Phylogeny of Lyophyllum section Difformia Does hon-shimeji (L. shimeji) occur in Sweden? Henrik Sundberg Degree project for Master of Science in Botanical Systematics and Evolution 30 hec Department of Plant and Environmental Sciences University of Gothenburg ABSTRACT ........................................................................................................................................................... 2 1. INTRODUCTION ............................................................................................................................................. 3 1.1. BACKGROUND ............................................................................................................................................. 3 1.2. WHAT IS HON-SHIMEJI? ............................................................................................................................. 3 2. PROBLEMS & OBJECTIVES ........................................................................................................................ 4 3. LITERATURE REVIEW ................................................................................................................................. 4 3.1. GENERAL ECOLOGY AND DISTRIBUTION OF THE GENUS LYOPHYLLUM P. KARST. ................................... 4 3.2. TAXONOMY ................................................................................................................................................. 4 3.2.1. Traditional classification of the genus Lyophyllum .......................................................................... 4 3.2.2. Old school - Problems and confusion ................................................................................................ 5 3.2.3. Non-molecular classification on other grounds than morphology ................................................... 6 3.2.4. The molecular revolution ................................................................................................................... 6 3.2.5. Impact of molecular phylogenetics on the genus Lyophyllum .......................................................... 6 3.2.6. Classification and naming within the genus Lyophyllum ................................................................. 6 3.2.7. The section Difformia (Fr.) Kühner .................................................................................................. 7 3.3. ECOLOGY AND DISTRIBUTION OF THE SPECIES IN SECTION DIFFORMIA .................................................. 8 3.3.1. Worldwide ........................................................................................................................................... 8 3.3.2. Habitat types in which the L. decastes/Pinus is known to occur and its distribution ....................... 9 3.3.3. Mycorrhiza or not? ............................................................................................................................. 9 4. MATERIAL & METHODS ........................................................................................................................... 10 4.1. GATHERING OF MATERIAL ....................................................................................................................... 10 4.2. DNA EXTRACTION, AMPLIFICATION & SEQUENCING.............................................................................. 10 4.3. ALIGNMENTS, ANALYSES & TREE RECONSTRUCTION ............................................................................. 10 5. RESULTS ........................................................................................................................................................ 11 5.1. TAXON SAMPLING ..................................................................................................................................... 11 5.2. RESULTS FROM THE ANALYSIS ................................................................................................................. 11 6. DISCUSSION .................................................................................................................................................. 12 6.1. THE CLADES .............................................................................................................................................. 12 6.2. WHICH SPECIES IS WHICH? ...................................................................................................................... 13 6.3. CONCLUSION ............................................................................................................................................. 13 6.4. FURTHER INVESTIGATIONS ...................................................................................................................... 13 ACKNOWLEDGEMENTS ................................................................................................................................ 14 REFERENCES .................................................................................................................................................... 14 APPENDIX .......................................................................................................................................................... 18 1 Abstract The taxonomy of the genus Lyophyllum and especially the section Difformia has been controversial for a long time. There are currently five species grouped in the section but none of them have been typified according to taxonomical standards so therefore no one knows which name refers to which species. There are more than 20 synonyms applied to these five species. In this study we tried to elucidate the phylogeny of the species in the section, with focus on Fennoscandia and Japan. It was especially interesting to see whether the mushroom Hon-shimeji (L.shimeji), a very important Japanese delicacy, occurs in Fennoscandia. The ribosomal DNA analysis showed that there are four major clades within the decastes complex. What we denote as the decastes clade split into three supported subclades; one Japanese, one from Fennoscandian deciduous forests and one from Fennoscandian coniferous forests. The clade denoted as L. fumosum also split into three subclades one unsupported from deciduous forest and two supported; Japanese and coniferous. In the clade we named shimeji the Japanese L. shimeji was identified as a supported subclade but it only differed by two substitutions from the Fennoscandian subclade. There was also a supported major clade consisting of a putative Norwegian fumosum and a Japanese decastes cultivar. The conclusion was that the Japanese L.shimeji and the Fennoscandian L. decastes from pine moorland were the same species and that the section Difformia (decastes complex) consists of (at least) seven distinct species. Furthermore it was concluded that ecology is an important and underused character in the determining of species in the section Difformia. 2 1. Introduction 1.1. Background This investigation would not have been done were it not for the attention that Swedish matsutake (Tricholoma matsutake (S. Ito & S. Imai) Sing.) has attracted in the latest decade. In August 2008 the Japanese researcher in mycology Etsuko Harada (Iwade Research Institute of Mycology) found what she judged to be Hon-shimeji (Lyophyllum shimeji (Kawam.) Hongo) during a field excursion in the matsutake forests around Skellefteå in Northern Sweden. Considering that Niclas Bergius in 1998 showed that the very much sought after T. matsutake is indeed the same species as T. nauseosum (A. Blytt) Kytöv. described from Scandinavia it was not so far-fetched to believe that this could be a parallel case. After studying Etsuko Harada’s picture of the suspected shimeji and subsequent consultation with Professor Iwase (Tottori University) and Dr. Yamada (Shinshu University) our suspicions were reinforced that it de facto could be another Japanese gourmet mushroom dwelling in the Swedish forests. Later, skilled amateur mycologists in Northern Sweden were asked if they had seen similar mushrooms in pine moorland. The answer was that they had encountered specimens to which the closest match was Lyophyllum decastes (Fr.: Fr.) Singer) but it seemed an unsatisfactory identification. The following year Niclas Bergius and Henrik Sundberg went looking for “shimeji” in the matsutake forests of Northern Sweden and to their surprise they found some similar fruiting bodies in various localities. As a consequence a molecular study was undertaken investigating the relationship between the “suspicious” mushroom and Japanese L. shimeji. 1.2. What is Hon-shimeji? Shimeji together with different prefixes denotes around twenty edible mushroom species in Japan (Yamanaka 2009). The word means roughly that the fungus has a nice “mushroomish” appearance (Yamada personal comm.). Hon-shimeji (meaning real-shimeji) is the common name of Lyophyllum shimeji, which after matsutake is the next most valuable and sought after mushroom in Japan. Similar to matsutake, there has been an ongoing decline in Hon-shimeji production since the mid 20th century. There are several reasons such as loss of suitable forests due to infections by the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) and because of changed ways of forest management (Ohta 1994; Kawai 1997; Kosaka 2001; Yamanaka 2009). Today wild grown Hon-shimeji is almost only served in high-class restaurants and is only available through a few dealers specializing in delicacy mushrooms (Kippo News, Vol.3 No.114 Tuesday, November