Ghent University Faculty of Sciences, Department of Biology Mycology Research Group Members of the examination committee: Chairman: Prof. dr. Koen Sabbe, Faculty of Sciences, Ghent University Dr. Kobeke Van de Putte, Faculty of Sciences, Ghent University Prof. dr. Paul Goetghebeur, Faculty of Sciences, Ghent University Dr. Ursula Eberhardt, Staatliches Museum Naturkunde Stuttgart Dr. Sunita Chamyuang, Scool of Sciences, Mae Fah Luang University Promoter: Prof. dr. Annemieke Verbeken, Faculty of Sciences, Ghent University Prof. dr. Kevin D Hyde, School of Sciences, Mae Fah Luang University Dr. Jorinde Nuytinck, Naturalis Biodiversity Center Research funded by: Bijzonder Onderzoeksfonds Ghent University (BOF) Thesis submitted in fulfillment of the requirement for the degree of Doctor (PhD) of Sciences: Biology Proefschrif voorgedragen tot het behalen van de grad van Doctor in de Wetenschappen: Biologie Titel van het doctoraat in het Nederlands: Diversiteit en fylogenie van Lactarius subgenus Russularia in Zuidoost-Azië To refer to this thesis: Wisitrassameewong, K. (2015). Diversity and phylogeny of Lactarius subgenus Russularia in Southeast Asia. Thesis submitted in fulfillment of the requirements of the degree of Doctor (PhD) of Sciences: Biology. Faculty of Sciences, Ghent University. Cover illustration: Komsit Wisitrassameewong The author and the promoters give the authorization to consult and to copy parts of this work for personal use only. Any other use is subject to the restrictions of author’s rights. Permission to reproduce any material contained in this work should be obtained from the author. Ghent, 9 December 2015 Author, Komsit Wisitrassameewong Promoter Prof. dr. Annemieke Verbeken Prof. dr. Kevin D Hyde Dr. Jorinde Nuytinck ACKNOWLEDGEMENTS First of all, I wish to thank my supervisor in Thailand, Asst. Prof. Dr. Kevin D Hyde, the first supervisor in my PhD life. He gave me the great opportunity to start my PhD study in 2010. Thanks for being so patient with me because I did not have any experience in fungal research and at that moment my English was not so good. I experienced that international networking and collaboration are essential for scientific research. I also learnt how to deal with writing and publishing in international scientific journals. Secondly, I am very grateful for the opportunity to work in a joint PhD scholarship offered by my supervisor in Belgium, Prof. Dr. Annemieke Verbeken. Many thanks for your constant support, hospitality during my stay in Belgium and cooperation in fieldwork in Thailand! Especially thanks to Dr. Jorinde Nuytinck for the first guidance in fieldwork in Vietnam, for the warm welcome during my first visit to Belgium, the first accommodations in Belgium, city tours in the city of Ghent, and innumerable valuable suggestions in molecular and phylogenetic approaches! During my Ph.D. study, many colleagues have been very cooperative, both in Thailand and overseas. Without their support and collaboration, it would have been tough to succeed in this research project. Regarding fieldwork, I must start by mentioning Dr. Huyen Thanh Le for organizing expeditions in Vietnam and for her contribution of additional Thai specimens. Prof. Dr. Steve L. Miller, Prof. Dr. Steve L. Stephenson and Dr. Philippe Callac, thanks for organizing mycological excursions in Thailand. During those trips I collected several interesting specimens that influenced my study. Thanks to Dr. Olivier Raspé for inviting me to join his collecting trips and for his support in finding accommodation during these trips. Thanks to Dr. Slavomir Adamcik for organizing the Russulales workshop in 2014 and for being a nice host in Slovakia. Thanks for the people who contributed dried materials, DNA sequences and assistance. Thanks to Dr. Dirk Stubbe for providing material for the two new Malaysian species. A warm appreciation to Gao Jiayu, Dr. Kanad Das and Dr. Kobeke Van de Putte for allowing me to study and include their specimens in some of my publications. Dr. Ursula Eberhardt, thanks for your DNA sequences, professional phylogenetic knowledge and advices. Jan Hackel, thanks for being a such nice mentor in dating phylogeny. Brian Looney, I am much appreciate your valuable suggestions in dating phylogeny. Felix Hampe, thanks for supporting me in molecular works and joining me in fieldwork in Thailand. Viki Vandomme, thanks for all assistance in molecular works. Kristof de Vos, I would like to thank you all assistance and support. I appreciated very much all your advices on life in Belgium and your corrections on English grammar in some of the thesis chapters! Eske De Crop, thanks for your hospitality and all your advice in phylogenetic analyses. I will certainly miss our office! Thanks to Marleen Vlaeminck for helping me with all paperwork at Ghent University. Thanks to Olivier Leroux and Rosette Heynderickx for all their support and advice during my stay at Ghent University. Phongeun Sysouphanthong and Samantha Karunarathna, thanks for being my mentors on general knowledge of basidiomycetes and fieldwork management at the beginning of my study. We had a great time together in the field! Jie Chen, thanks for all favours and for all suggestions on field trips and for the interesting conversations on basidiomycetes and molecular phylogeny. Mingkwan Doilom and Benjarong Thongbai, thanks for your help with paperwork and phylogenetic analyses. Other colleagues, Anan Thawthong, Naritsada Thongklang, Thatsanee Luangharn and Kritsana Jatuwong for friendship and favours. Jian Kui Liu, thanks for your hospitality and the interesting conversations on phylogeny. Thanks for all the support in fieldwork, lab work and in supplied materials from all staff at Ghent University and institute of excellence in fungal research, Mae Fah Luang University For all paperwork, including bilateral agreement between Mae Fah Luang University and Ghent University, certainly, I wish to thank the division of international affairs and the graduate division and school of science Mae Fah Luang University, for all facilities and advice. I was travelling between Thailand and Belgium several times during my PhD study so I wish to thank all my good friends in Thailand, they always helped me on document submissions during my stay in Belgium. They also provide accommodation and facilities at arrival in Chiang Rai: Nattawan, Saowanee, Supaluk, Ravee and Yothino. Ultimately, I will never forget all encouragement and all support from my parents and relatives. Thank you so much! TABLE OF CONTENTS CHAPTER 1 GENERAL INTRODUCTION Page 1.1 Rationale of study 1 1.2 Milkcaps in a new generic landscape 2 1.3 Morphology of Lactarius and Lactifluus 4 1.4 Lactarius subgenus Russularia: a short taxonomical and nomenclatural 12 overview of Lactarius subg. Russularia (Fr. ex Burl.) Kauffman. 1.5 Species delimitation and previous phylogenetic studies 16 1.6 Ecology and biogeography 19 1.7 Uses 21 1.8 Objectives 22 1.9 Outline of this thesis 25 2 SPECIES WITH VERY DISTANT GILLS 33 Wisitrassameewong, K. et al. (2014a). Lactarius subgenus Russularia in Southeast Asia: 1. Species with very distant gills. Phytotaxa 158(1): 23−42. 3 SPECIES WITH REMARKABLY SMALL BASIDIOCARPS 61 Wisitrassameewong et al. (2014b). Lactarius subgenus Russularia (Russulaceae) in South- East Asia: 2. Species with remarkably small basidiocarps. Phytotaxa 188(4): 181−197. 4 NEW DIVERSITY FROM THAILAND AND VIETNAM 87 Wisitrassameewong et al. (2015a). Lactarius subgenus Russularia (Russulaceae) in South- East Asia: 3. New diversity in Thailand and Vietnam. Phytotaxa 207(3): 215−241. 5 TWO NEW SPECIES FROM THAILAND 131 Liu et al. 2015. Fungal diversity notes 1−110: taxonomic and phylogenetic contributions to fungal species. Fungal Diversity DOI 10.1007/s13225-015-0324-y. TABLE OF CONTENTS Continued. Page CHAPTER 6 BIODIVERSITY, MOLECULAR PHYLOGENY AND EVOLUTIONARY 149 RELATIONSHIPS OF LACTARIUS SUBGENUS RUSSULARIA Wisitrassameewong et al. Lactarius subgenus Russularia: biodiversity and an overview of molecular phylogeny and evolutionary relationships (unpublished) 7 GENERAL DISCUSSION 203 7.1 Species concept and cryptic species 203 7.2 Biodiversity 207 7.3 Classification and evolutionary relationships 212 7.4 Phylogenetic signal of the morphological characters 214 7.5 Intercontinental conspecificity 217 7.6 Future perspectives 218 7.7 General conclusion 220 SUMMARY 225 APPENDICES 231 CURRICULUM VITAE 273 CHAPTER 1 GENERAL INTRODUCTION 1.1 Rationale of the study Fungi are a large and diverse group of organisms, which are estimated to be the second largest group of organisms after the group of insects. However, the diversity of fungi remains understudied. The real number of fungal species on earth might be higher than previously hypothesized (O’Brien et al. 2005). This actual number has been estimated to be up to 1.5 million species (Hawksworth 1991, Hawksworth & Rossmann 1997) or even 5.1 million species (Blackwell 2011), whereas about 100,000 species are described so far (Kirk et al. 2008). Numerous new species are still waiting to be explored. Species delimitation of fungi mainly relies on the morphological species concept (MSC) up to the middle of the 20th century. This species concept is based on the similarity and difference of morphological characteristics. However, fungal taxonomists encounter difficulty in delimiting fungal species because many groups of fungi are morphologically rather simple. Furthermore groups with a lot of morphological variation or morphological differences may not have evolved