Master thesis submitted in partial fulfilment of the requirements for the degree of Master in Biology Evolution and Behaviour Biology Toxin variation in Alpine salamander populations Gilles De Meester © Friedrich Böhringer Promotor Prof. Dr. Raoul Van Damme Faculty of Science Daily supervisor Department of Biology Emina Šunje Academic year 2016-2017 i Table of Contents Abstract .................................................................................................................................................. 1 Samenvatting .......................................................................................................................................... 1 Summary in layman’s terms ................................................................................................................... 2 1. Introduction ........................................................................................................................................ 4 1.1. Poisons of amphibians ................................................................................................................. 4 1.1.1. General ................................................................................................................................. 4 1.1.2. Functions of amphibian poisons ........................................................................................... 5 1.2. Variation in toxin composition .................................................................................................... 6 1.3. The alpine salamander (Salamandra atra) .................................................................................... 8 1.3.1. General description ............................................................................................................... 8 1.3.2. Samandarines........................................................................................................................ 9 1.4. Research questions .................................................................................................................... 11 2. Material and methods ....................................................................................................................... 13 2.1 Fieldwork ................................................................................................................................... 13 2.1.1. Locations ............................................................................................................................ 13 2.1.2. Toxin sampling ................................................................................................................... 15 2.1.3. Soil sampling ...................................................................................................................... 15 2.2. Lab work ................................................................................................................................... 16 2.2.1. Toxin analysis..................................................................................................................... 16 2.2.2. Test degradation of toxins .................................................................................................. 17 2.2.3. Cultivation of soil samples ................................................................................................. 17 2.2.4. Identification of soil fungi .................................................................................................. 18 2.2.5. Predation pressure .............................................................................................................. 20 2.3. Data analysis ............................................................................................................................. 21 2.3.1. Toxins ................................................................................................................................. 21 2.3.2. Test degradation of toxins .................................................................................................. 23 2.3.3. Morphology ........................................................................................................................ 23 2.3.4. Bacterial concentrations...................................................................................................... 23 2.3.5. Soil fungi ............................................................................................................................ 24 3. Results .............................................................................................................................................. 25 3.1. Toxins........................................................................................................................................ 25 3.1.1. Descriptive statistics ........................................................................................................... 25 3.1.2. Total amount of toxins ........................................................................................................ 25 3.1.3. Relative contributions ......................................................................................................... 27 i 3.2. Degradation of toxins ................................................................................................................ 32 3.3. Morphology ............................................................................................................................... 32 3.4. Bacterial densities...................................................................................................................... 36 3.5. Soil fungi ................................................................................................................................... 36 3.6. Predation pressure ..................................................................................................................... 39 4. Discussion ........................................................................................................................................ 41 4.1. Variation in toxin composition .................................................................................................. 41 4.2. Total amount of toxins and predation pressure .......................................................................... 43 4.3. Toxin variation and predation pressure ...................................................................................... 45 4.4. Toxin variation and infection risk .............................................................................................. 47 4.5. Differences between sexes......................................................................................................... 50 4.6. Peptides? ................................................................................................................................... 51 5. Conclusion ....................................................................................................................................... 51 6. Acknowledgements .......................................................................................................................... 52 7. References ........................................................................................................................................ 53 8. Appendix .......................................................................................................................................... 60 ii Abstract Amphibian poisons provide protection against predators and against infections by micro-organisms. It might be expected that toxin composition will be driven by adaptation to local predators and pathogens. Nevertheless, studies on geographic variation in toxins and its underlying causes are sparse. In this thesis, toxin variation and the possible reasons for this variation were studied in four populations of the alpine salamander (Salamandra atra) in the Dinaric Alps. The hypothesis was that salamanders would secrete higher amounts of toxins and more samandarine in populations with higher predation pressure. In populations with higher infection risk, higher concentrations of samandarone were expected. Toxin samples were collected in the field and analysed using Ultra Performance Liquid Chromatography – tandem Mass Spectrometry. Local predator communities were characterized from literature sources to estimate predation pressure. Soil samples were collected for 1) cultivation of soil bacteria and 2) DNA- sequencing of soil fungi. This way, the abundance and diversity of micro-organisms to which S. atra is exposed per population could be estimated. Considerable among-population variation in toxin composition was detected. Toxin variation did not match variation in predation pressure, although it was found that body size and poison gland size were larger in populations with snake predators. Toxins contained more samandarone in populations with higher infection risks, however, contributions of samandarone were far lower than expected from literature. Males and females did not differ in toxin composition. In conclusion, there was only limited support for the hypothesis that toxin variation is driven by variation in predation pressure and infection risk. This is one of few studies to look at the underlying reasons for toxin variation, and the first one to incorporate the effect of microbial variation in the environment. Keywords: amphibian toxins, geographic variation, samandarines, predation