, Schwartz B, Schwenk K, Simoes N, Stanković D, Stoch F, Taylor JD, Van Der Heyde M, Vehovar Ž, Zakšek V, White NE, Allentoft ME, Nevill P, Korbel KL, Guzik MT (2025) Advancing subterranean conservation through Global Research on eDNA in Groundwaters (GReG). Subterranean Biology 53: 31–40. https://doi.org/10.3897/subtbiol.53.165710

If water is life, then groundwater is its lifeblood—essential, but hidden, it forms the largest reservoir of unfrozen freshwater globally. Populated by highly specialized, often relictual lineages across the tree of life, groundwater ecosystems not only support remarkable taxonomic, functional and phylogenetic diversity (Griebler and Lueders 2009; Martínez et al. 2018; Marmonier et al. 2023), but also play a crucial role in sustaining the functioning of the global water cycle (Griebler and Avramov 2015; Saccò et al. 2024). Consequently, the protection of groundwaters as ecosystems, which can be affected by overextraction, warming or pollution, is essential. Despite this, groundwater is largely absent from policy frameworks and overlooked by large initiatives such as the European Water Framework Directive (Di Lorenzo et al. 2024) that are designed to protect the environment (Fiser et al. 2022, 2025; Griebler et al. 2023). While the human impact on above-ground global biodiversity has triggered widespread conservation responses, subterranean ecosystems continue to suffer from a lack of visibility, research attention, and regulation (Saccò et. al., 2025a). Current global knowledge on groundwater biodiversity and ecological dynamics is far from complete, hindering effective assessment and conservation efforts (Mammola et al. 2024). Regrettably, groundwater species are facing escalating cumulative threats including climate and land use change, contamination, over-extraction, heating and salinization, amongst many others (Manenti et al. 2021; Becher et al. 2022; Couton et al. 2023a; Nanni et al. 2023; Vaccarelli et al. 2023). Left unaddressed, these threats compromise not only biodiversity but also the vast number of services that groundwater ecosystems unnoticeably provide to both nature and human society (Boulton et al. 2008; Griebler and Avramov 2015; Mammola et al. 2025; Saccò et al. 2025b).

The development of conservation strategies for groundwater and groundwater-dependent ecosystems (GDE, ecosystems that rely on groundwater for some or all of their water needs) requires an improved understanding of subterranean ecological dynamics, particularly under ongoing climatic changes (Boulton et al. 2023; Griebler et al. 2023; Vaccarelli et al. 2023). To address these challenges, increased collaborative efforts based on new technologies, scientific rigour, interdisciplinarity and inclusivity are required (Saccò et al. 2025b). In this regard, the analysis of environmental DNA (eDNA) is a technique proving to be an effective bridge connecting researchers,
stakeholders and citizens over a myriad of aquatic ecosystems (Deiner et al. 2017; Takahashi et al. 2023; Blackman et al. 2024). Broadly defined as the total pool of DNA isolated from environmental samples (Pawlowski et al. 2020), eDNA has been successful at unveiling biodiversity patterns from regional (Nakagawa et al. 2018) to global scales (Ficetola et al. 2024), and has been applied in taxonomy-free (Apothéloz-Perret-Gentil et al. 2017) and functional (Condachou et al. 2023; Cantera et al. 2025) approaches. Moreover, eDNA is being increasingly combined with other large-scale monitoring tools (e.g., satellite data, Zong et al. 2024) and broader biodiversity datasets (Abarenkov et al. 2023; Takahashi et al. 2025) to advance a rich, diverse research agenda. Concerning groundwater, eDNA research is gaining similar momentum (e.g., Koch et al. 2022; Saccò et al. 2022; Couton et al. 2023a, b; Al-Rashidi et al. 2025; Guzik et al. 2025a, b), despite a lag in protocol validation, application, and DNA-assay development compared to other biomes (e.g., oceans or rivers - Altermatt et al. 2025).

With the intention of disrupting the current inertia in the global conservation of groundwater ecosystems - where many calls for action have gone unheeded (e.g., Sánchez-Fernández et al. 2021; Wynnee et al. 2021; Di Lorenzo et al. 2024) - we present here a research initiative defined by the acronym of GReG: Global Research on eDNA in Groundwaters. The first pitch for this collaborative global eDNA study in groundwater ecosystems was proposed during the 26th International Conference on Subterranean Biology (ICSB) held in Sardinia in September 2024. Nine months later, on the 5th of June 2025, GReG was launched *via* an online seminar, led by Dr Mattia Saccò, Dr Michelle Guzik and Dr Kathryn Korbel. A gathering of 56 representative researchers and regulators across the globe took place, and guest speakers provided insights regarding global eDNA studies in oceans (Citizens of the Sea; Pochon et al. 2025), open and FAIR (Findable, Accessible, Interoperable and Reusable) data practices for sharing eDNA data (Takahashi et al. 2025), taxon-free eDNA approaches in groundwater studies, and how to utilize eDNA to address global biodiversity targets (Altermatt et al. 2025). The second part of the seminar was dedicated to the planning of a self-funded project in collaboration with partners such as Biodiversa+ projects (DarCO - Mammola et al. 2024; Sub-BioMon - Zagmajster et al. 2024), research laboratories (TrEnD Lab - https://research.curtin.edu.au/scieng/research/trend-lab/; Altermatt Lab - https://www.altermattlab.ch/) and global datasets (Stygofauna Mundi - Martínez et al. 2018).

Planned to be carried out in two phases, GReG is conceived as a unifying platform for researchers and diverse stakeholders from government and industry to meet and significantly advance groundwater science primarily through eDNA and genomics. Phase 1 (June 2025–June 2026) will involve fieldwork at over 50 well-known sites (karst, alluviums and anchialine caves) across four continents, using standardized sampling and analytical protocols targeting biodiversity across the entire Tree of Life. In addition, a survey on methodological approaches in groundwater eDNA research has been circulated to all the collaborators, aimed at understanding current practices and limitations for eDNA analysis in groundwater environments. Further projected research outputs include peer-reviewed papers on groundwater
eDNA protocols and publicly available DNA barcode quality assessments for stygofauna. Phase 2 (from July 2026) will build on the lessons learned from Phase 1 to expand the project to the Global South and other under-investigated areas of the world *via* participatory science approaches. This Forum Paper also serves as an opportunity to make a public call to the researchers and stakeholders from those regions, with an ambition of widening novel groundwater-based research beyond well-known global biodiversity hotspots. The outcomes derived from this collaborative effort will involve scientific publications, data sharing following the FAIR eDNA guidelines, workshops and media articles, with the objective to advance subterranean aquatic eDNA research. Last but not least, GReG also aims to raise awareness regarding the importance of groundwater ecosystems - a notoriously arduous task for an out of sight, out of mind environment (Boulton et al. 2023).

A shared vision amongst GReG collaborators is that the key to effectively preserving unique subterranean biota while using groundwater resources sustainably is not yet lost; we just need the right team of locksmiths (researchers, regulators, stakeholders, citizens) to come together and cut the right set of biomonitoring keys and conservation guidelines. GReG and its multidisciplinary approach provide the first step towards preserving subterranean biota at the global level, and thus utilising groundwater resources sustainably—an ambitious but vital goal for future water security.

## Acknowledgements

M.S., M.T., A.E., and M.B. acknowledges support from the School of Molecular and Life Sciences at Curtin University. We acknowledge support from Biodiversa+ (Projects “DarCo” and “Sub-BioMon”), the European Biodiversity Partnership under the 2021–2022 BiodivProtect joint call for research proposals, co-funded by the European Commission (GA N°101052342) and with the funding organisations Ministry of Universities and Research (Italy), Agencia Estatal de Investigación – Fundación Biodiversidad (Spain), Fundo Regional para a Ciência e Tecnologia (Azores, Portugal), Suomen Akatemia – Ministry of the Environment (Finland), Belgian Science Policy Office (Belgium), Agence Nationale de la Recherche (France), Deutsche Forschungsgemeinschaft e.V. (Germany), Bundesministerium für Forschung, Technologie und Raumfahrt (BMFTR, Germany), Schweizerischer Nationalfonds (Grant N° 31BD30_209583, Switzerland), Fonds zur Förderung der Wissenschaftlichen Forschung (Austria), Ministry of Higher Education, Science and Innovation (Slovenia), the Australian Research Council (Linkage grant N° LP190100555) and the Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania). Financial support from the Austrian Biodiversity Fonds (Biodiversitätsfonds BMLUK) to C.G. for the project Stygofauna Austria and Slovenian Research and Innovation Agency (P-0255 to N.M., D.S., and Ž.V.) is acknowledged. We further acknowledge financial support from the Joint Danube Survey 5 (JDS5) to C.G. supporting groundwater eDNA analyses.
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