MOLECULAR AND MORPHOLOGICAL INVESTIGATIONS OF PEZIZALEAN MYCORRHIZAE by Keith James Williams B .Sc. (Agr.), The Nova Scotia Agricultural College, 1996 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE m BIOLOGY © Keith James Williams, 1999 THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA December 1999 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or any other means, without the permission of the author Abstract A phylogeny of the Pezizales was reconstructed using 18S rDNA sequence data. The data set consisted of 117 taxa. Several approaches were used to generate the phylogenetic trees, including Neighbor Joining consensus (ML algorithm), Neighbor Joining with bootstrap analysis (ML algorithm) and Maximum likelihood using quartet puzzling. The clades resulting from this analysis roughly corresponded to both the traditionally recognized families within the order Pezizales and to the results reported from recent phylogenetic work on this group. The clades delineated by this study included the Helvellaceae, Pezizaceae, Morchellaceae, Discinaceae, Sarcosomataceae, Otideaceae and Sarcoscyphaceae. Ofthese, only the Sarcoscyphaceae had no suspected or proven associates. The Otideaceae and Tuberaceae contained the highest number of proven ectomycorrhizal and biotrophic fungi. The Morchellaceae and Helvellaceae had the second highest number of proven associates. Pezizalean fruitbodies and nearby root tips were collected for analysis with particular attention paid to members of the Otideaceae, Tuberaceae, Morchellaceae and Helvellaceae. The root tips were morphotyped and the the fruitbodies were identified. DNA was extracted from the fruitbodies and the root tips that were potentially ascomycetous. A portion of the nuclear ribosomal DNA was amplified spanning the two internally transcribed spacer regions (ITS 1 and ITS2) in addition to a portion of the 28S gene. This was then digested using the restriction enzymes Alu I, Hinf I and Rsa I. The resulting restriction patterns were analysed, and a match between fruitbody and root tip iii indicated an association (it being ectomycorrhizal depended upon the presence of a Hartig net in the root tip) . No matches were found. Potentially ascomycetous root tip DNA and restriction patterns, in addition to herbarium samples of other Pezizalean fruitbodies were obtained from other sources in an attempt to find matches. Matches were found between Helvella leucomelaena and He/vella latispora fruitbody RFLP patterns and root tips morphotyped as MRA, E-strain and 'lightly colonized' under Abies lasiocarpa, Picea glauca X engelmanni and Pinus contorta var. latifolia. In addition, a match was found between a Trichophaea hemisphaerioides fruitbody RFLP pattern and an E-strain morphotyped tip under Abies lasiocarpa. lV Table of Contents Approval Abstract Table of Contents List of Tables List of Figures Acknowledgement Dedication 11 lll v Vlll IX XI Xll Prologue I. Rationale II. Research objectives III. Project procedural outline References 1 2 3 4 Chapter 1 Literature Review Abstract 1.0 The Pezizales 1.1a Ectomycorrhizal ecology and function 1.1b Ectomycorrhizal structure 1.2 Molecular systematics 1.3 References 5 5 6 9 10 11 13 Chapter 2 Phylogenetic distribution of ectomycorrhizal Pezizales 2.0 Introduction 2.1 Materials and Methods 2.la Sequence acquisition 2.1b Molecular protocols 2.2 Results 2.2a Tree topology 2.2b Agreement between reconstruction methods 2.3 Discussion 2.3a Phylogenetic relationships within the Pezizales 2.3b The evolution of the mycorrhizal habit 2.3c The origins of mutualism 2.3d New ectomycorrhizal taxa since Maia et al. 1996 2.3e Proof of an ectomycorrhizal association 2.3fThe symbiotic continuum 2.4 References v 17 18 20 20 20 25 25 26 44 44 48 51 51 52 53 54 Chapter 3 Morphological and molecular comparisons of fruitbody and root tip collections Abstract 3.0 Introduction 3.1 Methods 3.1a Sampling 3.1 b DNA extraction 3.1c Amplification 3.1d Restriction digestion 3.1e RFLP analysis 3.1 f Trouble shooting techniques 3.2 Results 3.3 Discussion 3.3a Recalcitrant samples 3.3b Lack ofRFLP matches between fruitbodies and root tips 3.4 References Chapter 4 Comparison of RFLP patterns from various databases Abstract 4.0 Introduction 4.1 Materials and Methods 4.1a Sporocarp collection 4.1b Ectomycorrhizal root tip collection 4.1c DNA extraction, amplification, restriction digestion and RFLP analysis 4.1d Trouble shooting techniques 4.2 Results 4.2a Taxa analyzed in this study 4.2b PCR trouble-shooting 4.2c Trichophaea hemisphaerioides as a below-ground associate 4.2d Helvella leucomelaena as a below-ground associate 4.2e He/vella latispora as a below-ground associate 4.2f Wilcoxina rehmii as a below-ground associate 4.2g Wilcoxina mikolae as a below-ground associate 4.3 Discussion 4.3a Morpho type as a function of factors other than mycobiont 4.3b Preferential amplification 4.3c Concerted evolution 4.3d Trichophaea hemisphaerioides 4.3e Root inhabiting members of the genus Helvella 4.3fWilcoxina spp. 4.3g RFLP band match tolerance: guideline or rule? 4.3h Fruitbodies that did not match with root tips 4.4 References Vl 57 57 58 61 61 68 69 71 71 72 73 82 82 84 87 90 90 91 91 91 95 98 98 98 98 100 100 101 106 107 109 112 112 113 114 115 117 119 120 121 123 Chapter 5 Conclusions 5.0 Efficacy of the techniques used for ectomycorrhizal identification 5.1 The use of phylogenetics for identifying ectomycorrhizal fungi 5.2 Future directions 5.3 References 127 127 128 129 131 Glossary 132 Appendix 1: Species of basidiomycete fruitbodies used in Chapter 4 and their RFLP accession codes Appendix II: Morphotype descriptions from Chapter 3 Appendix III: RFLP patterns 135 138 148 Vll List of Tables Table 1 Information on taxa used in phylogenetic analysis Table 2 Fruitbody and root tip type accession codes and habitat information Table 3 Ectomycorrhizal root tip morphotype descriptions Table 4 Fruitbody DNA extraction/ PCR trouble shooting techniques Table 5 Root tip DNA extraction/ PCR trouble shooting techniques Table 6 Fruitbody/ culture reference samples analyzed with collection data and source of material Table 7 ECM root tip RFLP database morphotype and location information Table 8 ECM root tip morphotype, host and habitat information Table 9 Results of ascomycete fruitbody/ root tip RFLP comparisons Table 10 Banding topologies of Trichophaea hemisphaerioides (thd) and two E-stain root tips Table 11 Banding topologies ofHelvella leucomelaena (14u) and several 'MRA' root tips Table 12 Banding topologies ofHelvella leucomelaena (14u) and several E-strain root tips Table 13 Banding topologies ofHelvella leucomelaena (14u) and a type 0 ro~~ Table 14 Banding topologies ofHelvella leucomelaena (14u) and several type 5 root tips Table 15 Banding topologies of Helvella latispora ( 13p) and some MRA type root tips Table 16 Banding topologies ofHelvella latispora (13p) and root tip types 3 and4 Table 17 Banding topologies ofWilcoxina rehmii (a1436) and some 'MRA' root tips Table 18 Banding topologies ofWilcoxina rehmii (a1436) and some E-strain root tips Table 19 Banding topologies ofWilcoxina rehmii (a1436) and two Type 5 root tips Table 20 Banding topologies ofWilcoxina mikolae (a1789) and some MRA root tips Table 21 Banding topologies ofWilcoxina mikolae (al789) and E-strain root tips Table 22 Banding topologies ofWilcoxina mikolae (a1789) and Type 5 root tips Table 23 Banding topologies ofWilcoxina mikolae (a1789) and a Type K root tip Vlll 22 64 67 76 77 93 96 96 99 101 103 104 1M 104 107 107 108 108 109 110 110 111 111 List of Figures Fig. 1 Neighbour Joining consensus tree Fig. 2 Neighbour Joining bootstrap tree (100 replicates) Fig. 3 Maximum Likelihood (quartet puzzling) tree Fig. 4a The Neighbour Joining consensus tree's Morchellaceae clade Fig. 4b The Neighbour Joining bootstrap tree's Morchellaceae clade Fig. 4c The Maximum Likelihood quartet puzzling tree's Morchellaceae clade Fig. Sa The Neighbour Joining consensus tree's Discinaceae clade Fig. 5b The Neighbour Joining bootstrap tree's Discinaceae clade Fig. 5c The Maximum Likelihood quartet puzzling tree's Discinaceae clade Fig. 6a The Neighbour Joining consensus Tree's Tuberaceae clade Fig. 6b The first Neighbour Joining bootstrap tree's Tuberaceae clade Fig. 6c The second Neighbour Joining boostrap tree's Tuberaceae clade Fig. 6d The Maximum Likelihood quartet puzzling tree's Tuberaceae clade Fig. 7a The Neighbour Joining consensus tree's Helvellaceae clade Fig. 7b The Neighbour Joining bootstrap tree's Helvellaceae clade Fig. 7c The Maximum Likelihood quartet puzzling tree's Helvellaceae clade Fig. 8a The Neighbour Joining consensus tree's Sarcoscyphaceae clade Fig. 8b The Neighbour Joining bootstrap tree's Sarcoscyphaceae clade Fig. 8c.The Maximum Likelihood tree's Sarcoscypaceae clade Fig. 9a The Neighbour Joining consensus tree's Sarcosomataceae clade Fig. 9b The Neighbour Joining bootstrap tree's Sarcosomataceae clade Fig. 9c The Maximum Likelihood quartet puzzling tree's Sarcosomataceae clade Fig. lOa The Neighbor Joining bootstrap tree's Otideaceae clade IX 29 30 31 32 32 32 33 33 33 34 34 34 35 36 36 36 37 37 37 38 38 38 39 Fig. lOb The Neighbour Joining bootstrap tree ' s Otideaceae clade Fig. lOc The Maximum Likelihood quartet puzzling tree's Otideaceae clade Fig. lOd The Maximum Likelihood quartet puzzling tree's second Otideaceae clade Fig. lla The Neighbour Joining consensus Pezizaceace clade Fig. llb The Neighbour Joining bootstrap tree's Pezizaceae clade Fig. llc The Maximum Likelihood quartet puzzling tree ' s Pezizaceae clade Fig. 12a The Neighbor Joining consensus tree's Ascobolaceae clade Fig. 12b The Neighbour Joining bootstrap tree's Ascobolaceae clade Fig. 12c The Maximum Likelihood quartet puzzling tree's Ascobolaceae clade Fig. 13 Collection sites in the pacific northwest Fig. 14 Collection areas near Prince George Fig. 15 Root tip system collected under Trichophaea hemisphaerioides (thrt) Fig. 16 Root tip system collected under Sarcosphaera coronaria (scrt2) Fig. 17 Senescent monopodia! pyramidal system collected under Sarcosphaera coronaria (scrt2) Fig. 18 MRA like tips under He/vella elastica (hert) Fig. 19 A net synenchymal mantle found under an Otidea sp. fruitbody (olrt1) Fig. 20 An interlocking irregular synenchymal mantle found under a Trichophaea hemisphaerioides fruitbody (thrt) Fig. 21 A non interlocking irregular synenchymal mantle found under a Pseudorhizina sphaerospora fruitbody (gb3rtl) Fig. 22 An irregular synenchymal mantle found under a P. sphaerospora fruitbody (gb3rt1) X 39 40 40 41 41 42 43 43 43 62 63 80 80 80 80 81 81 81 81 Acknowledgement I wish to thank Dr. Keith Egger for his tireless guidance through the course of this thesis . I would also like to thank Dr. Hugues Massicotte for his unfailing enthusiasm towards fungi and all other things. Dr. Kathy Lewis was of invaluable help with the thesis revisions and Linda Tackaberry for her aid in morphotyping and for those afternoon conversations. I would like to thank the members of my family for their years of support and encouragement; my uncle Alec Lindsay, my partner Michelle and our two dogs Magee and Dougal, my mum, Elspeth and dad, Keith, in addition to my sisters Kathleen and Miss Sarah-bear. Although many people dislike Prince George, I like it. I think the main reason why is because of the warm friendships and challenging rivalries I experienced here. Thank you: Thierry Betrand Clavel. . .'death to idle kings', Monsignor (Shawn) Nelson, Yumi Kanaoka, William 'dancing-vegetarian-showgirl' Scherk, Aniko 'chocolat' Varga, Kevin James Ward and Mark 'watch me go' Thompson. Without the help and camaraderie of the people in the lab, I would be lost, thanks Quentin, Brent, Linda, Tamara, Joanne and everyone else. I would also like to thank the Gaelic group for providing me with an important distraction. Michael O'Mallaig, Nuala dePuer, Maureen MacMoran, Marc Mc'Thomais, Derrick McCandless, Shawn Nelson and Mauriah .. ... .go raibh maith agat. Festina tarde! XI Dedication This thesis is dedicated to my gran, Margaret Hamilton Lindsay. I appreciate the close relationship we have. Xll PROLOGUE I. RATIONALE Ectomycorrhizae (ECM) are mutualistic associations between the roots of most tree species and some higher fungi (Basidiomycotina and Ascomycotina) (Harley and Smith 1983 ). Ectomycorrhizae are essential components of the boreal forest ecosystem (Kimmins 1997) and aid in nutrient uptake, pathogen resistance and drought tolerance (Read 1991). The majority of documented ectomycorrhizal fungi belong to the subdivision Basidiomycotina (Trappe 1971). The lack of documented ascomycetous ECM could be due to a few factors . First, many ascomycetes are asexual and therefore produce no discernible fruiting bodies, such as the ECM Cenococcum geophilum and Phialocephala fortinii. Second, the sexual fruiting bodies of many ascomycetes are inconspicuous, described somewhat disparagingly by David Arora (1986) as 'small, dingy cups '. Finally, the order of ascomycetes containing the most known ECM fungi, the Pezizales, are not always found under potential host trees. This has led researches in the past to the assumption that many of these fungi are only capable of a saprotrophic lifestyle (Petersen 1985). Since the late eighties, species of fungi within the order Pezizales have been demonstrated to be parasitic, biotrophic, ectomycorrhizal or saprotrophic depending upon the environmental conditions (Egger 1986, Egger and Paden 1986, Scales and Peterson 1991 b). This discovery has established the Pezizales as a legitimate target group for ECM investigations. Only 6 genera ofPezizales have been confirmed as ECM, but other research suggests that others may form ECM (Egger 1986). Clearly, more work needs to be done to identify and characterize Pezizalean ectomycorrhizae. II. RESEARCH OBJECTIVES The overall objective of this thesis was to document previously undescribed ectomycorrhizal Pezizales. The objective of Chapter 2 was to identify 'ECM-rich' clades within the Pezizales from a phylogenetic reconstruction of the order Pezizales. Fungi from these ECM rich clades were the target in chapter 3, in which the objective was to document ECM associations between fruitbodies and nearby host trees. Field collections of these ' target' fungi and ECM root tips from nearby hosts were compared using both morphological and molecular techniques (PCR-RFLP) in an attempt to find a match, thus demonstrating an association. The objective of chapter 4 was to find RFLP pattern matches between both the fruitbodies and root tips collected as part of the last study and those acquired from other sources, which are represented in databases ofPCRRFLP patterns at UNBC. 2 III. PROJECT PROCEDURAL OUTLINE Three approaches were used to identify and characterize Pezizalean ectomycorrhizae. For the first approach, an 18S rDNA sequence based phylogeny of the Pezizales was constructed using both distance (neighbour joining (NJ) and NJ with bootstrapping) and probabilistic (maximum likelihood with quartet puzzling) approaches. These phylogenies provided an evolutionary framework from which to base the selection of potentially mycorrhizal taxa. This analysis is presented in Chapter 2. The second approach consisted of the collection of fruitbodies of suspected taxa, along with a root tip sample from under or nearby the fruitbody. These collections were then processed, fruitbodies were identified and root tips were described both macro- and microscopically. Finally, DNA was extracted from both root tips and fruitbodies and amplified using the primers ITS 1 and NL6Bmun which targets a region encompassing the ITSl , 5.8S gene, ITS2 and part of the 28S gene of the nuclear ribosomal RNA gene repeat. This amplified product was then cut with a series of restriction enzymes (Alui, Hinfi., Rsai), each enzyme recognising a specific sequence. Restriction digests were then run on a high viscosity electrophoretic agarose based gel. Ideally, the banding pattern for each enzyme will be different but unique to that species. Banding patterns were compared between root tips and the fruitbodies of suspected ectomycorrhizal taxa. Identical patterns indicated that the fungus examined was either colonizing or growing nearby the root tip and different patterns indicated that there was either no association or another fungus growing on or near the root tip under question preferentially amplified. The results of this study are presented in Chapter 3. 3 Finally, the third approach involved the merging ofPCR-RFLP patterns from fruitbodies and root tips from other sources (other thesis research studies and on going research projects), into my database ofPezizalean fruitbody and suspected pezizalean root tip PCR-RFLP patterns. These were compared in the manner listed above, with an identical match suggesting an association. The results of this study are presented in Chapter 4. REFERENCES Arora, D. 1986. Mushrooms Demystified: A comprehensive guide to the fleshy fungi . 2nd ed. Ten Speed Press, Berkeley. Egger, K.N. 1986. Substrate hydrolysis patterns of post-fire ascomycetes (Pezizales). Mycologia 78: 771-780. Egger, K.N. and J.W. Paden. 1986. Biotrophic associations between lodgepole pine seedlings and postfire ascomycetes (Pezizales) in monoxenic culture. Can. J. Bot. 64: 2719-2725. Harley, J.L. and S.E. Smith. 1983. Mycorrhizal Symbiosis. Academic Press, London. Kimmins, J.P. 1997. Forest Ecology. 2nd ed. Prentice Hall, London. Petersen, P.M. 1985. The ecology of Danish soil inhabiting Pezizales with emphasis on edaphic conditions. Opera Bot. 77:1-38. Read, D.J. 1991. Mycorrhizas in ecosystems. Experientia 47: 376-391. Trappe, J.M. 1971. Mycorrhiza forming ascomycetes. In: Mycorrhizae (ed. E . Hacskaylo), pp. 19-37. USDA, Forest Service, Miscellaneous Publication 1189. 4 Chapter 1 Literature review Abstract The Pezizales are an order of operculate, ascomycetous cup fungi . Pezizalean taxa occupy varied ecological niches; some are saprotrophic, some are parasitic, and some members of the Pezizales have been confirmed as ectomycorrhizal. This thesis proposes to document other ectomycorrhizal members of the Pezizales. Ectomycorrhizal associations are mutualistic symbioses that occur between various members of the higher fungi (Basidiomycotina and Ascomycotina) and woody host plants, usually coniferous or deciduous trees. The woody host may gain increased nutrient uptake, drought tolerance and pathogen resistance. The fungal symbiont gains photosynthates. The one morphological feature used to positively identify an ectomycorrhizal root tip is the Hartig net. This is the labyrinth-like fungal structure that surrounds the cortical or epidermal cells of the host and acts as the organ of exchange between the two symbionts. Molecular approaches, such as PCR-based RFLP or sequence analysis are common methods in the fungal systematist's tool kit. Restriction Fragment Length Polymorphism, or RFLP, analysis utilizes restriction endonucleases which cut the amplified DNA at certain recognition sites. These RFLP patterns are then used to compare fungal isolates. While not a good means of determining precise phylogenetic relationships, RFLP patterns can be effectively used to identify samples (via comparison of patterns). DNA sequences are the primary form of data used for phylogenetic reconstruction. 5 1.0 The Pezizales The Pezizales are an order of the ascomycete subclass Discomycetidae. The pioneering French mycologist, Emil Boudier, at the end ofthe 19th century, separated discomycetous fungi as operculate or inoperculate groups based on the presence or absence of an operculum, which is a hinged lid like opening on top of the ascus, or spore sac which allows for forcible spore discharge (Harrington 1999). Further refining this concept, Rifai (1968) divided the Pezizales into two suborders, the Sarcoscyphineae and the Pezizinae based on the presence of sub-operculate asci in the former and operculate asci in the latter. Although not fully accepted until years later, this taxonomic character is still in current usage (Landvik 1996). Further microanatomical perspectives were offered by Eckblad (1968) who focused on the morphology of the apothecial excipulum and the excipular hairs in taxonomic delimitation instead of the placement of the operculum, which he viewed as questionable. The discomycetes are traditionally recognised by the presence of a disc-shaped, or cupshaped apothecium, but there is considerable variation in apothecial shape. Korf(1973) illustrated the different apothecial forms present in the Pezizales ranging from the gyrose pileus as seen in Gy romitra and some species of Discina to the pitted ascocarp (e.g. Morchella) to the semi-hypogean apothecium opening by splitting as seen in species of Sarcosphaera and Geopora. In a landmark publication, Korf (1973) erected a new family, the Sarcosomataceae, merged the Thelebolaceae with the Ascobolaceae and 6 merged the Humariaceae with the Pyronemataceae in addition to introducing 6 new tribes. Prior to 1979, the orders Pezizales and Tuberales, for epigeous and hypogeous taxa respectively, were separate. Based upon morphological similarities, Trappe (1979) emended the Pezizales to include the Tuberales under several familial designations. This was based on several lines of evidence. The main difference between the two orders was forcible spore discharge in the Pezizales versus no spore discharge in the Tuberales. The first line of evidence argued that this difference is artificial and difficult to apply in some taxa. For example, Burdsall ( 1965) discovered that Geopora cooperi, then classified in the Tuberales, exhibited forcible spore discharge, unlike its hypogeous relatives. He reassigned Geopora spp. to the Pezizales. Secondly, Korf (1973) suggested that members of the Tuberales were derived from members ofthe Pezizales. Finally, Trappe ( 1979) based this emendation on his own studies, which demonstrated microscopic similarities between epigeous and hypogeous taxa. New families were erected to accommodate those Tuberales that he could not place within the Pezizales including Balsamiaceae, Geneaceae, Terfeziaceae, and Tuberaceae. Since then, more morphological evidence has accumulated supporting this transfer. Kimbrough et al. (1991) suggested that the hypogeous Hydnobolites cerebriformis Tul., at the time in the Terfeziaceae, belonged in the Pezizaceae. Characters suggesting that H. cerebriformis belonged in the Pezizaceae included the presence of electron-dense, biconvex bands in septal pores of asci, weak bluing of asci in iodine, and similar spore wall deposition. In 1984, Pfister linked the epigeous Japhneadelphus (Otideaceae), to the hypogeous Genea. 7 Another paper (Li-Tzu and Kimbrough 1994) supports this by providing evidence for a link between Genea gardnerii and the Otideaceae. The proposal of this relationship is based on the presence of a fan-shaped septal plugging structure with a double translucent band at each side ofthe plug and a similar spore wall deposition. With the development of molecular phylogenetics, studies continue to link hypogeous fungi to epigeous families (O'Donnell et al. 1997, Landvik et al. 1998, Norman and Egger 1999). The Pezizales exhibit a wide range oflifestyle modes. The majority ofPezizales is considered saprotrophic, such as members of the genera Peziza, Gyromitra , Ascobolus, Py ronema, and Discina (Petersen 1985). Several members of the Pezizales are adapted to degrade post-fire debris (Egger 1986). Other members ofthis group are plant pathogens, such as Caloscypha fulgens, which is a conifer seed pathogen (Paden et al. 1978) and Rhizina undulata, which causes minor damage to conifer seedlings (Tylutki 1979). Some Pezizales are confirmed biotrophs, forming undetermined associations with host plants, examples include He/vella aestivalis (Weidemann et al. 1999) and Geopyxis carbonaria (Vdilstad et al. 1998). Finally, some Pezizales are ectomycorrhizal, meaning that they form mutualistic associations, such as Sphaerosporella brunnea (Danielson 1984), Wilcoxina spp. (Yang and Korf 1985), Tuber spp., Genea hispidula, Leucangium carthusianum and Balsamia alba (Agerer 1987 -1998). 8 l.la Ectomycorrhizal ecology and function Mycorrhizae are segregated into classes based on morphology of the symbiosis and the specific partners involved (Molina and Amaranthus 1990). The eight main classes of mycorrhizae include arbuscular mycorrhizae, ectomycorrhizae (EM), ectendomycorrhizae, cistoid, arbutoid, monotropoid, ericoid and orchidaceous (Harley and Smith 1983). Ectomycorrhizae occur primarily in forest ecosystems at intermediate altitudes and latitudes characterized by surface litter accumulation, generally in the boreal forest (Read 1991 ). ECM are found in the slow decomposing, acidic surface layers of the soil. Soils with adequate moisture and a seasonally fluctuating supply of nitrogen and phosphorous have greater ECM populations (Read 1991). The main hosts are found in the families Pinaceae, Betulaceae and Fagaceae (Molina and Amaranthus 1990). ECM fungi serve to mobilize previously unavailable elements essential for plant growth (Read 1991 ). Some ectomycorrhizal fungi produce polyphenol oxidase, cellulase and phosphatase (Giltrap 1982; Linkins and Antibus 1981; Alexander and Hardy 1981). EM roots capture and store both phosphate ions (Harley and Smith 1983) and organic nitrogen from protein (Abuzinadah and Read 1989). This increase in nutrient uptake results in increased plant photosynthetic capacity (Vogt et al. 1982). In addition, the hyphae of mycorrhizal fungi, which extend through the soil, have a greater surface area to volume ratio than the plant feeder roots (Read 1991). This gives them the ability to access water in soil pores normally inaccessible to the uncolonized roots, thus they aid in 9 drought tolerance (Nelsen and Safir 1982). Some other less examined benefits attributable to mycorrhizal infection include the tolerance of heavy metals, protection against pathogens, lengthening of root life, and finally resistance to high soil temperatures, soil toxins and extreme pH (Harley and Smith 1983; Molina and Arnaranthus 1990). 1.1 b Ectomycorrhizal structure The primary means of ectomycorrhizal characterization is through examination of the ECM root tip macro- and micro-morphology. There are two main features which define EM. First is the hyphal mantle or sheath which surrounds the root. Emanating hyphae extends from the mantle and penetrates the substrate acting as the primary organ of absorption (Harley and Smith 1983). This is what is typically observed as the ' mycorrhizal root tip', which macroscopically appears as a swollen, sometimes coloured root tip, often with a prominent hyphal mantle and emanating hyphae. Second, the hyphae which form the mantle also grow inward forming labyrinth-like projections against the root cell wall. This structure, called the Hartig net, forms around the epidermis of angiospermous hosts and the cortical layer in gymnosperms (Harley and Smith 1983). The Hartig net represents the interface of exchange between the plant and fungus. The presence of a Hartig net is the feature defining an association as ectomycorrhizal. The Hartig net will not penetrate the cell wall of cortical cells, hence the prefix ecto- (Barbour et al. 1987). 10 A variant of the ectomycorrhizal association is the ectendomycorrhizal association, in which the fungal associate penetrates the cortical cell walls of the host. The association between Sphaerosporella brunnea and Pinus banksiana was described by Danielson ( 1984) as ectomycorrhizal, but the same fungus was described as ectendomycorrhizal by Egger (1986). This supports Wilcox's (1983) concept of gradations in the form and function of symbioses between plants and fungi , with the association shifting on different hosts, or under different environmental conditions. 1.1 b Molecular systematics PCR, the polymerase chain reaction (Mullis and Faloona 1986), according to Palumbi (1996) is 'one of the standard colours on the systematist's palette'. Since the advent of the polymerase chain reaction (Mullis and Faloona 1986), there has been a flurry of research using this powerful tool with applications in population genetics, phylogenetics, phylogeography, ecology and developmental biology (Harvey et al. 1996). The polymerase chain reaction is based on the ubiquitous process of DNA replication. Enzymes which copy DNA are referred to as DNA polymerases. The DNA polymerase enzyme used in PCR is called Taq polymerase after the thermophilic bacteria from which it was isolated, Thermus aquaticus. DNA polymerases recognize single stranded DNA as a template. Double stranded DNA is copied, or amplified, during a series of heating and cooling reactions . These steps are divided in three: denaturation, annealing and extension (Palumbi 1996). 11 DNA fragment and sequence data were not used for examining relatedness in the higher fungi until the early 1990's (Bruns et. a!. 1990, White et al. 1990, Egger et al. 1991 ). This was met with a certain degree of skepticism by traditional alpha-level taxonomists who, some suggest, felt insecure because the terminology and techniques associated with molecular systematics were previously the province ofbiochemists (Seifert 1996). Sequence based analysis not only presented more characters for taxonomic investigation and helped factor out the effects of environment on taxonomic characters but reduced the chance of homoplasy, which is a similarity in character states not due to common ancestry (Seifert 1996). The algorithmic approaches used in this study were all created before the PCR breakthrough. The two main types of phylogenetic inference algorithms are qualitative (character state) and quantitative (distance). Character state algorithms assign a character state for each character in each sample. Maximum Likelihood is an example of a character state approach. Maximum Likelihood inference is a probabilistic approach evaluating an evolutionary hypothesis based on the probability that, under the model of evolution assumed, the hypothesized data matches the observed data. This approach has been attributed to Fisher in the early 1920's (Fisher 1921 in Goldman 1990). The second, and last approach used in this study, Neighbour Joining is a distance method, meaning that the analysis begins with a pairwise comparison matrix of estimated genetic distances between taxa (Swofford et al. 1996). It differs from traditional cluster analysis in that, by adjusting branch lengths between nodes based on a mean divergence from other nodes, it accounts for differing rates of molecular evolution (Swofford et al. 1996). 12 1.3 References Abuzinadah, R.A. and D.J. Read. 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. V. Nitrogen transfer in Birch grown in association with mycorrhizal and non-mycorrhizal fungi. New Phytol. 112: 61-68. Agerer, R. 1987-1998. Colour Atlas ofEctomycorrhizae. Schwabisch Gmund, Germany. Einhorn-Verlag Eduard Dietenberger. Alexander, I.J. and K. Hardy. 1981. Surface phosphatase activity of Sitka spruce mycorrhizas from a Serpentine site. Soil Bioi. Biochem. 13: 301-305 Arora, D. 1986. Mushrooms Demystified: A comprehensive guide to the fleshy fungi. 2nd ed. Ten Speed Press, Berkeley. Barbour, M.G., J.H. Burk and W. Pitts. 1987. Terrestrial Plant Ecology. Benjamin Cummings Publishing Co., CA. Bruns, T. D., R. Fogel, J. W. Taylor. 1990. Amplification and sequencing of DNA from fungal herbarium specimens. Mycologia 82:175-184. Burdsall, H.H. 1965. Operculate asci and puffing of ascospores in Geopora (Tuberales ). Mycologia, 57: 485-488. Danielson, R.M. 1984. Ectomycorrhiza formation by the operculate Discomycete Sphaerosporella brunnea (Pezizales). Mycologia 76(3) :454-461. Eckblad, F.-E. 1968. The genera of the operculate Discomycetes: a reevalutation of their taxonomy, phylogeny and nomenclature. Nytt. Mag. Bot. 15: 1-191. Egger, K.N. 1986. Substrate hydrolysis Patterns ofPost-fire Ascomycetes (Pezizales). Mycologia 78(5): 771-780. Egger, K.N. and J.W. Paden. 1986. Biotrophic associations between lodgepole pine seedlings and postfire ascomycetes (Pezizales) in monoxenic culture. Can. J. Bot. 64: 2719-2725. Egger, K.N., R.M. Danielson and J.A. Fortin. 1991. Taxonomy and population structure of E-strain mycorrhizal fungi inferred from ribosomal and mitochondrial DNA polymorphisms. Mycological Research 95: 866-872. Giltrap, N.J. 1982. Production ofpolyphenol oxidases ectomycorrhizal fungi with special reference to Lactarius species. Trans. Brit. Mycol. Soc. 78- 75-81. 13 Goldman, N. 1990. Maximum likelihood inference of phylogenetic trees with special reference to a Poisson process model of DNA substitution and to Parsimony analysis. Syst. Zool. 39(4): 345-361. Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, London. Harvey, P.H. , E.C. Holmes, A.0. Mooers and S. Nee. 1996. Inferring evolutionary processes from molecular phylogenies, pp. 313-333. In: R.W. Scotland, D.J. Siebert, D.J. Williams (eds.), Models in Phylogeny Reconstruction. Systematics Association Special Volume 52, Oxford. Harrington, F.A. , D.H. Pfister, D. Potter and M.J. Donoghue. 1999. Phylogenetic studies within the Pezizales. I. 18S rRNA sequence data and classification. Mycologia, 91: 4150. Kimbrough, J.W., C.G. Wu and J.L. Gibson. 1991. Ultrastructural evidence for a phylogenetic linkage of the truffle genus Hydnobolites to the Pezizaceae. Bot. Gaz. 152(4): 408-420. Kimmins, J.P. 1997. Forest Ecology. 2nd ed. Prentice Hall, London. Korf, R.P. 1973 . Discomycetes and Tuberales. In: The Fungi, A Taxonomic Review with Keys: Ascomycetes and Fungi Imperfecti. Pp. 249-307. Vol. IV A. Ainsworth, G.C., F.K. Sparrow, A.S . Sussman (eds.). Academic Press, New York. Landvik, S. 1996. Phylogenetic rDNA studies ofDiscomycetes (Ascomycota). Dept. of Ecological Botany, Umea University. PhD dissertation. Landvik, S., K.N. Egger and T. Schumacher. 1998. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analysis of SSU rDNA sequences. Nordic J. Bot. 17:403-418. Linkins, A. E. and Antibus, R.K. 1981. Mycorrhizae of Salix rotundiflora in coastal arctic tundra, in:Arctic and Alpine Mycology, pp. 509-531. Eds. G.A. Laursen and J.F. Ammirati. University of Washington Press, Washington. Li-Tzu, L. and J.W. Kimbrough. 1994. Ultrastructural evidence for a relationship of the truffle genus Genea to Otideaceae (Pezizales). Int. J. Plant Sci. 155 : 235-243. Molina, R. and M. Amaranthus. 1990. Rhizosphere biology: ecological linkages between soil processes, plant growth and community dynamics. Symposium on Management and Productivity ofWestem-Montane forest soils, Boise, Idaho, April10-12, 1990. Mullis, K.B. and F.A. Faloona. 1987. Specific synthesis ofDNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol. 155 :335-350. 14 Norman, J.E. and K.N. Egger. 1999. Molecular phylogenetic analysis of Peziza and related genera. Mycologia, 91(5):820-829. Nelsen, C.E. and G.R. Safir. 1982. Increased drought tolerance of mycorrhizal onion plants caused by improved phosphorous nutrition. Planta 154: 407-413. O'Donnell, D., E. Cigelnik, N.S. Weber. 1997. Phylogenetic relationships among ascomycetous truffles and true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48-65. Paden, J.W., J.R. Sutherland and T.A.D. Woods. 1978. Caloscyphafulgens (Ascomycetidae: Pezizales): the perfect state of the conifer seed pathogen Geniculodendron pyriforme (Deuteromycotina, hyphomycetes). Can. J. Bot. 56: 23752379. Palumbi, S. 1996. Nucleic Acids II: The Polymerase Chain Reaction. In Molecular Systematics. Hillis, D.M., Moritz, C and Mable, B.K. Sinauer Associates, Sunderland, Mass. pp. 205-245. Petersen, P.M. 1985. The ecology ofDanish soil inhabiting Pezizales with emphasis on edaphic conditions. -Opera Bot. 77:1-38. Copenhagen. Pfister, D.H. 1984. Genea-Jafneadelphus- A Tuberalean- Pezizalean connection. Mycologia 76(1): 170-172. Read, D.J. 1991. Mycorrhizas in ecosystems. Experientia 47: 376-391. Rifai, M.A. 1968. The Australasian Pezizales in the herbarium of the Royal Botanic Gardens Kew. Verh. Kon. Ned. Akad. Wetensch., Afd. Naturrk., Tweede Sect. 57: 1-295 . Scales, P.F. and R.L. Peterson. 1991b. Structure of ectomycorrhizae formed by Wilcoxina mikolae var. mikolae with Picea mariana and Betula alleghaniensis. Can. J. Bot. 69: 2149-2157. Seifert, K.A., B.D. Wingfield and M.J. Wingfield. 1995. A critique ofDNA sequence analysis in the taxonomy of filamentous Ascomycetes and ascomycete anamorphs. Can. J. Bot. 73 (Suppl. 1):S760-S767. Smith, S.E. and D.J. Read. 1997. Mycorrhizal Symbiosis. 2nd ed. Academic Press, San Diego, California. Swofford, D.L. G.J. Olsen, P.J. Waddell and D.M. Hillis. 1996. Phylogenetic Inference. In: Molecular Systematics 2nd ed. eds: D.M. Hillis, C. Moritz and B.K. Mable. Sinauer Associates, Sunderland, Mass. 15 Trappe, J.M. 1971a. Mycorrrhiza forming ascomycetes . In: Mycorrhizae (ed. E. Hacskaylo), pp. 19-37. USDA, Forest Service, Miscellaneous Publication 1189. Trappe, J.M . 1979. The orders, families and genera ofhypogeous Ascomycotina (Truffles and their relatives). Mycotaxon IX(1): 297-340. Tylutki, E.E. 1979. Mushrooms ofldaho and the Pacific Northwest. VI. Discomycetes. Northwest Naturalist. University ofldaho Press, Moscow, Idaho. Vogt, K.A., C.C. Grier, C.E. Meir and R.L. Edmonds. 1982. Mycorrhizal role in net production and nutrient cycling in Abies amabilis ecosystems in western Washington . Ecology 63: 370-380. White, T. J. , T. D. Bruns, S. B. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. Pp 315-322 In: PCRProtocols and Applications- A Laboratory Manual. Eds, N. Innis, D. Gelfand, J. Sninsky, and T. White. Academic Press, New York. Wilcox, H.E. 1983. Fungal parasitism ofwoody plant roots from mycorrhizal relationships to plant disease. Ann. Rev. Phytopath. 21:221-242. Yang, C.S. and R.P. Korf. 1985 . A monograph of the genus Tricharina and of a new segregate genus Wilcoxina . Mycotaxon, 24 : 467-531. 16 Chapter 2 Phylogenetic distribution of ectomycorrhizal Pezizales Abstract Partial 18S rDNA sequences from 117 Pezizalean taxa were aligned and analysed with Phylogenetic Analysis Using Parsimony (PAUP) 4.1. Neighbor Joining and Maximum Likelihood search algorithms were employed for tree reconstruction. The selection of taxa was based on available sequences and previous reports as ECM symbionts or suspected symbionts. The trees that were reconstructed were very similar irrespective of algorithm. Three sub-ordinal clades were identified. The first contained members ofthe families Morchellaceae, Discinaceae, Helvellaceae and Tuberaceae. The second contained members of the Otideaceae, Sarcoscyphaceae and Sarcosomataceae. Finally, the third contained members ofthe Pezizaceae and Ascobolaceae. A concentration of confirmed ECM taxa is found in the Otideaceae and Tuberaceae. The Morchellaceae and Helvellaceae have the next greatest number of both suspected and confirmed ECM symbionts. 17 2.0 Introduction Phylogenetic studies on the Pezizales most often involve the ribosomal RNA gene repeat (rDNA), which includes the 18S gene, two non-coding regions, ITS 1 and ITS 2, the 5.8S gene, and the 28S gene. This region is of particular importance because the coding and spacer regions evolve at different rates, thus allowing for different levels of taxonomic resolution (Egger 1995). The first sequence-based phylogenetic study of the Pezizales dealt with the relationship of the saprotrophic genus Tricharina to a segregate genus, the ectomycorrhizal Wilcoxina spp. (Egger 1996). Other studies include the relationship of Plicaria to Peziza (Norman and Egger 1996), relationships between truffles and morels (0 'Donnell et al. 1997), sub-ordinal classification within the Pezizales (Landvik et al. 1998), the relationship between Pindara and Helvella (Landvik et al. 1999), and finally relationships within the Sarcoscyphinae (Harrington et al. 1999). Interestingly, the first phylogenetic study on the Pezizales (Egger 1996) is the only study dealing directly with the ecological roles of these fungi . A fungal nutrition study by Egger ( 1986) revealed the versatile nature of the Pezizales. Several members of this group produce a wide array of substrate degrading enzymes, thus suggesting varied ecological roles . This was further substantiated by Egger and Paden (1986) in which Pinus contorta seedlings were inoculated with various fungi and observed for signs ofbiotrophy (meaning various types of associations). Associations observed ranged through endophytic, weakly pathogenic, mycorrhizal and aggressively pathogenic. E-strain fungi (Wilcoxina spp.) are the 'textbook' examples of ectendotrophic mycorrhizae, which are distinguished from ectomycorrhizae on the basis 18 of intracellular penetration in the fom1er and cellular invagination in the latter (Harley and Smith 1983 ). According to Wilcox ( 1983 ), ectendomycorrhizal associations are one step away from parasitism. Wilcoxina mikolae var. mikolae, forms ectendomycorrhizae with P. banksiana and ectomycorrhizae with Picea mariana and Betula alleghanensis, suggesting a range of possible interactions (Scales and Peterson 1991b). Sphaerosporella brunnea exhibits a similar phenomenon; it is ectomycorrhizal with Pinus banksiana (Danielson 1984) and ectendomycorrhizal with Pinus contorta (Egger and Paden 1986). The objective of this study is to identify clades within the Pezizales likely to contain hitherto unknown ECM fungi, which will then serve as a basis for sampling potential ECM taxa for identification and characterization. This will be accomplished through the reconstruction of a DNA sequence-based phylogeny of the Pezizales with all the confirmed and many suspected ectomycorrhizal genera included. This work builds upon results by Agerer ( 1987 -1998) who recently described several ECM Pezizales and Maia et al. ( 1996), who compiled a list of members of the Ascomycotina suspected of forming ectomycorrhizae. Finally, several new biotrophic, and possibly ectomycorrhizal, Pezizales have been identified since Maia et al. (1996), including Geopyxis carbonaria (Vralstad et al. 1998), He/vella corium, H. aestivalis, H. clovrensis (Weidemann et al. 1999), and Morchella esculenta and M. elata (Smith et al. 1999). 19 2.1 Materials and Methods 2.1a Sequence acquisition Partial 18S rDNA sequences were obtained using GenBank's Entrez Browser, maintained 1 by the National Centre for Biotechnology Information , with the exception of Rhodotarzetta rosea, Selenaspora guernisascii, Sclerotinia sclerotiorum and Neolecta vitellina. Table 1 lists all ofthe samples used in this study, including, when known, their original accession numbers, location of origin and GenBank accession number. 2.1 b Molecular protocols DNA extraction, amplification and sequencing protocols follow those outlined in Landvik et al. (1998), otherwise they conform to those listed in the papers specified in GenBank under each species accession number. All sequences were aligned visually, because data file size (117 taxa) precluded computer-based alignment. Sequences gaps were indicated with a"-" and missing sequence segments, i.e. when one sequence is shorter than the rest, were filled in with an "N" . The data set was analysed using the Maximum Likelihood (ML) option combined with a quartet puzzling algorithm and both Neighbour Joining (NJ) consensus and bootstrap analyses with a Maximum Likelihood distance algorithm in the P AUP 4.1 software package (Swofford 1999). Both Sclerotinia sp. and Eleutherascus p eruvianus were chosen as the outgroup taxa because they are both discomycetes and are distinct from the Pezizales. ' (http://www .ncbi.n lrn.ni h. gov/Taxonom v/tax.htrn l) 20 Two NJ and one ML analysis were generated from the aligned data set. The first NJ analysis used a Maximum Likelihood algorithm for distance calculation with the rate of transitions to transversions set to 2 and with the assumption that all sites evolved at equal rates. Neighbor Joining is an algorithmic approach to phylogenetic reconstruction that groups taxa according to overall similarity, expressed as measure of distance. It begins with the construction of a distance matrix, then combines taxa starting with the smallest distance between two taxa in the set. NJ is similar to cluster analysis in the above ways, but differs in that it accounts for rate variation of molecular change between branches. The second NJ analysis used the same options, but with a bootstrap analysis ( 100 replicates) (Fig. 2). Bootstrapping is a type of numerical resampling in which n number (typically 100 or 1000) of alternate data sets are generated by randomly sampling the original data set, with replacement. The number of times a branch appears in the population oftrees generated by the analysis algorithm is an expression of the overall statistical support for the branch. One ML analysis was conducted with default values and 1000 puzzling steps using the quartet puzzling algorithm option (Strimmer and von Haeseler 1996) in PAUP 4.1. Maximum likelihood is an approach which assesses the probability of generating a particular tree based on hypothesised models of character change. ML tends to outperfonn other methods of generating phylogenies (e.g. Maximum Parsimony or additive distances) because it is least affected by sampling error and can accommodate many violations of the assumptions used in its models (Swofford et al. 1996). Quartet puzzling is a ML approach in which ML trees for all possible combinations of quartets 21 (sets of four sequences) are constructed. Each ofthese quartet trees are combined to generate a majority-rule, consensus tree (Strimmer and von Haeseler 1996). Table 1 Information on taxa used in phylogenetic analysis Location Collection Species Aleuria aurantia (Fr.) Fuckel Ascobolus lineolatus Brumm. Ascodesmis sphaerospora Obrist Balsamia magnata Harkn. Balsamia vulgaris Vitt. Barssia oregonensis Gilkey Boudiera acanthospora Dissing & Schumach. Byssonectria aggregata P. Karst. Byssonectria terrestris (Alb. & Schw.) D. Pfister Caloscypha fulgens (Pers.) Boud. A Caloscyphafulgens (Pers.) Boud. B Cazia flexiascus Trappe Chalazion helveticum Dissing Cheilymenia stercorea (Pers.) Boud. Chorioactis geaster Eckblad Choiromyces venosus (Fr.) Th.Fr. Cookeina sulcipes (Berk.) Kuntze Cookeina tricholoma Kuntze Desmazierella acicola Lib. Dingleya verrucosa Trappe Discina macrospora Bubak Disciotis venosa (Pers.:Fr.) Amault Donadinia sp. Bellemere & MelendezHowell Eleutherascus peruvianus v. Arx Fischerula subcaulis Trappe Galiella rufa Nanf. & Korf Geopyxis carbonaria (Alb. & Schw.) Sacc. Glischroderma sp. Gyromitra esculenta (Pers. :Fr.) Fr. Gyromitra montana Harmaja ARON2167 Sweden Unknown Norway California Unknown Oregon, U.S.A. Norway GenBank accession No. U53371 L37533 U53372 U42656 AF054905 U42657 U53373 Unknown Unknown Sweden Unknown Z30241 Z30241 UME31196 KNE2192 JMT12993 Unknown AR02241 Unknown JMT7014 AR02242 Unknown UME30708 JMT12617 MICH4498 NRRL22213 Unknown Sweden Canada California, U.S .A. Unknown Norway Texas, U.S.A. Oregon, U.S.A. Indonesia Puerto Rico Norway New Zealand Michigan Idaho New York, U.S.A. U53374 U62009 U42666 AF061716 U53375 AF104340 U42661 U53376 AF006311 U53377 U42659 U42651 U42643 AF104342 Unknown JMT1889 Unknown DAOM19888 7 CUP62651 NRRL20925 NSW6137 Unknown Washington, U.S.A. Georgia, U.S.A. Canada U63553 U42646 AF004948 U62011 New York, U.S.A. Finland Califomia, U.S.A. AF133141 U42648 U42652 UME29770 Unknown UME31258 JMT13020 Unknown osc 22 Gyromitra melaleucoides (Seaver) Pfister Hefvella lacunosa Afz.:Fr. Helvella capucina Quel. Helvella corium (Web.) Massee Helvella silvicola (Beck) Harmaja Helvella terrestris (Velen.) Landvik Hydnotrya tulasnei (Berkeley) Berk. & Broome lodophanus carneus (Pers.) Korf Kimbropezia campestris Korf & W.-y. Zhuang Kompsoscypha chudei Pfister Labyrinthomyces varius (Rodway) Trappe Lasiobolus papillatus Pers. (Sacc.) Leucangium carthusianum (Quel.) Paol. Leucoscypha oroarctica nomen ined. Microstoma protracta (Fr.) Kanouse Microstoma jloccosum Berenstein Morchella elata Fr. :Fr. Morchella esculenta (L.:Fr.) Pers. Morchella sp. Nanoscypha tetraspora Denison Neolecta vitellina (Bres.) Korf & J.K. Rogers Neournula pouchetii Paden & Tylutki Neotiella rutilans Fr. (Dennis) Otidea leporina (Batch.) Fuckel Otidea onotica (Pers.) Bonord. Pachyella clypeata (Schw.) LeGal Pachyphloeus melanoxanthus (Tul.) Tul. & Tul. Paurocotylis pila Berk. Peziza badia Pers.: Fr. Peziza ostracoderma Korf Peziza petersii Berk. Peziza praetervisa Bres. Peziza quelepidotia Korf & O'Donnell Peziza succosa Berk. NSW7196 Oregon, U.S.A. U42653 Unknown ARON2193 ARON2177 NSW6219 ARON2666 UME29682 Oregon, U.S.A. Unknown Unknown Idaho, U.S.A. Unknown Sweden AF06717 AF046218 AF046226 U42655 AF046216 U53379 02102 CUPMM276 1 Unknown JMT14825 Norway Canary Islands U53380 AF133147 Uganda Australia AF006316 U42662 Unknown JMT7205 Unknown Oregon, U.S.A. AF010588 U42647 Unknown Unknown Unknown AF061724 U53395 AF006313 NRRL25405 NRRL22335 UC1475091 Unknown Unknown Unknown Unknown Pennsylvania, U.S.A. Michigan, U.S.A. Oregon, U.S .A. Michigan, U.S.A. Puerto Rico Unknown U42641 U42642 U42668 AF006314 Z27408 Unknown Unknown Unknown Unknown ALTA9069 Unknown Oregon, U.S.A. Unknown Unknown MA, U.S.A. Alberta Unknown AF104666 AF061720 U53381 AF006308 U40686 AF054899 UME30230 UC1475104 DAOM19960 8 DAOM19579 6 DAOM19581 6 Unknown UME29567 New Zealand Sweden Quebec U53382 L37539 U40678 British Columbia AF133152 British Columbia U40684 Unknown Sweden U42665 U53383 23 Peziza vesiculosa Bulliard Peziza griseo-rosea Gerard Peziza sylvestris Fr. Peziza aff. merdae Donadini Peziza aff. brunneoatra Desm. Phillipsia domingensis Berk. Pithya cupressina Fuckel Plectania sp. Plectania rhy tidia (Berk. in Hooker) Nannf. & Korf Plicaria endocarpioides (Berk.) Rifai Pseudopithyella minuscula Seaver Pseudoplectania nigrella Fuckel Pseudorhizina californica (Phillips) Harmaja Pulvinula archeri (Berk.) Rifai Pyronema dom esticum (Sow.) Sacc. Reddellomyces donkii (Malen<;on) Trappe et al. Rhizina undulata Fr. :Fr. Rhodotarzetta rosea Dissing & Sivertsen Saccobolus sp. Sarcoscypha austriaca (Fr.) Boud. Sarcosoma globosum (Schmidel) Rehm. Sarcosphaera coronaria (Jacq.) Boud. Scabropezia scabrosa (Cooke) Dissing & Pfister Sclerotinia sp. Scutellinia torrentis (Rehm.) T. Schumach. Scutellinia scutellata (L.) Lamb Selenaspora guernisascii Weber Sphaerosporella brunnea (Alb. & Schw.) Svrcek & Kubicka Strobiloscypha keliae Weber & Denison Tarzetra catinus (Holmskj.) Korf & J.D. Rogers Terfezia arenaria (Moris) Trappe Terfezia terfezioides (Matt.) Trappe AR02243 CUP62472 Unknown CUP RPK206 KNE2143 Unknown Unknown Unknown Unknown DAOM19908 9 Unknown Unknown NSW7300 Norway MA, U.S.A. New York, U.S.A. U53384 U40682 AF006309 AF133151 Quebec Puerto Rico Oregon, U.S.A. Puerto Rico Unknown AF133146 AF006315 AF023613 AF006134 AF061 723 British Columbia U40676 California, U.S.A. Japan Oregon, U.S.A. AF006317 AF104345 U42650 ! New York, U.S.A. DAOM19592 8 01766 JMT13292 British Columbia U62012 Norway California, U.S.A. U53385 U42660 NRRL22168 Unknown Netherlands Unknown U42664 Unknown 02131 UME29449 UME30189 Norway Sweden Sweden U53393 U53392 U53386 SA289 FH:Pfister Unknown Maine, U.S.A. U62013 AF133158 Unknown UME31146 Unknown Sweden Unknown U86069 02188 Unknown UME31147 Norway Unknown Sweden U53387 Unknown U53388 Unknown Oregon, U.S.A. AF006310 UME29731 Sweden U53389 Unknown Unknown Unknown Unknown AF054898 AF054900 24 Unknown Unknown AFOl 0589 CSY104 Greenland U38577 UME29738 Sweden U53390 AR02222 Norway U53391 Unknown Unknown Unknown Unknown Unknown UME29400 MICH1951 Unknown Unknown Unknown Unknown Unknown Unknown Sweden Michigan, U.S.A. New Hampshire, U.S.A. Sweden Germany Netherlands Unknown Ohio, U.S.A. Japan AF054902 X98089 L37001 AF054901 AF054903 Z49755 U42658 AF104347 Thecotheus holmskjoldii (E.C. Hansen) Chenant Tricharina groenlandica (Dissing) Yang & Korf Trichophaea hybrida (Sow.) Schumach. Trichophaeopsis bicuspis (Boud.) Korf &Erb. Tub er borchii Vitt. Tub er excavatum Vitt. Tuber melanosporum Vitt. Tuber magnatum Pico Tuber panniferum Tul. & C. Tul. Tuber cf rapaeodorum Tul. Underwoodia columnaris Peck Urnula craterium Fr. Urnu la hiemalis Nannf. Verpa bohemica (Krombh.) SchrOt. Verpa conica (Mtill.:Fr.) Swartz Wilcoxina mikolae Yang & Korf Wolfina aurantiopsis Eckblad Wynnea sp. Berk. & M .A. Curtis UME30174 NRRL20858 NRRL20856 Unknown Unknown Unknown Z49754 U42648 U42644 U62014 AF104664 AF006319 2.2 Results 2.2a Tree topology Three trees were generated from the data. A Neighbour Joining consensus tree (Fig. 2), a Neighbor Joining bootstrap tree (100 reps.) (Fig. 3) and a Maximum Likelihood quartet puzzling tree (Fig. 4). The different clades on each tree roughly corresponded to the traditionally classified families in the Pezizales as emended by Trappe (1979) and 0 'Donnell et al. ( 1997). These clades are indicated on each tree and are accompanied by a colour code regarding the confirmed or suspected associates present in that clade. Confirmed ECM genera were highlighted in red, confirmed biotrophs were highlighted in blue, suspected ECM genera were highlighted in green, genera that contain species either 25 suspected of being ectomycorrhizal/ biotrophic or proven so were highlighted in orange, and finally, genera that are either pathogenic or saprotrophic are listed in black. 2.2b Agreement between recon struction methods All trees consisted of very similar, sub-ordinal level clades, these clades are split up into individual family-level clades and are presented separately after the three complete trees for individual consideration. The Morchellaceae, Discinaceae, Tuberaceae and Helvellaceae consistently group together. The NJ consensus (Fig. Sa), NJ bootstrap (Fig. 5b) and ML (quartet puzzling) (Fig. 5c) Morchellaceae trees are similar with the exception of the ML tree, which includes Hydnotrya tulasnei as a basal taxon with 57% support. The NJ consensus (Fig. 6a), NJ bootstrap (Fig. 6b ), and ML (quartet puzzling) (Fig. 6c) Discinaceae trees are identical with respect to taxa included with the exception of the exclusion of Hydnotrya tulasnei in the ML tree. Another contentious clade was the Tuberaceae. The NJ bootstrap tree separates the Tuberaceae into sister clades, all the Tuber spp. cluster together and receive 97% support (Fig. 3, Fig. 7b ). The other taxa: Dingleya venosus, Reddellomyces donkii, Labyrinthomyces varius and Choiromyces meandriformis cluster together with 92% support (Fig. 3, Fig. 7c). The NJ consensus tree (Fig. 2, Fig. 7a) groups the above taxa in one clade, also ML (quartet puzzling) analysis groups them together with 39% support (Fig. 4, Fig. 7d). The NJ consensus (Fig. 8a), NJ bootstrap 26 (Fig. 8b ), and ML (quartet puzzling) (Fig. 8c) Helvellaceae trees are identical with respect to taxa included. The Sarcoscyphaceae, Sarcosomataceae and Otideaceae cluster together in all the analyses. The Sarcoscyphaceae clade is consistent between the NJ consensus (Fig. 9a), NJ bootstrap (Fig. 9b) and ML (Fig. 9c) trees. The NJ bootstrap tree (Fig. 3, Fig. 1Ob) divided the Sarcosomataceae into two sister clades, the first receiving 96% support and consisting of Sarcosoma mexicana and S. globosum, Urnula hiemalis and U. craterium, Pseudoplectania nigrella and Pseudoplectania sp., Plectania sp. and P. rhytidia, Galiella rufa and Donadinia sp. The second and less strongly supported clade (78%) consisted of Desmazierella acicola, Neournula pouchetii, Chorioactis geaster and Wolfina aurantiopsis (Fig. 1Oc ). Both the NJ consensus (Fig. 2, Fig. 1Oa) and the ML (quartet puzzling) (Fig. 4, Fig. 10d) trees grouped the taxa from both ofthe above clades together. The NJ consensus (Fig. 2, Fig. 11 a) and the NJ bootstrap (Fig. 3, Fig. 11 b) trees for the Otideaceae are identical with respect to taxa included, however, the Otideaceae is divided in two by the ML (quartet puzzling) analysis (Fig. 4). One clade (receiving 72% support) consists of Leucoscypha oroartica, Byssonectria terrestris, B. aggregata, Tricharina groenlandica, Trichophaea hybrida, Wilcoxina mikolae, Aleuria aurantia , Trichophaeopsis bicuspis, Scutellinia torrentis and S. scutellata, Cheilymenia stercorea, Pyronema domesticum, Sphaerosporella brunnea, Selenaspora guernisascii, Otidea leporina, 0. onotica and Rhodotarzetta rosea (Fig. 11 c). The other clade, which only received 20% support, consisted of Geopyxis carbonaria, Paurocotylis pila, Pulvinula archeri, Ascodesmis sphaerospora, Chalazion helveticum, Glaziella aurantiaca and 27 Tarzetta catinus (Fig. 11 d) . Both the NJ consensus tree and the NJ bootstrap tree group the two aforementioned clades together. The Pezizaceae groups out as a sister clade to the Ascobolaceae using NJ, NJ (bootstrap), and ML. The NJ consensus (Fig. 2, Fig. 12a), NJ bootstrap (Fig. 3, Fig. 12b), and ML (quartet puzzling) (Fig. 4, Fig. 12c) Pezizaceae trees are identical with respect to taxa included. Finally, The NJ consensus (Fig. 2, Fig. 13a), NJ bootstrap (Fig. 3, Fig. 13b), and ML (quartet puzzling) (Fig. 4, Fig. 13c) Ascobolaceae trees are identical with respect to taxa included. 28 NJ Scletollnla • . ~ Asc:obolac:eae • Pezizac:eae ••• •••• Disc:inac:eae ••• Tuberac:eae ••• J Helvellac:eae ••••• Otideac:eae ••••• ll Sarc:osc:yphac:eae • Sarc:osomatac:eae Bootslrlp Sc/eroflnia sp. Nooltctl valine n 96 L1alobolu1 """''" PulvlnullmtNti S1111010m1 globo.um 52 96 Gtltllnda S1111010m1 mtx/cene Pltdlnia sp. 92 74 78 78 93 I I Sarcosomataceae • 98 72 76 100 78 63 lclcoll 90 54 91 N ~~ Sarcoscyphaceae • 98 55 100 100 55 Otideaceae ••• • 84 95 57 99 55 96 100 58 Ascobolaceae • 79 52 100 99 Pezizaceae 69 57 70 85 I I n 99 68 I Morellellaeeae 69 62 100 •• 57 I I n 84 -- I Disciuceae Puzzlo Sarcoaomataceae •• 13 Otideaceae 97 I l l I 10 76 J Taberaceae 42 I II I Helvellaceae I l l I Morchella elata Morchella sp. Morchella esculenta Disciotis venosa Verpa conics Verpa bohemica Fischerula subcaulis ' - - - - - - - Leucangium carthusianum c____ Fig. 4a The Neighbour Joining consensus tree's Morchellaceae clade 85 elata 1 - - - c = = = = Morchella MorcneTia sp. 99 r------f 70 ' - - - - - - - - - - Morchella esculenta r - - - ' - ' ' - - - - 1 - - - - -- - - - - - - - - - Disciotis venosa ~ ~~~ 69 ~~ ~ o VefP.a bohemica Fischerula subcaulis Leucangium carthusianum Fig. 4b The Neighbour Joining bootstrap tree's Morchellaceae clade 82 57 96 85 1 I 99 11~~ 1 1 92 ~~ 57 ~~ Morchella elata Morchella sp. Morchella esculenta Disciotis venosa Verpa conica Verpa bohemica Fischerula subcaulis Leu.canqium cartbusianum Rjtanotrya tU1asne1 Fig. 4c The Maximum Likelihood quartet puzzling tree's Morchellaceae clade 32 rl Gy_rom.itra esculenta . n'----- - Gyrom1tra melaTeucoJdes Pseudorhizina californica Discina macrospora L _ Gyromitra montana '---H- d_c_ HydnolfJ!a c.erebriformis ~ notrya tulasne1 L___r- Fig. 5a The Neighbour Joining consensus tree's Discinaceae clade 64 _ 77 --{==== Gyromitra esculenta (lyromitra melaleucoides 68 PSeudorhizina californica - -=-=-- - - - 1 - - - - - - - - - - - - - Hydnotrya cerebriformis 78 fjydnotrya tulasnei ~ r Discina macrospora ' - - - - - - Gyromitra montana r--------11 _!_!__ Fig. 5b The Neighbour Joining bootstrap tree's Discinaceae clade 63 94 99 esculenta 94 1.------1'---- Gyromitra ffyromitra melaleucoides Pseudorhizina californica Discina macros ora Gvromitra mon ana ffydnotrya cerebriformis 1 99 Fig. 5c The Maximum Likelihood quartet puzzling tree's Discinaceae clade 33 Tuber magnatum Tuber melanosporum Tuber panniferum Tuber borchii Tuber cf. rapaeodorum '------- Tuber excavatum Din_qleya verrucosa Redaellomyces donkii Labvrinthomvces varius C'170tromyces venosus L_____ - Fig.6a The Neighbour Joining consensus Tree's Tuberaceae clade 89 88 97 Tuber maanatum Tuber meTanosporum Tuber borchii Tuber cf. rapaeodorum Tuber panniferum Tuber excavatum I l Fig. 6b The first Neighbour Joining bootstrap tree's Tuberaceae clade 100 Dingleya verrucosa Reddellomyces donkii Labyrinthomyces varius ' - - - - - - - - - - - - - - Choiromyces venosus ~ ~~ 1 Fig. 6c The second Neighbour Joining boostrap tree's Tuberaceae clade 34 51 I l 80 39 91 I I 56 I 90 Tuber magnatum Tuber melanosporum Tuber panniferum Tuber borchii Tuber cf. rapaeodorum Tuber excavatum Dingleya verrucosa Reddellomyces donkii Labynntnomyces vanus Choiromyces venosus I 96 94 1 I 95 Fig. 6d The Maximum Likelihood quartet puzzling tree's Tuberaceae clade 35 Balsa mja magna fa Bs rssis oregon ensis Balsa mia vulgaris I I ~ ~ ~ ~~ o -----Jl He/Vella ~ ~ corium Undetwoodla column• Fig. 7a The Neighbour Joining consensus tree's Helvellaceae clade 97 -l ~ 100 I 100 I 99 I 58 ~~~~ Balsamia magnata Barssia oregonensis . - - - - - , - - - - - - - Balsamia vulgaris 100 Helvell!llacunosa ~ He/vella corium _ _ Helvella capucina He/vella terrestris - - - - - - - - - - - - He/vella silvicola - - - - - - - - - - - - Underwoodia columnaris _ _ _ _ _ _ _ _ __ Fig. 7b The Neighbour Joining bootstrap tree's Helvellaceae clade 99 94 88 94 I I 1 99 99 98 96 Balsamia magnata Barssia oregonensis Balsamia vulgaris He/vella Jacunosa He/vella terrestris He/vella silvicola He/vella corium He/vella cap_ucina Underwoodia columnaris Fig. 7c The Maximum Likelihood quartet puzzling tree's Helvellaceae clade 36 Microstoma protracta Microstoma floccosum J - - Cookeina su/cipes -Cookeina tricholoma Nanoscypha tetraspora Pithya cupressina o ~ ch.udei . Phtlltpsta dommaensts Pseudopithyella minuscu/a Sarcoscypha austriaca Wynnea sp. Fig. Sa The Neighbor Joining consensus tree's Sarcoscyphaceae clade 90 54 98 72 76 100 78 63 Microstoma protracta Microstoma floccosum Cookeina sulcipes Cookeina tricholoma Nanoscypha tetraspora Pithya cupressina Kompsoscypha chudei Phif11psia domingensis Pseudopithyella minuscula Sarcoscypfia austriaca Wynnea sp . Fig. 8b The Neighbor Joining bootstrap tree's Sarcoscyphaceae clade 99 M(crostoma o.rotracta Mtcrostoma 'floccosum Cookeina sulcipes Cookeina tricholoma Nanoscypha tetraspora Pithya cupressina Kompsoscypha chudei Philltpsia domingensis Pseudopithyella minuscula Sarcoscypha austriaca Wynnea sp . 100 57 53 53 98 59 98 ~ Fig. 8c The Maximum Likelihood tree's Sarcoscyphaceae clade 37 ~ osorn.a H{ot1 .sum 8 rfrfl8!B cr'1imrfflm Umula hiemalis Galiella rufa '---- Plectania sp. Sarcosoma mexicana .___ Donadinia sp. PseudoP.Iectania sp. Pseudoplectania n_igrella Desmazier,ella acicola ._____ Neoumwa poucnetu Chorioactis geaster ' - - - - - - Wolfina aurantiopsis Strobiloscypha keliae Fig. 9a The Neighbour Joining consensus tree's Sarcosomataceae clade r o o globosum mula c;raterium mula hiemalis ~ 96 52 96 74 78 ~~~r Plectama rflytfd1a Galiella rufa Sarcosoma mexicana Pf8ctanl4 sp. Donadlma SP..· Desmazleniffa aclcgJa Nf}OU_mu/a pouchetll ChOrioactis aeaster Wolfina aurantiopsis N?oti.ella rutilans RIIOdOtarzetta rosea Chalazion helveticum Leucoscypha oroarctica 92 78 Fig. 9b The Neighbour Joining bootstrap tree's Sarcosomataceae clade Sarcosoma globosum Plectania rhVtidia craterium mula hiemalis ialiella rofa lectama sp. ' - - - - - Sarcosoma mexicana Donadinia sp. Pseudoplecfania sp. Pseudop.Jectania nigrella Desmazierella acicola , ____ Neoumula pouchetii Chorioactis geaster Wolfina aurantiopsis ' - - - - - - - - - Strobiloscypha Reliae ~ 13 Fig. 9c The Maximum Likelihood quartet puzzling tree's Sarcosomataceae clade 38 =a==s=iobolus paP.illatus L - - ---=L Pulvmula archeri '------- Ascodesmis sphaerospQra _ _ _ _ _ __:Taaetta catinus . ,_ C:ieopyx1s cafbonana Paurocotylis pi/a Chalazion he/veticum ' - - - - - - - - - - - Glaziel/a aurantiaca ~~ Neotiella I· Rhodotarzetta rosearutilans Leucoscypha oroarctica '---------r- Byssonectria terrestris . Bvssonectria ~ r Tnchanna groenlandlca Trichophaea hybrida Wilcoxina mikolae Otidea lef.?.orina Otidea onotica Aleuria aur.antia . . I ncnophBBOQSIS biCUSpiS .____ _ _ Pyronema domesticum Sp haerosporella ~ ' - - - - - - - - Selenaspora guem1sascu Scutellinia torrentis Scutellinia scutellata Cheilymenia stercorea Fig. lOa The Neighbor Joining consensus tree's Otideaceae clade 72 Fig. lOb The Neighbor Joining bootstrap tree's Otideaceae clade 39 55 ~~ - - - - - --=-=-- - - - - - - - - - - - i _ __ _ _ _:==== Tricharina groentandica Trichophaea hybrids - - - - - - - - - - - - - - - - - - - - - - - - - - Wilcoxina mikolae Tanetta.catin.us . -_-_-_-_-_-_-_-_-_-_-_-_-_-_- 1~oo~ ~ ~~ ~~~ Byssonectria Geopyxts carbonarta terrestris Byssonectria aggregate Otidea Otidea leporina onotica ~r r - - - - - - - - - - - - - - - - - - - - - - - - - - Trichophaeopsis bicuspis Gtaziellaaurentiaca - - - - - - - - - - - - - - - - - - - - - - - - - - Ascodesmis sphaerospore 84 Paurocotylis pita ~~ Sphaerosporel/a brunnea - - - - - - - - - - - - - - - - - - - - - - - - - - Se/enaspore guemlsascii 95 Cheilymenia stercorea ~~ Scute/linla torrentis cutellinia ~ yronema uomesttcum - - - - - - - - - - - - - - - - - - - - - - - - - - Strobiloscypha keliae Fig. lOc The Maximum Likelihood quartet puzzling tree's Otideaceae clade Fig.lOd The Maximum Likelihood quartet puzzling tree's second Otideaceae clade 40 Peziza queletii .------ Terfezia terfezioides Kimbro ezia. campestris Pez1za pr. eterv1sa Peziza P.etersii Peziza aft. merdae Peziza vesiculosa Peziza sylvestris lodophanus cameus_ ------f"_:__-c=-Gj Pachyphloeus melanoxanthus .Glischro(Jerma sp. Scabropezia scabrosa Pachyel/a c/ypeata PTicaria endocarpioides Sarcosphaera coronaria Terfezia arenaria Cazia flexiascus Pez1za gnseo-rosea Peziza ostracoderma Peziza succosa Peziza aft. brunneoatra L___ ~ ~ ~ Boudiera acanthospora Fig. lla The Neighbour Joining consensus Pezizaceace clade . - - - - - - - - - - - - - - , : : - - . , - - - - - - - - - Peziza queletii 52 ~ ~ ~~ ro o L___ ~ ~~~ ~ o F;.ez· a aft. runn oatra 1 - - - - - - - - - - - - - - - - - rae yet/a CIJipea a 1 - - - - - - - - - - - - - - - - - Peziza bad1a t----------=c-:------------ Peziza ostracoderma ~~ Plicaria endocarpioides Sarcosphaera coronaria 1 - - - - - - - - - - - - - - - - - - - - - - Boudiera acanthospora 1 - - - - - - - - - - - - - - - - - - - - - - Terfezia terfezioides ~ lodophanuscameus . - -=-- - - - - - - Pez1za aft. merdae 57 Peziza vesiculosa Pr;ziza sylvestris 1 - - - - - - - - - - - - - - - - - - - - - - K1m/)ropezia ~ r Pez1za petersu Peziza praetervisa 1 99 ~ ~~ Glisch'roderma SP.. Scabropezia scabrosa 1--------=7=-=9- - - - - - - - - - - Terfez1a arenaria ~ Cazia flexiascus rPeziza griseo-rosea 1OO .- Fig. llb The Neighbour Joining bootstrap tree's Pezizaceae clade 41 ~ 97 Fig. llc The Maximum Likelihood quartet puzzling tree's Pezizaceae clade 42 Saccobolus sp. Ascobolus lineolatus Thecotheus holmskjoldii Fig. 12a The Neighbor Joining consensus tree's Ascobolaceae clade 57 Saccobolus sp. Ascobolus lineolatus Thecotheus holmskjoldii Fig. 12b The Neighbour Joining bootstrap tree's Ascobolaceae clade 79 89 Saccobolus sp. Ascobolus lineolatus Thecotheus holmskjoldii Fig. 12c The Maximum Likelihood quartet puzzling tree's Ascobolaceae clade 43 2.3 Discussion 2.3a Phylogenetic relationships with in the Pezizales The data set was consistently divided into several large clades irrespective of reconstruction algorithm. These include a Pezizaceae/Ascobolaceae clade, a Morchellaceae/Discinaceae/Tuberaceae/Helvellaceae clade, and an Otideaceae/Sarcosomataceae/Sarcoscyphaceae clade. These large clades were further divided into smaller clades which are discussed below. Support for each clade was mentioned in section 2.2b, clades receiving less than 50% support were considered unsupported, and are not discussed. The majority of the sequences in the Sarcosomataceae clade and the Sarcoscyphaceae clade were obtained from Harrington et al. (1999). That study found (using unweighted parsimony) that the Sarcosomataceae sensu lata was a paraphyletic group (meaning that at least one of the taxa or clades arose from a different ancestral taxon than the rest) and suggested that the Sarcosomataceae be emended to include only Galiella, Plectania, Urnula, Pseudoplectania, Donadinia and Sarcosoma. In our study, the Sarcoscyphaceae clade clustered out as identical to that in the Harrington et al. ( 1999) paper in the NJ consensus tree (Fig. 1) with Sarcoscypha austriaca, Pseudopithyella minuscula, Nanoscypha tetraspora, Pithya cupressina, Kompsoscypha chudei, Phillipsia domingensis, Microstoma jloccosum and M. protracta, Cookeina tricholoma and C. sulcipes and Wynnea sp. This clade was also found using NJ (bootstrap) and ML (quartet puzzling), receiving 72% support and 96% support respectively. Harrington et al. (1999) reported Byssonectria aggregata and Otidea onotica as in-group taxa of the 44 Sarcoscyphaceae clade. The MP and NJ trees from our study placed Byssonectria aggregata and Otidea onotica as clearly members of the Otideacean clade. Since the analyses reported in this chapter distinguish between the Otideaceae and the Sarcoscyphinae, and there were many more representatives of the family Otideaceae, the inclusion of B. aggregata and 0. onotica in the Sarcoscyphaceae (Harrington et al. 1999) was likely due to the small sample number of Otideaceaous fungi in that study. Using a bootstrapped NJ analysis, the Otideacean clade was collapsed, however, several of the smaller clades within the Otideaceae received support. According to the ML (quartet puzzling) tree, the Otideacean clade was paraphyletic, however, this was not found in the other analyses. A phylogenetic study by Landvik et al. (1998) using Maximum Likelihood, Maximum Parsimony and Neighbor Joining reported the Otideaceae as monophyletic, and as a sister clade to the Sarcosomataceae and Sarcoscyphaceae. The placement of the ECM genera within the Otideaceae is similar between that reported in this chapter and the Landvik et al. (1998) study. Wilcoxina mikolae, the confirmed E-strain ectomycorrhizal fungus, clusters out with Trichophaea hybrida, an ECM suspect. In addition, Geopyxis carbonaria, a confirmed biotroph (Vralstad et al. 1998) is a sister taxon to a hypogeous, and therefore suspect ECM, Paurocotylis pila according to the Maximum Parsimony, ML and NJ trees presented in Landvik et al. ( 1997) and both the NJ and ML trees presented in this chapter. 45 The Morchellaceae clade was similar in topology to that reported by O'Donnell et al. (1997) with the exception of the inclusion of Leucangium carthusianum and Fischerula subcaulis. O'Donnell et al. (1997) reported these taxa as incertae cedis (ofuncertain phylogenetic placement). L. carthusianum and F. subcaulis are both hypogeous fungi and L. carthusianum has been documented as ECM (Agerer 1996). The ML tree's Morchellaceae clade differed by the inclusion of Hydnotrya tulasnei, however since it only received 57% support, it is likely an artifact of the approach. The NJ consensus tree's Morchellaceae clade agreed with the NJ bootstrap tree's Morchellaceae clade. The Discinaceae clade is similar in topology to that reported by O'Donnell et al. (1997). Using NJ with bootstrapping, this clade received 68% support, however, branches leading to both H. tulasnei and H. cerebriformis were collapsed suggesting uncertain relationships. Using ML (quartet puzzling), this clade received 63% support, but lacked H. tulasnei which grouped out in the Morchellaceae clade. The NJ consensus Tuberaceae clade is identical to the ML Tuberaceae clade. The NJ bootstrap tree lacks the following taxa: Dingleya verrucosa, Choiromyces venosus, Labyrinthomyces varius and Reddellomyces donkii. The analyses of O'Donnell et al. (1997) include D. verrusosa, L. varius , R. donkii and C. venosus with the genus Tuber. The genus Tuber, represented by six taxa in our analysis, is monophyletic. The phylogenetic arrangement of species of He/vella within the Helvellaceae clade follows that presented in Landvik et al. (1999). Hypogeous members of this clade, 46 Balsamia and Barssia were resolved as a sister group to the genus He/vella (with Wynella) in our study and in O'Donnell et al. (1997). A study by Weidemann et al. (1999) demonstrated the biotrophic nature of a fungus with affinities to He/vella corium suggesting that it was potentially ectomycorrhizal. Also, Agerer (1996) described the ECM association between Balsamia alba and Pinus jeffreyi. Finally, the Helvellaceae clade consistently groups out as a sister clade to the Tuberaceae clade, which is in agreement with Landvik et al. (1998). This link between the largely ECM Tuberaceae and the Helvellaceae, in addition to the above biotrophic and mycorrhizal members of the Helvellaceae clade, provides more evidence for the ectomycorrhizal status of some members ofthe genus He/vella. Two Caloscypha fulgens sequences included formed a separate clade using NJ and ML. The branch leading to the Caloscypha clade was collapsed using NJ (bootstrap) and was shown as a sister clade to the Sarcoscyphaceae/Sarcosomataceae/Otideaceae clade. This runs contrary to Landvik et al. (1998) whose work places C. fulgens with Tuber spp. and He/vella spp. Using all three reconstruction techniques, Ascobolus lineolatus and Saccobolus sp. both cluster with Thecotheus holmskjoldii as a sister clade of the Pezizaceae. This is in agreement with Landvik et al. (1998). 47 2.3b The evolution of the mycorrhizal habit Burdsall's (1965) observation of puffing ascospores in Geopora cooperi and Trappe's (1979) emendation of the order Pezizales to include the former order Tuberales facilitated the phylogenetic approach to the discovery of novel ectomycorrhizal taxa. At that time, all hypogeous taxa were assumed ectomycorrhizal (Trappe 1971). These ECM hypogeous taxa were distributed throughout the order Pezizales, some amongst primarily epigeous clades thus leading to the suspicion ofwidespread biotrophy in the order Pezizales (Trappe 1971). The Otideacean clade contains the most biotrophic and EM fungi, including the biotroph G. carbonaria (Vralstad et al. 1998) in addition to the EM fungi W. mikolae and S. brunnea. Paurocotylis pila, the hypogeous and therefore presumed mycorrhizal fungus, also occurs in this clade. Trichophaea hybrida is also in this clade. All members of the Tuberaceae clade are considered mycorrhizal, six species were included in this analysis: T magnatum, T melanosporum, T panniferum, T borchii, T cf rapaeodorum and T excavatum. Only two of these have been documented as mycorrhizal: T melanosporum and T borchii (Agerer 1987-1998). The Morchellaceae clade contains two biotrophic representatives, M. esculenta and M. elata (Smith et al. 1999). Hypogeous members of this clade include Fischerula subcaulis and Leucangium carthusianum, a related species Leucangium vulgare was described as ectomycorrhizal by Agerer (1987-1998). 48 The Helvellaceae clade contains the biotrophic H. corium (Weidemann et al. 1999) and likely other proven biotrophic Helvella species not analysed here including: H. aestivalis, H. dovrensis (Weidemann et al. 1999). In addition to the above taxa, the hypogeous fungi Balsamia magnata, B. vulgaris and Barssia oregonensis are included in this clade. Agerer (1996) described Balsamia alba as ectomycon·hizal. There are two well-known pathogenic members of the Pezizales: Rhizina undulata which attacks conifer seedlings and Caloscypha fulgens which parasitizes conifer seeds. Both fungi grouped separately from any other Pezizalean clade. R. undulata forms a sister clade to the clade that contains the Pezizaceae, Morchellaceae, Discinaceae, Helvellaceae and the Tuberaceae clades. C. fulgens appears divergent from the Sarcoscyphinae/ Otideaceae clades. Neither of the documented pathogens are phylogenetically wellresolved. An interesting future study could involve a closer look at the relationship between the pathogenic Caloscypha and ectomycorrhizal members of the Otideaceae. Finally, several clades are poorly represented or conspicuously lacking in biotrophic fungi. The Pezizaceae contains few known biotrophs although Pachyphloeus and Cazia are suspected mycorrhizal by virtue of their hypogeous habit. In addition, Awamah et al. ( 1979) reported the synthesis of mycorrhizae between Terfezia boudieri and T claveryi and Helianthemum ledifolium and H. salicifolium. The Sarcosomataceae, Sarcoscyphaceae and the Ascobolaceae clades also lack both documented biotrophs and hypogeous fungi. 49 There are two possible scenarios that could describe this distribution. First, the Pezizales' most recent common ancestor was myconhizal and this mode of living was lost by most contemporary taxa (with the exception of those outlined in this chapter and other undiscovered symbionts). Second, the ectomycorrhizallifestyle evolved independently in several lineages within the Pezizales. Although neither of the above hypotheses can be verified with the data presented, the most parsimonious hypothesis is the latter, since it requires the fewest character state changes and the ectomycorrhizal association has evolved several times in different lineages in both ascomycetes and basidiomycetes (LoBuglio et al. 1996; Bruns 1995). 50 2.3c The origins of mutualism Several theories have been presented regarding the origins of fungal/ plant mutualism. Pirozynski and Malloch (1975) suggested that fungal biotrophism arose from saprophytic associations between fungi and green algae in the sea. An article by Clay (1988) suggests that mutualistic endophytes in the family Clavicipitaceae (Balansia spp. and Epichloe spp.) evolved with their grass hosts from a prior parasitic association (vis a vis Claviceps purpurea on rye). This follows the amelioration model of coevolution (Boucher et al. 1982) which holds that the negative effects of parasitism on the host decrease over evolutionary time until the host benefits from the infection. Clay's (1988) argument is more compelling than Pirozynski and Malloch's (1975), being based on observation and experimentation. It is interesting to note that on all of the phylogenetic reconstructions presented in this chapter, both of the pathogens within the Pezizales, Rhizina undulata and Caloscypha fulgens are basal taxa, suggesting ancestral status. R. undulata is basal to all the major clades of the Pezizales and C.fulgens is basal to the Sarcoscyphinae/ Otideaceae. Although based upon few taxa, this suggests that mutualistic mycorrhizal associations could follow the 'amelioration model' with ancestral taxa being pathogenic. 2.3d New ectomycorrhizal taxa since Maia et al. 1996 Since the Maia et al. (1996) paper, several new taxa have been added to the biotrophic /EM list. Smith et al. (1999) reported putative ectomycorrhizal associations between both Morchella esculenta and M. elata and various members of the Pinaceae using pure culture synthesis. Vralstad et al. (1998) reported Geopyxis carbonaria as biotrophic on Picea abies . This was shown by comparison ofiTS1 and ITS2 sequences from both G. 51 carbonaria fru itbodies and P. abies root tips collected near G. carbonaria fruitbodies. In addition, cultural isolates from both root tips and fruitbodies were morphologically similar. Finally, Weidemann et al. (1999) demonstrated the association between He/veLla aestivalis and Dryas octopetala and He/vella aff. corium and He/vella aff. dovrensis with Salix reticulata on the basis ofiTS 1 sequence (rDNA) similarity between He/vella fruitbodies and host root tips . Since no morphological studies were conducted, the association could not be described as ectomycorrhizal, only root inhabiting. 2.3e Proof of an ectomycorrhizal association Proof of an ectomycorrhizal association relies upon a combination of approaches. The fungus observed must be properly identified. This can be accomplished (in some cases) through morphological examination, although many fungi produce such similar vegetative structures that PCR-based analysis may be a more reliable approach for identification. Second, the association observed must be confirmed as ectomycorrhizal. This involves either observation of a Hartig net (the functional component of an ECM association) (Harley and Smith 1983) or through radio-labelled isotope studies, in which labelled nutrients are typically transferred from fungus to host. Many of the articles quoted in Maia et al. (1996) use none ofthese approaches. Three of the main articles cited (Trappe 1971, Moreno et al. 1991 , Alvarez et al. 1993) suggest that a fungus is ectomycorrhi zal if it is exclusively found under a potential host. This, as mentioned above, is not strong enough evidence. As the importance of ectomycorrhizal associations is increasingly realized, a demand for more rigorous establishment of ECM status is required. 52 2.3f Clades with potential ectomycorrhizal fungi This study identified the Tuberaceae and Otideaceae clades as having the greatest number of confirmed ECM taxa. The Morchellaceae and Helvellaceae had the greatest number of suspected ECM fungi and confirmed biotrophs, respectively. The next study attempted to document an ECM associations in these clades by comparing rDNA from fruitbodies and ECM root tips found directly beneath them in the field. 53 2.4 References Agerer, R. 1987-1998. Colour Atlas ofEctomycorrhizae. Schwabisch Gmund, Germany. Einhorn-Verlag Eduard Dietenberger. Agerer, R. 1996. Concise descriptions of ectomycorrhizae. Schwabisch Gmund, Germany. Einhorn-Verlag Eduard Dietenberger. Alvarez, I. , J. Parlade, J. M. Trappe and M. Castellano. 1993. Hypogeous mycorrhizal fungi of Spain. Mycotaxon 47: 201-217. Awamah, M.S ., A. Alsheikh and S. Al-Ghawas. 1979. Mycorrhizal synthesis between Terfezia boudieri and T. clavery i and H elianthemum . Abstract No. 23, In: 4 1h North American Conference on Mycorrhizae, June 24-28, Colorado State Uni versity, Fort Collins, Colorado. p.23 Boucher, D.H., S. James and J.H. Keeler. 1982. The ecology of mutualism. Annu . Rev. Ecol. Syst. 13 : 315-347. Bruns, T.D . 1995. Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil 170: 63-73. Burdsall, H.H . 1965. Operculate asci and puffing of ascospores in Geopora (Tuberales ). Mycologia 57: 485-488. Clay, K. 1988. Clavicipitaceous fungal endophytes of grasses: coevolution and the change from parasitism to mutualism pp. 79-105. In: Coevolution of fungi with plants and animals. (eds.) Pirozynski , K.A. and D.L. Hawksworth, Academic Press Ltd. , London. Danielson, R.M. 1984. Ectomycorrhiza formation by the operculate discomycete Sphaerosporella brunnea (Pezizales) 76:454-461. Egger, K.N. 1986. Substrate hydrolysis patterns of post-fire ascomycetes (Pezizales). Mycologia 78: 771-780. Egger, K.N. 1995. Molecular analysis of ectomycorrhizal fungal communities. Can. J. Bot. 73 (Suppl. 1): S1415-1422. Egger, K.N. 1996. Molecular systematics ofE-strain mycorrhizal fungi : Wilcoxina and its relationship to Tricharina (Pezizales). Can. J. Bot. 74:773-779. Egger, K.N. and J. W. Paden. 1986. Biotrophic associations between lodgepole pine seedlings and postfire ascomycetes (Pezizales) in monoxenic culture. Can. J. Bot. 64: 2719-2725. 54 Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, London. Harrington, F.A., D.H. Pfister, D. Potter and M.J. Donoghue. 1999. Phylogenetic studies within the Pezizales. I. 18S rRNA sequence data and classification. Mycologia 91:41-50. Landvik, S., K.N. Egger and T. Schumacher. 1998. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analysis of SSU rDNA sequences. Nordic J. Bot. 17: 403-418. Landvik, S., R. Kristiansen, T. Schumacher. 1999. Pinclara: a miniature He/vella. Mycologia 91: 278-285. LoBuglio, K.F., M.L. Berbee, J.W. Taylor. 1996. Phylogenetic origins ofthe asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes. Molec. Phylogen. Evol. 6: 287-294. Maia, L.C., A.M. Yanno and J.W. Kimbrough. 1996. Species ofascomycota forming ectomycorrhizae. Mycotaxon, 57: 371-390. Moreno, G., R. Galan and A. Montecchi. 1991. Hypogeous fungi from peninsular Spain. II. Mycotaxon 62: 201-238. National Centre for Biotechnology Information, GenBank. 1999. created by S. Federhen, C. Hotton, D. Leipe, V. Soussov. at: http ://www .ncbi .nlm.nih.gov/W eb/Genbank/index. html Norman, J.E. and K.N. Egger. 1996. Phylogeny ofthe genus Plicaria and its relationship to Peziza inferred from ribosomal DNA sequence analysis. Mycologia 88: 986-995. O'Donnell, D., E. Cigelnik, N.S. Weber. 1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48-65. Pirozynski, K.A. and D.W. Malloch. 1975. The origin of land plants: A matter of mycotrophism. Biosystems 6: 153-164. Scales, P.F. and R.L. Peterson. 1991b. Structure ofectomyconhizae formed by Wilcoxina mikolae var mikolae with Picea mariana and Betula alleghaniensis. Can. J. Bot. 69: 2149-2157. Smith, I.E., J. Dahlstrom, Y. Valachovic, N.S. Weber, C. Barro, D. McKay and K. Fuj imura. 1999. Morchella : More challenges to understanding its ecosystem function. Abstract from: Second International Conference on Mycorrhiza, July 5-10, Uppsala, Sweden. 55 Strimmer, K. and A. von Haeseler. 1996. Quartet Puzzling: a quartet maximumlikelihood method for reconstructing tree topologies. Mol. Bioi. Evol. 13:964-969. Swofford, D.L. 1999. PAUP: phylogenetic analysis using parsimony, version 4.0b2 . Distributed by the Smithsonian Insitute, Washington. Swofford, D.L., G.J. Olsen, P.J. Waddell and D.M. Hillis. 1996. Phylogenetic Inference. in: Molecular Systematics 2nd ed. eds: D.M. Hillis, C. Moritz and B.K. Mable. Sinauer Associates, Sunderland, Mass. Trappe, J.M. 1979. The orders, families and genera ofhypogeous Ascomycotina (truffles and their relatives) . Mycotaxon, 9: 297-340. Trappe, J.M. 1971. Mycorrrhiza forming ascomycetes. In: Mycorrhizae (ed. E. Hacskaylo), pp. 19-37. USDA, Forest Service, Misc. Publ. 1189. VdHstad, T., A. Holst-Jensen and T. Schumacher. 1998. The post-fire discomycete Geopyxis carbonaria (Ascomycota) is abiotrophic root associate with Norway spruce (Picea abies) in nature. Molec. Ecol. 7: 609-616. Weidemann; H.M., A. Holst-Jensen and T. Schumacher. 1999. Demonstration of Helvella ectomycorrhizae on Dryas and Salix hosts by means of taxon-selective Helvella -based nrDNA-primers. Abstract from: Second International Conference on Mycorrhiza, July 510, Uppsala, Sweden. Wilcox, H.E. 1983. Fungal parasitism ofwoody plant roots from mycorrhizal relationships to plant disease. Annu. Rev. Phytopathol. 21 :221-242. 56 Chapter 3 Morphological and molecular comparisons fruitbody and root tip collections Abstract To date, several ECM Pezizales have been identified, however the number of unconfirmed ectomycorrhizae in this order likely remains large. The purpose of this investigation was to document previously unknown Pezizalean ECM. Fruitbodies of suspected ectomycorrhizal taxa were collected, along with root tip samples from nearby potential hosts. These fruitbodies were identified and the root tips were morphologically characterized. DNA was extracted from ectomycorrhizal root tips that passed a set of criteria identifying them as potentially ascomycetous. Fruitbody DNA was also extracted. PCR-RFLP analysis then followed, the ITS region of the rDNA was amplified and digested using the restriction enzymes Alu I, Hinfi, Rsa I. A match between the fruitbodies and root tips using these three enzymes was taken to indicate that the fungus amplified on the root tips was the same as the fruitbody. If the root tip that matched with the fruitbody was confirmed ECM (i.e. by the observation of a Hartig net), the inference was that the particular taxon is ectomycorrhizal. All of the root tips observed had Hartig net, however there were no RFLP matches between root tips and fruitbodies . 57 3.0 Introduction Ectomycorrhizae (ECM) are mutualistic associations between the roots of some woody and herbaceous plants and ascomycete or basidiomycete fungi. In exchange, the host gains increased nutrient uptake, drought tolerance and pathogen resistance. The fungal associate gains photosynthates (Harley and Smith 1983). The main feature defining an ectomycorrhizal association is the Hartig net, which is a labyrinth-like fungal structure formed between the cortical and epidermal cells of the host and allows for the exchange of nutrients, water and photosynthates (Harley and Smith 1983). The first report of ectomycorrhizal associations involves ascomycetes. Vittadini ( 1831 in Maia et al. 1996) noticed that species of Elaphomyces and other fungi also placed in the Tuberales grew near the roots of some vascular plants. He later asserted that these roots were not parasitized, but likely nourished by the fungus (Vittadini 1842 in Maia et al. 1996). The term mycorrhiza, meaning 'fungus-root' was introduced by Frank (1885 cited in Maia et al. 1996) who described Tuber aestivum as mycorrhizal with Fagus sy lvatica. Frank used mantle surface view as a primary descriptive character. Research did not progress further until the beginning ofthis century when both Mangin (1910) and Melin (1923) in Agerer (1987-1998) described ECM in some anatomical detail. Another 'ground breaking' development in the history of morphological description of mycorrhizae (i.e. morphotyping) was Dominik's (1969) creation of a dichotomous key for ECM identification based on root cross section anatomy/morphology. Mantle surface view was re-considered a valid character by Chilvers ( 1968) who used this to 58 differentiate between a number of different Eucalyptus ECM. Ingleby et al. (1990) published a manual on the identification of ECM based on mantle characters and other morphological features, e.g. cystidia presence, shape and size. Agerer (1987-1998) continued to work on a compendium of descriptions and techniques called the 'Colour Atlas of Ectomycorrhizae', which is very detailed in regards to both taxa described and characters observed. In 1996, Agerer initiated 'Descriptions of Ectomycorrhizae', following up on his Colour Atlas. Most recently, a 'Concise Description of North American Ectomycorrhizae' was published in 1996 by Goodman and collaborators outlining protocols for ECM morphotyping and DNA analysis. According to Danielson ( 1982), one of the best methods of characterising ectomycorrhizae is by direct observation of root tip gross morphology under low magnification. This view has been contested by several researchers (Egli et al. 1993; Karen 1997), who suggest that many morphotypes cannot be distinguished from one another based on gross morphology alone. While morphological techniques are inexpensive and not dependent upon an extensive laboratory, molecular methods are superior in that they are independent of host variation and environment (Egger 1995) plus they do not require as skilled an investigator as does morphotyping (Nylund et al. 1995). Polymerase chain reaction/ restriction fragment length polymorphism (PCR-RFLP) based analysis has increased the confidence with which non-fruiting mycorrhizal taxa can be identified (Egger 1995). The nuclear ribosomal RNA gene region (rDNA), particularly the internal transcribed spacer (ITS) 59 region, is often used in both ecological and phylogenetic studies of the higher fungi . Different portions of the rDNA are used for different levels of resolution. The region used in this study encompasses the 5' end ofthe 18S gene, ITS 1, 5.8S gene, ITS 2 and a portion of the 28S gene, including divergent domain Dl. Divergent domains are loops in the DNA secondary structure that have undergone large variations in size and are ideal for species to genus level phylogenetic studies (Michot et al. 1984). Restriction site and sequence data have been used to identify ECM fungi in the genera Wilcoxina (Egger and Fortin 1990, Egger 1996), Geopyxis (Vralstad et al. 1998) and He/vella (Weidemann et al. 1998). PCR-RFLP analysis has become accepted to the point that manuals on ECM identification also include PCR/RFLP banding patterns (Goodman et al. 1996; Agerer 1987-1998). The objective of this study was to identify and describe previously unknown Pezizalean ECM. RFLP banding patterns from putatively ascomycetous root tips, diagnosed for Hartig net presence, were compared to patterns from fruit bodies of suspected ectomycorrhizal members of the order Pezizales. Identical patterns for three restriction enzymes indicate an association, identical patterns between two, with the third enzyme differing suggest an intra-generic relationship between the root tip and fruitbody (Karen 1997). 60 3.1 Methods 3.1 a Sampling The sampling of taxa was based on two factors: previous reports of potential as mycorrhizal symbionts, and phylogenetic relationships to known or suspected symbionts both illustrated in Chapter two of this thesis and other phylogenetic papers. Published species descriptions (Abbott and Currah 1988; Danielson 1979) especially of those species found in northern B.C. or in a similar climate served as an identification guide. Sources describing cosmopolitan taxa were also used to identify ascomycete fungi (e.g. Fogel and Trappe 1976; Stewart and Heblack 1979; Weber 1972; Weber 1975; Yao and Spooner 1996). Fruitbodies ofthe suspected mycobionts were collected along with a soil sample containing roots of the suspected host from directly beneath the fruitbody. If sufficient fruitbodies were present, at least four fruitbodies were collected, one for identification and DNA work and three for herbarium reference material which will be deposited at a later date. Figure 13 is a map of the Pacific Northwest, indicating collection sites with an arrow. Figure 14 is a more detailed map of the Prince George region with collection sites (i.e. where fruitbody/ root tip samples were found) there indicated by red coloured letters. Table 2 lists the fruitbodies collected and includes habitat information. 61 Prince George collection site Vancouver Island Collection site Oregon Collection site Fig. 13 Collection sites in the pacific northwest (1 " = 158 mi) * map courtesy of Geomantics Canada, National Atlas of Canada base map series 62 Table 2 Fruitbody and root tip type accession codes and habitat information Fruitbody accession (Root tip accession) DBS1 * EM (emrt) GBl (gb3rt) GB2 (gc 1rt, gc3 rt2, gcrt 1) GSl (gslrtl, gs1rt2) 1 GS2 (gs2rt1 ) GS4 (gs4rtl , gs4rt2) GS5 \ (gs5rt1) HE (hert) HYD (hrtl' hrt2) I 01 (o1rt1) 02 (o2rtl) 03 (o3rtl , o3rt2) oc (ort) Identity Discina sp. Elaphomy ces muricatus Fr. Pseudorhizina sphaerospora (Phillips) Harmaja Pseudorhizina sp. Location and suspected host (fig. #, map location letter) under Abies lasiocarpa near Aleza lake, B.C. (13, A; 14, A) under Tsuga heterophylla at the China Beach Park, Vancouver Island, B .C. (13 , B) under Abies lasiocarpa, near Aleza lake, B.C. (13, A; 14, A) under Abies lasiocarpa, Crescent Spur, Robson Valley, B.C. (13 , A; 14, F) under Picea mariana on the Cranbrook Hill Greenway, Prince George, B.C. (13 , A; 14, B) under Picea mariana on the Cranbrook Hill Gyromitra esculenta Greenway, Prince George, B .C. (13 , A; 14, (Pers. :Fr. )Fr. B) under Picea mariana on the Cranbrook Hill Gyromitra esculenta Greenway, Prince George, B.C. (13 , A; 14, (Pers. :Fr. )Fr. B) Gy romitra esculenta I near Picea mariana and Abies lasiocarpa on (Pers.:Fr. )Fr. I the Cranbrook Hill Greenway, Prince George, B.C. (1 3, A; 14, B) He/vella elastica (Bull.) under Picea mariana and Tsuga Baud. heterophylla, near Port Renfrew, Vancouver Island, B.C. (13 , C) under Pseudotsuga menziesii, Viking Ridge, Hydnotrya variiformis B.C. (13, A; 14, E) Gilkey trailside under Populus tremuloides, Abies Otidea sp. (Cke.) Mass. Grev. lasiocarpa and Betula papyrifera on the Cranbrook Hill Greenway, Prince George, B.C. (13, A; 14, B) trailside near Abies lasiocarpa, Populus Otidea onotica (Pers.) Bonord. tremuloides and Betula papyrifera on the Cranbrook Hill Greenway, Prince George, B.C. (13 , A; 14, B) Otidea cochleata FuckeL trailside near Abies lasiocarpa , Populus tremuioides and Betula papy rifera on the Cranbrook Hill Greenway, Prince George, B.C. (13, A; 14, B) Otidea leporina var. near Pseudotsuga menziesii at the HJ. Andrews experimental Forest near Eugene, I minor (Batsch) Fuckel I OR. (13, D) Gyromitra esculenta (Pers. :Fr. )Fr. 64 PE* Peziza echinospora Karst. sc Sarcosphaera coronaria (Jacq.) Boud. (scrt2) SF (sfrt) Spathularia flavida Pers.:Fr. Trailside near Populus tremuloides, Forests for the World, Prince George, B.C. (13, A; 14, B) under Pice a mariana at the L. C. Gunn municipal park, Prince George, B.C. (13, A; 14, C) Abies lasiocarpa stand in the Forests For the World, Prince George, B.C. (13, A; 14, B) trailside near Abies lasiocarpa, Populus tremuloides and Betula papyrifera on the Cranbrook Hill Greenway, Prince George, B.C (13, A; 14, B) Neolecta vitellina (Bres.) under Pseudotsuga menziesii, West Lake, UND B.C. (13, A; 14, D) Korf ex J.K. Rogers *frmtbod1es lackmg root tip accessiOns were not collected near a potential host TH (thrt) Trichophaea hemisphaerioides (Mouton) Graddon The quantity of root tips sampled from the soil block depended upon their density in the soil, with an average collection being about 40 tips. Root samples collected were soaked in cold tap water for 30 minutes to 2 hours to loosen soil and non-potential host root systems. The roots were gently rinsed and then were transferred, in small aliquots to a petri plate filled with water. The roots in the petri plate were then further cleaned of debris with the use of forceps under a dissection microscope. Up to 10 of these were mounted permanently for morphological examination and up to 5 were stored for DNA work. Permanent mounts included whole mounts and squashes. The protocol for morphological characterization of the root tips follows that of Ingle by et al. ( 1990), Agerer (1987-1 998), and Goodman et al. (1996). The characters used to describe the ECM included: shape, dimensions, colour and texture of the ECM system, shape and colour of the emanating hyphae. Mantle structure, size and finally presence or absence of cystidia were all noted using the 10, 40, and 100 power lenses on an Olympus compound 65 m1croscope. The designation of morpho types as potentially ascomycetous followed these guidelines : a root tip was considered if clamp connections and rhi zomorphs (two features characteristic ofbasidiomycetes) were absent. Stronger support came if the root tip morphology resembled that of an already documented ascomycete ECM (i.e. thin mantle, broad mantle cells and Tuber-like cystidia). Some ofthe ECM root tips, such as those near the fruitbodies of N. vitellina , P. sphaerospora and H. elastica, did not resemble other already described Pezizalean ECM, but were collected if they were the only clampless root tips present. Certain problems arise using these criteria. A number of ectomycorrhizal basidiomycetes have inconspicuous clamp connections or lack them completely, including but not limited to: Amanita muscaria, Lactarius rufus, Lactarius glyciosmus, Th elephora spp. , Lactarius pubescens and Leccinum spp. (Ingleby et al. 1990). This makes the absence of clamps a non-diagnostic character when trying to ascertain if an ECM root tip is ascomycetous. Also, numerous basidiomycete ECM lack rhizomorphs (Ingleby et al. 1990). Finally, only a few ascomycetes have been documented as ectomycorrhizal, resulting in only a few morphotype descriptions for comparison (Agerer 1987-1998; Ingleby et al. 1990). A list of all the root tips that properly amplified, a briefmorphotype description and some habitat information are included in Table 3 . 66 Table 3 Ectomycorrhizal root tip morphotype descriptions Fruitbody and study accession Host Gross morphology Mantle characters Additional info. E. muricatus T heterophylla straight to club shaped, unbranched inner: net synenchyma outer: felt prosenchyma emanating hyphae rare and tortuous Picea sp. non-branched and bent inner mantle: interlocking irregular synenchyma Outer mantle: net prosenchyma Emanating hyphae common, septate, no clamps, hyaline Otidea sp. Olrtl A. lasiocarpa, P. tremuloides, B. papyrifera straight and monopodia] pinnate net synenchyma emanating hyphae common, light brown, verrucose, septate. 0. onotica 02rtl A. lasiocarpa, P. tremuloides, B. papyrifera Tips bent and non branched 0. cochleata 03til A. lasiocarpa, P. tremuloides, B. papyrifera Tips bent and monopodia! pinnate noninterlocking irregular synenchyma many hyphae present near the root tip surface A. lasiocarpa, P. tremuloides, B. papyrifera Tips bent and monopodia] pinnate felt prosenchyma emanating hyphae are curved and common P. glauca bent and monopodia! pinnate net prosenchyma emanating hyphae common and tortuous S. coronaria scrt l P. glauca bent and monopodia] pyramidal net prosenchyma this morphotype seemed to blacken with senescence T hemisphaeriaides A. lasiocarpa, Tortuous and P. tremuloides, · monopodia! B. papyrifera pyramidal interlocking irregular synenchyma emanating hyphae rare, clear emrt H. elastica hert 0. cochleata 03rt2 S. coronaria scrt2 THrt 67 netsynenchyma none noted 3.1 b DNA extraction The primary DNA extraction technique used was a CTAB based protocol modified from Zolan and Pukkila (1986) (Baldwin and Egger 1996). In addition, an SDS based extraction procedure was used on samples that did not extract with the CT AB protocol (Sambrook et al. 1989). Frozen or dried fruitbodies or frozen (-40°C) root tips (a 0.5 cm 2 piece of fruitbody or one root tip) were crushed with either micro or macro pestles (Mandel scientific) for root tips and fruitbodies respectively. A 2x CT AB buffer consisting of 1.4M NaCl, 100 mM Tris-HCl (pH 8.0) (Sigma Chern. Co.), 20 mM EDTA (pH 8.0), 2% CTAB (Sigma Chern. Co.) and 0.2% beta-mercaptoethanol was added. Fruitbodies required 700 f.!L of buffer whereas root tips required 350 f.!L. Tissue was ground again in the buffer and incubated in a 60°C water bath (VWR Scientific) for 45 minutes, then 350 f.!L of 24:1 chloroform: isoamyl alcohol was added and thoroughly mixed. This mixture was centrifuged for 10 minutes at 13000 rpm, the aqueous layer removed to a clean tube, and the DNA was precipitated by adding an equal volume of cold absolute isopropanol. At this point, one of two procedures was undertaken. Either the mixture was inverted repeatedly for 5 min., then incubated in a -20°C freezer for 5 minutes, or the mixture was incubated overnight at -40°C 1 (Baldwin and Egger 1996). The mixture was then centrifuged at 13000 rpm for 10 minutes, after which the isopropanol supernatant was removed and the DNA pellet was washed with cold 70% ethanol and centrifuged for 5 minutes at 13000 rpm. The washing step was repeated twice and the excess ethanol was allowed to evaporate overnight. The pellet was resuspended in 50 ~ of either NaOH for the root tips or dH 20 for the fruitbodies . The SDS protocol involved an overnight incubation at 36°C in a mixture of 475 f.!L proteinase K buffer 68 (5ml, 0.01M, pH 7.8 Tris HCI, 20 ml , 0.01M EDTA, 25 ml, 0.5% SDS and 450 ml H20) 2 and 251J.L proteinase K. As in the previous protocol, roughly 0.5 cm of fruitbody tissue or one root tip was used. After incubation, 550 iJ.L PCI (Phenol: Chloroform: Isoamyl alcohol, 25:24 :1)(volume plus 10%) was added, mixed by inversion for five minutes, then centrifuged at 13, 000 rpm for 15 minutes. The top layer (400 iJ.l) was removed and 440 iJ.l PCI (volume plus 10 %) was added. This mixture was centrifuged at 13000 rpm for 15 minutes. The top layer (250 !J.L) was removed, 25 iJ.l of 3M NaOAc (pH 5.5) (sodium acetate) and 165 iJ.l isopropanol was added. This solution was mixed by inversion for 5 minutes, then centrifuged at 13000 rpm for 10 minutes. The remaining liquid was removed and 400 iJ.L of 70% ethanol was added. This was refrigerated (4 °C) overnight. The mixture was then centrifuged at 13000 rpm for 10 minutes, drained and vacuum centrifuged (Speedvac) at 60°C for 3 minutes. Finally 100 iJ.l TE ( 99ml H20, 1000 iJ.l Tris base, 200 iJ.l EDTA, pH 8.0) was added and the extract was incubated at 36°C for one hour (Sambrook et al. 1989). 3.1c Amplification The PCR cocktail consisted ofDNA template, H 20 , primers, MgCh, dNTPs, Tag DNA polymerase and Tag buffer. The concentrations and relative formulation of the buffer differ depending on the Tag used. Three different types of Tag were used: Ultratherm Tag (BioCan Scientific), Platinum Tag and Recombinant Tag (Life Technologies). 1 Dr. Keith Egger, Mycologist and Biology Professor at the University of Northern British Columbia. Tel: 250-960-5860 69 Ultratherm amplification cocktail contained: 17.2 ~ dH20, 3 ~ 1Ox Taq buffer, 3 ~ dNTPs (2mM stock), 2.4 ~ MgCh (25mM), 1.2 ~ primer ITSl (10 ~ NL6Bmun (10 ~ and 0.08 ~ Ultrathem1 Taq DNA polymerase (1:1). Platinum Taq amplification cocktail contained: 17.8 ~ dH 20, 3 ~ of lOx buffer, 3 ~ stock), 1.8 ~ MgCh (25 mM), 1.2 ~ primer ITS 1 (1 0 ~ (1 0 ~ 1.2 ~ primer dNTPs (2 mM 1.2 ~ primer NL6Bmun and 0.2 ~ Taq DNA polymerase. Recombinant Taq amplification cocktail contained: 17.8 ~ dH20, 3 ~ primer ITS 1 (10 ~ lOx buffer, ~ 1.2 ~ primer NL6Bmun dNTPs, 1.8 ~ MgCh (25 mM), 1.2 ~ ~ and 0.25 ~ Taq DNA polymerase. Two different thermal cyclers were used, a Model480 Perkin-Elmer-Cetus them1al cycler and a PTC-1 00 thermocycler from MJ Research Inc. The run consisted of 35 cycles with a denaturation step of94°C for 46 seconds, an annealing step of 44°C for 1 minute, 30 seconds and an extension step of 72°C for 3 minutes and 5 seconds increased by one second per cycle. Various annealing temperatures were tested both above and below 70 3.1 d Restriction digestion The restriction endonucleases A lui (AG!CT ), I Jmj[ ( u / ANTC) and Rsal (GTI AC) were used to generate restriction patterns for each of the ;:;anlples. , separate digestion master mix was required for each enzyme. Bctvvcen 7 and 17.. uL nfPCR product was added to each digestion master mix consisting of 5.0 ~ (Pharmacia), and 0.5 ~ H 20, 2 FL of 1Ox 'One-Phor-All' buffer Alul, 0.3 ~ of Hinjl and or 0.37 ilL Rsal (equaling 2 units for each enzyme). The mixture was incubated overnight at 36°C. These samples were then run through a 2.5% agarose gel consisting of300 ml 0.5x TBE (108g Tris base, 55 .0 g boric acid, 45 ml 0.5M EDTA (pH 8.0), 3 g agarose and 4.5 g ofNusieve agarose). Ethidium bromide was added to the gel, which binds to the DNA and fluoresces under uv light. 3.1e RFLP analysis Photos of electrophoretic gels containing digested samples were taken using a Gelprint2000 photo-documentation system (BioPhotonics) . The software package, RFLPscan Plus (Scanalytics) was used to analyse both band size and relationship between samples. The Log Piecewise Linear Curve fitting option was used to calibrate band sizes. These bands were then matched within each gel at a 2% match tolerance. Databases were created using RFLPscan database manager 3.1. The band sizes were calibrated at a 6% match tolerance between gels using the Pairwise Method . A cluster analysis using the Neighbor module ofPHYLIP version 3.5c (Felsenstein 1993) was used to determine relatedness between pairs of samples. The genetic similarity between individuals was calculated using Dice's index (Chew et al. 1997): 71 Dice Index = [2x common bands/(2x common bands+polymorphic bands)] Since the PHYLIP program requires pairwise comparisons to be distances, the distance (i .e. 1-Dice Index) was calculated as follows: Distance = Le[polymorphic bands] I ([shared bands]+ [total bands]) Where i = restriction endonucleases Phylograms based on these distances were created using the UPGMA (unweighted pair group methods using arithmetic means) option ofPHYLIP. These phylograms were then viewed using Treeview 1.3 (Page 1996). 3.1f Trouble shooting techniques A series of steps were taken when amplifications failed. These steps did not necessarily conform to the order stated. 1. Pure Ultratherm Taq was tried instead of 1:1 Ultratherm Taq. 2. Platinum and Recombinant Taq (Life Technologies) were tried as an alternative to Ultratherm Taq. Recombinant Taq optimizes PCR by not only transcribing from DNA, but mRNA also (Higuchi 1990). Platinum Taq is a recombinant Taq polymerase with associated antibodies that render the polymerase inactive until the temperature reaches 94 °C. This inhibits non-specific amplification resulting from polymerase activation before the denaturation step. The PCR protocols using these Tag's were similar to Ultratherm (see previous section). 3. A template dilution series ranging from 1: 10 to 1:1000 was used. Dilutions reduce the amounts of inhibitory compounds, such as pigments and proteins to tolerable levels while leaving enough DNA template to successfully amplify (Palumbi 1996). 72 4. If the dilution series failed, the DNA extract was run on an agarose gel as an attempt to check for the presence of DNA. Large quantities of DNA appear as smears. The downfall of 'checking' for DNA on gels is its inability to detect small but amplifiable quantities ofDNA (Palumbi 1996). 5. "Smeared" samples from step four were run through PCR wizard (Promega) columns to purify the DNA. This protocols should eliminate other compounds that may interfere with the amplification process. 3.2 Results Twenty-four root tip types from under different hosts were collected and processed. Of those, ten were amplified, digested and analysed. The remaining types failed to amplify, their descriptions are therefore not included. None of the restriction patterns from the root tips collected matched any fruitbody restriction patterns. In addition to failed ECM root tip amplifications, a number of fruitbodies failed to amplify and others only amplified after a series of 'trouble shooting' techniques were attempted, the success and failure of these techniques are listed below in reference to each sample. Table 4 documents fruitbody 'trouble-shooting' data and Table 5 deals with root tip 'troubleshooting' data. Sixteen fruitbody taxa, represented by 19 collections, were studied. Of those 19, 16 fruitbodies amplified properly. Fifteen amplified with 1:1 ultratherrn Taq, which was the taken directly from the modified Zolan and Pukkila protocol (Baldwin and Egger 1996). One of the 19 analysed fruitbodies, Hydnotrya variiformis, amplified with Recombinant Taq and a dilution series. Of the 15 that amplified with 1:1 ultratherrn Taq, 7 fungi in the genera Gyromitra, Pseudorhizina, Otidea, Trichophaea and Neolecta required a dilution series for successful amplification. Of the 8 remaining, one, Peziza 73 echinospora, amplified with a doubling of the MgC}z, and another, Sarcosphaera coronaria, amplified with half the dNTP concentration. The final 6, belonging to the genera Gyromitra, Otidea and Spathularia amplified according to the standard protocol (as discussed above). The fruitbodies that did not amplify were Discina spp., Elaphomyces muricatus and He/vella elastica. An SDS protocol, dilutions, platinum Taq, recombinant Taq and a column extraction kit (Promega PCR wizard) were all tried, but to no avail. Interestingly, some Gyromitra and Otidea collections amplified with a dilution series, and others did not require dilution for proper amplification. In the case of Gyromitra, it was different collections of the same species that required different protocol modifications. Of the 24 root tip types collected, 10 successfully amplified. Of those 10, 6 worked with 1:1 ultratherm Taq. Four ofthose 6 worked only when another aspect ofthe protocol was modified, e.g. the root tip, (emrt), collected under Elaphomyces muricatus worked only when the annealing temperature was reduced to 44°C, the root tip, (gcrt1), collected under Pseudorhizina sp., amplified with a 1:50 extract dilution and the root tip, (sfrt), collected under Spathularia jlavida, amplified with a 20 ).lM MgC}z concentration, and 40 cycles on the thermocycler (as opposed to the standard 10)-lM MgC}z and 36 cycles). Finally, the root tip collected under H. elastica (hrt) amplified only when the extract was cleaned with the PCR Wizard column extraction kit (Promega). Five root tips amplified without 1:1 ultrathem1 Taq, one of those, (scrt2), collected under Sarcosphaera coronaria , amplified with pure ultratherm Taq. The remaining four, collected under 74 Pseudorhizina sphaerospora (gb3rt), P. californica (gc3rt2), and Hydnotrya variiformis (hrtl, hrt2) amplified with recombinant Taq. Six root tip morphotypes did not amplify at all, these included all the root tips collected under Otidea (olrtl, o2rtl, o3rtl) and those collected under Trichophaea hemisphaerioides (thrt) and Neolecta vitellina (unkrtl, unltcol.) . An SDS extraction, a dilution series, column extraction were tried on these root tips, but nothing worked. Table 3 is a summary ofthe root tip morphotypes. Root tips collected under some of the samples from Table 2 were not described in Table 3 because they were found, either upon collection or later examination to belong to non-Pezizalean morphotypes (due to the presence of clamp-connections). 75 Table 4 Fruitbody DNA extraction/ PCR trouble shooting techniques sample DBS1 EM --- ·- - pure ultra therm Taq X X 1:1 ultra therm Taq X X alternate Taq - X X - X (P, R) SDS extraction protocol X X GBl GB2 GS1 GS2 GS4 GS5 HE X + + + + + + X HYD X X + (R) - 01 02 03 - X - + + - - - - - PE - + + + + + sc - + oc - X - - - - - - Extract dilution X 1: 25 (X) 1:50 (X) , 1:100 (X) - 1:25 (+) 1:50 (+) - 1:50 (+) 1:50 (+) 1:25 (X), 1:50 (X). 1:100 (X) 1:25 (+), 1:50 (+) Promega PCR wizard other technique X - - - - - - - - - - - X - - - - - - - - 1:100 (+) - - - - + - - - - 2x MgCh cone. 0.5X DNTP cone. + + SF + 1:50 (+) TH 1:25 (+) + UND a - md1cates no attempt, an X md1cates a failed attempt and a + md1cates a successful attempt. A row without a'+' indicates a failed amplification. A 'P' under the column 'alternate Taq' indicates Platinum Taq was used, an 'R' indicates Recombinant Taq was used . Results in the last four columns complement a successful Taq amplification. When not otherwise noted, the extraction method follows a modified Zolan and Pukkila (Baldwin and Egger 1996). 76 Table 5 Root tip DNA extraction/ PCR trouble shooting techniques extract SDS alternate sample pure ultra 1:1 ultra extraction dilution Taq therm therm protocol Taq Taq + emrt gb3rt gc1rt gc3rt2 gcrtl gs4rt hert hrt1 hrt2 o1rtl o2rt1 o3rt1 scrt2 sfrt - - - - - - 1:50 (+) - - - - X X X X X + +(R) +(R) X X X - - + - - - +(P,R) - annealing temp . of 44° c - X X X - - - - other technique - - +(R) X + X + + + X X X X X X X X Prom ega PCR wizard - - X X X + - X X X - - - - - - - - 42 ° c annealing temp 20f.1M MgCb and a 40x cycle 1:25 (X), X 1:50 (X), 1: 100 (X) unkrtl X X X X X X unltcol. X X X X X X a - md1cates no attempt, an X md1cates a failed attempt and a + md1cates a successful attempt. A row without a '+' indicates a failed amplification. A 'P' under the column 'alternate Taq' indicates Platinum Taq was used, an 'R' indicates Recombinant Taq was used. Results in the last four columns complement a successful Taq. When not otherwise noted, the extraction method follows a modified Zolan and Pukkila (Baldwin and Egger 1996). thrt - X X X 77 Justification for the selection of tips varied from a combination of the above characteristics to lack of other tips in the root collection . No tips with definite clamp connections were included. However, tips with hyphae growing near them but not obviously attached and with clamp connections were sometimes included. The root tips collected surrounding the E. muricatus fruitbody were identical to Agerer's (1987-1998) description of E. muricatus ectomycon·hizae, unfortunately fruitbody DNA could not be isolated to confirm this. Two of the root tip types collected under Sarcosphaera coronaria were light orange yellow and consisted of a net prosenchyma inner mantle (Fig. 16) which resembled the Ingleby et al. (1990) Humaria hemisphaerica description. Ingleby et al. (1990) described the mantle surface as net prosenchyma which, as it ages, thickens and shortens to form a net synenchyma inner mantle. The root tips collected under Trichophaea hemisphaerioides resembled Tuber spp. morphotypes (Ingleby et al. 1990) with the exception of a lack of cystidia in the former. T. hemisphaerioides tips were described as medium yellow brown with a yellow apex and tortuous (Figs. 15, 20), whereas Tuber spp. tips are described as buff coloured, short and stubby (Ingleby et al. 1990). The mantles of both morphotypes are of the interlocking irregular synenchymal variety (Fig. 20). Otidea sp. and 0. onotica both resemble Ingleby's Humaria hemisphaerioides and Estrain (what he calls Tricharina gilva) in that they both have a net synenchymal mantle. The host ambiguity (Abies lasiocarpa, Populus tremuloides or Betula papyrifera) of the aforementioned Otidea spp. makes it difficult to compare morphotypes in terms of 78 branching pattern, tip colour and surface features. The irregular synenchymal inner mantle ofthe ECM root tip (rtl) found under Otidea cochleata resembles the inner mantle of many Tuber spp. and Genea verrucosa (Agcrer 1987-1998). The morpho types of all ECM root tips collected under Neolecta vitellina, Pseudorhizina sphaerospora (Figs. 21 , 22) and Helvella elastica (Fig. 18) fruitbodies did not resemble other already described Pezizalean ECM in terms ofboth mantle characteristics and distinctive emanating hyphae. 79 Fig. 15 Root tip system collected under Trichophaea hemisphaerioides (thrt) Fig. 16 Root tip system collected under Sarcosphaera coronaria (scrt2) - l mm Fig. 17 Senescent monopodia! pyramidal system collected under Sarcosphaera coronaria (scrt2) Fig. 18 MRA like tips under He/vella elastica (hert) Fig. 19 A net synenchymal mantle found under an Otidea sp. fruitbody (o1rt1) Fig. 20 An interlocking irregular mantle found under a Trichophaea hemisphaerioides fruit body (thrt) Fig. 21 A non interlocking irregular synenchymal mantle found under a Pseudorhizina sphaerospora fruitbody (gb3rt1) Fig. 22 An irregular synenchymal mantle found under a Pseudorhizina sphaerospora fruit body (gb3rt 1) 81 3.3 Discussion 3.3a Recalcitrant samples A number of root tips and fruitbody samples failed to amplify. Many fungi, especially some ascomycetes, contain high levels of polysaccharides which, due to their structural similarity to DNA, inhibit PCR (Arrnaleo and Clerc 1991). Pezizalean fruitbodies , specifically members of the tribe Sarcoscyphinae and those in the families Helvellaceae and Otideaceae, contain high levels of pigments, which can also inhibit PCR (Landvik 1996). In addition, root tips consist of a minute quantity of fungal tissue and therefore a minute quantity of DNA, making careful extraction and amplification critical for successful PCR. The requirement of a dilution series for amplification implies an inhibiting factor such as pigments or polysaccharides that, when reduced by a dilution factor, no longer limited PCR. Several fruitbody samples, including Gyromitra esculenta, Pseudorhizina sphaerospora, Otidea cochleata, Trichophaea hemisphaerioides and Neolecta vitellina and one root tip sample (gcrtl) found under Pseudorhizina sphaerospora required DNA extract dilutions for successful amplification. Interestingly, different collections (meaning different populations) of the same species of Gyromitra esculenta and Pseudorhizina sphaerospora required different amplification protocols. One collection of each required a dilution factor of 1:50 and 1:25 respectively and the other two collections of each species did not require dilution. This requirement difference could be due to varying levels of polysaccharides or pigments in the different populations or it could be a result of tissue selection for extraction. Samples chosen for extraction, 82 exclusively from less pigmented regions of the hymenium may require either a smaller dilution factor than those samples taken from more heavily pigmented regions of the hymenium, or no dilution factor at all. The modified Zolan and Pukkila protocol (Baldwin and Egger 1996) was efficacious on most fruitbody samples. Protocol modifications that had the greatest effect on recalcitrant fruitbody samples included extract dilutions and PCR reagent concentration changes. Discina sp., Elaphomyces muricatus, and He/vella elastica did not amplify. Many of the root tip samples amplified using the modified Zolan and Pukkila protocol (Baldwin and Egger 1996). The majority of the remaining root tips amplified using recombinant Taq. Root tip morphotypes that did not amplify consistently were those found under the genus Otidea and Neolecta. Unlike the fruitbody samples, a dilution series did not increase the amplification rate of samples that did not previously amplify. This suggests that many of the inhibiting compounds found in fruitbodies, such as pigments or polysaccharides are either not present in root tips or not present in concentrations sufficient to prevent amplification. 83 3.3b Lack ofRFLP matches between fruitbodies and root tips There are several possible explanations for the lack of success in finding fruitbody/ root tips matches. First, the fungus in question is not mycorrhizal or root associated , thus any attempts at finding matches inevitably end in failure . Second, the fungus is root associated, but none ofthe tips was collected. A study by Karen (1997) reported onl y 14 fruitbody/ root tip matches out of 43 different RFLP patterns generated from Pinus sylvestris ECM root tips (33 % matched). In a similar study, Varga (1998) reported only 1 fruitbody/ root tip match out of 68 fruitbody samples and 305 ECM alder and pine root tips reinforcing Karen's (1997) results. Finally, Gardes and Bruns (1996) pointed out that there was an incongruence between the number of ECM fruitbodies at a site and the number of root tips colonized by those fungi, i.e. the predominant ECM fruitbody at a site may not produce the most EM tips and vice versa. This study, Gardes and Bruns (1996) also showed that root tips colonized by a specific fungus are not necessarily found immediately beneath the fruitbody. This is logical since the fungal thallus can be meters in length (Alexopolous et al. 1996). The approach used in my study was to collect a soil and root sample from beneath the fruitbody . If only a few root tips were colonized by the sample fungus or if they occurred beyond 12 em from the fruitbody, they may not have been collected. Third, the target fungus may have amplified, but due to intra-specific variation in the ITS region, the RFLP patterns between fruitbody and host colonising fungus did not match. A paper by Cullings et al. (1996) reported that a collection of the ECM fungus, Rhizopogon subcaerulescens from a Pterospora andromedea root ball yielded a different ITS-RFLP pattern from three other collections of R. subcaerulescens found within 15 em of the P. andromedea. A study by Karen et al. (1997) revealed intra- 84 specific variation in the ITS region of 13 species of basidiomycetes out of a total of 44 examined. This variation took the form ofboth length polymorphisms and base pair mutations at endonuclease recognition sites. The above two studies have focused on basidiomycetes, but according to Seifert et al. (1995), intra-specific ITS variation differs in species of ascomycetes and has been shown to range from 0% to 15 .8%. Fourth, the fungus is root associated, but the DNA extraction or the PCR simply failed as with the case of the tips collected under the Otidea spp. (see Table 4) or the DNA extraction/ PCR failed on the fruitbodies, as with the case of Helvella elastica (see Table 5) thus preventing possible root tip/ fruitbody RFLP pattern matching. Even if an ECM root tip and fruitbody RFLP pattern had matched, the association could not be called ECM without the presence of a Hartig-net which is one of the characters that distinguishes a mycorrhizal association from a parasitic one (Harley and Smith 1983). On the colonized roots examined during the course of this study, the majority of the Hartig nets examined were of the 'coarse with broad and infrequently ramified lobes' type (Agerer 1987-1998). This structure can be very difficult to distinguish in squashed root cells. Also, the difference between the classically described 'labyrinthic' type Hartignet and an interlocking irregular synenchymal inner mantle is often slight. This leads to difficulties when trying to define the trophic status ofthe association examined. Due to the present study's failure and the relative failures of others in matching fruitbody RFLP patterns with potential EM root tip patterns (Karen 1997, Cullings et al. 1995, Varga 1998), I suggest that this technique is best used in conjunction with other 85 approaches, such as pure culture synthesis, radio-labelled isotope studies, or comparison to a large database of potentially ECM fmitbodies. The next chapter, following the latter suggestion, details the comparison of the data set generated from this study with a data set of fmitbodies and ECM root tips accumulated from other sources in an attempt to find some RFLP pattern matches. 86 3.4 References Abbott, S.P., and R.S. Currah. 1988. The Genus He/vella in Alberta. Mycotaxon 33:229250. Agerer, R. 1987-1998. Colour Atlas ofEctomycorrhizae. Schwabisch Gmund, Germany. Einhorn-Verlag Eduard Dietenberger. Agerer, R . 1996. Concise descriptions of ectomycotThizae. Schwabisch Gmund, Germany. Einhorn-Verlag Eduard Dietenberger. Alexopoulos, C. Mims, and M. Blackwell. 1996. Introductory Mycology (4th Edition), Wiley & Sons, New York. Armaleo, D. and P. Clerc. 1991. Lichen chimeras: DNA analysis suggest that one fungus forms two morphotypes. Exp. Mycol. 15:1-10. Baldwin, Q. and K.N. Egger. 1996. Protocols for analysis of DNA from mycorrhizal roots . In Concise descriptions ofNorth American Ectomycorrhizae. Goodman, D.M., Durall, D.M., Trofymow, J.A. , Berch, S.M. (Eds.). Mycologue Publications and CanadaB.C. Forest Resource Development Agreement, Canadian Forest Service, Victoria, B .C. Chew, J.S.K. , D.B . Strongman, and R.M. MacKay. 1997. RFLP analysis ofrRNA intergenic spacer regions of 23 isolates of Paecilomyces farinosus. Can. J. Bot. 7 5: 203 82044. Chilvers, G.A. 1968. Some distinctive types of eucalypt mycorrhiza. Aust. J. Bot. 16: 49. Cullings, K.W. , T.M. Szaro and T.D. Bruns. 1996. Evolution of extreme specialization within a lineage of ectomycorrhizal epiparisites. Nature, 379: 63-66 . Danielson, R.M. 1979. Hypogeous ascomycetes in Alberta, Canada with two new North American records. Mycotaxon 9: 445-450. Danielson, R.M. 1982. Taxonomic affinities and criteria for identification of the common ectendomycorrhizal symbiont ofpine. Can. J. Bot. 60: 7-18. Dominik, T .1969. Key to ectotrophic mycorrhizae. Fol. Forest. Polon. Ser. A 15 :309-328 . Egger, K.N. 1995 . Molecular analysis of ectomycorrhizal fungal communities. Can. J. Bot. 73 (suppl.): s1415-s1422. Egger, K.N. 1996. Molecular systematics of E-strain mycorrhizal fungi: Wilcoxina and its relationship to Tricharina (Pezizales). Can. J. Bot. 74: 773-779. 87 Egger, K.N. and J.A. Fortin. 1990. Identification of taxa of E-strain myconhizal fungi by restriction fragment analysis. Can. J. Bot. 68 : 1482-1488. Egli, S., R. Amiet, M. Zollinger, and B. Schneider. 1993. Classification of Picea abies (L.) Karst. ectomycorrhiza: discrepancy between classification according to macroscopic versus microscopic features . Trees 7: 123-129. Fogel, R. and J.M. Trappe. 1976. Additions to the hypogeous mycoflora of Colorado . I. Ascomycetes. Mycotaxon 4: 211-217. Felsenstein, J. 1993 . Phylip version 3.5c. Distributed by the author. Dept. of Genetics, University of Washington, Seattle. Gardes, M., T.D. Bruns. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above and below ground views. Can. J. Bot. 74: 1572-1583 . Goodman, D.M. , D.M. Durall, J.A. Trofymow, and S.M. Berch. 1996. Concise descriptions of North American Ectomycorrhizae. Goodman, D.M. , Durall, D.M., Trofymow, J.A. , Berch, S.M. (Eds.). Mycologue Publications and Canada-B.C. Forest Resource Development Agreement, Canadian Forest Service, Victoria, B.C. Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, London. Ingleby, K., P.A. Mason, F.T. Last and L.V. Fleming. 1990. Identification of ectomycorrhizas. Institute of Terrestrial Ecology, Midlothian, Scotland. Karen, 0. 1997. Effects of air pollution and forest regeneration methods on the community structure of ectomycorrhizal fungi . Doctoral Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. Landvik, S. 1996. Phylogenetic rDNA studies of discomycetes (Ascomycota). Ph.D. thesis, Umea University, Umea, Sweden. Maia, L.C. , A.M. Yano and J.W. Kimbrough. 1996. Species ofascomycota forming ectomycorrhizae. Mycotaxon 217 : 371-390. Michot, B., N. Hassouna and J.-P . Bachellerie. 1984. Secondary structure ofmouse 28S rRNA and general model for the folding of the large rRNA in eukaryotes. Nucleic Acids Res. 12: 4259-4279. Nylund, J.-E ., A. Dahlberg, N. Hogberg, 0. Karen, K. Grip, and L. Jonsson. 1995. Methods for studying species composition of myconhizal fungal communities in ecological studies and environmental monitoring. In: Biotechnology of ectomyconhizae, Stocci, V., Bonfante, P. , Nuti, M. (Eds.). Plenum Press, New York, pp 229-239. 88 Page, R.D.M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer applications in the Biosciences 12: 357-358 Palumbi, S. 1996. Nucleic Acids II: The Polymerase Chain Reaction. In Molecular Systematics. Hillis, D.M., Moritz, C and Mable, B.K. Sinauer Associates, Sunderland, Mass. pp . 205-245. Sambrook, J. , E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a lab manual. 2nd ed. Cold Spring Harbour Lab Press, New York Seifert, K.A. , B.D. Wingfield, M.J. Wingfield. 1995 . A critique of DNA sequence analysis in the taxonomy of filamentous Ascomycetes and ascomycete anamorphs . Can. J. Bot. 73(suppl.) :s760-s767 . Stewart, E.L. and R.K. Heblack. 1979. Hypogeous fungi ofMinnesota: Genea anthracina sp . nov. Mycotaxon 9: 451-458 . Varga, A. 1998. Characterization and seasonal ecology of ectomycorrhizae associated with Sitka Alder and Lodgepole pine from naturally regenerating young and mature spruce forests in the Sub-Boreal Spruce zone ofBritish Columbia. M.Sc. thesis, University of Northern British Columbia. Vralstad, T. , Holst-Jensen, A and T. Schumacher. 1998. The post-fire discomycete Geopyxis carbonaria (Ascomycota) is abiotrophic root associate with Norway Spruce (Picea abies) in nature. Mol. Ecol. 7: 609-616. Weber, N.S. 1972. The genus He/vella in Michigan. The Michigan Botanist 11 : 147-201. Weber, N.S. 1975. Notes on western species of He/vella I. In: Studies on higher fungi: a collection of papers dedicated to Dr. A. H. Smith on the occasion of his 701h birthday. H.E. Bigelow and H.D. Thiers. (eds.) Vaduz-Cramer, Germany. Weidemann, H.M. , A. Holst-Jensen and T. Schumacher. 1998. Demonstration of He/vella ectomycorrhizae on Dryas and Salix hosts by means of taxon-selective He/vella based nrDNA primers. Proceedings of the 2nd International Conference on Mycorrhizae, Uppsala, Sweden. Yao, Y.-J. , and B.M. Spooner. 1996. Notes on British species of Trichophaea. Mycol. Res. 100: 798-800. Zolan, M. and P.J. Pukkila. 1986. Inheritance ofDNA methylation in Coprinus cinerus. Mol. Cell Biol. 6: 195-200. 89 Chapter 4 Comparison of RFLP patterns from various databases Abstract PCR-RFLP based analysis of ectomycorrhizal fungi has become accepted to the point where it is used as standard protocol, often in conjunction with morphological characterization, in the identification of ectomycorrhizae. The purpose of this study was to compare the RFLP patterns of ECM root tips and fruit bodies from chapter 3 of this thesis along with Pezizalean fruitbodies and ECM root tips from various herbaria and collections with larger databases of ECM patterns. A positive match between fruitbody RFLP patterns and ECM root tip patterns provides evidence for an association, however, confirmation of ECM status would require more research which is out of the scope of this chapter. Some RFLP patterns from several fruitbodies and root tips matched. He/vella leucomelaena was amplified from 34 root tips morphotyped as MRA, E-strain, uncolonized/ lightly colonized and Thelephora-like . He/vella latispora was amplified from 11 root tips morphotyped as MRA. Wilcoxina rehmii was amplified from 6 root tips morphotyped as MRA, E-strain and uncolonized/ lightly colonized. Wilcoxina mikolae was amplified from 5 root tips morphotyped as MRA, E-strain and uncolonized/ lightly colonized. 90 4.0 Introduction PCR, combined with RFLP analysis, has provided researchers with a powerful tool to identify fungal associates of plants (Egger 1995, Karen et al. 1997, Kernaghan et al. 1997, Varga 1998). PCR-RFLP analysis complements morphological characterization of ectomycorrhizal and biotrophic (root-inhabiting) associations in that it is independent of host variation and environment (Egger 1995). Ectomycorrhizal associations occur between most woody (and some herbaceous) plants and some higher fungi. Ectomycorrhizae are mutualistic associations in which the host plant benefits primarily through increased nutrient uptake ability (Harley and Smith 1983). A morphological feature distinguishing ECM from other associations is the presence of a Hartig-net. The Hartig-net represents the interface between plant and fungus and is where nutrient exchange takes place. The purpose of this study was to document root inhabiting Pezizales by comparing fruitbody RFLP patterns from Chapter 3 with newly acquired fruitbody and root tip RFLP patterns. Identical RFLP patterns between root tip associate and fruitbody suggested the fungus examined was root inhabiting. 91 4.1 Materials and Methods 4.1a Sporocarp collection One hundred and eighteen sporocarps or cultures were used in this study. Thirty-five of the 49 were newly acquired for this study (Table 6). Forty-nine of the 118 belong to the order Pezizales (collected for the study reported in chapter 3, from various herbaria and from reference cultures) along with one member of the Leotiales and one member of the Neolectales. In addition, RFLP patterns of22 members ofthe Agaricales from Varga (1998) were included to see if any of the previously unidentifiable root tips matched with known basidiomycetes (Appendix 1). 92 Table 6 Fruitbody/ culture reference samples analyzed with collection data and source of material Location Reference Herbarium Identity number accessiOn number Renfrew Co. , Ont. , Can. Anthracobia maurilabra DAOM 322 (Cooke) Boud. 198886 (C) Syracuse, N.Y., U.S .A. Chloridium paucisporum a1786 BDD22 Wang & Wilcox Portland, OR, U.S .A. Geopora cooperi Harkn. DAOM ll a 144788 University of Alberta SA478, Geopora cooperi Harkn. Sal Campus, Alta., Can . ALTA909 0 Mt. Robson, Jasper SA402, Geopora cooperi Harkn. 6kl National Park, Alta. , Can. ALTA 9078 Geopora cooperi Harkn. ? 7k2 ? Yamnuska area, Alta. , Geopora cooperi Harkn. 9al DAOM Can. 167783 Mt. Work Trail, lOal DAOM Geopora sp. Vancouver Island, B.C. , 199517 Can. Geopyxis carbonaria (Alb. Renfrew Co., Ont., Can. 330 DAOM 198887 & Schwein.) Sacc. (C) 2007 DAOM Geopyxis vulcanalis (Peck) Mt. Douglas Park, 199532 Sacc. Vancouver Island, B .C., Can. (C) Cranbrook Hill Greenway gs3fb Gyromitra esculenta Trail, near Prince George, (Pers.) Fr B.C. , Can. gblfb Gy romitra sp. Crescent Spur, near Prince George, B.C., Can. Mt. Ste. Anne provincial DAOM He/vella lacunosa Afz. 2082 park, P.Q. Can. 199599 (C) DAVP He/vella latispora Boud. soil and duff, Bamberton, 13p 25038 VI, B .C., Can. 14u DAOM He/vella leucomelaena under Pinus contorta 9257 (Pers.) Nannf. near Edson, AB , Can. hydl Hydnotrya variiformis Viking Ridge, near Prince George, B .C., Can. Harkn. Morchella elata Fr. ? 2006 JWP 749 93 Source ofD A DNA from culture DNA from culture fruitbod y DNA from fruitbod y DNA from fruitbod y ? DNA from fruitbod y fruitbod y DNA from culture DNA from culture fruitbod y fruit body DNA from culture fruit body fruitbody fruit body ? a2202 FAP 7 Phialocephala fortinii U.S.A. a1474 DAOM 180366 DAOM 199541 (C) DAOM 199749 (C) ? Plectania campylospora Auckland dist., NZ Plectania milleri Paden & Lightning Lakes, B.C., Can. DNA from culture DNA from fruitbody DNA from culture Peziza echinospora Karst. Duchesnay Forest, P.Q ., Can. DNA from culture Peziza petersii Berk. & ? ? DAOM 199673 (C) DAOM 199561 (C) Peziza violacea Pers. B.C., Can. DNA from culture Peziza violacea Pers. Lightning Lakes, B.C., Can. DNA from culture Pseudoplectania melaena Sugar Bowl Trail, near Prince George, B.C. , Can. fruitbody Pseudorhizina californica fruitbody (Bull.) Fuckel Aleza Lake, near Prince George, B.C., Can. Hoh Valley Rd., WA, U.S.A. Sphaerosporella brunnea Oregon, U.S.A. DNA from fruitbod y Sandy Lake, Alta., Can. DNA from fruitbody DNA from fruitbody 2018 2136 1074 1074 2040 pm1 - Gca - 2048 DAOM 199569 (C) NSW 6793 a2245 15a1 a1804 a1808 Trich thd Wang & Wilcox (B erk.) Nann£ Tylutki Curtis (Pers. : Fr.) Sacc. ALTA667 4 CSY 98DAOM 193027 CSY 102DAOM 191896 - (Phillips) Harmaja Pyronema omphalodes (Alb. & Schwein.) Svrcek & Kubicka Tarzetta bronca (Peck) Kanouse Tricharina gilva (Baud. ex Cooke) Eckblad Norway DNA from culture Tricharina ochroleuca Nordland, Norway DNA from fruitbody Trichophaea hemisphaerioides Mouton Cranbrook Hill Greenway Trail, near Prince George, B.C., Can. trailside under Abies fruit body Bres. (Eckblad) (Graddon) Trichophaea hemisphaerioides (Mouton) Graddon 94 lasiocarpa, Populus tremuloides and Betula papyrifera, B.C., Can. fruitbody al436 RMD 2338 Wilcox ina rehmii Yang and Korf Alberta, Can. DNA from culture Al789 BDGMISSb Wilcoxina mikolae Yang and Korf U.S.A. DNA from culture 4.1 b Ectomycorrhizal root tip collection Four hundred and forty six putative ascomycete root tips were used ; 10 were collected by the author and 436 were procured from other sources. In addition to the root tips discussed in chapter 3, several RFLP pattern databases not created by the author were used in this study. This includes five RFLP pattern databases from the Forest Renewal British Columbia (FRBC) project of Drs. K. Egger and H. Massicotte (with some collected and processed by K. Mah (1999)), including root tips that were morphotyped as E-strain (E-strain), MRA (MRA and types 3 &4), uncolonized or lightly colonized (type 5), and finally a database which includes 'unidentified' root tips, designated 'KMOR'. ine root tips from the Okanagan University College that were morphotyped as MRA and E-strain were also examined. Ten root tips morphotyped as ascomycetous from researcher Jacqueline Barr's doctoral work (U.C. Berkeley) were also included. Inf01mation on these databases is presented in Table 7. Only the samples that matched with fruitbodies are listed in Table 8 with their experimental identification, their source and their geographic origin. 95 Table 7 ECM root tip RFLP database morphotype and location information Database code/ description E-strain (FRBC) Morphotype (sample codes) location of origin E-strain (ZBIRnn2n) MRA (FRBC) MRA (ZSXnnn1n) Type 3 (FRBC) MRA (ZSXP 1134) Type 5 (FRBC) uncolonized or lightly colonized (ZSXPnn5n, ZBIRnn5n, ZPIPnn5n) Type: K=E-strain-like (ZPIP23K1) O=Tomentella-like (ZSXP2301) Eagle fire site, adjacent to the Aleza lake research forest, Northern B.C. Eagle fire site, adjacent to the Aleza lake research forest, Northern B.C. Eagle fire site, adjacent to the Aleza lake research forest, Northern B.C. Eagle fire site, adjacent to the Aleza lake research forest, Northern B.C. KMOR(FRBC) Jacqueline Barr's root tips OUC database 'unknown' morphotype, possibly E-strain (30) Both MRA and E-strain morphotyped root tips (rnra2, fbnnn, afnnn) Eagle fire site, adjacent to the Aleza lake research forest, Northern B.C. California Okanagan area, B.C. Table 8 ECM root tip morphotype, host and habitat info Sample ZBIR2225, ZBIR6526, ZBIR6527, ZBIR6529, ZBIR652F ZBIR4421 , ZBIR1121, ZBIR1124, ZPIP4559 ZSXP1356, ZSXP455F, ZSXP1256, ZSXP1256, ZSXP1255 , ZSXP1251 ZPIP6456 morphotype (source database code) E-strain (FRBC, Mah ( 1999) database) E-strain (FRBC) 5 (FRBC) 5 (FRBC) 5 (FRBC) 96 host species habitat Abies lasiocarpa (regenerated) burned and salvaged site, B.C. Abies lasiocarpa (regenerated) Pinus contorta var. latifolia (planted) Picea glauca X engelmanni (planted) Pinus contorta var. latifolia (planted) burned and unsalvaged site, B.C burned and unsalvaged site, B.C. burned and unsalvaged, B.C. burned and salvaged, B.C. ZBIR6551, ZBIR6154, ZBIR2355, ZBIR2351, ZBIR2352 5 (FRBC) Abies lasiocarpa (regenerated) burned and salvaged, B.C. ZSXP1254, ZSXP1252, ZSXP425J, ZSXP4151, ZSXP135H, ZSXP1456 ZPIP455H 5 (FRBC) burned and unsalvaged, B.C. ZSXP425J, ZSXP4151, ZSXP135H, ZSXP1456 5 (FRBC) ZBIR1151 5 (FRBC) ZBIR1152 5 (FRBC) ZSXR2115, ZSXR261B, ZSXR2619, ZSXR2618, ZSXR2617, ZSXR2613, ZSXR2614 ZSXP2119, ZSXP2115 MRA (FRBC) Picea glauca X engelmanni (planted) Pinus contorta var. latifolia (planted) Picea glauca X engelmanni (planted) Abies lasiocarpa (regenerated) Abies lasiocarpa (regenerated) Picea glauca X engelmanni (regenerated) 5 (FRBC) MRA (FRBC) MRA(FRBC) ZSXP1414, ZSXP1413, ZSXP1412, ZSXP1312, ZSXP1311, ZSXP1216, ZSXR2615 MRA (FRBC) ZSXP1134 3 (FRBC) ZSXP2301 0 (FRBC) ZPIP23Kl < K (FRBC) af90b MRA (OUC database) mra2 MRA (OUC database) fb 120-5 MRA (OUC database) 97 Picea glauca X engelmanni (planted) Picea glauca X engelmanni (planted) Picea glauca X engelmanni (regenerated) Picea glauca X engelmanni Picea glauca X engelmanni Pinus contorta var. latifolia Pseudotsuga menziesii Picea glauca X engelmanni Picea glauca X engelmanni burned and unsalvaged, B.C. burned and unsalvaged, B.C. burned and unsalvaged, B.C. burned and unsalvaged, B.C. burned and salvaged, B.C. burned and salvaged, B.C. burned and unsalvaged, B.C. planted, burned and salvaged, B.C. planted, burned and unsalvaged, B.C. planted, burned and salvaged, B.C. planted, burned and salvaged, B.C. Adams Lake, Okanagan, B.C. Sicamous Creek, Okanagan, B.C. Sicamous Creek, Okanagan, B.C. fb63-4 fb63-7 30 E-strain (OUC database) E-strain (OUC database) ascomycete (J. Barr's root tips) Picea glauca X engelmanni Picea glauca X engelmanni ? Sicamous Creek, Okanagan, B.C. Sicamous Creek, Okanagan, B.C. California, U.S.A. 4.1c DNA extraction, amplification, restriction digestion and RFLP analysis The molecular protocols used followed those described in Chapter 3 (sections 3.1c to 3.1£). 4.1d Trouble shooting techniques Trouble shooting methods follow those outlined in 3.1g (chapter 3) with the exception that lithium chloride (LiCl) was also used to purify recalcitrant samples. LiCl acts to precipitate contaminating RNA. RNA subunits can act as primers resulting in misamplification (Jobes 1995; Palumbi 1996). 4.2 Results 4.2a Taxa analyzed in this study The fruitbody taxa used as reference for root tip RFLP comparison are listed below, in Table 9. A 100% match at 3 enzymes suggests that the fungus listed is associated with an EM root tip and is indicated by a "+" in the left hand column. Fungal taxa that are imperfectly matched or not matched with any examined EM root tips are indicated by a ff ff 98 Table 9 Results of ascomycete fruitbody/ root tip RFLP comparison RFLP match Species RFLP ID with root tip 1074 Peziza petersii Berk. & Curtis 10a1 Geopora sp. llal Geopora cooperi Harkn. 13p He/vella latispora Peck + 14u He/vella leucomelaena (Pers.) Nannf. + 15al Tarzetta bronca (Peck) Kanouse 2006 Morchella elata Fr. Geopyxis vulcanalis (Peck) Sacc. 2007 2018 Plectania milleri Paden & Tylutki 2040 Peziza violacea Pers. 2048 Pyronema omphalodes (Bull.) Fuckel 2082 He/vella lacunosa Afz. Peziza echinospora Karst. 2136 Anthracobia maurilabra (Cooke) Boud. 322 330 Geopyxis carbonaria (Alb. & Schw.) Sacc. Sal Geopora cooperi Harkn. 6kl Geopora cooperi Harkn. 7k2 Geopora cooperi Harkn. 9al Geopora cooperi Harkn. a1436 Wilcoxina rehmii Yang and Korf + a1474 Plectania sp. al786 Chloridium paucisporum Wang and Wilcox a1789 Wilcoxina mikolae Yang and Korf + a1804 Tricharina gilva (Boud. ex Cooke) Eckblad al808 Tricharina ochroleuca Bres. Bres. (Eckblad) a2202 Phialocephala fortinii Wang and Wilcox a2245 Sphaerosporella brunnea (Alb. & Schwein.) Svrcek & Kubicka Gyromitra sp. gblfb Gca Pseudorhizina californica (Phillips) Harmaja gs3fb Gyromitra esculenta (Pers.) Fr. hydl Hydnotrya variiformis Harkn. pml Pseudoplectania melaena (Pers. : Fr.) Sacc. trich Trichophaea hemishaerioides (Mouton) Graddon + 99 · 4.2b PCR trouble-shooting A sequence of troubleshooting techniques was followed. Most fruitbody samples were in the form ofDNA extracts or RFLP patterns either from chapter 3 of this thesis, or from Dr. Keith Egger's personal database and as such presented minimal amplification problems, however, a number of fruitbody samples in the genus He/vella were acquired from various herbaria and did not amplify regardless of trouble-shooting techniques tried . 4.2c Trichophaea hemisphaerioides as a below-ground associate Two root tip RFLP patterns corresponded with the RFLP pattern for a Trichophaea hemisphaerioides fruitbody (Table 10). One root tip, ZBIR 1121 , matched with T hemisphaerioides (100% similarity with cluster analysis). The other root tip, ZBIR 1124, matched with Alul and Hinjl but not with Rsal (75 % similarity with cluster analysis) . Both root tips were morphotyped as E-strain like, both were Abies lasiocarpa root tips and both came from burned and unsalvaged sites. The fruitbody grew on a trailside near Abies lasiocarpa, Populus tremuloides and Betula papyrifera. 100 '· T able 10 Banding topologies of Trichophaea llemisplzaerioides (thd) and two E-strain root tips * indicates bands which fall outside the 6% tolerance range I% Rsai Samp1e I Alui Hinfi % 1 similarity I similarity 1 simil arity I I thd 383 , 254, 180, 107 100 ZBIR1124 382, 258, 179, 106 100 ZBIR1121 382, 252, 179, 108 100 + 1% I 509, 18o, 100 968 100 508, 180, 164, 132 II 100 970 100 507, 180, 163 , 129 100 1037* 0 I 164, 132 i 4.2d He/vella leucomelaena biotroph RFLP patterns from 34 root tips matched with the H. leucomelaena fru itbody RFLP pattern. Of the aforementioned 34 root tips, morpho types ranged from MRA, E-strain and uncolonized or lightly colonized. The H. leucomelaena fruitbody used was found under Pinus contorta. Fourteen MRA morphotyped root tips from the FRBC database matched identicall y (100%) at three enzymes with H. leucomelaena (Table 11 ). All of the FRBC roo t tips were from Picea glauca X engelmanni in burned and either planted or naturally regenerated sites. One MRA morphotyped Picea glauca X eng elmann i root tip from the Okanagan University College Sicamous Creek site matched identically with H. leucomelaena at the three enzymes. 101 ~ One E-strain root tip from the FRBC database matched identically (1 00%) with H. leucomelaena (Table 12). This root tip was from Abies lasiocarpa in a burned and naturally regenerated site. One root tip from the KMOR database matched with the H. leucomelaena fruitbody (Table 13). It was 33% similar at A lui and 100% at both Hinji and Rsal. This root tip, a type 0 root tip, was morphotyped as an 'unknown', possibly a member of the Thelephoraceae. It was a Picea glauca X engelmanni root tip from a burned and salvaged site that had been planted. Seventeen type 5 (uncolonized or lightly colonized) root tips from the FRBC database were identified as H. leucomelaena (Table 14). Of those, 15 were identical and 2 were close (A lui and Hinji 100% and Rsal 80% similarity). The root tips come from Pinus contorta, Picea glauca X engelmanni and Abies lasiocarpa. All root tips come from burned and either planted or naturally regenerating sites. 102 Table 11 Band topologies of He/vella leucomelaena (14u) and several 'MRA' root tips * indicates bands which fall outside the 6% tolerance range Sample Hinjl % A lui Rsal % similarity similarity 100 425, 247, 100 580, 181 14u 648, 147,115 163 ZSXP2119 643, 151, 100 434, 245, 100 569, 178 119 167 ZSXP2115 ZSXP1414 ZSXP1413 ZSXP1412 ZSXP1312 644, 150, 119 625, 151 , 114 626, 151 , 115 627, 150, 115 618, 147, 114 100 100 100 100 100 427,246, 168 444,252, 165 444,252, 165 444, 251, 165 436, 247, 163 % sim il arity 100 100 100 567, 177 100 100 567, 181 100 100 567, 180 100 100 567, 179 100 100 559, 177 100 ZSXP1311 620, 148, 113 100 436,248, 161 100 560, 175 100 ZSXP1216 650, 147, 116 632, 144, 112 628, 143, 111 630, 143, 110 631, 144, 112 620, 145, 111 628, 144, 111 667, 155, 115 100 434, 252, 165 432,246, 163 433,246, 163 433 , 246, 163 434,247, 164 435,245 , 164 435 , 245, 163 433 , 249, 161 100 575, 182 100 100 575 , 190 100 100 579, 188 100 100 581, 189 100 100 574, 189 100 100 580, 189 100 100 579, 189 100 100 552, 173 100 ZSXR261B ZSXR2619 ZSXR2618 ZSXR2617 ZSXR2613 ZSXR2614 MRA2 100 100 100 100 *100 100 100 103 Table 12 Banding topologies of He/vella leucomelaena (14u) and several E-strain root tips * indicates bands which fall outside the 6% tolerance range Samples A lui Hinfl Rsal % % % similarity similarity similarity 648, 147, 580, 181 425, 247, 100 14u 100 100 115 163 ZBIR6529 625, 144, 575, 182 100 426,249, 100 100 110 162 Table 13 Band topologies of He/vella leucomelaena (14u) and a type 0 root tip * indicates bands which fall outside the 6% tolerance range sample Alul Hinfl Rsal % % similarity similarity 14u 648, 147, 425,247, 100 100 580, 181 115 163 33* ZSXP2301 640, 157, 100 569, 182 427,250, 129 163 % similarity 100 100 Table 14 Band topologies of He/vella leucomelaena (14u) and several Type 5 root tips * indicates bands which fall outside the 6% tolerance range sample Alu I Hinfl % Rsal % % similarity similarity similarity 14u 648, 147, 100 425, 247, 100 580, 181 100 115 163 ZSXP1251 650, 149, 100 439, 250, 100 572, 181 100 116 163 ZSXP1255 651 , 153, 100 438, 253 , 100 566, 179 100 122 172 ZSXP1256 649, 146, 436,252, 569, 182 100 100 100 116 164 ZSXP455F 624, 150, 100 435, 253, 100 569, 177 100 121 172 ZPIP6456 656, 148, 100 448, 252, 100 100 564, 182 115 166 ZBIR6551 643, 154, 452, 260, 100 100 567, 182 100 121 170 ZBIR6154 625, 150, 436,253, 100 100 569, 177 100 122 172 ZBIR2351 67* 612, 150, 100 428, 257, 549, 176 100 118 179 104 ZBIR2352 ZBIR2355 ZPIP455H ZSXP425J ZSXP4151 ZSXP135H ZSXP1456 ZSXP1252 ZSXP1254 614, 149, 117 615, 150, 116 625, 150, 109 631, 150, 112 621, 147, 112 622, 147, 114 623, 149, 114 647, 154, 123 641, 153, 123 100 100 100 100 100 100 100 67* 67* 431,256, 177 433, 257, 177 451, 257, 167 432,251, 161 426, 242, 157 437, 249, 162 436, 250, 163 445, 256, 170 438,253, 170 105 67* 551,176 100 67* 553, 177 100 100 558, 179 80 100 555 , 175 80 100 557, 183 100 100 562, 176 100 100 557, 179 100 100 572, 183 100 100 568, 180 100 4.2e He/vella latispora biotroph RFLP patterns from four root tips, all morphotyped as MRA, matched with the RFLP pattern from the H. latispora fruitbody. The He/vella latispora fruitbody used was found in soil and duff on Vancouver Island, B.C. and was borrowed from the herbarium at the Pacific Forestry Centre in Victoria. Interestingly, no potential host was mentioned on the herbarium label. Two MRA morphotyped root tips, one from the FRBC database (Picea glauca X engelmanni), and one from the OUC collection (Pseudotsuga menziesii) matched identically with He/vella latispora (Table 15) using Alui, Hinjl and Rsal. Another ECM root tip from the OUC collection (Picea glauca X engelmanni) matched the H. latispora fruitbody RFLP pattern identically at Alui, Hinjl but differed by an absent band in the Rsai digest. One root tip from the FRBC Type 3 database matched with He/vella latispora (Table 16). The ECM root tip RFLP pattern matched the H. latispora fruit body pattern 100% at Hinjl and Rsal. This Type 3 & 4 ECM root tip matched 50% with the He/vella latispora fruitbody at Alul. This root tip was from a burned site planted with hybrid spruce (Picea glauca X engelmanni). 106 Table 15 Banding topologies of He/vella latispora (13p) and some MRA type root tips Rsal Sample Alul % Hinjl % % similarity similarity similarity 427, 248, 648, 145 100 100 593, 184 13p 100 170 af90b 687, 154 100 433, 248, 100 571, 177 100 161 tb120-5 675, 159 100 443,249, 100 50 567 164 ZSXR2115 641, 146 100 436, 247, 100 559, 180 100 163 Table 16 Band topologies of He/vella latispora (13p) and root tip types 3 and 4 * indicates bands which fall outside the 6% tolerance range Samples Alul % Hinjl % Rsal % similarity similarity similarity 427, 248, 13p 648, 145 100 100 593, 184 100 170 ZSXP1134 642, 128 50* 434, 245, 100 579, 175 100 165 4.2f Wilcoxina rehmii biotroph Several root tip types were identified as Wilcoxina rehmii, including those morphotyped as MRA, E-strain and uncolonized or lightly colonized. The W rehmii reference culture (RMD 2338) used was isolated from a root tip found in Alberta, Canada and identified as W rehmii by molecular analysis (Egger 1996). One MRA morphotyped root tip from the FRBC database matched identically at Alul and Hinjl and 80% at Rsal with Wilcoxina rehmii (Table 17). This root tip was from hybrid spruce (Picea glauca X englemanni) sampled at a burned site. 107 Three root tips morphotyped as E-strain matched identically at three enzymes with W rehmii (Table 18). All of the root tips were from Abies lasiocarpa on burned, naturally regenerating sites. Two uncolonized or lightly colonized (type 5) root tips were identified as closely related to W rehmii (Table 19). There was 100% similarity between samples at Alul, 75 % at Hinji and 80% at Rsal. Both root tips were from Abies lasiocarpa on burned and naturally regenerating sites. Table 17 Banding topologies for Wilcoxina reltmii and some 'MRA' root tips Sample Alul % Hinjl % Rsal similarity similarity 706, 179, 386, 259, 100 W rehmii 100 516, 181 , 84 166, 130 183, 111 723 , 180 ZSXR211 390, 255 , 100 100 499, 176, 2 182, 109 159, 125 Table 18 Banding topologies for Wilcoxina reltmii and some E-strain root tips sample Alul Hinjl % Rsal % similarity similarity 386, 259, 516, 181, 706, 179, W rehmii 100 100 166, 130 84 183, 111 486, 175, 100 707, 173 , 100 ZBIR442H 377, 249, 174, 109 159,131 84 ZBIR4427 389, 254, 508 , 182, 100 701 , 182, 100 182, 115 160, 133 85 ZBIR642D 386, 254, 100 517, 181, 100 719, 177, 186, 114 166, 131 86 108 % similarity 100 80 % simil arity 100 100 100 100 Tahle 19 Banding topologies for Wilcoxina relunii and two Type 5 root tips * indicates bands which fall outside the o% tolerance range I Sample Alul I% II-iinjl ____0Yo ..::: _ _ _--.-I_R_s_a_l___ , _o/c _ ____~ 0 !I a i436ZBIR1151 ZBIR1152 386,259, 183,111 383 , 251, 181, 109 382, 253, 180, 108 similarity 100 100 100 I similarit 1 516, 18L-k100 ---f706, 179, 166,130 84 ~ 510, 183, 75* 711, 175 166, 139 75* 711,174 510, 183, 166, 139 simi larity ~ 100 ~ 80 80 L __ _ _ _ _L __ _ _ _L __ _ _ _ _ _ _ L __ _ 4.2g Wilcoxilw mikolae biotroph Seven root tips were identified as Wilcoxina mikolae, includ .ng those morphotyped as MRA, E-strain and uncolonized or lightly colonized. On e MRA morphotyped root tip from the FRBC database matched 100% at A lui and Hinjl with W. mikolae (Table 20). It matched 67% at Rsai. This root tip was from a burned site naturally regenerating with hybrid spruce (Picea glauca X eng elmanni). Two root tips morphotyped as uncolonized or lightly colonized from the FRBC type 5 database matched with W. mikolae (Table 21 ). The first, ZSXP2151 , matched 100% at Alu l, and 67% at both Hinfl and Rsa I. The second, ZBIR2354 matched 67% at Alu I, 100% at Hinf I and 50% at Rsa I. Both were from a burned site, the first was from a planted sta1d of Picea glauca X engelmanni and the second was from a naturally regenerating stand of Abies lasiocarpa . IU9 ~ One E-strain morphotyped root tip from the FRBC database matched identically at three enzymes (Alui, Rsai, Hinfl) with Wilcoxina mikolae and one root tip matched identically from Jaqueline Barr's doctoral work (Table 22). The root tip was from a burned site naturally regenerating with Abies lasiocarpa . Two E-strain morphotyped root tips from the OUC database matched the W mikolae RFLP pattern at A lui and Hinfl but were missing the lowest molecular weight band in the Rsai digest. Both of these ECM root tips were from Picea glauca X engelmanni. One root tip morphotyped as Thelephora-like from the FRBC KMOR database matched with W mikolae, 100% at Alu I and Hinfl and 80% at Rsa I (Table 23). This root tip was from a burned site planted with Pinus contorta var. latifolia. Table 20 Banding topologies for Wilcoxina mikolae and some MRA root tips Rsai Sample A lui Hinfl % % similarity similarity 902, 87 669, 181, 498, 160, 100 A1789 100 144 107 502, 163, ZSXR2615 664, 178, 100 100 893 108 150 Table 21 Banding topologies for Wilcoxina mikolae and some E-strain root tips A lui Hinfl % Rsai Sample % similarity similarity a1789 ZBIR442I FB63-4 FB63-7 30 669, 181, 107 636, 177, 107 709, 184, 110 736, 190, 115 665, 182, 112 100 100 100 33 * 100 498 , 160, 144 484, 161 ' 146 506, 161 , 146 506, 163, 148 508, 163, 148 110 % similarity 100 67 % similarity 100 902, 87 100 100 855 , 83 100 100 914 67 100 924 67 100 863 , 89 100 Table 22 Banding topologies for Wilcoxina mikolae and some Type 5 root tips * indicates bands which fall outside the 6% tolerance range Rsal Hinjl Alul % Sample % similarity similarity 902, 87 498, 160, 100 a1789 669, 181, 100 144 107 499, 178, 67* 930 100 ZSXP2151 688 , 183, 143 113 877,81 483 , 164, 100 669, 184, 67* ZBIR2354 152 115 % similarity 100 67 50 Table 23 Banding topologies for Wilcoxina mikolae (a1789) and a type K root tip * indicates bands which fall outside the 6% tolerance range Rsal Hinjl % % Alul % Sample similarity similarity similarity 498, 160, 100 100 902,87 a1789 669, 181, 100 144 107 80 692, 183, 514, 164, 100 889 ZPIP23K1 100 147 109 111 4.3 Discussion 4.3a Morphotype as a function of factors other than mycobiont Some researchers have speculated that morphotype based fungal identification is valid at the genus level but not the species level (Godbout and Fortin 1985 ; Scales and Peterson 1991 a) . Host influences on morpho type include size, branching pattern and Hartig net location (Godbout and Fortin 1985). The host also dictates the fonn of the symbiosis: studies by both Egger and Paden (1986) and Scales and Peterson (1991b) demonstrate that a fungus which forms ectomycorrhizae on one host may form ectendomycorrhizae on another. Evidence suggests that both Helvella and Wilcoxina root inhabiting fungi appeared on several root tip morphotypes, including MRA, E-strain and uncolonized or lightly colonized root tips . Morphotype seems to correlate more with host than with mycobiont. All of the 'MRA' root tips that matched with fruitbodies were from hybrid spruce (Picea engelmanii X glauca) with the exception of one (rnra2) from Pseudotsuga menziesii and all of the 'E-strain' root tips that matched with fruitbodies were from true fir (Abies lasiocarpa) with the exception of two (fb63-7, fb63-4) that were from Picea glauca X engelmanii. All three exceptions are from the OUC database. Of the five RFLP patterns from the OUC database that matched with fruitbodies , two (MRA on Picea engelmanii X glauca) followed suit with the majority of collections in regards to host associated with morphotype. The uncolonized or lightly colonized root tips that matched with fruitbodies were from a variety of hosts . This could represent the initial stages of colonization, which later develop into what is called 'MRA' or 'E-strain'. This could also represent 112 surface and not root inhabiting mycelium, however this is unlikely since the roots are washed with water before the DNA is extracted . Interestingly, the study by Scales and Peterson (1991b) reports that the morphology of the mycorrhiza forn1ed between Betula alleghaniensis and Phialophorafinlandia appears to be very similar to that formed between Betula alleghaniensis and Wilcoxina mikola e var. mikolae. In our study, RFLP patterns from root tips morphotyped as E-strain and MRA matched RFLP patterns from W mikolae and W rehmii. This is supporting evidence for the theory that host plays more of a role in morphotype determination than does mycobiont. 4.3b Preferential amplification An alternate possibility to the one above is that He/vella leucomelaena, He/vella latispora and Wilcoxina mikolae hyphae or spores all are ubiquitous in the soil and amplify more effectively than 'MRA' root tips, 'E-strain' root tips, uncolonized or lightly colonized root tips and type 3 root tips. Edwards et al. (1997) discussed 'competitive' PCR as resulting from two PCR templates with identical primer sites present in the DNA extract. According to that study, the template present in the greatest amount is preferentially amplified. Since the Pezizalean genus He/vella is considered difficult to amplify (Egger 1998 pers. comm.i, the above scenario seems unlikely. 2 Dr. Keith Egger, Mycologist and Biology Professor at the University of Northern British Columbia. Tel: 250-960-5860 11 3 4.3c Concerted evolution A proportion of the eukaryotic genome consists of repe;ncd DNA sequences (Li 1997). Gene repeats sometimes function as ' back-up' in case the activated gene mutates. In some cases, one gene does not code for sufficient quantities of a protein, so the repeats are all active in contributing protein (Ridley 1996). Interestingly, all the copies of a gene evo lve ' in concert' , the mechanisms for concerted evolution are poorly understood (Elder and Turner 1995). Most highly repetitive sequences (as opposed to unique sequences or middle repetitive sequences) are non-transcribed regions (Elder and Turner 1995). The DNA region (ITS) that was examined in both chapters 3 and 4 of this thesis included two transcribed, noncoding regions (ITS 1 and ITS2). Sometimes, the mechanisms that allow for concerted evolution fail resulting in two or more distinct sequence variants for the same gene or non-transcribed region (Li 1997). If this were the case with some of the fungi examined in chapters 3 or 4, a different ITS variant of one of the samples might have been amplified resulting in failure to match RFLP patterns from two different samples ofthe same species. This would depend on three things. First, failure of the mechanisms allowing for concerted evolution. Second, sufficient change at the primer annealing site on one of the variants resulting in either mis-priming or no amplification (of one variant). Third, the ITS variant selectively amplified was also different at one or more of the restriction sites resulting in a different banding pattern from the other sample (whether fungal fruitbody or fungus isolated from root tip). 114 4.3d Trichophaea hemisphaerioides Rifai (1968) distinguishes Humaria from Trichophaea based on presence of coarsely warted spores with two drops in the former and finely punctate or rough spores and one drop, in the latter. Ingleby (1990) lists Humaria hemispherica as ectomycorrhizal and includes a morphotype description ofthis species. Unfortunately, none of the references he lists as the origin of this information effectively demonstrate H. hemisphaerica's status as ectomycorrhizal (Danielson 1982; Danielson 1984; Dennis 1968; Thomas et at. 1983 ; Wilson et al. 1987; Yang and Wilcox 1984). The taxonomic history of Trichophaea places it in the category of ectomycorrhizal suspect. When the genus Patella was dissolved, its members were reassigned to various genera including Trichophaea, Humaria, Scutellinia, Leucoscypha, Tricharina, Anthracobia and Cheilymenia (Rifai 1968). The ectomycorrhizal genus Sphaerosporella was erected to accommodate the spherical spores present in this genus which is the only feature distinguishing it from Trichophaea (which has ellipsoid spores) (Wu and Kimbrough 1994). Korf(1973) suggests that spore size alone is not sufficient for its separate placement. The phylogenetic trees presented earlier in this thesis (section 1.1 a) support the notion of the interrelatedness of the members of the former genus Patella. Chapter 2 of this thesis presents analysis in which Trichophaea hybrida clusters out with Tricharina groenlandica and Wilcoxina mikolae, which is one of theE-strain ectomycorrhizal fungi. Sphaerosporella brunnea, another ectomycorrhizal fungus, is more closely related to Selenaspora, Cheilymenia and Scutellinia than the Trichophaea / Wilcoxina /Tricharina clade based on the results presented in Chapter 2. The knowledge of Trichophaea's past assignment in the genus Patella (along with ECM taxa that are now Wilcoxina and 115 Sphaerosporella) provides more evidence for its status as root inhabiting, if not ECM and serves to shed light on the evolution of the ECM habit in the Otideaceae. T hemisphaerioides was the least abundant of the identified root tips. Out of 446 root tips examined, with 158 ofthem being E-strain, only 2 root tips from the same seedling were identified as T hemisphaerioides. In addition to the above phylogenetic evidence for the ectomycorrhizal status ofT hemisphaerioides, Egger and Paden ( 1986) conducted some in vitro studies of the associations produced between various Pezizales and Pinus contorta. Conditions symptomatic of infection were exhibited with Pinus contorta and T hemisphaerioides, such as tannin deposition in cortical cells and lignification of cortical and epidermal cells. The fungus did not invade the vascular cylinder thus suggesting a root inhabiting status (Egger and Paden 1986). Whether or not T hemisphaerioides is ectomycorrhizal remains unknown. It is root inhabiting (Egger and Paden 1986), but until a Hartig net has been observed, no definitive concltisions can be drawn. 116 4.3e Root inhabiting members of the genus He/vella He/vella spp. have been suspected mycorrhizal fungi since 1936 (see Maia et al. 1996). A floristic study on the Pezizales conducted by Petersen (1985) suggests that He/vella corium forms an association with Salix species. He also observed a number of other members of the genus He/vella fruiting exclusively under ectomycorrhiza-forming trees and cautiously concluded that this does not necessarily indicate an association, but possibly coincident habitat requirements. Martinez-Amore et al. (1991) inoculated Pinus patula and Pinus radiata seedlings with He/vella lacunosa spores; both hosts produced ectomycorrhizae. Their confirmation of the H. lacunosa ECM was hasty considering that the seedlings were not grown in an aseptic environment, there was no molecular confirmation and the morphotyping was not detailed. Until recently, no conclusive proof has surfaced regarding the trophic status of He/vella spp. Weidematm et al. (1999) isolated DNA from rootlets of Dryas octopetala and Salix reticulata. The rDNA ITS 1 genotype from the Dryas isolate was identical to the rDNA ITS1 genotype from He/vella aestivalis. The two Salix ITS1 genotypes were similar to He/vella corium and He/vella dovrensis. This supports, but does not prove, Petersen's (1985) earlier hypothesis regarding the ectomycorrhizal association between H. corium and Salix spp. since no Hartig net was observed. The genus He/vella clusters out with well known ectomycorrhizal members of the Pezizales such as Tuber spp. with both 18s and 28s rDNA based analyses (O'Donnell et 11 7 al. 1997; Landvik et al. 1998). Their placement in a monophyletic group that contains other known mycorrhizal taxa supports the findings of Weidemann et al. (1999). Our study found Helvella species forming a variety of'morphotypes' including E-strain, MRA, uncolonized or lightly colonized and Tomentella like. Both E-strain and MRA fungi represent a complex of fungi; since E-strain fungi have been identified as Wilcoxina spp., it is not surprising that their relatives Helvella spp. form these associations also. It is surprising that some MRA fungi appear to be Helvella spp. MRA's have always been thought of as belonging to taxonomic groups basal (ancestral) to the Pezizales (Harney et al. 1997). Root inhabiting Helvella spp. fall into the subgenera Cupuliforme (H. corium), Elasticae (H. latispora) and Leucomelaenae (H. leucomelaena, H. aestivalis, H. dovrensis). H. dovrensis is considered an extralimital species, but most closely related to the Leucomelaenae (Abbott and Currah 1988). There seems to be a concentration of biotrophs in the subgenus Leucomelaenae. Examining the functional diversity in this subgenus, as well as the systematics, could reveal the identities and relationships of some unknown ascomycetous ectomycorrhizae. Helvella leucomelaena was the root inhabiting fungus found in the greatest abundance over all other identified root inhabiters. Of 446 root tip RFLP patterns, 34 patterns matched with the H. leucomelaena fruitbody pattern. He/vella latispora was considerably less common, with 4 RFLP patterns matching the H. latispora fruitbody 118 RFLP pattern. Petersen ( 1985) noted that H. leucomelaena proliferated in calcareous soils. The Aleza lake region, where many H. leucomelaena root tips were collected does not have calcareous soils in the root zone 3 (P. Sanborn pers. comm. 1999). 4.3f Wilcoxina spp. E-strain fungi were first isolated and described from Finnish nurseries on Pinus spp. (Mikola 1965; Laiho 1965). Yang and Wilcox (1984) described the first E-strain fungus, Tricharina mikolae, based on fruitbodies that appeared in pot culture of an E-strain inoculated red pine seedling. A year later, Yang and Korf (1985) erected a new genus, Wilcoxina, to accommodate the E-strain fungi. Wilcoxina was separated from Tricharina due to differing anamorphic states (Complexipes spp. for the forn1er and Ascorhizoctonia spp. for the latter), differing excipular structure ascospore germination, apothecial hair morphology, and its habit as an ectomycorrhizal associate. This segregation was later confirmed by phylogenetic analysis of ribosomal ITS DNA (Egger 1996). The use of Wilcoxina' s ECM habit as a character for taxonomic placement is validated by the aforementioned evidence. This further supports the notion of an evolutionary basis for the ectomycorrhizal habit. 3 Dr. Paul Sanborn, Regional Soil Scientist, Prince George Forest District. Tel : 250-5657100 119 E-strain mycorrhizae are typically found in conifer nurseries and burned sites (Laiho and Mikola 1964; Danielson 1982). According to Danielson (1982), E-strain mycorrhizae are widely distributed in nature. Abundance's for Wilcoxina ectomycorrhi zae were unexpectedly low. Of the 158 root tips morphotyped as E-strain in theE-strain database, three matched identically at Alu I, Hinfi and Rsa I with W rehmii and one matched identically with W mikolae. Since Wilcoxina and Sphaerosporella are the only confirmed E-strain fungi, this suggests that there are many other ectomycorrhizal fungi that can form E-strain morphotypes, but whether these are ascomycetes or basidiomycetes cannot be ascertained without examining the septal ultrastructure (Woronin bodies, dolipore septum), or conducting a Benomyl test (Danielson 1982). Morphotyping is conducted at a level whereby a morphotype is assigned to the Ascomycotina based on a lack of clamp connections and a similarity to other ascomycetous morpho types (Ingleby et a!. 1990; Agerer 1987 -1998) . Clamp connections are absent in all ascomycetes and some basidiomycetes (Hanlin and Ulloa 1988) and morphotype similarities may have a lot more to do with host than fungus (Egger 1995) thus rendering the placement of unknown morpho types based on the above criteria unreliable. 4.3g RFLP band match tolerance: guidelin e or rule? When analyzing bands in RFLPscan, bands between samples are recognized as separate when there is greater than 6% variation in base pair number. Anything above or below this cut off point is considered a different band. I suggest that this 6% match tolerance be used more as a guideline rather than a rule, because the difference between a band that is 120 5.9% greater or smaller and a band that is 6.1% greater may not be biologically significant. There are a number of root tip samples examined that are identical to a fruitbody in all but one band. Most ofthese fall just outside the 6% limit, an example is: FRBC Type 5, uncolonized or lightly colonized, root tips ZBIR1151 and ZBIR1152 match identically with W. rehmii at Alu I and Rsa I but are 1.2 bp off the 6% tolerance limit for the Hinfi digest. 4.3h Fruitbodies th at did not match with root tips Surprisingly, very few root tips and fruitbodies matched. This could be a result of one of three things: First, there is no association, root inhabiting or ectomycorrhizal between the fungus and any host. In that case, RFLP patterns of fruit bodies and root tips will only match if the fungus is growing near or on the surface of the root. However, this seems unlikely due to the processing procedure which involves washing the root tips before amplification. Second, the fungus is root associated, but none of the root tips were collected. Gardes and Bruns (1996) noticed a discrepancy between fruitbody abundance and ECM root tip abundance at a particular site, i.e. an abundant fruitbody producing species does not necessarily colonize many root tips. Similar work (matching fruitbody RFLP ' s with root tip fungal RFLP's) by Karen et al. (1997) and Varga (1998) reported difficulty finding RFLP pattern matches between ECM fruitbodies and root tips . Third , the target fungus did amplify, but due to intra-specific variation in the ITS region, the RFLP patterns between fruitbody and host colonising fungus did not match. This possibility, and the two before it are discussed in chapter 3, section 3.3b of this thesis. 121 Representatives from all the families of the Pezizales were included in the analysis (with the exception of the Ascobolaceae, a non-mycorrhizal group). None of the root tips matched with Sphaerosporella brunnea, which is a documented Pezizalean ECM. This was surprising since S. brunnea occurs on burned sites, which were the source of the FRBC databases. It is also surprising that very few matched with Wilcoxina mikolae or W. rehmii, since these are two of the three documented E-strain fungi . Finally, it is interesting that none of the root tips matched any basidiomycetous fruitbodies, although this is not surprising, since the basidiomycete database is not a comprehensive one, consisting of only a few dozen species. The fact that few of the Pezizales and few of the Agaricales matched with root tips suggests that there are a lot of unidentified root inhabiting or ectomycorrhizal fungi in this order. 122 4.4 References Abbott, S.P. , and R.S. Currah. 1988. The Genus He/vella in Alberta. Mycotaxon. 33 :229250. Danielson, R.M. 1982. Taxonomic affinities and criteria for identification of the common ectendomycorrizal symbiont ofpine. Can. J. Bot. 60: 7-18 . Danielson, R.M. 1984. Ectomycorrhiza formation by the operculate discomycete Sphaerosporella brunnea (Pezizales). Mycologia 76, 454-461. Deru1is, R.W.G. 1968. British Ascomycetes. J. Cramer. Lehre. 455p . Edwards, S.G. , A.H. Fitter, J.P .W. Young. 1997. Quantification of an arbuscular mycorrhizal fungus Glomus mosseae, within plant roots by competitive polymerase chain reaction. Mycol. Res. 101: 1440-1444. Egger, K.N. 1995. Molecular analysis of ectomycorrhizal fungal communities. Can. J. Bot. 73 (suppl.): s1415-s1422. Egger, K.N. 1996. Molecular systematics ofE-strain mycorrhizal fungi: Wilcoxina and its relationship to Tricharina (Pezizales). Can. J. Bot. 74: 773-779. Egger, K.N. and J.W. Paden. 1986. Pathogenicity ofpostfire ascomycetes (Pezizales) on seeds and germinants of lodgepole pine. Can. J. Bot. 64: 2368-2371. Elder, J.F. and B.J. Turner. 1995. Concerted evolution of repetitive DNA sequences in eukaryotes. The Quarterly Review ofBiology 70: 297-320. Gardes, M. , T.D. Bruns. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above and below ground views. Can. J. Bot. 74: 1572-1583 . Godbout, C. and J.A. Fortin. 1985 . Synthesized ectomycorrhizae of aspen: fungal genus level of structural characterization. Can. J. Bot. 63: 252-262 . Hanlin, R.T. and M. Ulloa. 1988. Atlas oflntroductory Mycology. 2nd ed. Hunter Textbooks, North Carolina. Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, London. Harney, S.K. , S.O. Rogers and C.J.K. Wang. 1997. Molecular characterization of dematiaceous root endophytes. Mycol. Res. 101: 1397-1404. Ingleby, K., P.A. Mason, F.T. Last and L.V. Fleming. 1990. Identification of Ectomycorrhizas. Institute ofTerrestrial Ecology, Midlothian, Scotland. 123 Jobes, D.V., D.L. Hurley and L.B. Thien. 1995. Plant DNA isolation: a method to efficiently remove polyphenolics, polysaccharides and RNA. Taxon 44: 379-386. Karen, 0., N. Hogberg, A. Dahlberg, L. Jonsson, J.E. Nylund. 1997. Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytol. 136: 313-325. Kernaghan, G., R.S. Currah and R.J. Bayer. 1997. Russulaceous ectomycorrhizae of Abies lasiocarpa and Picea engelmannii. Can. J. Bot. 75: 1843-1850. Korf. , R.P. 1973. Discomycetes and tuberales In: G.C. Ainsworth, F.K. Sparrow, A.S. Sussman, ed. The Fungi. Vol IVa. Academic Press, New York, pp. 249-319. Laiho, 0. 1965. Further studies on the ectendotrophic mycorrhiza. Acta For. Fenn. 79(3): 1-35. Laiho, 0. and P. Mikola. 1964. Studies on the effect of some eradicants on mycorrhizal development in forest nurseries. Acta Forest. Fenn. 77: 3-34. Landvik, S., K.N. Egger and T. Schumacher. 1998. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analyses of SSU rDNA sequences. Nord. J. of Bot. 17: 403-418. Li, W.-H. 1997. Molecular Evolution. Sinauer Associates, Inc. Mass., U.S.A. Mah, K. 1999. The effect of broadcast burning after clearcutting on the diversity of ectomycorrhizal fungi associated with hybrid spruce and subalpine fir seedlings in the central interior of B.C. M.Sc. thesis, University ofNorthem British Columbia. Maia, L.C., A.M. Yano and J.W. Kimbrough. 1996. Species ofascomycota forming ectomycorrhizae. Mycotaxon 217: 371-390. Martinez-Arnores, E., M. Valdes, and M. Quintos. 1991. Seedling growth and ectomycorrhizal colonization of Pinus patula and P. radiata inoculated with spores of He/vella lacunosa, Russula brevipes or Lycoperdon perlatum. New Forests 4: 237-245. Mikola, P. 1965 . Studies on the ectendotrophic mycorrhiza on pine. Acta For. Fenn. 79(2) : 1-56. O' Donnell, D., E. Cigelnik and N.S. Weber.1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48-65 . 124 Palumbi, S. 1996. Nucleic Acids II: The Polymerase Chain Reaction. In Molecular Systematics. Hillis, D.M., Moritz, C and Mable, B.K. Sinauer Associates, Sunderland, Mass. pp. 205-245 . Petersen, P.M. 1985. The ecology of Danish soil inhabiting Pezizales with emphasis on edaphic conditions. Opera Bot. 77 :1-38. Copenhagen. Ridl ey, M. 1996. Evolution. Blackwell Science, Cambridge, Mass. Rifai, M.A. 1968. The Australasian Pezizales in the Herbarium of the Royal Botanic Gardens, Kew. Verh K Ned Akad Wet Afd Natuurkd Tweede Sect, 2, 57:1-295 . Seifert, K.A. , B.D. Wingfield, M.J. Wingfield. 1995. A critique ofDNA sequence analysis in the taxonomy of filamentous Ascomycetes and ascomycete anamorphs. Can. J. Bot. 73(suppl.):s760-s767. Scales, P.F. , and R.L. Peterson. 1991a. Structure and development of Pinus banksianaWilcoxina ectendomycorrhizae. Can. J. Bot. 69:2135-2148 . Scales, P .F. and R.L. Peterson. 1991 b. Structure of ectomycorrhizae formed by Wilcoxina mikolae var mikolae with Picea mariana and Betula alleghaniensis. Can. J. Bot. 69: 2149-2157. Thomas, G.W., D. Rogers and R.M . Jackson. 1983. Changes in the mycorrhizal status of Sitka spruce following outplanting. Plant Soil 71: 219-232 . Varga, A.M. 1998. Sitka alder and lodgepole pine ectomycorrhizae. M.Sc. thesis, University of Northern British Columbia. Vralstad, T., Holst-Jensen, A and T. Schumacher. 1998. The post-fire discomycete Geopyxis carbonaria (Ascomycota) is abiotrophic root associate with Norway Spruce (Picea abies) in nature. Mol. Ecol. 7: 609-616. Weidemann, H.M., Holst-Jensen, A. and T. Schumacher. 1999. Demonstration of He/vella ectomycorrhizae on Dryas and Salix hosts by means of taxon-selective He/vella based nrDNA primers. Proceedings of the 2nd International Conference on Mycorrhizae, Uppsala, Sweden. Wilson, J. , P.A. Mason, F.T. Last, K. Ingleby and R.C. Munro. 1987. Ectomycorrhiza fonnation and growth of Sitka Spruce seedlings on first-rotation forest sites in Northern Britain. Can. J. For. Res. 17: 957- 963 . Wu, Chi-Guang, and J.W. Kimbrough. 1994. Ultrastructure of spore ontogeny in Trichophaea brunnea (Pezizales). Int. J. Plant Sci. 155: 453 -459. 125 Yang, C.S. and H.E. Wilcox. 1984. An E-strain ectendomycorrhizae formed by a new species, Tricharina mikolae. Mycologia 76: 675-684. Yang, C.S. and R.P. Korf. 1985. A monograph of the genus Tricharina and of a new segregate genus Wilcoxina. Mycotaxon 24: 467-531. 126 Chapter 5 Conclusions 5.0 Efficacy of the techniques used for identification of Pezizalean ectomycorrhizae Morphological and molecular approaches to ECM identification have their strengths and weaknesses. The results presented in chapter 4 suggest that the identity of the host could play more of a role in determining the morphotype than the identity of the fungus. RFLP patterns from He/vella leucomelaena , He/vella latispora, Wilcoxina mikolae and Wilcoxina rehmii fruitbodies were identical to those generated from root tips morphotyped as E-strain and MRA. Also, a study by Scales and Peterson (1991) found that the morphology ofthe association between Wilcoxina mikolae var. mikolae (classic E-strain fungus) and Betula alleghaniensis was very similar to the morphology of the association between Phialophora finlandia and B. alleghaniensis. This may not be the case for all ectomycorrhizal fungi, some fungi, such as Amanita muscaria, Russula spp., Lactarius spp. , Tuber spp., Amphinema byssoides, etc. produce very distinctive morphotypes (Ingleby et al. 1990). Perhaps morphotyping studies need to be conducted in greater detail with some of these 'problem' groups (He/vella leucomelaena, H. latispora, Wilcoxina spp.) in order to tease out some conserved morphological features that can be used to identify these morphotypes. Molecular approaches to fungal identification are independent of host variation and environment (Egger 1995), however, rates of variation and therefore the efficacy of certain gene regions for identification at any taxonomic level can vary within a genus, species or even population (Seifert et al. 1995). This means that identification of taxa without corroborating evidence is suspect. As more information accumulates regarding 127 rates of genetic change, regions that evolve at predictable and quantifiable rates for specific taxa are targeted thus increasing the robustness of molecular based identifications. DNA sequences or RFLP patterns provide the systematist or ecologist with more tools to investigate questions of relevance. The dichotomy between morphological and molecular approaches is unnecessary. 5.1 The use of phylogenetics for identifying ectomycorrhizal fungi Ectomycorrhizal fungi occur in many ascomycetous and basidiomycetous clades suggesting that the mycorrhizal association evolved independently in several fungal lineages (Bruns 1995, LoBuglio et al. 1996). This makes phylogenetics a powerful tool for the investigation of potentially ectomycorrhizal taxa. As the phylogenetic trees from chapter 2 indicated, confirmed ectomycorrhizal and root inhabiting fungi only occurred in a few clades. These clades served as focal points for our investigation and, not surprisingly, the fungi identified as root inhabiting in this thesis (H. leucomelaena, H. latispora, W rehmii, W mikolae, T hemisphaerioides) were found in two (the Helvellaceae and the Otideaceae) of the four clades containing confirmed associates. Future phylogenetic work on ectomycorrhizal Pezizales could include an investigation into the origins of the ectornycorrhizal habit within the Pezizales. Were the progenitors of the ectomycorrhizal Pezizales pathogens or saprotrophs? Did the association evolve independently in several lineages, or did it arise once and was subsequently lost by 128 several groups? Follow up studies from the work presented in this thesis could include phylogenetic re-examinations of the Trichophaea/ Humaria / Sphaerosporella complex and the cupulate species of He/vella. Studies like the two previously mentioned would not only help to clarify relationships among those groups, but could help to answer the former questions about the origins of the ectomycorrhizal habit among the Pezizales. 5.2 Future directions The two significant findings presented in this thesis are that He/vella leucomelaena and H. latispora are root inhabiting and possibly mycorrhizal and that the morphotype of the ECM or root inhabiting associations formed between Wilcoxina spp. and He/vella spp. varies considerably depending upon the host, Evidence from this thesis strongly suggests that He/vella leucomelaena and H. latispora are root inhabiting. Other work suggests that other species of He/vella are root inhabiting (Weidemann eta!. 1999). Several options could be taken in order to further explore the ecological roles of He/vella leucomelaena and H. latispora. First, DNA could be sequenced from both the He/vella fruitbodies and root tips that matched (RFLP) with them. DNA sequencing is a more accurate means of identifying taxa than RFLP analysis (Bruns 1995). Once established that the root tip is inhabited by He/vella sp., the next step is to determine the nature of the association. Approaching the aforementioned question could be done in a few ways. Pure culture synthesis along with detailed morphological descriptions of the association between the He/vella in question and various hosts would 129 determine the type(s) of associations He/vella could form under artificial conditions. The morphology of these associations could be compared to field collected samples to gauge the validity of pure culture synthesis for examining the morphology of He/vella associations. Another and complementary way of determining the ecological role that He/vella plays would be to conduct radio-labelled isotope studies with some of the pure culture trials between He/vella and various hosts. This would determine ifthere was any nutrient transfer between He/vella and the host in question and if it was reciprocal (thus indicating mycorrhizal status). The second significant finding from this thesis, that morphotype seems to correlate more with fungus than host, could also be further examined in a few ways . Again, pure culture synthesis of both Wilcoxina spp. and He/vella spp. with various hosts accompanied by detailed morphological descriptions could help reveal the circumstances under which these fungi form different morphotypes with the same hosts. It is interesting to note that out of the five root tips examined from the OUC database, three did not fit the MRA found on Picea glauca X engelmanii and E-strain found on Abies lasiocarpa irrespective of mycobiont scenario. This suggests that environment could also play a role in determining morpho type and a careful examination of the properties of the collection sites involved could reveal interesting trends. 130 5.3 References Bruns, T.D. 1995 . Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi . Plant Soil 170: 63-73 . Egger, K.N. 1995 . Molecular analysis of ectomycorrhizal fungal communities. Can. J. Bot. 73 (suppl. ): s1415-s1422. Ingleby, K. , P.A. Mason, F.T. Last and L.V. Fleming. 1990. Identification of Ectomycorrhizas. Institute of Terrestrial Ecology, Midlothian, Scotland. LoBuglio, K.F. , M.L. Berbee and J.W. Taylor. 1996. Phylogenetic origins ofthe asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes. Molec. Phylogen. and Evol. 6: 287-294. Scales, P .F. and R.L. Peterson. 1991. Structure of ectomycorrhizae formed by Wilcox ina mikolae var mikolae with Picea mariana and Betula alleghaniensis . Can. J. Bot. 69 : 2149-2157. Seifert, K.A. , B.D. Wingfield, M.J. Wingfield. 1995. A critique of DNA sequence analysis in the taxonomy of filamentous Ascomycetes and ascomycete anamorphs . Can . J. Bot. 73(suppl.) :s760-s767. Weidemann, H.M. , Holst-Jensen, A. and T. Schumacher. 1999. Demonstration of Helvella ectomycorrhizae on Dryas and Salix hosts by means of taxon-selective Helvella based nrDNA primers. Proceedings of the 2nd International Conference on Mycorrhizae, Uppsala, Sweden. 131 Glosssary Alignment- The juxtaposition ofnucleotides in homologous molecules to maximize similarity. Alignment is used to infer positional homology prior to phylogenetic analysis . Anamorph- the asexual stage in the life cycle of a fungus. Apothecium- an open and often cupulate ascocarp; found only in ascomycetes. Ascocarp- a spore-producing body containing asci (sing. ascus) ; found only in ascomycetes. Ascomycete- a synonym for the sub-division ascomycota, which are characterized by septate hyphae and the production of ascospores in an ascus. Ascus- a sac-like cell typically containing eight ascospores; found only in ascomycetes. Asexual- reproduction not involving karyogamy or meiosis. Biotroph- a fungus which obtains it's nutrients from a living host; an undetermined relationship (trophically). Bootstrapping- a statistical method based on repeat random sampling with replacement from an original sample to provide a collection of new pseudoreplicate samples, from which sampling variance can be estimated. Clamp connection- a bridge-like hyphal connection characteristic of the secondary mycelium of many basidiomycetes. Cluster analysis- a rapid method of hierarchically grouping taxa or sequences on the basis of similarity Ectomycorrhiza- a symbiotic association between higher fungi and the roots of many vascular plants in which the fungal hyphae do not penetrate the root cell walls. Epigean- fruiting above-ground. Facultative ectomycorrhiza- a saprotrophic or pathogenic fungus that is also capable, under certain conditions, of forming ectomycorrhizal associations. Fruitbody- any fungal structure that contains or bears sexual spores; also called a sporocarp . Heurisitic method- any analysis procedure that does not guarantee finding the optimal solution to a problem (much faster than other, more exact methods). 132 Host- a living organism (plant) that harbours a symbiont (fungus) in a parasitic, mutualistic or commensalistic relationship. Hypha- (pl. hyphae) the unit of structure ofmost fungi; usually filamentous in shape. Hypogeous- growing below the ground (truffles are hypogeous). Maximum Liklihood- A criterion for estimating a parameter from observed data under an explicit model. In phylogenetic analysis, the optimal tree under the maximum likelihood criterion is the one most likely to have occurred given the observed data and under the assumed model of evoluton. Monophyletic group- of a single line of descent. Mycelium- mass ofhyphae constituting the body (thallus) of a fungus . Mycorrhiza- a mutualistic association between the roots of a plant and the hyphae of some fungi. Neighbor Joining- An heuristic method for obtaining a point estimate of minimum evolution. Operculum- a hinged lid on the ascus allowing for forcible spore discharge; a defining characteristic of the Pezizales. Outgroup- one or more taxa considered to be outside the monophyletic group of interest (in this case the Pezizales). Phylogenetics- study of the natural groups (monophyletic) as evidenced by shared derived characters. Polymerase Chain Reaction (PCR)- A process for amplifying a target DNA sequence manyfold, in which a series of thermal cycles each result in denaturation of a doublestranded target, annealing of oligonucleotide primers to the resulting single strands, and primer extension catalyzed by a thermostable DNA polymerase. Primers- Oligonucleotides used to initiate synthesis of DNA by a DNA polymerase or reverse transcriptase. RFLP (Restriction fragment length polymorphism)- A polymorphism in an individual or species defined by restriction fragments of a distinctive length. Usually caused by gain or loss of a restriction site, but may result from an insertion or deletion of a fragment of DNA between two conserved restriction sites. Rhizomorph- a thick strand of vegetative hyphae present only in some Basidiomycetes. 133 Saprotroph- an organism that uses dead organic matter for food. Septum (pl. septa)- a cross-wall in the hypha that develops centripetally. Woronin body- an electron-dense, sphaerical body found in the hyphae of Ascomycota, usually near the septa. References Alexopoulos, C.J., C.W. Mims and M. Blackwell. 1996. Introductory Mycology. John Wiley and Sons, New York. Molecular Systematics. 211 d ed. eds. Hillis, D.M., Moritz, C. and Mable, B.K. Sinauer Associates, Mass., U.S.A. 134 APPENDIX I: Species ofbasidiomycete fruitbodies used in Chapter 4 and their RFLP accession codes 135 RFLP ID ALDIPIB ALDIPlC ALDIPID ALDIP2B ALDIP2C CHRUT18M CHRUT28M CHRUT38M CHVIN19C CHVIN29C CHVIN39C CHVIN49C CHVIN59C CHVIN68C CHVINF3 CODERM8M CORT19M CORT29C CORT39M CORT48M CORT58M CORT68M CORT78M CORT88M CORT98M COTELA HEBEL29M HEBEL38M HEBEL59M HEBEL6?8 HEBEL7?8 ININOC9M INRAIN19 LACCA18M LACT19M LACT28M LACT29M Species Alpova diplophloeus (Zeller & Dodge) Trappe & Smith Alpova diplophloeus (Zeller & Dodge) Trappe & Smith Alpova diplophloeus (Zeller & Dodge) Trappe & Smith Alp ova diplophloeus (Zeller & Dodge) Trappe & Smith Alpova diplophloeus (Zeller & Dodge) Trappe & Smith Chroogomphus rutilus (Schaef. ex Fr.) Miller Chroogomphus rutilus (Schaef. ex Fr.) Miller Chroogomphus rutilus (Schaef. ex Fr.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Chroogomphus vinicolor (Pk.) Miller Cortinarius subgen. Dermocybe Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius sp. Cortinarius subgen. Telamonia Hebeloma sp. Hebeloma sp Hebeloma sp Hebeloma sp Hebeloma sp Inocybe cf rainierensis Stuntz Inocybe sp. cf subgenus Inocibium Laccaria sp. Lactarius sp. Lactarius sp. Lactarius sp. 136 LACT49M LACT58M LALACFFl LARUFF LARUFFl LARUFF5 LARUFU19 LARUFU29 LARUFU38 LARUFU49 LEBYSF4 LEPT028M RUDEC028 RUDEC038 RUDEC048 RUSSU19M RUSSU29M SUBORF2 SUGRAF13 SUILL19M SUILL28M SUILL39M SUILL49M SUILL68C SUTOME18 SUTOME29 SUTOME38 SUTOMF14 SUTOMF9 UNIDIFD8 Lactarius sp. Lactarius sp. Laccaria laccata Fr. (Berk. & Broome) Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lactarius rufus (Fr.) Fr. Lentaria cf byssiseda Leptonia sp. Russula decolorans Fr. Russula decolorans Fr. Russula decolorans Fr. Russula sp. Russula sp Suillus borealis Smith, Thiers and Miller Suillus granulatus (Fr.) Kuntze Suillus sp. Suillus sp. Suillus sp. Suillus sp. Suillus sp. Suillus tomentosus (Kauff.) Singer, Thiers and Miller Suillus tomentosus (Kauff.) Singer, Thiers and Miller Suillus tomentosus (Kauff.) Singer, Thiers and Miller Suillus tomentosus (Kauff.) Singer, Thiers and Miller Suillus tomentosus (Kauff.) Singer, Thiers and Miller Unknown 137 APPENDIX II: Morphotype descriptions from Chapter 3 138 Elaphomyces muricatus emrt + Tsuga heterophy lla Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips straight to club shaped, unbranched; up to 13 mm long. Colour and texture: orange yellow, turning deep orange yellow with age, apices brilliant orange yellow; both felty and shiny. Emanating elements: Mycelial strands: none present. Hyphae: rare, tortuous. Anatomy (Compound microscope): Mantle in plan view: mantle is thin, Hartig net present, specialized cells not seen . Outer layer: felt prosenchyma, cells 4(2-6) urn wide, clear contents, septa are common. Inner layer: net synenchyma; cells 4(2-6) urn wide; cell contents clear. Emanating Hyphae: rare, 4 urn wide, clear, no ornamentation, granular contents, rare septa, hypha! junctions are common. Other Features: none noted 139 He/vella elastica bert + Picea sp. Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent, unbranched; up to 13 mm long. Colour and texture: dark brown with a lighter apex. Emanating elements: Mycelial strands: none present. Hyphae: common. Anatomy (Compound microscope): Mantle in plan view: mantle is thin, Hartig net present, specialized cells not seen. Outer layer: net prosenchyma, cells 4(2-6) urn wide, clear contents, septa are common. Inner layer: interlocking irregular synenchyma; cells 4(2-6) urn wide; cell contents clear. Emanating Hyphae: common, septate, clampless and hyaline (1-2 J...l.ffi) wide. Other Features: none noted 140 Otidea sp. ol rtl + Abies lasiocarpa, Populus tremuloides or Betula papyrifera Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: straight and monopodia! pinnate; system up to 8 mm long. Colour and texture: tips orange brown with a brilliant yellow apex; finely grainy and shiny. Emanating elements: Mycelial strands: none observed Anatomy (Compound microscope): Mantle in plan view:mantle is thin, Hartig net present, specialized cells not seen. -net synenchyma; cells 4(2-6) urn wide; cell contents clear, septa rare, no clamp connections. Emanating Hyphae: common, 4(3-4) urn wide, light brown, verrucose, septa rare, no clamp connections. Cystidia: rare, median width of 15 urn, hyaline, no ornamentation, clear contents, no septa, no clamp connections. Other features: none noted 141 Otidea onotica o2rtl +Populus tremuloides, Abies lasiocarpa orBetula papyrifera Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent and non branched; system up to 8 mm long. Colour and texture: tips orangish brown, smooth and matte. Emanating elements: Mycelial strands: none present. Hyphae: none present. Anatomy (Compound microscope): Mantle in plan view: thin mantle, Hartig net, specialized cells not seen. -net synenchyma, cells from (2-8) urn wide, clear with no septa, no clamp connections; no hypha] junctions. Other Features: none noted 142 Otidea cochleata 03 rtl +Populus tremuloides, Abies lasiocarpa or Betula papyrifera Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent and monopodia] pinnate; system 2(1-6) mm long. Colour and texture: tips orange brown and finely grainy. Emanating elements: Mycelial strands: none present. Hyphae: none present. Anatomy (Compound microscope): Mantle in plan view: thin mantle, Hartig net, specialized cells not seen. Outer layer: net synenchyma; cells 4(2-6) urn wide; cell contents clear, septa rare, no clamp connections. Inner layer: same as outer layer. Other Features: none noted 143 Otidea cochleata 03 rt2 +Populus tremuloides, Abies lasiocarpa or Betula papyrifera Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent and monopodia! pinnate; system 2(1-6) mm long. Colour and texture: orange yellow; both felty and shiny. Emanating elements: Mycelial strands: none present. Hyphae: common and curved. Anatomy (Compound microscope): Mantle in plan view: thin mantle, Hartig net, specialized cells not seen. Outer layer: felt prosenchyma, cells 1 urn wide, clear contents, no septa, no clamp connections, no hyphal junctions. Inner layer: same as outer layer. Emanating Hyphae: hyaline, cells 2 urn wide, no ornamentation, clear contents, no clamp connections, no septa, no hyphal junctions. Other Features: none noted 144 Sarcosphaera coronaria scrtl + Picea glauca Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent and monopodia! pinnate; system up to 6 mm long. Colour and texture: Black, matte and coarsely grainy. Emanating elements: Mycelial strands: none present. Hyphae: common and straight. Anatomy (Compound microscope): Mantle in plan view: Thin mantle, Hartig net, specialized cells not seen. Outer layer: net prosenchyma, cells from 3-5 urn wide, clear with neither septa nor clamp connections; hyphal junctions frequent at a 30 angle Inner layer: same as outer layer. Other Features: none noted 145 Sarcosphaera coronaria scrt2 + Picea glauca Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tips bent and monopodia! pinnate; system up to 11 mm long. Colour and texture: tips light orange yellow, slightly felty and matte. Emanating elements: Mycelial strands: none present. Hyphae: common and tortuous. Anatomy (Compound microscope): Mantle in plan view: medium, Hartig net, specialized cells not seen. Outer layer: net prosenchyma, cells from 3-5 urn wide, clear with neither septa nor clamp connections; hyphal junctions frequent at a 30 angle. Inner layer: same as outer mantle. Emanating Hyphae: common, tortuous; cell width 1(1-2) urn; septa common; common verrucose ornamentation; clear contents. Other features: none present 146 Trichophaea hemisphaerioides thrt + Abies lasiocarpa, Populus tremuloides or Betula papyrifera Distinguishing features: Morphology (Dissection Microscope): Ectomycorrhizal system: Shape and dimensions: tortuous and monopodia] pyramidal; system up to 5 mm long. Colour and texture: tips medium yellow brown with a yellow orange apex; smooth, slightly velvety and shiny. Emanating elements: Mycelial strands: none observed Hyphae: rare, curved. Anatomy (Compound microscope): Mantle in plan view: mantle is thick, Hartig net present, specialized cells not seen. Outer layer: interlocking irregular synenchyma; cells 8( 4-1 0) urn wide; cell contents clear, no septa, no clamp connections. Inner layer: same as outer layer. Emanating Hyphae: rare, 2.5(2.5-4) urn wide, clear, no ornamentation, septa common, no clamp connections. Other features: none noted 147 Appendix III: RFLP patterns 148 ... ..."'"'Oi ...~ "' .e0 CD CD N <'> u 0> (") "'C N 0 .e<'> 0 w 0.. . 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