Unravelling an enigma: Ecology of waxcaps (Hygrocybe: Agaricomycetes) Master in Science Thesis Patricia A. Silva-Flores Munich Graduate Program for Evolution, Ecology and Systematics Ludwig-Maximilians-Universität München Supervisor Prof. Dr. Reinhard Agerer Ludwig-Maximilians-Universität München Department Biology I and GeoBio-Center Organismic Biology: Mycology September 2011 dedicated to Horacio Acknowledgements I would like to thanks to my supervisor Prof. Dr. Agerer, for showing me the amazing and wonderful world of fungi and for teaching me about it with much enthusiasm. Thanks also for accepting me in his research group, for guiding my work so carefully and patiently, for funding this Master Thesis and for teaching me to be patience and dedicated with my work. I am deeply grateful with many people who helped me in the development of this thesis, without their help this thesis would not have been possible: Dr. Derek Persoh who kindly guide me in the molecular work of this thesis and also contribute with many ideas, suggestions, methods and specially the design of primers. Dr. Arthur Schüssler and his PhD student Matthias Ellerbeck, they provide all the primary material for the arbuscular mycorrhiza cultures and were very kind in showing me the methods, borrowing literature and answer all the questions I made. Mr. Hans Halbwachs, who cordially received me for the samples collection, shared his knowledge of waxcaps, shared literature and waxcaps spores. Sofia Tello, a Master Student who performed the “Plant-AM” experiment of this thesis as a part of her individual research training, she spent many days (and nights) in the lab working and sharing with me her happiness and enthusiasm during long work mornings and evenings. Dr Christian Mayer for performing the isotopic analysis of arbuscular mycorrhiza spores. Sara Troxell, also a Master Student who performed the patient task of collecting spores of arbuscular mycorrhiza for the quantification of isotopic signatures as an individual research training, she also spent many days (and nights) in the lab working and sharing her lovely enthusiasm when she looked at the spores. Claudia Bubenzer, for being always available for me for any question that I could have, always helping me and showing me were was all the material to work. Anina Neumann, for her kindly and patient training in the molecular lab, and also for being always available for any question that I could have. Sebastian Gardt, for being always available for any question and always with a smile. To the actual and former members of the mycology group, Rita Verma, Dr. Philomena Bodensteiner, Dr. Norou Yorou and Dr. Yie Wei for make me feel very cozy in the lab sharing a meal, a talk or a smile. Also special thanks to Dr. Rosemarie Weigt, for the kindly correction of this manuscript and nice lunches with nice talks. To the biology bachelors Rebecca Rogers, Dagmar Judenmann and Jennifer Diehm, for the nice and fun time working in parallel in the lab. Dr. Martina Silber for helping me with methods and logistic in the molecular work. Special thanks to my Yogui Jane, for her love, peace and happiness. Infinite thanks also to Mónica Arias, Adriana Dorado, Hsuan Ju Chen and Fernanda Antunez for her friendship, laughs, meals and love. Thanks to Gigi, Francisco, Andrés, Sinem, Yusuf, Jeaninne and Amanda for the fun in the classroom and outside as well. Deeply thankful of Catalina, Ricardo, Pablo, Meike and Francesco for being such cool friends and being always there supporting me. Without all of them being so far from home and write this thesis would not have been possible. I also want to thanks to my biggest fans: my mom, my dad, my sister, my nephew, my grandmothers and my Paulina for always being there, for loving me and for supporting me in this quest from so far and being always so close at the same time. Finally I want to thank to my Gonzalo which always support me no matter what and care about me and mostly for loving me unconditionally, you are my inspiration. Abstract Waxcaps are the most distinctive and visible components of grassland funga, however many aspects of their ecology are poorly understood. Indirect evidence of the trophic status of waxcaps has being obtained with the use of stable isotopes pointing out waxcaps to be in a biotrophic relation with an unknown partner. In the present study it was aimed to find first hints of waxcaps being in a biotrophic relation with four possible general partners: bryophytes, roots of grasses and herbs, arbuscular mycorrhizal (AM) fungi and moulds. In order to do so, roots of herbs and grasses, AM fungi (mycelia and spores), bryophyte structures, and isolated moulds were studied with molecular, anatomical and experimental approaches. From the analysis of a possible relation of waxcaps with roots and bryophytes, specific primers for the study of Hygrocybe virginea, Hygrocybe pratensis and Hygrocybe coccinea were designed, no DNA or hyphae of waxcaps was found in relation with the moss Rhytidiadelphus squarrosus or with roots of grasses and herbs, no evident germination of spores of waxcaps was detected within an AM-Plant symbiotic system. From the analysis of a possible relation of AM fungi with waxcaps, the isotopic signature of nitrogen (δ15N = 1.96 ‰) and carbon (δ13C = -28.04 ‰) of AM fungi were obtained and cultures of AM fungi in relation with waxcaps spores were made with no evident germination. Also a fungal infection of whitish, sepate and no clamped hyphae was found inside of AM spores. This fungi was not identified, however here is hypothesised that this infection may come from Hygrocybe or from Microdochium sp, which is a mould isolated in this study from fruit bodies of H. coccinea and posteriorly was used to test a possible relation with waxcaps placing spores of H. virginea and H. coccinea with the mould, however no germination occurred. Although no hyphae or DNA of waxcaps was found in the suggested partners, the hypothesis of Hygrocybe as a biotrophic fungi cannot be rejected, as there were still many methodological constrains which should be considered in future. Improvements in the sampling design as well as in the time span of the experiments and studies of the fungal infection of spores of AM fungi are urgently needed in order to clearly help in solving the enigma of the ecology of waxcaps. Table of contents List of figures....................................................................................................................................... 1 List of tables......................................................................................................................................... 2 1. Introduction...................................................................................................................................... 3 1.1 The genus Hygrocybe................................................................................................................. 3 1.2 Hygrocybe ecology..................................................................................................................... 3 1.3 Objectives of the study............................................................................................................... 6 2. Materials and methods......................................................................................................................7 2.1 Collection and isolation of samples............................................................................................7 2.2 Primer design..............................................................................................................................9 2.3 Analysis of bryophyte, roots of grasses and herbs and AM fungi within roots........................10 2.4 “Plant-AM-waxcaps” experiment.............................................................................................11 2.5 Collection and anatomical analysis of AM fungi spores......................................................... 13 2.6 “Waxcaps-AM”experiment in root organ culture (ROC) plates..............................................14 2.7 Quantification of the isotopic signature of AM fungi spores................................................... 16 2.8 Isolation and molecular identification of moulds from Hygrocybe fruit bodies......................17 3. Results ........................................................................................................................................... 19 3.1 Primer design............................................................................................................................19 3.2 Molecular analysis of Rhytidiadelphus squarrosus, roots of grasses and herbs and AM fungi within roots.........................................................................................................................................21 3.3Anatomical analysis of R. squarrosus, roots of grasses and herbs and AM fungi within roots 22 3.4 “Plant-AM-waxcaps” experiment............................................................................................ 24 3.5 Anatomical analysis of AM fungi spores.................................................................................24 3.6 “Waxcaps-AM”experiment in ROC plates..............................................................................26 3.7 Quantification of the isotopic signature of AM fungi spores................................................... 26 3.8 Isolation and molecular identification of moulds from Hygrocybe fruit bodies......................27