Environmental DNA and Karst Systems: Advancing Groundwater Tracing, Aquifer Connectivity, and Ecosystem Research – Mike Buchanan 2025

Abstract

Karst systems are complex dynamic environments that present significant challenges to hydrological and ecological research. Traditional methods, such as dye tracing, provide limited spatial and temporal resolution in groundwater studies. Recent advances in environmental DNA (eDNA) analysis have opened new frontiers, offering powerful, non-invasive tools for exploring species distribution, ecosystem functioning, groundwater flow, and aquifer connectivity. This paper synthesises current knowledge on the use of eDNA in karstological research and groundwater tracing, highlighting its advantages, limitations, and future research needs. By integrating both ecological and hydrological applications, this review demonstrates the transformative potential of eDNA in advancing karst science.

Introduction

Karst systems represent some of the most intricate and least accessible carbonate landscapes, shaped by the dissolution of soluble rocks such as limestone and dolomite. They host diverse ecosystems and provide critical groundwater resources for human consumption, agriculture and biodiversity conservation. Understanding groundwater flow, aquifer connectivity, and ecosystem dynamics in karst systems is vital, yet traditional methodologies, including dye tracing and direct biological surveys, remain constrained by scale, invasiveness, and limited temporal coverage (Goldscheider & Drew, 2007).

Environmental DNA (eDNA) has emerged as a promising alternative. eDNA refers to genetic material shed by organisms into their environment through skin cells, faeces, mucous, or other biological processes (Taberlet et al., 2012). Its application in karst research allows for sensitive, non-invasive detection of species and provides new opportunities for hydrological tracing and ecological monitoring. This paper integrates insights from groundwater tracing and karstological studies to evaluate the potential of eDNA for advancing knowledge on karst ecosystems.

The Use of eDNA in Karst Systems

eDNA offers unique opportunities for research in karst environments due to its versatility across multiple scientific domains. Applications include:

  • Species detection and identification: eDNA enables the detection of rare, cryptic, or inaccessible organisms, providing an advantage over conventional survey techniques (Deiner et al., 2017).
  • Mapping species distribution: Through systematic sampling, eDNA allows the creation of detailed species distribution maps across inaccessible subterranean environments.
  • Studying ecosystem functioning: eDNA metabarcoding can reveal information on community composition, nutrient cycling, and decomposition processes, offering insights into karst ecosystem dynamics.
  • Groundwater tracing and aquifer connectivity: eDNA can act as a natural tracer, revealing the movement of biological material within aquifers, thus complementing or surpassing dye tracing in certain contexts.

Advantages of eDNA Approaches

The adoption of eDNA in karst and groundwater studies offers several benefits:

  • Non-invasive sampling: Unlike physical surveys, eDNA sampling requires minimal disturbance to ecosystems.
  • High sensitivity and specificity: eDNA techniques can detect very small amounts of DNA, enabling identification of rare or low-abundance species.
  • Cost-effectiveness: Compared to long-term monitoring or invasive sampling campaigns, eDNA offers a relatively low-cost approach to biodiversity and hydrological assessments (Thomsen & Willerslev, 2015).
  • Multidisciplinary applications: eDNA provides a single platform for both ecological and hydrological investigations, enhancing its research value.

Limitations and Challenges

Despite its promise, the use of eDNA in karst and aquifer studies faces several challenges:

  • Uncertainty in eDNA dynamics: The processes governing DNA shedding, transport, and degradation in groundwater remain poorly understood (Barnes & Turner, 2016).
  • False positives and negatives: Contamination, persistence of legacy DNA, and detection thresholds can complicate interpretation.
  • Standardisation issues: Lack of universal protocols for eDNA sampling and analysis limits comparability across studies and ecosystems.

 
Case Studies and Applications

Several studies have demonstrated the utility of eDNA in karst and aquifer research:

  • Species detection in groundwater: eDNA has been employed to reveal subterranean biodiversity, highlighting previously undocumented species presence in groundwater habitats.
  • Tracing groundwater flow: Research has shown that eDNA can trace biological material through karst conduits, offering insights into flow dynamics and aquifer connectivity. These applications demonstrate the potential of eDNA to serve as both an ecological and hydrological tracer.

Future Directions

For eDNA to reach its full potential in karstological and groundwater studies, future research should prioritise:

  1. Improving understanding of eDNA dynamics – Controlled laboratory and field experiments are needed to quantify shedding, persistence, and transport in karst groundwater.
  2. Developing standardised protocols – Harmonisation of methodologies will improve reproducibility and comparability across studies.
  3. Integration with other methods – Combining eDNA with traditional techniques, such as dye tracing, geophysics, and hydrochemistry, can provide a holistic understanding of karst processes.
  4. Expanding ecological applications – Beyond hydrology, eDNA could offer insights into ecosystem resilience, responses to climate change, and conservation planning in karst environments.

Conclusion

Environmental DNA has emerged as a transformative tool in karst research, bridging hydrology and ecology. Its application in groundwater tracing and aquifer connectivity complements and extends traditional methods, while its capacity for species detection and ecosystem analysis enriches understanding of subterranean biodiversity. Despite limitations, continued methodological refinement and interdisciplinary integration will cement eDNA as a cornerstone of future karst science.

References

Barnes, M.A. & Turner, C.R. (2016) The ecology of environmental DNA and implications for conservation genetics. Conservation Genetics, 17(1), 1–17.


Deiner, K., Bik, H.M., Mächler, E., Seymour, M., Lacoursière-Roussel, A., Altermatt, F., Creer, S., Bista, I., Lodge, D.M., de Vere, N., Pfrender, M.E. & Bernatchez, L. (2017) Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26(21), 5872–5895.


Goldscheider, N. & Drew, D. (2007) Methods in Karst Hydrogeology. London: Taylor & Francis.


Taberlet, P., Coissac, E., Hajibabaei, M. & Rieseberg, L.H. (2012) Environmental DNA. Molecular Ecology, 21(8), 1789–1793.


Thomsen, P.F. & Willerslev, E. (2015) Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation, 183, 4–18.

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