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 19, 1996; Ueda personal comm.) Great effort has been undertaken in trying to cultivate Hon-shimeji, and in the last decade there has been a breakthrough (Ohta 1994; Yamanaka 2009) but the yields have been relatively low and wild harvested mushrooms are still considered much more tasty and with price levels accordingly much higher. Some confusion is caused by the fact that a similar mushroom called Buna-shimeji (Hypsizygus tessullatus (Bull.) Singer) is often presented to the market as Hon-shimeji by unscrupulous merchants, an act that is prohibited by the authorities (Ueda personal comm.). However, even professional mycologists sometimes mistake H. tessullatus for Hon-shimeji.

3 2. Problems & Objectives

My initial objective was to answer the question whether the highly esteemed fungus Lyophyllum shimeji (Kawam.) Hongo do occur in Sweden. The study was subsequently enlarged to encompass the section Difformia (Fr.) Kühner of the genus Lyophyllum as a whole, regardless of habitat preferences and also to attempt to partly elucidate its taxonomy both in Sweden and in Japan. I will also consider the ecology of the section and its significance to classification. The taxonomy of the section is interesting because there is some confusion about which species there are hiding behind the rich flora of synonymous names. The genus Lyophyllum in Northern Europe is also quite poorly studied from taxonomical and distributional points of view since the scientists dedicated to its study mostly reside in Central Europe and North America (Kalamees 2004). The section is here considered in the sense of (Moncalvo, Toriola & Clémençon 1991, Moncalvo, Rehner & Vilgalys 1993, Moncalvo & Clémençon 1994) thus delimiting the section to the species in the decastes complex, in this study initially presumed to be L. decastes (Fr.: Fr.) Singer, L. fumosum (Pers.) P.D. Orton, L. loricatum (Fr.) Kühner and L. shimeji (Kawam.) Hongo.

3. Literature Review

3.1. General ecology and distribution of the genus Lyophyllum P. Karst.

The genus is represented in all continents except Antarctica although it is more widespread in boreal and temperate areas than in the tropics and subtropics. In 1986 Singer counted 40 species but with the remark that there was a great need for revision of the genus (Singer 1986). In the Index Fungorum (http://www.indexfungorum.org) 225 species names are listed, synonyms included. In the Nordic countries there are 34 species encountered (Knudsen & Vesterholt 2008). Some mycologists (e.g. Knudsen & Vesterholt 2008) argue that all Lyophyllum species are saprobic whereas others ascribe various nutritional strategies to the genus such as parasitism on mosses and fungi, saprobism and the formation of ectomycorrhiza (Matheny et al. 2006).

3.2. Taxonomy

3.2.1. Traditional classification of the genus Lyophyllum The genus Lyophyllum P. Karst. is the type genus of the family Lyophyllaceae Jülich (formerly included in Tricholomataceae R. Heim) and of the tribe Lyophylleae Kühner (see table 1). The Lyophyllaceae is a rather bothersome taxon when it comes to classification and naming since there are many different interpretations and attachments of importance to the morphological traits that define the taxa included in the family. This subjectivity has resulted in controversies over where different species and genera belong and it has pointed out the need for a classification that reflect the true phylogenetic relationships (Hofstetter et al. 2002; Kalamees 2004) The delimitation of the genus Lyophyllum has also been subject to controversy and the species have been placed under various taxonomical identities. Once (as many gilled mushrooms!) they were included in the genus Agaricus L. Since then they have been assigned to e.g.

4 Tricholoma (Fr.) Staude and Clitocybe (Fr.) Staude (Singer 1986; Knudsen & Vesterholt 2008; www.indexfungorum.org). Depending on different morphological traits the genus has been subdivided in sections Lyophyllum, Difformia and Tephrophana by Singer (1986). The last section has back and forth been considered as a genus (Tephrocybe) or even been designated a tribe of its own (Tephrocybeae). To make it even more complicated Kühner subordinated the genus Calocybe Kühner as a section under Lyophyllum (Hofstetter et al. 2002). In other words: great confusion! Before the use of molecular techniques, the discrimination of the tribe Lyophylleae was heavily dependent on the character of siderophilous granulation (i.e. the basidia being dyed blackish-violet in an acetocarmine reaction with metal ions), with the genus Lyophyllum separated from the rest of the tribe mainly by the cyanophilous (i.e. readily stainable with a blue dye) character of the spores. Within the genus, an important trait circumscribing the section Lyophyllum has been the staining of lamellae upon bruising (Singer 1986; Knudsen & Vesterholt 2008). However, the use of electron microscopy has made it clear that there are various types of siderophilous granules not delimited to Lyophylleae alone but found in more distantly related groups. So, fungi with these discriminating characters do not arrange into a natural clade after all. It must be pointed out though that on a higher taxonomic level siderophilous granulation is supported by molecular evidence as a trait indicating phylogenetic relationship (Moncalvo et al. 1993; Hofstetter et al. 2002).

Table 1.

Domain Eukaryota Kingdom Fungi Phylum Basidiomycota Class Basidiomycetes Order Agaricales Family Lyophyllaceae Tribe Lyophylleae Genus Lyophyllum Section Difformia

3.2.2. Old school - Problems and confusion In recent years it has been shown that classification by morphological characters could be severely influenced by homoplasies: “trait similarity due to evolutionary convergence, parallelism, or character reversals” (Avise & Robinson 2008), and that for example lack of gills and also mycorrhiza have been developed repeatedly within the agaricoid clade (Hibbett et al. 2000; Garnicia et al. 2007). Another thing that undermines the benefits of morphology as a base for classification is the fact that many species vary a lot in their appearance due to phenotypic plasticity, while still being the same species (Hofstetter et al. 2002). As an upshot, a lot of the genera and families as we know them today are non-natural (Moncalvo et al. 2000). In the case of Lyophyllum Buchalo & Diduk (2005) studied hyphae ornamentation under scanning electron microscopy (SEM) and found that they possibly could be good characters for taxonomic work. It does not seem though that their findings have received much attention, maybe due to the special skills needed and the high costs for SEM analysis.

5 3.2.3. Non-molecular classification on other grounds than morphology By tradition the species concept embraced by mycologists is the one based on morphological characters but also the biological concept of species has had importance in determining species status (Maeta et al. 2008). Problems attached to the former concept have been addressed above, but the latter is also problematic, it is for instance difficult in some species to do mating tests due to non-cultivability. A third concept is that of ecological species. Different habitat demands could for example be used to delimit species. Ecology has been, and still is, underused in species delimitation and for inferring relationships among natural groups (Matheny et al, 2006, p. 983). Support of this statement is given in a study by Hofstetter et al. (2002), where all the parasitic species studied cluster together with ammonia-dependent saprobic species. Thus they share a craving for ammonia, perhaps derived from a common ancestor. This under-use of ecology is therefore remarkable since overall it seems that ecology in many cases better demonstrates true relationships than morphology (Moncalvo et al. 2002).

3.2.4. The molecular revolution In the last twenty years or so the analysis of biomolecules has become increasingly important for the development of a supposedly correct taxonomy for all living things. As mentioned above it has been shown that the use of morphological characters could be misleading in the quest for correct relationships amongst basidiomycetes (Hofstetter et al. 2002). One example is that phylogenetic analyses using DNA has revealed relationships between gilled and non- gilled basidiomycetes (Matheny et al. 2006). However, there are some clouds in the molecular sky as well; you could get divergent results depending on which outgroup you choose, how you reconstruct the ancestral state (i.e. what evolutionary model you choose to estimate the state of characters of the earliest common ancestor of the species in your phylogeny), how character coding is performed (no problem when analysing DNA/RNA), if alignment is correct and whether adequate taxa have been sampled and so on (Matheny et al. 2006), but still molecular phylogenetics is a major leap forward in search for the true tree of life.

3.2.5. Impact of molecular phylogenetics on the genus Lyophyllum Phylogenies based on molecular data show that the tribe Lyophylleae consists of four major clades that do not correspond to the traditional classification. The main subject for this article, the Lyophyllum decastes complex in section Difformia of the genus Lyophyllum, lumps together in one of the clades with members from the two other sections (Lyophyllum and Tephrophana) of the genus. Former members of these sections are also found scattered in the other three clades of the Lyophylleae and showing closer relationship to other genera e.g. Clitocybe. It is evident that classification based on morphology at least in this case is artificial (Hofstetter et al. 2002). At present the impact of this revision in literature other than academical is minute and despite the attempts to circumscribe it the taxonomical status of the genus and its members the relationships and namings are still unclear.

3.2.6. Classification and naming within the genus Lyophyllum Singer (1986) subdivided the genus into three sections based on fruitbody stature and blackening upon bruising. The blackening of basidiocarps when bruised has been an important trait separating section Lyophyllum from the other sections. Quite recently it was demonstrated (Hofstetter et al. 2002) that the species in the Lyophyllum section are distributed through all three sections establishing that blackening is overestimated as a discriminating trait. Also the basidiocarp habitus is misleading since the collybioid (i.e. looks like a member of the genus Collybia (Fr.) Staude) species of section Tephrophana clusters both with the L.

6 decastes complex and with species outside the genus. Hence the genus is polyphyletic (Hofstetter et al. 2002; Montcalvo et al. 2002; Matheny et al. 2006). Even the type species L. leucophaeatum (P. Karst.) P. Karst. is not representative for Lyophyllum, as it is shown to be closely related to Calocybe. Therefore The International Committee of Nomenclature has recently proposed that L. semitale (Fr.) Kühner should serve as type species instead (Norvell 2010).

3.2.7. The section Difformia (Fr.) Kühner The Difformia is the most mind-bending of the three Lyophyllum sections when it comes to taxonomy. As Singer (1986) puts it: “the synonymy has reached such vast proportions that it seems almost impossible to attribute the right name to the right species”. There are many species described from Europe and North America (see below) but the descriptions are mostly too poor to be of substantial taxonomical value. The species’ polymorphic nature does not make things easier (Moncalvo et al. 1990). Typically the species has a fasciculate growth, rather dull coloration in ochre to dark greyish brown and +/- spherical smooth spores (Knudsen & Vesterholt, 2008). A series of subsequent studies in the early nineties encompassing rDNA analysis, comparative basidiocarp morphology, enzymic analysis and culture analysis (Moncalvo & Clémençon 1990; 1992; 1994; Moncalvo, Toriola & Clémençon 1990; Moncalvo, Rehner & Vilgalys 1993) tried to elucidate the species belonging to the section. The rDNA study alone could not completely resolve the relationship among these species, probably due to the use solely of LSU DNA: the opportunity to use the ITS-region as well would have given better resolution. Nevertheless, all these studies put together gave as a result five species in the section, denoted T1-T5 since their scientific names had not been based on type specimens but from drawings and old descriptions, a procedure not satisfying modern taxonomical standards. Accordingly there is a need for neo-typification, something that has not been done at the time of writing (Moncalvo personal comm.). Species T1, T4 and T5 are in Japan known under the common names Hatake-shimeji, Hon-shimeji and Shaka-shimeji respectively. T2 and T3 had only been encountered in Europe and T4 and T5 only in Japan. The only species common to Europe and Japan is T1. Another result of these studies is that L. connatum (Schumach.) Singer formerly considered a member of the section is now placed in the genus Clitocybe because of its staining of lamellae with iron sulphate, whitish colouration and ellipsoid spores (Clémençon & Moncalvo 1990; Moncalvo et al. 2002; Knudsen & Vesterholt 2008). The variety Lyophyllum aggregatum var. ovisporum (J.E. Lange) Kühner & Romagn. is now transferred to the genus Lepista as L. ovispora (Ryman & Holmåsen, 1984; Index Fungorum). Some darkening species from the Lyophyllum section that grows in clusters might also be transferred to Difformia (Moncalvo et al. 1993) as well as the species L. tucumanense Singer, L. multiforme (Peck) H.E. Bigelow, L. umbriniceps (Murrill) H.E. Bigelow, Clitocybe elephantina Murrill, C. tenebricosa Murrill, C. virgata A.H. Sm. and Melanoleuca submulticeps Murrill (Singer 1986), the last one belonging to a different family, the Limnoperdonaceae (Matheny et al. 2006). Accordingly the rank and naming of the species in the section has varied over time and depending on how different authors have interpreted the morphological characters. One problem has been the fact that no discriminating traits can be observed through microscopy and that you have to rely on habitus of the fruiting bodies, i.e. colour, texture, growth mode and so on. Because of this Breitenbach & Kränzlin (1991) claimed it useless to talk about different species based on morphology. As mentioned above there are (at present) five non-typified species belonging to Difformia. To these more than twenty names are applied (Montcalvo personal comm.). There is no point

7 in listing all of these but some of them can serve as examples: L. decastes has kept its status of species during the years although it once was called L. aggregatum (Schaeff.) Kühner and even L. fumosum (Kalamees 1994). Some authors interpret it as being one species while others subdivide it into several species (e.g. L. fumosum, L. loricatum) or varieties, hence the concept of the decastes complex. (Breitenbach & Kränzlin 1991). Lyophyllum fumosum currently have the rank of species but earlier it has been considered a subspecies under L. decastes as Lyophyllum aggregatum subsp. cinerescens (Bull.) Singer. It has also been designated as a non-valid name for L. decastes (Kalamees 1994) and as synonymous to Lyophyllum eustygium (Cooke) Clémençon, a blackening species (Trappe 1962). Lyophyllum loricatum is according to Kalamees (2004) “a very good species”, to compare with Knudsen & Vesterholt (2008) that treat it as a variety of L. decastes. It has also been confused with L. fumosum (Ingelström 1940). There has not been any problem with L. shimeji in case of synonyms and it has been considered a quite well defined species. However, just recently it has been revealed through mating tests that the one morphological species probably consists of two biological species (Maeta et al. 2008).

3.3. Ecology and distribution of the species in section Difformia

3.3.1. Worldwide Lyophyllum decastes sensu lato: The original description by Fries (1818) read: “In ericetosis ad radices Quercuum”. Interpreted as: “In heathland next to the roots of an oak”. In Japanese the common name hatake shimeji means roughly field shimeji, i.e. that it grows in arable land (Yamada personal comm.), which is also the habitat most commonly referred to in literature. But it is also said to be found in deciduous and coniferous forest, along roadsides, in parks, grassland etc. (e.g. Breitenbach & Kränzlin 1991; Kalamees 2004) and is seen as an indicator for nutrient rich soils (Gminder & Böhning 2008). It is distributed over much of the northern hemisphere and is common throughout Europe in the temperate to hemiboreal zones and occasional in the boreal to subalpine zones (Breitenbach & Kränzlin 1991; Gminder & Böhning 2008; Knudsen & Vesterholt 2008). According to Hallingbäck & Aronsson (1998) the species is found in Southern and Mid Sweden but not in the northernmost parts. Lyophyllum fumosum sensu lato: The original description by Persoon (1801) read: “Hab. passim in syluis, locis graminosis”, interpreted as: “Here and there in forest, on grassland”. Fries (1821) says: “In pinetis, betuletis similibusque, locis asperis”, interpreted as: “In pine forest, birch forest and similar (places), rough terrain”. More recently the species is described as being found in deciduous and coniferous forests, in parks, on bare soil, in grassland, roadsides, fat soil (Petersen & Vesterholt 1990; Breitenbach & Kränzlin 1991), wooded tundra (sic!) (Kalamees 2004), or as a species confined to coniferous forests (Hallingbäck & Aronsson 1998; Nylén 2001). On the other hand, Ingelström (1940) describes it as being a beech forest species. The species is said to be distributed over much of the northern hemisphere (Breitenbach & Kränzlin 1991), or as being found occasionally in the temperate to boreal zones (Knudsen & Vesterholt 2008). According to Kalamees (2004) it is widespread and common all over Northern Europe and “grows often en masse in the wooded tundra area in Finland and Norway”. In Sweden it is considered rare and is distributed in the southern and northern parts but not in between (Hallingbäck & Aronsson 1998). Lyophyllum loricatum: The original description by Fries (1838) read: “In coryletis umbrosis, montosis raro”, interpreted as: “In shady hazel groves, rare in mountainous areas”.

8 The species is described as being found in deciduous and mixed forests, along forest edges, in gardens, on bare soil, among grasses and herbs (Breitenbach & Kränzlin 1991), or in deciduous forest and also on burnt ground (Hallingbäck & Aronsson 1998). The distribution is reported as being over much of the northern hemisphere (Breitenbach & Kränzlin 1991), though with unknown distribution in Sweden (Hallingbäck & Aronsson 1998). In Japan, the species is said to be rare and possibly confined to Hokkaido (Yamada personal comm.). Lyophyllum shimeji: The species grows in (mixed) Pinus desiflora and Quercus serrata forests all over Japan and is also widely distributed in China and Korea (Fujita et al. 1982; Yamanaka 2009; http://www.gbif.org).

3.3.2. Habitat types in which the L. decastes/Pinus is known to occur and its distribution At present the Fennoscandian L. decastes/Pinus is known to occur in the same habitats as T. matsutake, i.e. sandy Scots pine moorland, and in one known case in Scots pine forest on rocky granite ground in Northern Finland (Ueda personal comm.). The sandy habitat consists mainly of sediment deposited by glacial streams during the latest ice age but also post glacial river sediments. The habitat is completely dominated by Scots pine up to almost 100% in some localities, based on measurements of the basal area (Risberg 2003). The forests are very oligotrophic and the ground cover is poor, mainly a mosaic consisting of lichens (mostly Cladonia sp. Hoffm.) and Vaccinium vitis-idaea L. and Calluna vulgaris (L.) Hull. In some localities ground cover is almost absent and the ground surface consists of needle litter. In the poorest woods the humus and litter layer is only about one or two centimetres in thickness. The Swedish distribution is poorly known and it has thus far only been found in the northern parts, except for one locality in Mid Sweden (Dalarna).

3.3.3. Mycorrhiza or not? According to Hibbett et al. (2000) the ancestor of the eugarics was ectomycorrhizal and the saprobic habit among many eugarics is accordingly derived. In their study (2002) Moncalvo et al. showed that there are several instances where it seems that a derived saprobic habit has once again transformed into an ectomycorrhizal one. Thus there seem to have been multiple reversals within the eugarics. The species in the genus Lyophyllum has traditionally been considered as saprotrophic (Singer 1986, Knudsen & Vesterholt 2008) with the exception of L. shimeji that has been described as forming facultative mycorrhiza (Ohta 1994; Moncalvo et al. 2002). According to other studies though, it does not seem that the mycorrhiza of L. shimeji is a unique trait within the genus Lyophyllum. Yamada et al. (2001) reported that L. semitale formed ectomycorrhiza in vitro with Pinus densiflora; and L. decastes did form ectomycorrhiza in vitro with Pinus pinaster according to Pera & Alvarez (1995). Also L. fumosum is considered as putatively ectomycorrhizal in Japan (Yamada et al. 2001). Trappe reported as early as 1962 that L. immundum (in the study considered as a synonym to L. fumosum) formed ectomycorrhiza with Pinus silvestris.

9 4. Material & Methods

4.1. Gathering of material The specimens analysed in this study were obtained through collecting in the field both in Sweden and in Japan, and through an appeal in the forum at Svampguiden.com. I also contacted skilled amateur mycologists in Northern Sweden and sent a written call to be distributed at a gathering of The Swedish Mycological Society (http://www.svampar.se). In the appeal specimens of Lyophyllum species found in pine moorland were requested. As a complement to the freshly collected specimens, collections were borrowed from the herbaria GB, L and O.

4.2. DNA extraction, amplification & sequencing Pieces of lamellae were picked from dried specimens and put in Eppendorf tubes. Between each sample the tweezers were cleaned with 70% ethanol and the paper surface was changed. The rDNA regions analysed are the complete ITS (ITS1, ITS2 and 5.8S) and 900 base pairs of the 5´ end of the LSU region. The DNA was extracted using DNeasy Plant Mini Kit (QIAGEN, July 2006) following the manufacturer’s recommendations. PCR was performed using illustra™ puReTaq Ready-To-Go PCR Beads (GE Healthcare). For DNA amplification the following primers were used: ITS1F (Gardes & Burns 1993), LR21, LR0R and LR7 (Hopple & Vilgalys 1999) and was carried out using Gen Amp® PCR System 9700 with the following program: 94°C: 5s, (94°C: 30s, 55°C: 30s, 72°C: 30s) * 35, 72°C: 7 min, 4°C: ∞ After amplification the products were purified using the QIAquick® PCR Purification Kit (QIAGEN, March 2008) according to the manufacturer’s recommendations. To see if there were satisfactory amounts of DNA the purified products were visualised on an agarose gel using ethidium bromide. The sequencing was performed by Macrogen in Seoul, South Korea , and the sequences were received as a data file. A list of the specimens sequenced is presented in table 2. Additional sequences were downloaded from GenBank® and the Japanese database NBRC (NITE Biological Research Center).

4.3. Alignments, analyses & tree reconstruction A crude alignment was performed using the online version of MAFFT ver.6 (http://mafft.cbrc.jp/alignment/server/) and the alignments were edited by hand in PAUP* Version 4.0b10. Thereafter dubious characters were checked and corrected as needed after studies of the chromatograms in Sequencher™ Version 4.1.2. (Ann Arbor, USA). In the final alignment there were 1867 characters of which 275 were excluded manually (see section 5.2). A heuristic search with 1000 random addition replicates for the most parsimonious trees were conducted in PAUP* Version 4.0b10 with the following settings: MaxTrees = 100 000, branch swapping algorithm = tree-bisection-reconnection (TBR), maximum 100 trees saved in each replicate, collapsing zero-branch lengths. The trees were rooted using the outgroup Lepista nebularis (Fr.) Harmaja = Clitocybe nebularis (Batsch) P. Kumm. which is closely related to but still outside the monophyletic ingroup clade according to other studies (Hofstetter et al. 2002). Gaps were treated as missing data and multistate taxa were interpreted as uncertainty. Support of topology was estimated via bootstrap analysis with 1000 heuristic replicates, each with 5 random addition sequence replicates and no more than 25 trees saved in each replicate, settings otherwise as above.

10 5. Results

5.1. Taxon sampling The result of the call for specimens was fairly good. Several people sent material after contacting me. Mostly responses came from Upper Norrland, very few from mid Sweden and none from the southern parts. The reason for this is probably that my appeal was for specimens from pine moorland and the fact that pine moors are predominantly found in the northern parts of Sweden. Few specimens of Lypohyllum were available from the Nordic herbaria, probably reflecting the lack of attention that the genus Lyophyllum has received. Sequences of Lyophyllum shimeji, L. decastes and L. fumosum available in GenBank® and the Japanese database NBRC were scarce, but 9 sequences were found relevant and were included in the data set. In total the final data set had 46 ingroup taxa. In addition seven sequences from earlier molecular phylogenetic studies of Lyophyllum and Agaricales (Hofstetter et al. 2002, Moncalvo et al. 2002, Matheny et al. 2006) representing Hypsizygus marmoreus, L. anthracophilum, L. semitale, L. sykosporum, Tephrocybe atrata, and T. boudieri were included and a sequence of Lepista nebularis selected as out-group.

5.2. Results from the analysis The aligned data set had 1867 characters and after exclusion of variable positions 1592 remained for analysis. Of these 1285 were constant, 125 variable and parsimony uninformative and 182 were parsimony informative. The maximum parsimony analysis yielded 99800 equally most parsimonious trees (length = 483, Consistency index = 0.7143, Retention index = 0.867). One of these equally most parsimonious trees is presented in Figure 1. The tree is presented as a phylogram to show character state changes on branches. The bootstrap analysis recovered Lyophyllum as monophyletic with 93 % support and section Difformia monophyletic with 100% support. Four major clades within Difformia were strongly supported, corresponding to L. decastes (100%), L. fumosum (79%), L. shimeji (90%) and Lyophyllum sp. (100%). Within the L. decastes clade two subclades are strongly supprted: Japanese decastes (93%), and European decastes from deciduous forest/grassland (81%). The Lyophyllum sp. clade consists of the cultivar of L. decastes, commercially grown in Japan, and a sequence from a Norwegian specimen named L. fumosum. Within the L. fumosum clade, one subclade (unsupported) includes sequences of L. fumosum specimens from deciduous forest. In this clade we also find the sequence of the culture of L. shimeji, originating from Japan. The other subclade (95%) includes sequences of Swedish specimens of L. fumosum from coniferous forest and three sequences downloaded from GenBank® and the Japanese database NBRC. These three sequences from Japan cluster within the subclades with 100% support indicating that they are genetically different from the Swedish specimens, but the ecology of these specimens could not be confirmed. Within the L. shimeji clade the Japanese specimens form a subclade with 66% support. These sequences differ in two substitutions from the specimens originating from Fennoscandia, determined to L. decastes and originating from Pinus forest. One sequence downloaded from the Japanese data base cluster among the sequences from Fennoscandia, but the ecology of the specimen yielding this sequence could not be confirmed. The sequences of the two specimens determined to L. loricatum cluster in different clades, one with deciduous forest L. decastes and the other in the deciduous forest L. fumosum subclades.

11 6. Discussion

6.1. The clades The species in the decastes complex are very similar in appearance regarding both macro- and micro-morphology, furthermore there is a great amount of intraspecific plasticity in terms of basidiocarp stature, gills being adnate or decurrent, colouration and so forth. This is probably the main reason why it is common that the species often are mixed up both in literature and in herbaria around the world (Kalamees, 2004; Yamanaka, 2009). This is clearly seen in the results of this study since the different specimen sequences (both sequenced solely for this study and downloaded ones) tend to be scattered to a great extent between the different clades, regardless of their original attribution. The results strongly (93%) support the genus Lyophyllum as being monophyletic since the new (Norvell 2010) type species L. semitale cluster within the clade. We can in this study also confirm the results by Hofstetter et al. (2002) that Tephrocybe atrata cluster within the Lyophyllum clade indicating that it should be moved to the genus Lyophyllum. The Lyophyllum sp. clade is a bit surprising. A sequence determined as fumosum clusters together with a commercial decastes cultivar from Japan (100 % support). The reason for this could be that they constitute a separate species in the decastes complex found both in Japan and Europe. But since it could not be determined that the cultivar is of Japanese origin it can only be concluded that the clade is well supported and that it probably consists of a discrete species. Moncalvo & Clémençon (1992) confirmed that the decastes complex in Europe and Japan consists of five separate species denoted T1-T5 and that T1 was the only one encountered in both regions. This study shows that the components in T1 (European deciduous decastes and hatake shimeji) are not identical, but form separate clades (81% and 93% respectively) within the decastes clade indicating that they might be separate species. In the major fumosum clade three subclades appear. The “deciduous” clade is not supported but is still separated from the other two. In this subclade we find a L. shimeji culture, probably due to a mix-up at the source. The other subclades denoted “coniferous” and “Japan” separate distinctly from each other (100%). Although the ecology of the Japanese clade could not be confirmed one might suspect that it is associated with conifers. The conclusion is that the fumosum clade probably holds three separate species. Finally, the core of the study: the shimeji clade that is separated from the decastes clade by a support of 90%. Within the major clade, the Japanese shimeji forms a separate subclade (66%). The difference from Fennoscandian decastes/Pinus is however only 2 substitutions and could be due to time and distance factors. Furthermore one of the Japanese shimeji arrange within the Fennoscandian subclade. These subclades may support the two biological species of shimeji suggested in the study of Maeta et al. (2008), but to confirm that more investigations, including mating tests, must be undertaken. As for the two putative L. loricatum specimens included in this study they apparently do not constitute a species of their own, and do not even belong to the same species. Instead one fits nicely into the “deciduous fumosum” clade and the other is obviously a “deciduous decastes”. This result does not say there is not a L. loricatum species, but merely that these two specimens do not belong to that species, and that we could not confirm the species with molecular methods in the present study.

12 6.2. Which species is which? In this study the sequences arrange neatly in different clades correlating to which habitat (as far as known) the specimens were found in, i.e. similar sequences cluster together showing a pattern tied to ecology (fig. 1). Thus ecology seems to be an important character in determining species affinity. To separate the species is one thing, to determine which name that applies to which species is another thing. This is the reason why the species T1-T5, determined by Moncalvo & Clémençon still have no names. To establish which is which you must go to the sources. Regarding the species L. decastes the original habitat description by Fries (1818) corresponds quite well with the ecology of the “deciduous decastes” clade and supposedly it represents what Fries originally intended with L. decastes, but this needs to be further addressed. The original description of L. fumosum by Persoon (1801) and the sanctioning citation by Fries (1821) together depicts a rather broad habitat range that suits both European subclades of fumosum, and the descriptions therefore give no clue to what the name originally referred to. Hence further studies are needed and if possible one should try to find out how the original habitats were constituted. Lyophyllum shimeji is morphologically quite easy to determine, but since the species is based on the original written description of Kawamura and some drawings and because there is no type specimen it is unclear what Kawamura originally intended. So here also there is a need for further investigations. Regarding L. loricatum its species status could not be confirmed in this study. In recent literature the species has been regarded as a variety of L. decastes, but also as a species in its own right. To establish whether L. loricatum constitute a species or not more specimens must be scrutinized with regard to morphological, ecological and molecular aspects.

6.3. Conclusion This study has supported the genus Lyophyllum and the section Difformia as being monophyletic in accordance with earlier studies. It has also been confirmed that the Lyophyllum species growing in pine moorland in northern Fennoscandia, previously determined as L. decastes is indeed the same species as the Japanese gourmet mushroom L. shimeji. It was also indicated that the decastes complex could comprise as much as seven distinct species. Furthermore it was evident that ecology is a very important factor in determining species affinity in the decastes complex and that it should be more widely used as a complement to morphology. The indication that the supported clades include specimens that are confirmed to grow in different forest types could also imply that they form ectomycorrhiza with specific host trees.

6.4. Further investigations It would be interesting to further investigate the ecology of the genus Lyophyllum, its importance in species determination and also how widespread the ectomycorrhizal habit is within the genus. Also it would be a challenge to try to confirm or disprove the affinity of Singers (1986) American species to Difformia.

13 Acknowledgements

First of all I which to thank my supervisor Dr. Ellen Larson at the Institution for Plant and Environmental Science at the University of Gothenburg for taking me on as a student, finding my idea for a thesis interesting and for being so patient with a me being out of practice in biology. Then I want to thank my good friend Niclas Bergius for your encouragement and enthusiasm. I am also grateful to Dr. Akiyoshi Yamada (Shinshu University) for answering my questions, taking me and Niclas out in the woods and providing me with material for my study. Thank you also to Professor Koji Iwase (Tottori University) for providing me with material. Great thanks to all those people that have helped me collecting mushrooms for the study. I hope no one is omitted. Thank you; Barbro Domeij, Annika Fällman, Per-Axel Karlsson, Lill Eilertsen, Jörgen Olausson, Irene Andersson, Nada Lipovac, Susanne Anttila, Peter Jägerbro, Gunhild Nyberg, Tove Vestlin and Katrin Elm Blåholtz, without you the study would have been hard to accomplish! Then, last but not least I want to thank my parents for always believing in me although my studies never seem to end.

References

Avise JC, Robinson TJ (2008) Hemiplasy: A New Term in the Lexicon of Phylogenetics. Syst Biol 57:503–507

Breitenbach J, Kränzlin F (eds) (1991) Fungi of Switzerland: a contribution to the knowledge of the fungal flora of Switzerland. Vol. 3, Boletes and agarics. Strobilomycetaceae and Boletaceae, Paxillaceae, Gomphidiaceae, Hygrophoraceae, Tricholomataceae, Polyporaceae (lamellate). Vlg Mykologia, Lucerne

Buchalo AS, Diduk MY (2005) Micromorphological Characteristics of Culinary-Medicinal Mushroom and Fungi Cultures. International Journal of Medicinal Mushrooms 7:249-261

Clémençon H, Moncalvo J-M (1990) Taxonomic analysis of cultural characters in the group Lyophyllum shimeji (Agaricales, Basidiomycetes) from Japan. Trans Mycol Soc Japan 31:479-488

Fries EM (1818) Observationes Mycologicae 2: I–XII. Köpenhamn, Gerhard Bonnier

Fries EM (1821) Systema Mycologicum Vol 1, Lundae

Fries EM (1838) Epicrisis systematis mycologici, seu synopsis hymenomycetum. Uppsala

Fujita H, Itoh T, Kobayashi F, Ogawa M (1982) Ecological studies of Lyophyllum shimeji in Pinus densiflora forest. Trans Mycol Soc Japan 23:391-403

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhizas and rusts. Molecular Ecology 2:113–118

Garnica S, Weiss M, Walther G, Oberwinkler F (2007) Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure. Mycol Res III:1019– 1029

14 Gminder A, Böhning T (2008) Mushrooms & Toadstools of Britain and Europe. A&C Black Publishers, London

Hallingbäck T, Aronsson G (eds.) (1998) Ekologisk katalog över storsvampar och myxomyceter. [Macrofungi and myxomycetes of Sweden and their ecology] ArtDatabanken, SLU, Uppsala. 2nd revised and extended printing

Hibbett DS, Gilbert LB, Donoghue MJ 2000. Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes. Nature 407:506–508

Hofstetter V, Clémençon H, Vilgalys R, Moncalvo J-M (2002) Phylogenetic analyses of the Lyophylleae (Agaricales, Basidiomycota) based on nuclear and mitochondrial rDNA sequences. Mycol Res 106:1043-1059

Hopple Jr JS, Vilgalys R (1999) Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly. Molecular Phylogenetics and Evolution 13:1–19

Ingelström EA (1940) Svampflora: med 126 svampfotografier. Nordisk rotogravyr, Stockholm

Kalamees K (1994) Verbreitungskonspekt der osteuropäischen und asiatischen Tricholomatales-Arten I. Die Gattung Lyophyllum P. Karst. Zeitschrift für Mykologie 60:13- 17

Kalamees K (2004) Palearctic Lyophyllaceae (Tricholomatales) in northern and eastern Europe and Asia : the genera: Lyophyllum s.str., Hypsizygus, Gerhardtia, Calocybe s.str., Tricholomella, Rugosomyces, Asterophora. Scripta mycologica: 18, Tartu, Estonian Agricultural University, Institute of Zoology and Botany

Kawai M (1997) Artificial ectomycorrhiza formation on roots of air-layered Pinus densiflora saplings by inoculation with Lyophyllum shimeji. Mycologia 89(2):228-232

Kosaka H, Aikawa T, Ogura N, Tabata K, Kiyohara T (2001) Pine wilt disease caused by the pine wood nematode: The induced resistance of pine trees by the avirulent isolate of nematode. European Journal of Plant Pathology 107:667–675

Kyoto scientists grow Hon-shimeji mushrooms (1996) Kippo News, Vol.3 No.114 http://www.kansai.gr.jp/e/news/kipponews_detail_114.html#20100308092723772

Maeta K, Koike A, Mukaiyama H, Ochi T, Terashita T, Kitamoto Y, Aimi T (2008) Preparation and crossing of mating-capable monokaryons via protoplasting of the dikaryotic mycelia of a mycorrhizal mushroom, Lyophyllum shimeji. J Wood Sci 54:337-340

Matheny PB, Curtis JC, Hofstter V, Aime MC, Moncalvo JM, Ge ZW, Yang ZL, Slot JC, Ammirati JF, Baroni TJ, Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW, Seidl MT, Aanen DK, DeNitis M, Daniele GM, Desjarden DE, Kropp BR, Norvell LL, Parker A, Vellinga EC, Vilgalys R, Hibbett DS (2006) Major clades of Agaricales: a multi-locus phylogenetic overview. Mycologia 98:982–995

15

Moncalvo J-M, Toriola D, Clémençon H (1990) Analyse taxonomique du complexe Lyophyllum decastes sensu lato (Agaricales, Basidiomycetes) sur la base des caractères culturaux. Mycologia Helvetica 3:397-415

Moncalvo, J-M, Clémençon H (1992) A comparative study of fruit body morphology and culture characters in the Lyophyllum decastes complex (Agaricales, Basidiomycetes) from Japan and Europe. Trans Mycol Soc Japan 33:3-11

Moncalvo J-M, Rehner SA, Vilgalys R (1993) Systematics of Lyophyllum section Difformia based on evidence from culture studies and ribosomal DNA sequences. Mycologia 85:788- 794

Moncalvo J-M, Clémençon H (1994) Enzymatic studies as an aid to the taxonomy of the Lyophyllum decastes complex. Mycological Research 98:375-383

Moncalvo J-M, Lutzoni F, Rehner SA, Johnson J, Vilgalys R (2000) Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Systematic Biology 49:278-305

Moncalvo J-M, Vilgalys R, Redhead SA, Johnson JE, James TY, Aime MC, Hofstetter V, Verduin S, Larsson E, Baroni TJ, Thorn RG, Jacobsson S, Clémençon H, Miller OK (2002) One hundred and seventeen clades of euagarics. Molecular Phylogenetics & Evolution 23: 357-400

Norvell LL (2010) Report of the Nomenclature Committee for Fungi: 15. TAXON 59:291– 293

Nylén B (2001) Svampar i Norden och Europa. Stockholm, Natur och kultur/LT

Ohta A (1994) Some cultural characteristics of mycelia of a mycorrhizal fungus, Lyophyllum shimeji. Mycoscience 35:83-87

Pera J, Alvarez IF (1995) Ectomycorrhizal fungi of Pinus pinaster. Mycorrhiza 5:193-200

Persoon CH (1801) Synopsis methodica fungorum. Sistens enumerationem omnium huc vsque detectarum specierum, cum brevibus descriptionibus nec non synonymis et observationibus selectis. Gottingae : Apud Henricum Dietrich

Petersen JH, Vesterholt J (eds) (1990) Danske storsvampe (basidiesvampe). Gyldendalske Boghandel, Nordisk Forlag A. S., Copenhagen

Risberg L (2003) Goliatmusseron (Tricholoma matsutake) : kräver den en kontinuitet av träd? Master Thesis, Institutionen för skoglig mykologi och patologi, Sveriges lantbruksuniversitet, Uppsala

Ryman S, Holmåsen I (1994) Svampar - en fälthandbok. Interpublishing AB, Stockholm

Singer R (1986) The Agaricales in Modern Taxonomy. 4th edn. Koeltz Scientific Books, Königstein

16

Swofford D (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods) Version 4. Sinauer Associates, Sunderland, Massachusetts

Trappe JM (1962) Fungus Associates of Ectotrophic Mycorrhizae. The Botanical Review 28:538-606

Vesterholt J, Ludvig E.. 2008. Lyophyllum P. Karst. In: Knudsen H, Vesterholt J. (Eds.) Funga Nordica, Agaricoid, Boletoid and Cyphelloid genera, Nordsvamp, Copenhagen, pp. 498–506.

Yamanaka K (2009) Commercial cultivation of Lyophyllum shimeji. Mushroom News. Presented at the 6th International Conference on Mushroom Biology & Mushroom Products Bonn, Germany Sept. 29 – Oct. 3, 2008

Yamada A, Ogura T, Ohmasa M (2001) Cultivation of mushrooms of edible ectomycorrhizal fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis; I. Primordium and basidiocarp formation in open-pot culture. Mycorrhiza 11:59–66

17 Appendix

Table 2. Data of specimens sequenced in this study.

Species Coll. ID. / Origin Ecology, substrate

Lyophyllum decastes Cultivar / Jpn L. decastes H. Sundberg 2009-10-07 a / Nagano Pref. Jpn Grass lawn under Pinus L. decastes H. Sundberg 2009-10-07 b / Nagano Pref. Jpn Grass lawn under Pinus L. decastes B. Brunsson 1992-09-24 / Sk, Swe Grass lawn L. decastes O. Andersson 1990-10-16 / Bl, Swe Fagus forest in deep tilth L. decastes C-G Bengtsson 1991-09-29 / Sk, Swe Grass in hardwood forest L. decastes LAS 00-144 / Öl, Swe In hardwood forest L. decastes KHL 02-006 / Ång, Swe Old moist Picea forest L. decastes A. Fällman 2009-09-27 / LyL, Swe Pinus moorland, sand L. decastes LAS 00-062 / Dl, Swe Pinus moorland, occ. Picea L. decastes N. Lipovac 2009-09-11 / Vb, Swe Pinus moorland, sand L. decastes L. Eilertsen 2009-09-08 / Vb, Swe Pinus moorland, sand L. decastes H. Sundberg 2009-08-13 a / Vb, Swe Pinus moorland, sand L. decastes H. Sundberg 2009-08-13 b / Vb, Swe Pinus moorland, sand L. decastes H. Sundberg 2009-08-13 c / Vb, Swe Pinus moorland, sand L. decastes S. Anttila 2009-09-21 / OP, Fin Pinus moorland, sand L. decastes P-A Karlsson 2009-09-15 / Nb, Swe Pinus moorland, sand L. decastes J. Olausson 2009-09-22 / LL, Swe Pinus moorland, sand L. decastes B. Domeij 2009-09-13 / Vb, Swe Pinus moorland, sand L. fumosum S. Aase 1981-10-14 / Vestf, Nor Ruderal ground in grass L. fumosum S. Aase 1982-10-31 / Vestf, Nor Pasture L. fumosum N. Lipovac 2009-09-03 / Vb, Swe Picea forest, occ. Pinus & Betula L. cf. fumosum S. Aase 1981-07-21 / Vestf, Nor Park in grass L. fumosum H. Sundberg 2009-08-13 / Vb, Swe Pinus moorland, sand L. cf. fumosum R. Haukebø 1982-??-?? / Rog, Nor ? L. cf. fumosum B. Olsen 1982-10-06 / Akh, Nor On path in forest L. loricatum LAS 06-152 / Vg, Swe Alongside compost heap L. loricatum SJ 93011 / Vg, Swe Under hardwood L. semitale EL 187-09 / Vg, Swe Ruderal ground, church yard L. shimeji H. Sundberg 2009-10-07 / Nagano Pref. Jpn Mixed Pinus / Quercus forest L. shimeji Cultivar / Jpn L. shimeji Culture / Jpn

18

Fig. 1. One of the most parsimonious trees generated from the analysis presented as a phylogram. Bootrap values are indicated on braches. Major clades of section Difformia have been named.

19 Appendix

Fig. 2. Young L. decastes/Pinus from Fennoscandia (Photo Niclas Bergius)

Fig. 3. Young L. decastes/Pinus from Fennoscandia (Photo Niclas Bergius)

Fig. 4. Japanese Lyophyllum decastes (Photo Akiyoshi Yamada)

Fig. 5. Outgrown Fennoscandian L. decastes/Pinus (Foto Niclas Bergius)

Fig. 6. L. fumosum from Swedish pine moorland (Foto Niclas Bergius)

Fig. 7. Typical habitat of the Fennoscandian L. decastes/Pinus (Foto Niclas Bergius)