Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi‐aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water‐borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus‐specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.

[1]  Arthur J. Sementelli,et al.  Reptilian Pathogens of the Florida Everglades: The Associated Costs of Burmese Pythons , 2007 .

[2]  François Pompanon,et al.  Persistence of Environmental DNA in Freshwater Ecosystems , 2011, PloS one.

[3]  B. Deagle,et al.  Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces , 2006, Frontiers in Zoology.

[4]  P Taberlet,et al.  Reliable genotyping of samples with very low DNA quantities using PCR. , 1996, Nucleic acids research.

[5]  W. L. Chadderton,et al.  “Sight‐unseen” detection of rare aquatic species using environmental DNA , 2011 .

[6]  S. Secor,et al.  Environmental temperatures, physiology and behavior limit the range expansion of invasive Burmese pythons in southeastern USA. , 2012, Integrative zoology.

[7]  Christina M. Romagosa,et al.  Severe mammal declines coincide with proliferation of invasive Burmese pythons in Everglades National Park , 2012, Proceedings of the National Academy of Sciences.

[8]  Robert S. Arkle,et al.  Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed Frogs and Idaho Giant Salamanders , 2011, PloS one.

[9]  David L. Wheeler,et al.  GenBank , 2015, Nucleic Acids Res..

[10]  W. P. Beach.  FOR THE SOUTH FLORIDA WATER MANAGEMENT DISTRICT , 2008 .

[11]  D. Rabosky,et al.  Python phylogenetics: inference from morphology and mitochondrial DNA , 2008 .

[12]  John A Darling,et al.  From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. , 2011, Environmental research.

[13]  W. E. Meshaka,et al.  An annotated inventory of the herpetofauna of Everglades National Park, Florida , 2000 .

[14]  W. E. Meshaka,et al.  A runaway train in the making: the exotic amphibians, reptiles, turtles, and crocodilians of Florida. , 2011 .

[15]  M. Thomas P. Gilbert,et al.  Investigating the Potential Use of Environmental DNA (eDNA) for Genetic Monitoring of Marine Mammals , 2012, PloS one.

[16]  C. Wiuf,et al.  Monitoring endangered freshwater biodiversity using environmental DNA. , 2012, Molecular ecology.

[17]  Gordon H. Rodda,et al.  What parts of the US mainland are climatically suitable for invasive alien pythons spreading from Everglades National Park? , 2009, Biological Invasions.

[18]  P. Taberlet,et al.  Species detection using environmental DNA from water samples , 2008, Biology Letters.

[19]  R. A. Pyron,et al.  Claims of Potential Expansion throughout the U.S. by Invasive Python Species Are Contradicted by Ecological Niche Models , 2008, PloS one.

[20]  Eske Willerslev,et al.  Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples , 2012, PloS one.

[21]  P. Taberlet,et al.  Urine collected in the field as a source of DNA for species and individual identification , 2000, Molecular ecology.

[22]  R. Snow,et al.  Dietary habits of the Burmese python, Python molurus bivittatus, in Everglades National Park, Florida , 2007 .

[23]  P. Shaw,et al.  Application of cytochrome b DNA sequences for the authentication of endangered snake species. , 2004, Forensic science international.

[24]  W. Böhme,et al.  Zur Taxonomie des Dunklen Tigerpythons, Python molurus bivittatus KUHL, 1820, speziell der Population von Sulawesi , 2009 .

[25]  Richard M. Engeman,et al.  The aggressive invasion of exotic reptiles in Florida with a focus on prominent species: A review , 2011 .