1. WYOMING EPSCOR PROGRAM FACTORS AFFECTING PROBABILITY OF AMPHIBIAN OCCURRENCE ON POLE MOUNTAIN: EVALUATING WATER QUALITY, DISEASE AND PREDATION Adrienne Elise Sulser Department of Ecosystem Science and Management with Dr. Melanie Murphy and Charlotte Gabrielsen

2. BACKGROUND Why do we care about amphibians? Freshwater ecosystem biodiversity decline Worldwide amphibian decline Indicator species Major Stressors: habitat loss, disease and invasive predators

3. AMPHIBIAN SPECIES ON POLE MOUNTAIN Tiger salamanders (Ambystoma mavortium) Leopard Frogs (Lithobates pipiens) Chorus Frogs (Pseudacris maculata)

4. SAMPLE SITES Samples from 60 sites were surveyed 4 times over the 2013 field season by Vicky Zero. Data collected Occurrence Visual eDNA Habitat distribution Vicky Zero

5. FACTORS Affecting Amphibians On pole mountain: Water Quality Disease Predation

6. ENVIRONMENTAL FACTOR: WATER QUALITY E. Coli is often used as an indicator of water quality due to feces contamination. Large permanent and ponds differ from seasonal/emergent wetlands.

7. AMPHIBIAN PRESENCE AND WATER QUALITY -Theoretical predictions -Dual hypothesis

8. ENVIRONMENTAL FACTOR: BATRACHOCHYTRIUM DENDROBATIDIS (BD) FUNGUS BD fungus causes Chytridomycosis disease in amphibian Contributing to a drastic decline in amphibians world wide

9. AMPHIBIAN PRESENCE AND DISEASE

10. ENVIRONMENTAL FACTOR: PREDATION Brook Trout pose as predators Stocked and non-native Prey on larval stages

11. AMPHIBIAN PRESENCE AND PREDATION BY BROOK TROUT

12. ENVIRONMENTAL DNA Nuclear and mitochondrial DNA releases through mucous, feces ect. Highly effective for detecting the presence of species in aquatic environments Quickly degraded in the environment within 7 days

13. PCR Polymerase Chain reaction used to amplify DNA Real time PCR is more accurate SYBER green technology. Primers are species specific

14. E. COLI PCR Standards from the lab Field samples Negative control Cycles (time) 75x 50x 20x 4x

15. BD PCR Cycles (time) Standards from the lab, Field samples, Negative control

16. BROOK TROUT PCR Cycles 6 trout species from Wyoming Game and Fish: Rainbow, Brown, Golden Yellowstone, Snake river and cutthroat -Brook Trout -Other Trout

17. MELT CURVES VS. BD melt curve; Highly Specific E. Coli Melt Curve Helps to determine whether primer is sequence specific

18. NEXT STEPS Add fluorescent probes to get a more qualitative measure of each factor Put all the pieces together Collect samples for the 2015 season Compare Seasons

19. ACKNOWLEDGEMENTS A huge Thank you to Murphy labs in Ecosystem Management for the support and advising! Dr. Melanie Murphy Charlotte Gabrielsen