CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Wildlife Research   
Wildlife Research
Journal Banner
  Ecology, management and conservation in natural and modified habitats
blank image Search
blank image blank image
blank image
  Advanced Search

Journal Home
About the Journal
Editorial Structure
Online Early
Current Issue
Just Accepted
All Issues
Special Issues
Sample Issue
For Authors
General Information
Submit Article
Author Instructions
Open Access
For Referees
Referee Guidelines
Review an Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates
Library Recommendation

blue arrow e-Alerts
blank image
Subscribe to our Email Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter logo LinkedIn

red arrow Submit Article
blank image
Use the online submission system to send us your paper.

red arrow CSIRO Wildlife Research
blank image
All volumes of CSIRO Wildlife Research are online and available to subscribers of Wildlife Research.


Article << Previous     |     Next >>   Contents Vol 39(7)

An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water

Zachary H. Olson A C, Jeffrey T. Briggler B and Rod N. Williams A

A Department of Forestry & Natural Resources, Purdue University, West Lafayette, IN 47907, USA.
B Missouri Department of Conservation, PO Box 180, Jefferson City, MO 65102, USA.
C Corresponding author. Email: olson.z.h@gmail.com

Wildlife Research 39(7) 629-636 http://dx.doi.org/10.1071/WR12114
Submitted: 26 June 2012  Accepted: 15 August 2012   Published: 17 September 2012

PDF (197 KB) $25
 Export Citation

Context: Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa.

Aims: We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments.

Methods: We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis.

Key results: Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control.

Conclusion: eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders.

Implications: eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.

Additional keywords: density, detection, DNA-based, monitoring, non-invasive, occupancy, presence.


Beja-Pereira, A., Oliveira, R., Alves, P. C., Schwartz, M. K., and Luikart, G. (2009). Advancing ecological understandings through technological transformations in noninvasive genetics. Molecular Ecology Resources 9, 1279–1301.
CrossRef |

Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., Slocombe, R., Ragan, M. A., Hyati, A. D., McDonald, K. R., Hines, H. B., Lips, K. R., Marantelli, G., and Parkes, H. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and central America. Proceedings of the National Academy of Sciences, USA 95, 9031–9036.
CrossRef | CAS |

Birky, C. W., Fuerst, P., and Maruyama, T. (1989). Organelle gene diversity under migration, mutation, and drift: equilibrium expectations, approach to equilibrium, effects of heteroplasmic cells, and comparison to nuclear genes. Genetics 121, 613–627.

Blaustein, A. R., Wake, D. B., and Sousa, W. P. (1994). Amphibian declines: judging stability, persistence, and susceptibility of populations to local and global extinctions. Conservation Biology 8, 60–71.
CrossRef |

Browne, R. K., Li, H., Mcginnity, D., Okada, S., Zhenghuan, W., Bodinof, C. M., Irwin, K. J., McMillan, A., and Briggler, J. T. (2011). Survey techniques for giant salamanders and other aquatic Caudata. Amphibian & Reptile Conservation 5, 1–16.

Burgmeier, N. G., Sutton, T. M., and Williams, R. N. (2011a). Spatial ecology of the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in Indiana. Herpetologica 67, 135–145.
CrossRef |

Burgmeier, N. G., Unger, S. D., Sutton, T. M., and Williams, R. N. (2011b). Population status of the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in Indiana. Journal of Herpetology 45, 195–201.
CrossRef |

Constable, J. L., Ashley, M. V., Goodall, J., and Pusey, A. E. (2001). Noninvasive paternity assignment in Gombe chimpanzees. Molecular Ecology 10, 1279–1300.
CrossRef | CAS |

Darling, J. A., and Mahon, A. R. (2011). From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. Environmental Research 111, 978–988.
CrossRef | CAS |

Deagle, B. E., Eveson, J. P., and Jarman, S. N. (2006). Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces. Frontiers in Zoology 3, 11.
CrossRef |

Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., and Miaud, C. (2011). Persistence of environmental DNA in freshwater ecosystems. PLoS ONE 6, e23398.
CrossRef | CAS |

Epps, C. W., Palsbøll, P. J., Weyhausen, J. D., Roderick, G. K., Ramey, R. R., and McCullough, D. R. (2005). Highways block gene flow and cause a rapid decline in genetic diversity of desert bighorn sheep. Ecology Letters 8, 1029–1038.
CrossRef |

Ficetola, G. F., Miaud, C., Pompanon, F., and Taberlet, P. (2008). Species detection using environmental DNA from water samples. Biology Letters 4, 423–425.
CrossRef |

Goldberg, C. S., Pilliod, D. S., Arkle, R. S., and Waits, L. P. (2011). Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders. PLoS ONE 6, e22746.
CrossRef | CAS |

Hausknecht, R., Gula, R., Pirga, B., and Kuehn, R. (2007). Urine – a source for noninvasive genetic monitoring in wildlife. Molecular Ecology Notes 7, 208–212.
CrossRef | CAS |

Howard, A. (1994). A detachment-limited model of drainage basin evolution. Water Resources Research 30, 2261–2285.
CrossRef |

Humphries, W. J., and Pauley, T. K. (2005). Life history of the hellbender, Cryptobranchus alleganiensis, in a West Virginia stream. American Midland Naturalist 154, 135–142.
CrossRef |

IUCN, Conservation International, and NatureServe (2008). ‘An Analysis of Amphibians on the 2008 IUCN Red List.’ Available at http://www.iucnredlist.org/amphibians [verified 30 May 2012].

Jerde, C. L., Mahon, A. R., Chadderton, W. L., and Lodge, D. M. (2011). ‘Sight-unseen’ detection of rare aquatic species using environmental DNA. Conservation Letters 4, 150–157.
CrossRef |

Kendall, K. C., Stetz, J. B., Roon, D. A., Waits, L. P., Boulanger, J. B., and Paetkau, D. (2008). Grizzly bear density in Glacier National Park, Montana. The Journal of Wildlife Management 72, 1693–1705.
CrossRef |

Kern, W. H. (1984). The hellbender, C. alleganiensis in Indiana. M.Sc. Thesis, Indiana State University, Terre Haute, IN.

Lips, K. R., Brem, F., Brenes, R., Reeve, J. D., Alford, R. A., Voyles, J., Carey, C., Livo, L., Pessier, A. P., and Collins, J. P. (2006). Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proceedings of the National Academy of Sciences, USA 103, 3165–3170.
CrossRef | CAS |

Marucco, F., Pletscher, D. H., Boitani, L., Schwartz, M. K., Pilgrim, K. L., and Lebreton, J.-D. (2009). Wolf survival and population trend using non-invasive capture-recapture techniques in the western Alps. Journal of Applied Ecology 46, 1003–1010.
CrossRef |

Minton, S. A., Jr (2001). ‘Amphibians and Reptiles of Indiana.’ (Indiana Academy of Science: Indianapolis, IN.)

Nickerson, M. A., and Briggler, J. T. (2007). Harvesting as a factor in population decline of a long-lived salamander; the Ozark hellbender, Cryptobranchus alleganiensis bishopi Grobman. Applied Herpetology 4, 207–216.
CrossRef |

Nickerson, M. A., and Mays, C. E. (1973). A study of the Ozark hellbender Cryptobranchus alleganiensis bishopi. Ecology 54, 1164–1165.
CrossRef |

Olson, Z. H., Burgmeier, N. G., Zollner, P. A., and Williams, R. N. (2013). Survival estimates for adult eastern hellbenders and their utility for conservation. Journal of Herpetology 47, .

Phillips, C. A., and Humphries, W. J. (2005). Cryptobranchus alleganiensis Daudin, 1803. In ‘Amphibian Declines: the Conservation Status of United States Species’. (Ed. M. Lannoo.) pp. 648–651. (University of California Press: Los Angeles, CA.)

Pietramellara, G., Ascher, J., Borgogni, F., Ceccherini, M. T., Guerri, G., and Nannipieri, P. (2009). Extracellular DNA in soil and sediment: fate and ecological relevance. Biology and Fertility of Soils 45, 219–235.
CrossRef | CAS |

Putman, R. J. (1995). Ethical considerations and animal welfare in ecological field studies. Biodiversity and Conservation 4, 903–915.
CrossRef |

Rosen, S., and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. In ‘Bioinformatics Methods and Protocols: Methods in Molecular Biology’. (Eds S. Misener and S. A. Krawetz.) pp. 365–386. (Humana Press Inc.: Totowa, NJ.)

Rudnick, J. A., Katzner, T. E., Bragin, E. A., Rhodes, O. E., and DeWoody, J. A. (2005). Using naturally shed feathers for individual identification, genetic parentage analyses, and population monitoring in an endangered Eastern imperial eagle (Aquila heliaca) population from Kazakhstan. Molecular Ecology 14, 2959–2967.
CrossRef | CAS |

Sabatino, S. J., and Routman, E. J. (2009). Phylogeography and conservation genetics of the hellbender salamander (Cryptobranchus alleganiensis). Conservation Genetics 10, 1235–1246.
CrossRef |

Sastre, N., Francino, O., Lampreave, G., Bologov, V. V., López-Martín, J. M., Sánchez, A., and Ramírez, O. (2009). Sex identification of wolf (Canis lupus) using non-invasive samples. Conservation Genetics 10, 555–558.
CrossRef | CAS |

Sawaya, M. A., Stetz, J. B., Clevenger, A. P., Gibeau, M. L., and Kalinowski, S. T. (2012). Estimating grizzly and black bear population abundance and trend in Banff National Park using noninvasive genetic sampling. PLoS ONE 7, e34777.
CrossRef | CAS |

Smyser, T. J., Beasley, J. C., Olson, Z. H., and Rhodes, O. E. (2010). Use of rhodamine b to reveal patterns of interspecific competition and bait acceptance in raccoons. The Journal of Wildlife Management 74, 1405–1416.

Solberg, K. H., Bellemain, E., Drageset, O.-M., Taberlet, P., and Swenson, J. E. (2006). An evaluation of field and non-invasive genetic methods to estimate brown bear (Ursus arctos) population size. Biological Conservation 128, 158–168.
CrossRef |

Taberlet, P., and Luikart, G. (1999). Non-invasive genetic sampling and individual identification. Biological Journal of the Linnean Society. Linnean Society of London 68, 41–55.
CrossRef |

Taberlet, P., Griffin, S., Goossens, B., Questiau, S., Manceau, V., Escaravage, N., Waits, L. P., and Bouvet, J. (1996). Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Research 24, 3189–3194.
CrossRef | CAS |

Taberlet, P., Prud’homme, S. M., Campione, E., Roy, J., Miquel, C., Shehzad, W., Gielly, L., Rioux, D., Choler, P., Clément, J.-C., Melodelima, C., Pompanon, F., and Coissac, E. (2012). Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies. Molecular Ecology 21, 1816–1820.
CrossRef | CAS |

Thomsen, P. F., Kielgast, J., Iversen, L. L., Wiuf, C., Rasmussen, M., Gilbert, M. T. P., Orlando, L., and Willerslev, E. (2012). Monitoring endangered freshwater biodiversity using environmental DNA. Molecular Ecology 21, 2565–2573.
CrossRef | CAS |

Unger, S. D., Fike, J. A., Sutton, T., Rhodes, O. E., and Williams, R. N. (2010). Isolation and development of 12 polymorphic tetranucleotide microsatellite markers for the eastern hellbender (Cryptobranchus alleganiensis alleganiensis). Conservation Genetics Resources 2, 89–91.
CrossRef |

Valière, N., Fumagalli, L., Gielly, L., Miquel, C., Lequette, B., Poulle, M.-L., Weber, J.-M., Arlettaz, R., and Taberlet, P. (2003). Long-distance wolf recolonization of France and Switzerland inferred from non-invasive genetic sampling over a period of 10 years. Animal Conservation 6, 83–92.
CrossRef |

Valsecchi, E., Glockner-Ferrari, D., Ferrari, M., and Amos, W. (1998). Molecular analysis of the efficiency of sloughed skin sampling in whale population genetics. Molecular Ecology 7, 1419–1422.
CrossRef | CAS |

Waits, L. P. (2004). Using noninvasive genetic sampling to detect and estimate abundance of rare wildlife species. In ‘Sampling Rare or Elusive Species: Concepts, Designs, and Techniques for Estimating Population Parameters’. (Ed. W. L. Thompson.) pp. 211–228. (Island Press: Washington, DC.)

Waits, L. P., and Paetkau, D. (2005). Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. The Journal of Wildlife Management 69, 1419–1433.
CrossRef |

Welsh, H. H., and Ollivier, L. M. (1998). Stream amphibians as indicators of ecosystem stress: a case study from California’s redwoods. Ecological Applications 8, 1118–1132.

Wheeler, B. A., Prosen, E., Mathis, A., and Wilkinson, R. F. (2003). Population declines of a long-lived salamander : a 20+-year study of hellbenders, Cryptobranchus alleganiensis. Biological Conservation 109, 151–156.
CrossRef |

Williams, R. D., Gates, J. E., Hocutt, C. H., and Taylor, G. J. (1981). The hellbender: a nongame species in need of management. Wildlife Society Bulletin 9, 94–100.

Subscriber Login

Legal & Privacy | Contact Us | Help


© CSIRO 1996-2016