Gretchen A. Hilt
Southeastern Louisiana University
November 2024
Background: The Georgia Blind Cave Salamander (Eurycea wallacei), is a completely aquatic, paedomorphic salamander endemic to the Floridan Aquifer System in Northwestern Florida and Southwestern Georgia. Initially described in 1939 by Archie Carr (Carr 1939), this salamander is quite unique compared to its counterparts. A cave-adapted species, this salamander has many stygobitic features that allow it to thrive in karst environments: reduced eyes, little to no pigmentation, and an enhanced lateral line system (Fenolio et al. 2013). The caves E. wallacei inhabit are typically spring-fed, high flow, submerged karst caves – with the occasional sighting in arid cave pools. Threats to this species are confounding, including habitat degradation, agricultural/industrial runoff influence, water level fluctuation, and more.
Little is known about the true distribution, life history, and ecology of E. wallacei. To make matters more complicated, sampling efforts to allow federal/state listing of the species are difficult to complete due to the complex and dangerous habitat of the karst cave systems. Historical surveys of this species are complex, dangerous, cost and time inefficient. Highly certified cave divers must enter these habitats to conduct surveys – and that’s if they’re lucky to see a few.
How can we monitor the presence, distribution, and ecological status of the Georgia Blind Cave Salamander? The solution: environmental DNA (eDNA). eDNA is any DNA shed by an organism in their surrounding environment through feces, skin shed, mucus secretion, etc. (Pilliod et al. 2014). These salamanders secrete mucus, release waste products, and shed skin cells – which collectively contribute to the presence of DNA in their surrounding aquatic environment.
Two main goals of this research include:
- Detect Eurycea wallacei in submerged cave systems using both active and passive eDNA techniques.
- Categorize a baseline metazoan framework of the cave system to get a better idea of the salamander’s ecological niche in this complex, understudied system.
Methods: Initial samples from a captive colony of E. wallacei were tested to confirm the validity of the metazoan primers in detecting salamander presence. I am happy to report that preliminary testing confirmed the presence of E. wallacei in captive colony samples. Though these preliminary tests were conducted using samples from a closed aquatic system, this was a positive sign that the lab protocol was effective in detecting the salamander in aquatic environments.
As previously mentioned, two types of eDNA data collection were performed. Active sampling refers to taking 1500mL samples of water and directly filtering the water using a Smith Root eDNA Backpack Filtration System. On the other hand, passive sampling is utilized by securing the same filters used in the active sampling in a submersible structure that is fixed to the cave system. Both types of samples were taken at three locations within each study site: surface, cavern (directly below surface, entrance to cave), and cave (300 ft. into cave system or at cave entrance sign).
Once all water samples were collected, they were processed in the lab for DNA sequencing. The DNA must first be extracted and separated from the glass microfiber filters. Then, these samples were subjected to PCR (polymerase chain reaction) in two rounds. The first round creates a substantial amount of DNA copies with metazoan specific primers (Leray et al. 2013). The second round adds a barcode sequence unique to each sample for ease of identification in the sequencing process. Samples were then sent off site for sequencing. Once the samples were sequenced, several steps of sequence cleaning, filtering, and processing were conducted using Python and other bioinformatic tools. Final taxonomic assignments and analyses are currently in progress.
Current and Future Work: Though I held PADI Divemaster status at the initiation of this research, highly specific safety protocols and training were required in order to conduct this work in these dangerous, unique karst caves. Funding from The Orianne Society’s Small Grant Program allowed me to get the necessary cave diving certifications and safety gear required for accessing these sites. I have successfully completed all required training and will be wrapping up the cave portion of field processing before the end of 2024.
There is still significant data processing and final data collection to be completed for this project, but I am elated to apply the preliminary study successes I have completed to the final field samples I have yet to collect. This study is a novel approach targeting this imperiled species in a contained environment, which could apply to many other stygobitic species detection assays upon its success. Results from this study show promise to optimize a sampling approach for future, long-term monitoring of Eurycea wallacei, as well as contribute to future species status assessments and protections.
Funding and support for this project was partially provided by The Orianne Society, Southeastern Louisiana University, Women Divers Hall of Fame, National Speleological Society, Association of Reptile and Amphibian Veterinarians, Beneath the Sea, Karst Waters Institute, American Society of Ichthyologists and Herpetologists, American Museum of Natural History, Cave Research Foundation, Editing Press, Friends of Sunset Zoo, the Cave Conservancy Foundation, and Adventure Outfitters Dive Center.
Literature cited
Carr, A. F. Jr. (1939). Haideotriton wallacei, a new subterranean salamander from Georgia. Occasional papers of the Boston Society of Natural History, 8, 333-336.
Fenolio, D., Niemiller, M., Levy, M., & Martinez, B. (2013). Conservation Status of the Georgia Blind Salamander (Eurycea wallacei) from the Floridan Aquifer of Florida and Georgia. Reptiles and Amphibians, 20, 97–111.
Leray, M., Yang, J. Y., Meyer, C. P., Mills, S. C., Agudelo, N., Ranwez, V., Boehm, J. T., & Machida, R. J. (2013). A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: Application for characterizing coral reef fish gut contents. Frontiers in Zoology, 10, 34.
Pilliod, D. S., Goldberg, C. S., Arkle, R. S., & Waits, L. P. (2014). Factors influencing detection of eDNA from a stream-dwelling amphibian. Molecular Ecology Resources, 14(1), 109–116.