. _ ., . . ,. H . .‘ ( “a. -... ...... ‘r1!’-$>II p. ' ' , a “488$ 1003 This is to certify that the dissertation entitled Studies In Amatoxin—Producing Genera Of Fungi: Phylogenetics & Toxin Distribution presented by Heather E. Hallen has been accepted towards fulfillment of the requirements for Ph.D. Botany & Plant Pathology degree in Major professor Date 081/2 2/2002 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/ClRC/DateDue.p65-p. 15 STUDIES IN AMATOXlN-PRODUCING GENERA OF FUNGI: PHYLOGENETICS AND TOXIN DISTRIBUTION By Heather E. Hallen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 2002 ABSTRACT STUDIES IN AMATOXIN-PRODUCING GENERA OF FUNGI: PHYLOGENETICS AND TOXICOLOGY By Heather E. Hallen The distribution of cyclic peptide amatoxins and the related phallotoxins were examined using HPLC and FAB mass spectrometry in three of the four genera that produce amatoxins. Amatoxins are responsible for 90% of fatal mushroom poisonings in humans. In a study of South African Amanita species, both types of toxins were found in members of Amanita section Phalloideae, and were not found in any other section of Amanita. Amatoxins and phallotoxins were detected in Amanita reidii for the first time. Phallotoxins were detected in the lawn mushroom Conocybe Iactea although amatoxins were absent. This is the first report of phallotoxins outside the genus Amanita. The phylogenetic relationships within Amanita and Conocybe were examined using PCR-RFLPs and DNA sequencing of the 5.88, 288 and ITS regions of the nuclear rDNA, and the large subunit of the mitochondrial rDNA. In Amanita, amatoxin producers (Amanita section Pha/Ioideae) formed a monophyletic clade in all analyses. Earlier reports of monophyly in Amanita sections Amanita, Caesareae and Vaginata based on 283 rDNA sequence data were supported by the ITS and mitochondrial large rDNA sequence data. Analysis of an extended 28$ rDNA sequence dataset placed three Amanita species basal to the outgroup genus Limacella. RF LPs and ITS sequence data from specimens of Amanita infected by the ascomycete mycoparasite Hypomyces hyalinus were compared to reference data from healthy Amanita species to identify the parasitized specimens. Hosts were identified as Amanita rubescens sensu lato, A. flavoconia and A. brunnescens in Amanita section Validae. Two parasitized specimens yielded DNA sequence matching that of mycorrhizal fungi outside of Amanita. Reports of parasitism of A. bispon'gera and A. muscan'a, both toxic, based on proximity to non-parasitized basidiocarps were not confirmed in this study. Systematic studies indicated that Conocybe lactea is not closely related to the amatoxin producer C. filaris, suggesting that toxin production has arisen independently in the two taxa. North American specimens of C. Iactea were found to be indistinguishable on the basis of DNA sequence from North American specimens of C. cn'spa. The European C. crispa was revealed to be a different species than North American C. crispa and C. Iactea. Gastrocybe Iaten'tia was placed in the genus Conocybe, closely related to North American C. Iactea. To identify the gene for amatoxin synthesis, Galen'na marginata was examined using PCR with degenerate primers designed to detect cyclic peptide synthetase (CPS) gene fragments. Attempts to amplify and sequence CPS genes were unsuccessful, due possibly to the use of ascomycete sequences to develop primers for use in a basidiomycete. Attempts to isolate amatoxin synthetase using ATP/pyrophosphate exchange assays are underway. ACKNOWLEDGMENTS Sincere and heartfelt thanks are due to many people for making this work possible. Special thanks are due to my advisor, Gerard Adams, who permitted me to choose my own project and work in his lab for five years. I must also thank the other members of my committee, Tao Sang, Frances Trail and Jonathan Walton, for all the help and advice they’ve given me in their areas of expertise. I owe a great debt to my collaborators Rodham Tulloss and Roy Watling, truly great taxonomists in Amanita (Tulloss) and Conocybe (Watling). Rod Tulloss provided me with the majority of my “destroying angel” Amanitas and - equally important - with the correct identifications and nomenclature for any Amanita I cared to send him. He has been very helpful in critiquing the Amanita chapters. Roy Watling provided the European and Asian Conocybe specimens, and notified me of several important references. I have carried on extensive e- mail discussions on nomenclature with both men. Joseph Leykam and the Macromolecular Structural Facility at Michigan State University were of great help with the HPLC, supplying the columns and troubleshooting. Thanks are also due to Ray Hammerschmidt, John Halloin and Jonathan Walton for permitting me to use their HPLC machines, and Alan Prather for permitting me to set up an HPLC machine in his lab. The Department of Botany and Plant Pathology, the Department of Plant Biology and the Department of Plant Pathology are thanked for providing me with an excellent working environment. Portions of this work have been supported by the A. L. Rogers Medical Mycology Scholarship and a grant from the International Association for Plant Taxonomy. iv TABLE OF CONTENTS LIST OF TABLES ................................................................................. viii LIST OF FIGURES ................................................................................ ix INTRODUCTION ................................................................................... 1 Mushroom poisoning - an overview ................................................... 1 Delayed-action poisoning syndromes ................................................ 3 Amatoxins ................................................................................... 5 Phallotoxins ............................................................................... 1 1 Detection of amatoxins and phallotoxins .......................................... 13 Taxonomy of amatoxin-producing fungi ............................................ 14 Overview of the following chapters .................................................. 16 References ................................................................................ 18 CHAPTER 1 MOLECULAR PHYLOGENETICS OF AMANITA, WITH A FOCUS ON SECTION PHALLOIDEAE .............................................................................. 22 Abstract ..................................................................................................... 22 Introduction ............................................................... - ................................ 22 Materials and methods .............................................................................. 27 DNA extraction, amplification and sequencing ............................... 27 Phylogenetic analyses .................................................................... 32 Results ...................................................................................................... 33 Discussion ................................................................................................. 42 Acknowledgements ..................................................................... 45 References ................................................................................................ 45 CHAPTER 2 IDENTIFICATION OF AMANITA SPECIES PARASITIZED BY HYPOMYCES HYALINUS ..................................................................................... 48 Abstract ..................................................................................................... 48 Introduction ............................................................................................... 48 Materials and methods .............................................................................. 51 DNA extraction, amplification and sequencing ............................... 54 Restriction fragment length polymorphisms ................................... 56 Phylogenetic analyses .................................................................... 56 Results ...................................................................................................... 57 Discussion ................................................................................................. 64 Acknowledgements ..................................................................... 67 References ................................................................................................ 67 CHAPTER3 AMATOXINS AND PHALLOTOXINS IN INDIGENOUS AND INTRODUCED SOUTH AFRICAN AMANITA SPECIES ........................................ 71 Abstract ..................................................................................................... 73 Introduction ............................................................................................... 74 Materials and methods .............................................................................. 76 Results and discussion ............................................................................. 80 Acknowledgements ................................................................................... 84 References ................................................................................................ 84 CHAPTER 4 TAXONOMY AND TOXICITY OF CONOCYBE LACTEA AND RELATED SPECIES ........................................................................................ 87 Abstract ..................................................................................................... 87 Introduction ............................................................................................... 88 Materials and methods .............................................................................. 91 HPLC and mass spectrometry ....................................................... 93 DNA extraction, amplification and sequencing ............................... 95 Phylogenetic analyses .................................................................... 96 Culture ............................................................................................ 97 Results ...................................................................................................... 98 HPLC and mass spectrometry ....................................................... 98 Phylogenetic analyses ................................................................... 98 Bacterial identification .................................................................... 99 Discussion ............................................................................................... 107 HPLC and mass spectrometry ..................................................... 107 Systematics .................................................................................. 1 08 References .............................................................................................. 112 CHAPTER 5 NON-RIBOSOMAL PEPTIDE SYNTHETASES AND GALERINA MARGINATA .......................................................................................................................... 1 15 Abstract .................................................................................................. 115 Introduction ............................................................................................. 116 Materials and methods ............................................................................ 123 Culture and HPLC ........................................................................ 123 Primer development and PCR ...................................................... 125 Pyrophosphate exchange assay .................................................. 127 Results .................................................................................................... 127 Discussion ............................................................................................... 129 Summary and future directions ..................................................... 129 References .............................................................................................. 130 APPENDICES .................................................................................... 134 Appendix 1. Aligned Amanita ITS sequence for Chapter 1 ................. 135 Appendix 2. Aligned Amanita 288 sequence for Chapter 1 ................. 140 vi Appendix 3. Aligned Amanita mitochondrial large rDNA sequence from Chapter 1 ........................................................................ 146 Appendix 4. Aligned ITS from control and parasitized Amanita specimens (Chapter 2) ...................................................................... 150 Appendix 5. Alignment of ITS 1 —5.83 — ITS 2 regions of the nuclear ribosomal RNA operon in Conocybe and related genera (Chapter 4) ..................................................................................... 158 Appendix 6. Alignment of the partial 28S region of the nuclear ribosomal RNA operon in Conocybe and related genera (Chapter 4) ......... 173 BIBLIOGRAPHY ................................................................................. 182 vii LIST OF TABLES Table 1. Specimens from which DNA was extracted and sequenced ................. 28 Table 2. Data from GenBank used in the 28S extended database ...................... 30 Table 3. Parasitized and non-parasitized Amanita specimens examined ........... 52 Table 4. Analysis of amatoxins and phallotoxins in Amanita species .................. 77 Table 5. Specimens of Conocybe and related genera examined ........................ 92 Table 6. Conserved regions in cyclic peptide synthetases ................................ 117 Table 7. Primers used in this study .................................................................... 125 Table 8. Estimated product size (bp) for each primer combination .................... 125 viii LIST OF FIGURES Figure 1. Structural formula for amatoxins. R1 = CHZOH in or- and B-amanitin and CH3 in y-amanitin. R2= NH2 in or- and y-amanitin and OH in B-amanitin. By convention, amino acids are numbered in a clockwise fashion, starting with asparagine ........................................................................................... 6 Figure 2. Structural comparison of principle amatoxins and phallotoxins. Atoms in black are identical in both toxin families, while those in gray differ ........... 12 Figure 3. Cladogram produced by maximum likelihood analysis of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA operon. Topology corresponds to one of nine equally most parsimonious trees (894 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree ................................ 34 Figure 4. Cladogram produced by maximum likelihood analysis of the 28S region of the nuclear ribosomal DNA operon. Topology corresponds to one of 132 equally most parsimonious trees (361 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%).Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree ................................................. 35 Figure 5. Cladogram produced by maximum likelihood analysis of the combined ITS and 288 regions of the nuclear ribosomal DNA operon. Topology corresponds to one of four equally most parsimonious trees (1338 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%).Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree. .............................. 36 Figure 6. Cladogram produced by maximum likelihood analysis of the mitochondrial large ribosomal DNA operon. Topology corresponds to one of 7 equally most parsimonious trees (156 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). * indicates branches that collapse on the strict consensus tree... 37 Figure 7a. Consensus based on 300 equally most parsimonious trees (length 1275) of the 28S region of the nuclear ribosomal DNA subunit. Names of sections in genus Amanita are given. Limacella (top) is the other genus in ix the Amanitaceae and was used as an outgroup. Arrow indicates node leading to amatoxin-producing taxa. * indicates Amanita manginiana and A. pseudoporphyn’a, traditionally placed in section Phalloideae ............... 38 Figure 7b. Section Phalloideae from the parsimony analysis of the extended 28S dataset. Numbers at nodes are bootstrap indices of support .................. 39 Figure 7c. Basal taxa from the parsimony analysis of the extended 28S dataset. Numbers at nodes are bootstrap indices of support ................................. 40 Figure 8. Amanita basidiocarps parasitized by Hypomyces hyalinus .................. 49 Figure 9. Ala l digest of reference Amanita specimens. Shapes indicate matches to the parasitized Amanita gel (Fig. 10) .................................................... 59 Figure 10. Alu I digest of parasitized Amanita specimens. Shapes indicate matches to the reference gel (Fig. 9) ........................................................ 59 Figure 11. Fnu 4HI digest of reference Amanita specimens. Stars indicate matches to the parasitized Amanita gel (Fig. 12) ...................................... 60 Figure 12. Fnu 4Hl digest of parasitized Amanita specimens. Stars indicate matches to the reference gel (Fig. 11) ...................................................... 60 Figure 13. Neighbor-joining tree of the ITS region of parasitized Amanita specimens and Amanita section Validae. Numbers at nodes are bootstrap indices of support (%). Branch lengths correspond to genetic distance (expected number of nucleotide substitutions per site) ............................. 62 Figure 14. Neighbor-joining tree of the 5.88 and partial ITS 1 and ITS 2 regions of parasitized Amanita specimens and representative Amanita species from other sections in the genus. Numbers at nodes are bootstrap indices of support (%). Branch lengths correspond to genetic distance (expected number of nucleotide substitutions per site) .............................................. 63 Figure 15. HPLC results for Amanita phalloides f. umbn’na (= A. reidii) PRE 48654. The dashed line indicates the percent acetonitrile. Solid line shows absorbance at 295 nm. Peak #1 represents B-amanitin, 3 a-amanitin, 5 phallacidin and 6 phalloidin. Peak 4 is likely y-amanitin; this could not be confirmed due to the lack of a y-amanitin standard ................................... 81 Figure 16. FAB mass spectra. Matrix = nitrobenzyl alcohol matrix. MH+ and M + Na = phalloidin + proton and phalloidin + sodium, respectively. A Phalloidin standard. B Conocybe Iactea ........................................... 100 Figure 17. Consensus based on 300 equally parsimonious trees (length 1090) of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA subunit. Numbers at nodes are bootstrap indices of support (%) ......................... 101 Figure 18. Cladogram of the maximum likelihood analysis of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA subunit. One of three trees with In likelihood = -5938.207. Branch lengths correspond to genetic distance (expected nucleotide substitutions per site) .............................. 102 Figure 19. Consensus based on 70 equally parsimonious trees (length 171) of the partial 28S ribosomal DNA subunit. Numbers at nodes are bootstrap indices of support (%) ............................................................................. 103 Figure 20. Cladogram of the maximum likelihood analysis of the partial 28S ribosomal DNA subunit. One of two trees with likelihood = -1833.145. Branch lengths correspond to genetic distance (expected nucleotide substitutions per site) ........................................................................ 104 Figure 21. Consensus based on 300 equally parsimonious trees (length 1122) of the combined ITS and partial 28S regions. Numbers at nodes are bootstrap indices of support (%) ............................................................. 105 Figure 22. Cladogram of the maximum likelihood analysis of the combined ITS and partial 288 regions. In likelihood = -7263.045. Branch lengths correspond to genetic distance (expected nucleotide substitutions per site) ......................................................................................................... 106 Figure 23. Structure of a typical cyclic peptide synthetase. A The entire synthetase for an eight amino acid cyclic peptide, as predicted for amatoxin synthetase. Shaded areas represent domains for the separate amino acids. Each domain shares conserved sequence motifs. B Enlargement of one domain, showing roughly where various activities are encoded (shaded areas). A-l encode adenylation functions, J encodes the acyl carrier, and K-O function in condensation. Some CPSs contain additional sequences P-Q, which function in epimerization, N-methylation and other modifications. After Kleinkauf & von Dohren (1996) and Panaccione (1996) ..................................................................... 1 19 Figure 24. Typical gel showing PCR products from G. marginata amplified by CPS primers. + = 1 kb+ ladder, a — s are lanes loaded with PCR products. Lane “d” is G. marginata amplified by primers JA1 and LGG; it contains no bands that are not also shown in lane ”,3 amplified by JA1 alone. Lane “n” was amplified by primer G alone ................................................... 128 xi INTRODUCTION Mushroom poisoning - an overview Mushroom poisoning, or mycetism, has occurred throughout human history. I follow Benjamin (1995) in defining mushroom poisoning as a state of intoxication that proceeds as a natural consequence of eating a fleshy macrofungus that produces a compound inherently toxic to a majority of humans upon consumption. This definition excludes idiosyncratic reactions, food allergies, poisoning by mycotoxins, pesticide or heavy metal poisoning resulting from contaminated mushrooms, and food poisoning resulting from spoilage. Mushroom poisoning cases reported to American Poison Control centers between 1989 and 2000 averaged 9,467 cases per year. Poison Control Center data are inflated due to the large number of reports of children eating unknown mushrooms. These are treated as poisoning cases until the identity of the mushroom can be established, even in the absence of symptoms. Fifty-three percent of the cases reported for the years 1991 - 2000 (for which a breakdown by toxin type is given) involved either known nontoxic or unidentified mushrooms producing no symptoms (data from the American Association of Poison Control Centers’ Toxic Exposure Surveillance System, ). Of 9,208 mushroom poisoning cases reported to Poison Control Centers in 1989, the fungus in 8,355, or 90.5% of the total, was classified as “unknown if toxic” (Trestrail 1991). In 6,046 of these cases the patient experienced no effect. An additional assessment of the mushroom poisoning situation in the United States can be obtained from the North American Mycological Association’s (NAMA’s) Mushroom Poisoning Case Registry. The Case Registry has the advantage that only cases resulting in adverse symptoms are reported; however, reporting is voluntary, and many people who encounter, diagnose and treat mushroom poisoning may not be aware of the Case Registry. One thousand eight hundred and eighty one cases of mushroom poisoning had been reported to the NAMA Case Registry between its initiation in 1984 and 2000, with a minimum of 44 cases reported in 1989, and a maximum of 174 in 1991 (Trestrail 1998; Cochran 1999; Cochran 2000; Cochran 2001). The majority of cases (approximately 73%) were in adults. Approximately 4% of known mushroom species are poisonous, 1.8% are popular edibles, and another 18% are “probably edible” (neither sought after nor likely to do harm) (Benjamin 1995). The remaining 75% of mushroom species are considered inedible; not dangerous but too woody, small, slimy, powdery, hairy, or otherwise undesirable. Of the poisonous mushroom species, the majority cause gastrointestinal distress within less than one hour of ingestion of the mushroom. Symptoms range from nausea to vomiting and/or diarrhea in varying degrees of severity. Hospitalization may be required to combat dehydration and pain in severe cases, but the poisoning is self-limiting and symptoms rapidly resolve once the offending mushroom has left the body. Several mushrooms elicit more severe symptoms, and some can be deadly. Fifteen potentially deadly species occur in Michigan, out of an estimated 2,500 species overall (Hallen & Adams 2002). Delayed-action poisoning syndromes Most serious are the delayed-action poisoning syndromes caused by amatoxins, Cortinan'us toxins and monomethylhydrazine (MMH). MMH poisoning occurs each spring when people gather and eat the toxic false morels, ascomycete fungi in the genus Gyromitra. These fungi contain gyromitrin, a hydrazine that is rapidly converted in the human body to monomethylhydrazine (MMH), a principle component of rocket fuel. Hydrazines interfere with enzyme systems that require a pyridoxine cofactor, leading to decreased GABA concentrations among other potential problems (Trestrail 1994; Michelot & Toth 1991). The metabolism of gyromitrin to form a series of different hydrazines, many of which are unstable and highly reactive, also contributes to toxicity. A hit- and-miss component of false morel toxicity is attributable to a wide range of variables. The amount of toxin present varies considerably between individual mushrooms, with samples from the western United States being considered less toxic than those from the eastern US or Europe (Benjamin 1995). The susceptibility of individual humans to the toxins varies as much as the quantity of the toxins themselves. The toxin is heat-labile, and can be largely removed by cooking, though care must be taken not to inhale any cooking vapors. The LD50 of gyromitrin is 20 - 50 mg/kg body weight in adults, and 10 - 30 mg/kg body weight in children, comparable to 1 - 5 cups of fresh mushrooms (Benjamin 1995) Cortinan'us toxins are present in certain mushrooms in the genus Cortinan'us, subgenus Leprocybe. There have been two competing theories about the toxic principle, one holding that the chemical is a bipyridal with the trivial name orellanine, and the other favoring the cyclic peptide cortinarin (also spelled “cortinarine”) (Benjamin 1995). The preponderance of research supports orellanine as the toxic principle (Bresinsky & Besl 1990; Danel, Saviuc & Garon 2001). Cortinan'us poisoning is particularly insidious in that a minimum of two days passes between ingestion of the mushroom and presentation of symptoms, and presentation can be delayed by up to three weeks (Danel, Saviuc & Garon 2001). Orellanine has a specific affinity for the kidneys. Animal experiments and biopsies of poisoned human kidneys have consistently shown damage to the epithelium of both the proximal and distal tubules. The glomeruli are not involved (Benjamin 1995). It has been suggested that orellanine inhibits protein synthesis in poisoned cells (Benjamin 1995). The human LD50 is unknown; oral LD50 in mice is 90 mg/kg body weight (12.5 mg/kg administered by intraperitoneal injection) (Danel, Saviuc & Garon 2001). The toxicity appears to be lower than that of the amatoxins; 3-10 mushroom caps “may be sufficient to produce irreversible kidney damage in an adult” (Benjamin 1995, p. 251), compared with one cap of the comparably-sized Amanita phalloides. No verified cases of Cortinan'us poisoning are on record for North America, despite the occurrence of orellanine-producing mushrooms (Keller-Dilitz et al. 1985). More than 200 cases have been reported in Europe since Grzymala’s first report of Cortinan'us poisoning, in 1965 (Danel and colleagues examined 245 cases in their 2001 review). Amatoxins The most common fungal toxins involved in serious to fatal human poisoning cases are the amatoxins. Four hundred and fifty-two cases of amatoxin poisoning were reported to American Poison Control Centers between 1991 and 2000. Three hundred and twenty of these cases were treated in a health care facility. In 134 cases, the poisoning was classified as moderate or major, and eight deaths resulted (). The relatively low death rate may be attributable to prompt treatment; emesis or lavage administered within two hours post ingestion can remove the majority of the toxin (Benjamin 1995). Amatoxins are bicyclic octapeptides (Fig. 1). Poisoning is characterized by a (6)12 - 18(36) hour delay before symptoms present. The initial symptoms are moderate to severe gastrointestinal distress accompanied by vomiting and diarrhea. This phase frequently requires hospitalization due to the severity of the symptoms. Within 12 - 36 hours symptoms subside (but see Wieland 1969 for a mention of death at the gastritis stage). There follows a remission period of 12 - 36 hours during which the patient feels better and in some cases may be discharged from the hospital. The third and final stage is characterized by liver failure and, rarely, additional organ damage. The mortality rate for amatoxin poisoning in humans is 10 - 30 %. Mortality rates from the 19705 and earlier of 50 - 90 % have been reduced by a more widespread recognition of the poisoning syndrome, aggressive treatment and the advent of liver transplantation. Liver failure is the common culmination of amatoxin poisoning and, indeed, liver cells actively import amatoxins (Kroncke et al. 1986). However, all eukaryotic cells are susceptible to amatoxins and the perceived sensitivity to the liver is due to its function as a detoxifying organ and the consequent high exposure to amatoxins (Benjamin 1995). The gastrointestinal tract encounters amatoxins before the liver does, and it is noteworthy that the first symptoms of poisoning are abdominal pain, vomiting and diarrhea. Severe poisoning cases exhibit kidney damage, and the heart may be affected in rare cases (Benjamin 1995) Figure 1. R1 FbC CHOH \ / CH 41 ~ 5 HN- -CH— CO- NH- CH- CO— NH— CH2— CO I i 00 3 H20 NH I ,2 I I6] /CH3 HOCCHCZ/V 03 N OH CH- CH l OC- CH- NH- CO CH- NH- CO- CH2— NH H2C- COR2 ,8‘ *7 Structural formula for amatoxins. R‘| = CHZOH in or— and B-amanitin and CH3 in y-amanitin. R2 = NH2 in or- and y-amanitin and OH in B-amanitin. By convention, amino acids are numbered in a clockwise fashion, starting with asparagine. There is no antidote for amatoxin poisoning. The only mushroom toxin to possess an antidote is muscarine, which occurs in lnocybe and Clitocybe, and can be treated with atropine (Benjamin 1995). Due to some unfortunate early history — muscarine is named for Amanita muscaria, in which it occurs in insignificant quantities - atropine has been used extensively in treating mushroom poisoning in general, and particularly Amanita poisoning. This practice is dangerous and has been discredited for all but muscarine poisoning. Despite over a century of research, amatoxin poisoning, and most other types of mushroom poisoning, must be treated symptomatically. Attempts to raise antibodies to amatoxins and thus produce an antiserum were thwarted by the fact that the conjugation of amatoxin to an antibody leads to a ten- to fifty- times increase in toxicity (Cessi & Fiume 1969; Faulstich, Kirchner & Derenzini 1988). Silibinin and penicillin G both block amatoxin uptake by hepatic cells in an experimental system, and show therapeutic promise (Jahn, Faulstich & Wieland 1980). Silibinin and the related silymarin, derivatives of the milk thistle Silybum man'anum, appear to be both safer and more effective than penicillin G (Faulstich & Zilker 1994; Benjamin 1995), but the effective intravenous form has not been approved for use in the United States. The primary form of treatment involves removing the toxins from the patient. Emesis or lavage are not effective by the time symptoms present, six hours or more post ingestion. Hemoperfusion and hemodialysis may be used, but are rarely effective by the time symptoms present. The most effective means of removing toxins at this stage is the administration of activated charcoal, which binds to the toxins, interrupting their enterohepatic circulation (Faulstich & Zilker 1994). Amatoxins were first characterized in the genus Amanita, from which they get their name (Wieland & Hallermayer, 1941). Amanita species have been known for their toxicity for at least 2000 years. In arguably the most celebrated case of mushroom poisoning, extracts from the death cap, Amanita phalloides, were used to poison the Roman Emperor Claudius (see Benjamin 1995, pp. 33- 34). Amanita species have been studied intensively for the production of toxic compounds since the 18605 (Wieland 1969). In addition to amatoxins, Amanita species produce muscimol (Benjamin 1995), muscarine in trace quantities (Wieland 1969; 1986), bufotenine (Seeger & Stijve 1980) and phallotoxins (Lynen & Wieland 1938; Wieland 1987). Amatoxins are poisonous in very low doses. The human LD50 is estimated at 0.1 mg toxin / kg body weight, or approximately 7 mg for an adult male. This is similar to the LD50 values for dogs and guinea pigs (Wieland 1986). One average sized fruiting body of Amanita phalloides can be estimated to contain 10 - 12 mg of amatoxins (Wieland 1986), more than a lethal dose. Amatoxins are poisonous in varying degrees to all eukaryotic organisms. The mode of action is the specific inhibition of RNA polymerase II (pol ll; RNA polymerase B) (Lindell et al. 1970). At high levels of amatoxins, RNA polymerase ”I may be inhibited (Horgen, Vaisius & Ammirati 1978); however, the levels at which this occurs are many times the mammalian lethal dose. Cochet-Meilhac and Chambon (1974) found a KD of 6.4 x 10'9 M for an a- amanitin-pol Il complex at physiological temperatures, similar to the inhibition constant, Ki, of 1.0 x 10'8 M. Pol II is the primary transcription enzyme and is responsible for messenger RNA (mRNA) synthesis. Amatoxins do not prevent DNA or dNTP binding, or release of the nascent RNA chain. Rather, the toxins cause a dramatic slowing in the translocation of the polymerase along the DNA template (Wieland 1986; Chafin, Guo & Price 1995). This mode of action accounts for the delay observed in all cases of amatoxin poisoning. Transcription comes to a halt in poisoned cells, followed by the cessation of translation as the pool of existing mRNA is used up. The ultimate result is cell death, caused when the supply of essential proteins has been exhausted. Sensitivity to amatoxins is correlated with taxonomic position. All eukaryotes possess some sensitivity to amatoxins. Mammals possess the highest observed sensitivities, although not all mammals are sensitive to ingested amatoxins. lngested amatoxins have no effect on mice, although or- amanitin has an LD50 of 0.4 - 0.8 mg/kg body weight when the toxin is injected (Wieland 1986). The same is true for many rodents, although not the guinea pig, which appears to be as sensitive as humans to oral amatoxins. Reptiles are less sensitive than mammals, and insects are less sensitive than reptiles. Plants are less sensitive than insects, and fungi are less sensitive than plants. At the bottom of the list are the amatoxin-producing fungi. Nuclei from Amanita phalloides experienced no inhibition of pol II when exposed to amatoxins at a concentration of 25 pg/ml, and showed 31% inhibition at 75 rig/ml. By comparison, pol II from Agan'cus bisporus showed 8% inhibition at 25 pg/ml amatoxin, and rabbit brain pol ll showed 63% inhibition at 0.25 ug/ml (Horgen, Vaisius & Ammirati 1978). Prokaryotic RNA polymerases are wholly insensitive to amatoxins. Amatoxins exist as a family of related chemicals, differing in the degree of hydroxylation of the individual amino acids. or-, B- and y-amanitins are the prevalent amatoxins in mushrooms, and are the primary chemicals responsible for poisonings. e-amanitin and amaninamide are other pol Il-inhibiting members of the amatoxin family, while amanullin, pro-amanullin and amanin are nontoxic. The ability of amatoxins to bind to pol II is based on structure. The OH- group in 4-trans-hydroxy-L-proline appears to be necessary, as its removal causes a great decline in toxicity (Wieland 1986). Some hydroxylation of the position 3 isoleucine is required for toxicity (Wieland 1980). or- and B-amanitin have dihydroxyisoleucine, while y- and e-amanitin possess hydroxyisoleucine, at position 3. Amanullin, a naturally occurring amatoxin lacking any hydroxylation of this isoleucine, is nontoxic, as are synthetic analogues. If the ring formed by the cysteine-to-tryptophan bridge is disrupted, toxicity is lost (Wieland 1986). The role of amatoxins as transcription inhibitors has led to their use as biochemical tools. Amatoxins have been used in elucidating biosynthetic pathways in which the involvement of mRNA transcription is suspected (Wieland 1986). They have been used in virus research to determine whether eukaryotic or viral RNA polymerases are being utilized; the viral polymerases are insensitive to amatoxins (Wieland 1986). The effects of a transcription inhibitor on rapidly 10 growing and dividing cells has led to the use of amatoxins in cancer research (Wieland & Faulstich 1991). Phallotoxins Structurally similar to amatoxins are phallotoxins, bicyclic heptapeptides (Figure 2). Phallotoxins were first isolated from Amanita phalloides, from which they receive their name. Like amatoxins, phallotoxins form a family of related chemicals. The first-described phallotoxin, phalloidin, was isolated and purified three years prior to the first amatoxin, a-amanitin (Lynen & Wieland 1938; Wieland 1986). For many years, phallotoxins were thought to be responsible for the initial, gastrointestinal phase of Amanita poisoning, with amatoxins contributing the terminal liver pathology. This belief has appeared in a review article as recently as 1993 (Koppel 1993). Now we know that phallotoxins are not absorbed by the digestive tract (Wieland & Faulstich 1978) and that gastrointestinal cells are insensitive to these toxins. There is no evidence for phallotoxin involvement in Amanita poisoning, and all symptoms can be explained by amatoxins alone (Benjamin 1995). Phallotoxins given parenterally to experimental animals cause liver necrosis and death. The toxins are readily taken into the liver, where they bind to and stabilize actin in the filamentous F- actin form (Wieland 1977; Wieland 1987). ll Figure 2. O \N/C‘\ H (I: H HI \ \\ / H3c-~»c-H 2 Ce /H H C H3C~/CH If ‘ \ c; “20’ rtIIOHzc If 0.8/C rle Q HO’ 3 // \ H2C . H 2 O H/N\ .I' -. fi/?\ C" "h N '\ O H II. l”: Amatoxins a-Amanitin R= CH2C(=O)NH2 B-Amanitin R= CH2C(=O)OH Phallotoxins Phalloidin R1: CH3, R2: CH3, R3: OH Phallacidin R1: CH(CH3)2, R2: OH, R3: COZH Structural comparison of principle amatoxins and phallotoxins. Atoms in black are identical in both toxin families, while those in gray differ. 12 Detection of amatoxins and phallotoxins Several methods for detecting amatoxins and phallotoxins have been developed over the past fifty years. The so-called Meixner test is a crude colorimetric method. A raw extract of a mushroom is applied to a lignin-rich paper, such as newsprint. Once the fungal extract has dried, a drop of concentrated hydrochloric acid is applied. A blue color developing within several minutes indicates the presence of 5-substituted tryptamines, as are present in amatoxins (Meixner 1979; Beutler & Der Marderosian 1981). However, Meixner false positives are common because the minor hallucinogen bufotenine, present in Amanita citn'na, and other, unidentified fungal compounds also contain 5- substituted tryptamines (Beutler 1980; Beutler & Vergeer 1980). Despite its limitations, the Meixner test is the quickest and easiest available test for amatoxins, and it is therefore of considerable value in a clinical situation. Other methods that have been used include the cinnamaldehyde method of paper chromatography that detects amatoxins (Block et al. 1955); thin-layer chromatography that measures amatoxins and phallotoxins (Palyza & Kulhanek 1970); and high performance thin-layer chromatography measuring amatoxins (Stijve & Seeger 1979). Additionally, Enjalbert et al. (1992) have developed a high performance liquid chromatography (HPLC) method that can distinguish between and quantify four amatoxins and four phallotoxins. The aforementioned methods have been developed for the detection of toxins in mushrooms. A 13 further area of interest is the detection of toxins in biological fluids from patients. Dorizzi et al. (1992) present a thorough review. While chromatographic methods predominate, radioimmunoassay (RIA) has been frequently used to detect toxins both in mushrooms (Faulstich & Cochet—Meilhac 1976) and in serum and bodily fluids, in the case of poisoning. The inhibition of RNA polymerase II, particularly in calf thymus, can serve as an amatoxin assay (Preston et al. 1982). Fast atom bombardment mass spectroscopy (FAB-mass spec) may be used in conjunction with chromatography or other methods to provide a positive identification. Taxonomy of amatoxin-producing fungi Amatoxins are produced by certain species in four unrelated genera of basidiomycete fungi: Amanita (family Amanitaceae, order Agaricales); Lepiota (family Agaricaceae, order Agaricales); Conocybe (family Bolbitiaceae, order Agaricales) and Galerina (family Cortinan'aceae, order Cortinan’ales). Toxin production is limited to a discrete, closely related group of species within each genus: section Phalloidae in Amanita, section Ovisporae in Lepiota, section Naucon'opsis in Galerina, and the single species Conocybe filan's in Conocybe. Reports of amatoxins occurring in a wide range of mushrooms, including edible species (Faulstich & Cochet-Meilhac 1976; Preston et al. 1982) have been made based on detection of toxins near the detection limit of the procedures being employed (Wieland 1986). These have not been substantiated when the same 14 species were tested using more sensitive HPLC methods (Enjalbert et al. 1993; Hallen, unpublished results). Within a species amatoxin production is disjunct, with individual mushrooms varying with regard to toxin production. Tyler and colleagues (1966) found individuals of the destroying angels Amanita virosa and A. vema that lacked amatoxin. Beutler (1980) identified one A. phalloides specimen, out of 205, that lacked detectable amatoxins and phallotoxins when evaluated with the Meixner test and thin-layer chromatography. The specimen was fresh, which suggests that the toxin was constitutively absent. Yocum and Simons (1976) detected no amatoxins or phallotoxins in three out of four specimens of A. vema. Beutler and Der Marderosian (1981) used thin-layer chromatography to split A. virosa into two chemotaxonomic types: Type A, which possesses both amatoxins and phallotoxins, and type B, which possesses phallotoxins but no detectable amatoxins. The “destroying angels”, white Amanita species in section Phalloidae containing amatoxins, are in a state of taxonomic flux. The number of species is not agreed upon. Species have been delimited on the bases of spore size, spore length-to—breadth ratio and number of spores per basidium, all of which are plastic characters. The accurate identification of white Amanitas in section Phalloidae is thus quite difficult (Jenkins 1986). It is uncertain whether Amanita vema is a distinct taxon, or whether it is simply a white form of A. phalloides. Amanita virosa sensu auct. amer. may be the same as the European taxon by that name, or may be a four-spored variant of A. bisporigera. Amanita bispongera 15 has the highest toxin levels of North American Amanita species (Tyler et al. 1966), with little variation in toxin content between different fruit bodies, whereas approximately half of A. virosa specimens test negative for amatoxins. The identities of these mushrooms are therefore of more than academic concern. Two major phylogenetic studies of Amanita have been conducted in the past five years. Weiss, Yang and Oberwinkler (1998) and Drehmel, Moncalvo and Vilgalys (1999) conducted independent studies of Amanita using a portion of the 288 nuclear ribosomal DNA (rDNA) operon. In both studies, the toxin- producing members of section Phalloidae form a monophyletic clade, but only a few (6) toxin-producing species are examined. The placement and circumscription of other sections in Amanita, and the monophyly of Amanita subgenus Amanita, differ between the two studies, due, in part, to the inclusion of different taxa. Further work by Moncalvo and colleagues (2000) demonstrates that, in the 28S region, Amanita shows much greater divergence than any other known basidiomycete genus, leading to ambiguities in alignment of DNA sequence. Overview of the following chapters The remainder of this thesis details several studies on amatoxin-producing fungi and related species. Chapters one, two and three cover research on Amanita. In chapter one, I use molecular phylogenetics to determine whether amatoxin production has arisen once or multiple times in Amanita. The 16 relationships within the morphologically similar “destroying angel” complex are examined and North American and European specimens called A. virosa are compared. The ITS1-5.8S-IT82 rDNA (ITS region), a portion of the mitochondrial large rDNA, and a portion of the 288 rDNA were sequenced for phylogenetic analysis. Amatoxin-producing Amanita species formed a well-supported, derived, monophyletic clade in all analyses. This result supports the hypothesis of a single origin of amatoxin synthesis within the genus. In chapter two, restriction fragment length polymorphisms (RFLPs) and sequence of PCR products of the ITS region are used to identify Amanita species aborted by the ascomycete parasite Hypomyces hyalinus. Chapter three presents an HPLC study on amatoxin and phallotoxin production in indigenous and introduced South African Amanita species, and contains the first report of amatoxins in the introduced species A. reidii. Chapter four covers taxonomic and toxicological studies of Conocybe, primarily section Candidae. It includes the discovery of phallotoxins in Conocybe Iactea, the first report of these toxins outside of the genus Amanita. Chapter 5 covers the potential of Galerina marginata, which produces amatoxins in culture, to serve as a model organism for amatoxin synthesis, and non-ribosomal peptide synthesis in basidiomycetes. Future directions and a brief summary conclude the chapter and the body of the thesis. l7 References Benjamin DR. 1995. Mushrooms: Poisons and Panaceas. New York, W. H. Freeman and Company. 422 pp. Beutler JA. 1980. Chemotaxonomy of Amanita: qualitative and quantitative evaluation of isoxazoles, tryptamines, and cyclopeptides as chemical traits. Ph.D. thesis, Philadelphia College of Pharmacy and Science. Beutler JA, H Der Marderosin. 1981. Chemical variation in Amanita. Journal of Natural Products 44(4): 422—431. Beutler JA, PP Vergeer. 1980. Amatoxins in American mushrooms: evaluation of the Meixner test. Mycologia 72: 1142-1149. Block SS, RL Stephens, A Barreto, WA Murrill. 1955. Chemical identification of the amanita toxin in mushrooms. Science 121: 505-506. Bresinsky A, H Besl. 1990. A Colour Atlas of Poisonous Fungi. London, Wolfe Publishing Ltd. 295 pp. Cessi C, L Fiume. 1969. Increased toxicity of B-amanitin when bound to a protein. Toxicon 6: 309-310. Chafin DR, H Guo, DH Price. 1995. Action of or-amanitin during pyrophosphorolysis and elongation by RNA polymerase II. Journal of Biological Chemistry 270: 19114-19119. Cochet-Meilhac M, P Chambon. 1974. Animal DNA-dependent RNA polymerases, 11. Mechanism of the inhibition of RNA-polymerases B by amatoxins. Biochimica et Biophysica Acta 353: 160-184. Cochran KW. 1999. 1998 annual report of he North American Mycological Association’s Mushroom Poisoning Case Registry. Mcllvainea 14(1): 93-98. Cochran KW. 2000. 1999 annual report of he North American Mycological Association’s Mushroom Poisoning Case Registry. Mcllvainea 14(2): 34-40. Cochran KW. 2001. 2000 annual report of he North American Mycological Association’s Mushroom Poisoning Case Registry. Mcllvainea 15(1): 87-91. Danel, VC, PF Saviuc, D Garon. 2001. Main features of Cortinan'us spp. poisoning: a literature review. Toxicon 39: 1053-1060. 18 Dorizzi R, D Michelot, F Tagliaro, S Ghielmi. 1992. Methods for chromatographic determination of amanitins and related toxins in biological samples. Journal of Chromatography 580: 279-291 . Drehmel D, J-M Moncalvo, R Vilgalys. 1999. Molecular phylogeny of Amanita based on large-subunit ribosomal DNA sequences: implications for taxonomy and character evolution. Mycologia 91(4): 610-618. Enjalbert F, C Gallion, F Jehl, H Monteil, H Faulstich. 1992. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography. Journal of Chromatography 598: 227-236. Enjalbert F, C Gallion, F Jehl, H Monteil. 1993. Toxin content, phallotoxin and amatoxin composition of Amanita phalloides tissues. Toxicon 31: 803-807 Faulstich H, K Kirchner, M Derenzini. 1988. Strongly enhanced toxicity of the mushroom toxin or-amanitin by an amatoxin-specific FAB or monoclonal antibody. Toxicon 26: 491-499. Faulstich H, TR Zilker. 1994. Amatoxins. . In: Handbook of Mushroom Poisoning: Diagnosis and Treatment. DG Spoerke & BH Rumack, eds. Boca Raton, FL, CRC Press, pp. 233-248. Grzymala S. 1965. Etude clinique des intoxications par les champignons du genre Cortinarius oral/anus. Bulletin Medecine Legale Toxicologie. 8: 60-70. Hallen HE, GC Adams. 2002. Don’t Pick Poison! When Collecting Mushrooms for Food in Michigan. Michigan State University Extension Bulletin MSUE E-2777. Horgen PA, AC Vaisius, JF Ammirati. 1978. The insensitivity of mushroom nuclear RNA polymerase activity to inhibition by amatoxins. Archives of Microbiology 118: 317-319. Jahn W, H Faulstich, T Wieland. 1980. Pharmacokinetics of [3H-]methyl- dehydroxymethy-a-amanitin in the isolated perfused rat liver, and the influence of several drugs. In: Amanita Toxins and Poisoning. H Faulstich, B Kommerell & T Wieland, eds. Baden-Baden, Verlag Gerhard Witzstrock, pp. 79-87. Jenkins DT. 1986. Amanita of North America. Eureka, CA, Mad River Press. 198 PP- Keller-Dilitz H, M Moser, JF Ammirati. 1985. Orellanine and other fluorescent compounds in the genus Cortinan'us, Section Orellani. Mycologia 77: 667-673. Koppel C. 1993. Clinical symptomatology and management of mushroom poisoning. Toxicon 31(12): 1513-1540. 19 Kroncke KD, G Fricker, PJ Meier, W Gerok, T Wieland, G Kurz. 1986. a-amanitin uptake into hepatocytes. The Journal of Biological Chemistry 261(27): 12562- 12567. Lindell TJ, F Weinberg, PW Morris, RG Roeder, WJ Rutter. 1970. Specific inhibition of nuclear RNA polymerase II by a-amanitin. Science 170: 447-448. Lynen F, U Wieland. 1938. Uber die Giftstoffe des Knollenblatterpilzes. IV. Justus Liebigs Anna/en der Chemie 533: 93-117. Meixner A. 1979. Amatoxin-Nachweis in Pilzen. Zeitschrift fr‘ir Mykologie 45: 137- 139. Michelot D, B Toth. 1991. Poisoning by Gyromitra esculenta - a review. Journal of Applied Toxicology 11(4): 235-243. Moncalvo J-M, D Drehmel, R Vilgalys. 2000. Variation in modes and rates of evolution in nuclear and mitochondrial ribosomal DNA in the mushroom genus Amanita (Agaricales, Basidiomycota): Phylogenetic implications. Molecular Phylogenetic Evolution 16(1): 48-63. Palyza V, V Kulhanek, 1970. Uber die chromatographische Analyse von Toxinen aus Amanita phalloides. Journal of Chromatography 53: 545-558. Preston JF, BEC Johnson, M Little, T Romeo, JH Stark, JE Mullersman. 1982. Investigations on the function of amatoxins in Amanita species: A case for amatoxins as potential regulators of transcription. In: H Kleinkauf & H von Gohten, eds. Peptide Antibiotics, Biosynthesis and Functions. Berlin, Gruyter, pp. 399-426. Seeger R, T Stijve. 1980. Occurrence of toxic Amanita species. In: Amanita Toxins and Poisoning. H Faulstich, B Kommerell & T Wieland, eds. Baden- Baden, Verlag Gerhard Witzstrock, pp. 3-17. Stijve T, R Seeger. 1979. Determination of or-, B-, and y-amanitin by high- perforrnance thin-layer chromatography of Amanita phalloides (Vaill. ex Fr.) Secr. from various origin. Zeitschrift fr‘ir Naturforschaft 34c: 1133-1138. Trestrail JH. 1991. Mushroom poisoning in the United States - an analysis of 1989 United States poison center data. Clinical Toxicology 29: 459-465. Trestrail JH. 1994. Monomethylhydrazine-containing mushrooms. In: Handbook of Mushroom Poisoning: Diagnosis and Treatment. DG Spoerke & BH Rumack, eds. Boca Raton, FL, CRC Press. pp. 279-287. 20 Trestrail JH. 1998. 1997 annual report of he North American Mycological Association’s Mushroom Poisoning Case Registry. Mcllvainea 13(2): 86-91. Tyler VE, Jr., RG Benedict, LR Brady, JE Robbers. 1966. Occurrence of amanita toxins in American collections of deadly Amanitas. Journal of Pharmaceutical Sciences 55(6): 590-593. Weiss M, Z-L Yang, F Oberwinkler. 1998. Molecular phylogenetic studies in the genus Amanita. Canadian Journal of Botany 76: 1170-1179. Wieland H, R Hallermayer. 1941. Uber die Giftstoffe des Knollenblatterpilzes. VI. Amanitin, das Hauptgift des Knollenblatterpilzes . Justus Liebigs Anna/en der Chemie 548: 1-18. Wieland T. 1969. Poisonous principles of mushrooms of the genus Amanita. Science 159: 946-952. Wieland T. 1977. Modification of actins by phallotoxins. Naturwissenschaften 64: 303-309. Wieland T. 1980. The chemistry of Amanita toxins - Amatoxins: structure and RNA polymerase B inhibition. In: Amanita Toxins and Poisoning. H Faulstich, B Kommerell & T Wieland, eds. Baden-Baden, Verlag Gerhard Witzstrock. pp. 22- 29. Wieland T. 1986. Peptides of Poisonous Amanita Mushrooms. New York, Springer-Verlag. 256 pp. Wieland T. 1987. 50 Jahre Phalloidin - Seine Entdeckung, Charakterisierung sowie gegenwartige und zukiinftige Anwendung in der Zellforschung. Naturwissenschaften 74: 367-373. Wieland T, H Faulstich. 1978. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. Critical Reviews in Biochemistry 260: 185-260. Wieland T, H Faulstich.1991. Fifty years of amanitin. Experimentia 47: 1186- 1193. Yocum RR, DM Simons. 1977. Amatoxins and phallotoxins in Amanita species of the northeastern United States. Lloydia 40:178-190. 21 CHAPTER 1 MOLECULAR PHYLOGENETICS OF AMANITA, WITH A FOCUS ON SECTION PHALLOIDEAE Abstract Portions of the nuclear large, internal transcribed spacers, and mitochondrial large rDNA were sequenced and used in parsimony and likelihood analyses of the genus Amanita. The validity of the circumscription of the sections of Amanita was examined. The mitochondrial large rDNA provided little resolution, but distinguished four sections of Amanita and will be of utility in identifying mycorrhizae. Sections Amanita and Caesareae were upheld as monophyletic in all analyses. Amatoxin-producing species in section Phalloideae also formed a monophyletic cluster in all analyses, suggesting a single gain of the capacity to produce these toxins. A parsimony analysis of nuclear large rDNA from 112 specimens showed the basal members of Amanita forming a polyphyletic group with the genus Limacella. Introduction Amanita Pers. is a common mycorrhizal inhabitant of temperate forests worldwide. The genus consists of at least 200 species (Hawksworth et al. 1995) 22 and is readily recognizable. Amanita species are medium to large mushrooms possessing white-to-Iight-colored free gills with white-to-light-colored spores and a universal veil. The universal veil envelope the entire immature fruit body, rupturing as the fruit body expands. Portions of the universal veil may remain on the pileus as patches or warts, and a volva at the base of the stipe formed of universal veil tissue is common. Most species possess a partial veil in addition to the universal veil. The partial veil covers and protects the developing gills, and remains after pileus expansion as an annulus surrounding the stipe. The only other agarics to form both universal and partial veils are in the genus Limacella Earle which belongs with Amanita in the family Amanitaceae R. Helm ex Pouzar (Hawksworth et al. 1995). Limacella has been separated from Amanita because it possesses a universal veil that is gelatinous rather than dry. Amanita section Vaginatae (Fr.) Quél possesses a universal veil only, in the form of a membranous, saccate volva at the base of the stipe. Section Vaginatae was at one time considered a separate genus, Amanitopsis (Roze) Konr. & Maubl., but microscopic characters permitted placement in Amanita (Bas 1969). This placement has been supported by molecular phylogenetics (Weill, Yang & Obenrvinkler 1998; Drehmel, Moncalvo & Vilgalys 1999; Oda, Tanaka & Tsuda 1999). Delimitation of the species and circumscription of subgeneric taxa are matters of debate (Bas 1969; Jenkins 1986; Singer 1986; Drehmel, Moncalvo & Vilgalys 1999). When Bas (1969) wrote his monograph, more than 50 sections of Amanita had been proposed based on morphology. Corner & Bas (1962; see 23 also Bas 1969) circumscribe the genus into two subgenera and six sections. Subgenus Lepidella (Gilbert) Vesely possesses spores that stain blue-black in iodine (amyloid), while subgenus Amanita has non-staining (inamyloid) spores. Subgenus Lepidella contains sections Amide/Ia (Gilbert) Konr. & Maubl., Lepidella, Phalloideae (Fr.) Quél. and Validae (Fr.) Quél. Subgenus Amanita contains sections Amanita and Vaginatae. The sections are delimited largely on the basis of universal and partial veil characters. For a key to the subgenera and sections of Amanita see Bas (1969). Singer’s (1986) classification differs from Corner & Bas (1962) by the addition of three sections and the renaming of a fourth. Singer (1986) splits section Phalloideae into sections Phalloideae and Mappae Gilbert, and renames section Lepidella as section Roanokensis Sing. He also transfers some taxa from sections Amanita and Vaginatae into section Ovigerae Sing, and further splits section Vaginatae into sections Vaginatae and Caesareae Sing. Several recent papers have addressed the subgeneric delimitation of Amanita using molecular phylogenies of the nuclear large ribosomal RNA gene (28$ region; WeiB, Yang & Oberwinkler 1998; Drehmel, Moncalvo & Vilgalys 1999) and the ITS 1 - 5.8S - ITS 2 regions of the nuclear ribosomal RNA operon (ITS region; Oda, Tanaka & Tsuda 1999). These studies have upheld sections Amidella, Caesareae and Vaginatae as distinct monophyletic groups. If section Ovigerae is discounted, section Amanita is monophyletic (each study includes one member of section Ovigerae, which falls within section Amanita in two studies (Weill, Yang & Oberwinkler 1998; Oda, Tanaka & Tsuda 1999), and 24 basal to section Amanita in the third (Drehmel, Moncalvo & Vilgalys 1999)). Sections Mappae, Phalloideae and Validae are problematic. Phalloideae is not monophyletic in any treatment. Phalloideae sensu Corner & Bas (1962) is polyphyletic, forming at least three distinct groups in the analysis of WeilS, Yang & Oberwinkler (1998): amatoxin-producing taxa, section Mappae sensu Singer (1986), and A. manginiana Har. & Pat. and A. pseudoporphyria Hongo. A. manginiana and A. pseudoporphyn’a are basal to the other Phalloideae, but their placement is otherwise unresolved. Section Mappae forms a sister group to section Validae. WeiB, Yang & Oberwinkler (1998) and Oda, Tanaka & Tsuda (1999) treat Mappae as part of a monophyletic section Validae. Drehmel, Moncalvo & Vilgalys (1999) treat Mappae as section Phalloideae, subsection Validae, series Mappae (sister to series Validae). Molecular phylogenetics have weakly supported section Lepidella and have been inconclusive in the placement of section Lepidella in relation to the other sections. Drehmel, Moncalvo & Vilgalys (1999) and Oda, Tanaka & Tsuda (1999) support the monophyly of the subgenera Amanita and Lepidella. WeiB, Yang & Oberwinkler (1998) found a paraphyletic subgenus Lepidella, but with low bootstrap support. Each of the three recent phylogenetic studies has taken a broad overview of the genus Amanita, sampling broadly across all sections. Our studies concentrate on section Phalloideae with more extensive sampling. Section Phalloideae contains the amatoxin-producing species that are responsible for the vast majority of serious mushroom poisonings in humans, in particular Amanita phalloides (Fr.:Fr.) Link and the “deadly white” or “destroying angel” complex 25 centered around A. bisporigera Atk. A. phalloides is distinct, but many of the destroying angels cannot be readily distinguished on the basis of morphology. Jenkins (1986) states, “(t)he difficulty of macroscopic differentiation of the several ‘white’ Amanitas is common knowledge among those professionals and amateurs who have made attempts at identification. Color, size, and texture are so similar that an accurate identification cannot be made using these features. The only taxa which appear to be ‘relatively’ distinct are A. vema, A. virosa, A. bisporigera and A. tenuifolia. As for the rest, the only difference appears to be spore size.” Several species of deadly white Amanitas have been circumscribed on the bases of spore size, spore length-to-breadth ratio, and the number of spores per basidium. The delimitation of these species requires reevaluation because individual specimens may exhibit variations or gradations in these characters. By examining the phylogenetic relationships in section Phalloideae, we were able to develop an evolutionary hypothesis for amatoxin production in Amanita. In this study, DNA sequencing and phylogenetic analyses of the ITS and partial 28S regions were used to clarify relationships among members of the A. bisporigera complex and circumscribe the section Phalloideae. Mitochondrial large rDNA sequence was examined for its utility in assessing the phylogeny of Amanita. Finally, the relationship between Amanita and its sister group, Limacella, the only two genera in the Amanitaceae, was examined. 26 Materials and Methods Specimens used are shown in Tables 1 & 2. Specimens denoted “PH-10” or “WH-#” (Table 1) were identified by and obtained from Dr. Rodham Tulloss, who has conducted extensive morphological studies on the genus Amanita. DNA extraction, amplification and sequencing DNA was extracted from gill and pileus tissue of Amanita following Raeder & Broda (1985). Approximately 1-20 ng of the total genomic DNA was used per 25 pl reaction mixture for polymerase chain reaction (PCR) amplification. Various brands of prepackaged buffers and polymerases were used for PCR amplification. Primer ITS 1F (Gardes & Bruns 1993) or ITS F (Glen et al. 2001) was used in combination with primer ITS 4B (Gardes & Bruns 1993) or ITS 4 (White et al. 1990) to amplify the ITS 1-5.8$-ITS 2 regions of the nuclear rDNA (ITS region). Primers CTB6 (Bruns & Li; cited in Hughey et al. 2000) and TW13 (White et al. 1990) were used to amplify approximately 600 bases of the 28S nuclear rDNA. Primers MLin 3 and CML 7.5 (Bruns & Li; cited in Hughey et al. 2000) were used to amplify approximately 500 bases of the mitochondrial large rDNA. 27 Tablg 1. Specimens from which DNA was extracted and sequenced. Taxon Section Locale , a Accessron # Amanita sp. SA 22 Lepidella South Africa PRUM 3611 Amanita sp. T27 (WH-30) Phalloidae Texas, USA RET 1-30-89-AWR Amanita arochae (WH-65) Phalloidae Costa Rica RET 6-25-95-K Amanita bisporigera Phalloidae Michigan, USA MSC 380551 Amanita bisporigera f. tetraspora Phalloidae Mexico F1118140 Amanita brunnescens Validae Maine, USA MSC 380552 Amanita citrina f. lavendula Validae Michigan, USA MSC 380550 Amanita citrina var. citrina sensu Validae Michigan, USA MSC 380559 auct. amer. Amanita cylindrispora (WH-28) Amide/la New Jersey, USA RET S. Tulloss 8-11- 96-8 Amanita flavoconia Validae Vermont, USA MSC 380548 Amanita fulva sensu auct. amer. Vaginatae Michigan, USA MSC 380554 Amanita gilbertii (WH-2) Amide/la France RET Massart 97013 Amanita magnive/aris (WH-1) Phalloidae Florida RET Kuechmann s.n. Amanita marmorata ssp. Phalloidae Hawaii, USA DED5845 myrtacearum Amanita muscaria orange Amanita Michigan, USA MSC 380556 Amanita muscaria var. alba Amanita Michigan, USA MSC 380555 Amanita muscaria var. Amanita Micihgan, USA MSC 380549 guessowii Amanita ocreata (WH-8) Phalloidae California, USA RET NAMA 98 s.n. Amanita phalloides Phalloidae Australia MEL 2028861 Amanita phalloides Phalloidae California, USA MSC 380564 Amanita phalloides (PH-10) Phalloidae Norway 0 Gulden 49/94 Amanita phalloides f. umbrina Phalloidae South Africa PREM 48618 Amanita phalloides var. alba Phalloidae France RET Massart 90041 (WH-22) Amanita p/eropus Lepidella South Africa PREM 47480 Amanita reidii Phalloidae South Africa PRUM 4306 Amanita cf. subpha/loides Validae Indiana, USA F1116789 Amanita thiersii Lepidella Illinois, USA F1127062 Amanita vaginata sensu auct. Vaginatae Minnesota, USA MIN 839788 amer. Amanita virosa China Phalloidae China F1121430 Amanita virosa France (WH-21) Phalloidae France RET Massart 98025 Amanita virosa sensu auct. Phalloidae New Jersey, USA RET Carlson 7-24-96- amer. (WH-44) K Amanita cf. virosa sensu auct. Unknown Mexico RET Montoya E. mexic. (WH-24) 1558 Table 1. Specimens from which DNA was extracted and sequenced. aDED = herbarium of Dennis Desjardin, San Francisco State University, San Francisco, CA, USA; F = Field Museum of Natural History, Chicago, USA; MIN = University of Minnesota Herbarium, St. Paul, MN, USA; MSC = Beal Darlington Herbarium, Michigan State University, East Lansing, MI, USA; PREM = National Herbarium, National Botanical Institute, Pretoria, South Africa; PRUM = RET = herbarium of Rodham E. Tulloss, Roosevelt, NJ, USA. Specimens were collected by Heather Hallen (all MSC specimens except A. phalloides from California), Gro Gulden (A. phalloides from Nonrvay); Sarah E. K. Tulloss and Rodham E. Tulloss (A. cylindrispora); the Texas Mycological Society (A. sp. T27); Roy E. Halling (A. arocheae); F. Massart (A. gilbertii, A. phalloides f. alba, A. virosa from France); Bruce Kuechmann (A. magnivelan's); Dennis E. Desjardin (A. marmorata ssp. Myrtacearum); the North American Mycological Association (A. ocreata); Fred Stevens (A. phalloides from California); Pat Leacock (A. vaginata); Britt Carlson & Rodham E. Tulloss (A. virosa sensu auct. Amer); A. Montoya Esquival & Rodham E. Tulloss (A. cf. virosa sensu auct. Mexic.) Table 2. Data from GenBank used in the 288 extended database. .1180" Amanita angustilamellata Amanita armillariiformis Amanita armillariiformis Amanita avellaneosquamosa Amanita bisporigera Amanita bisporigera Amanita brunneofuliginea Amanita brunnescens Amanita caesarea Amanita calyptrata Amanita ceciliae Amanita ceciliae Amanita chepangiana Amanita citrina Amanita citrina Amanita citrina Amanita citrina grisea Amanita clarisquamosa Amanita cokeri Amanita cokeri Amanita excelsa Amanita farinosa Amanita farinosa Amanita flavipes Amanita flavoconia Amanita flavorubescens Amanita franchetii Amanita franchetii Amanita fritillaria Amanita frostiana Amanita fuliginea Amanita fulva Amanita fulva Amanita aff fulva Amanita gemmata Amanita gemmata Amanita gemmata Amanita gemmata Amanita hemibapha ocracea Amanita incarnatifolia Amanita jacksonii Amanita japonica Amanita Iignitincta Amanita Iongistriata Amanita magniverrucata Amanita manginiana Accession # AF024440 AF261437 AF261436 AF024441 AF097384 AF097385 .AF024442 AF097379 AF024443 AD001545 AF097372 AF024444 AF024445 AF097377 AF097378 AF024446 AF024447 AF024448 AF097395 AF097398 AF024449 AF024450 AF097370 AF024451 AF042609 AF097380 AF097381 AF156915 AF024452 AF024453 AF024454 AF097373 AF024455 AF024456 AD001547 AF024457 AF097371 AF335440 AF 024458 AF024459 AF097376 AF024460 AF024461 AF024462 AD001548 AF024463 30 Reference Weiss, Yang 8 Oberwinkler 1998 Moncalvo et al. 2002 Moncalvo et al. 2002 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Obenivinkler 1998 Bruns et al. 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Obenrvinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Taylor 8 Bruns 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Bruns et al. 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Berbee, lnderbitzen 8 Zhang. Unpublished. Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Bruns et al. 1998 Weiss, Yang 8 Oberwinkler 1998 Table 2I cont. Amanita mira Amanita muscaria Amanita muscaria Amanita muscaria Amanita muscaria Amanita muscaria Amanita muscaria var. persicina Amanita nivalis Amanita pachycolea Amanita pantherina Amanita pantherina Amanita pantherina lutea Amanita peckiana Amanita peckiana Amanita phalloides Amanita phalloides Amanita phalloides Amanita pilosella Amanita pseudoporphyria Amanita pseudovaginata Amanita rhoadsii Amanita rhopalopus Amanita roseitincta Amanita rubescens Amanita rubescens Amanita rubescens Amanita rubrovolvata Amanita silvicola Amanita sinensis Amanita solitaria Amanita solitariiformis Amanita solitariiformis Amanita sp Lepidella Amanita strobiliformis Amanita subfrostiana Amanita subglobosa Amanita subjunquillea alba Amanita sychnopyramis Amanita umbrinolutea Amanita vaginata Amanita vaginata Amanita verrucosivolva Amanita virgineoides Amanita virosa Amanita virosa Amanita cf virosa Amanita volvata Amanita volvata AF024464 AD001549 AF042643 AF097368 AF024465 AJ406558 AF097367 AF024466 AD001550 AD001551 AF024467 AF024468 AF042608 AF097387 AD001548 AF024469 AF261435 AF024470 AF024471 AF024472 AF097391 AF097393 AF097369 AF042607 AF097382 AF097383 AF024473 AD001553 AF024474 AF024475 AF097389 AF097390 AF097392 AF024476 AF024477 AF024478 AF024479 AF024480 AF024481 AF097375 AF024482 AF024483 AF024484 AF097386 AF159086 AF024486 AF024485 AF097388 31 Weiss, Yang 8 Oberwinkler 1998 Bruns et al. 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Langer. Unpublished. Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Bruns et al. 1998 Bruns et al. 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Bruns et al. 1998 Weiss, Yang 8 Oberwinkler 1998 Moncalvo et al. 2002 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Bruns et al. 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Obenrvinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Moncalvo, Drehmel 8 Vilgalys 2000 Weiss, Yang 8 Oberwinkler 1998 Weiss, Yang 8 Oberwinkler 1998 Drehmel, Moncalvo 8 Vilgalys 1999 Table 2. cont. Amanita aff volvata AF024487 Weiss, Yang 8 Oberwinkler 1998 Amanita yuaniana AF024488 Weiss, Yang 8 Oberwinkler 1998 Limacella glioderma AF024489 Weiss, Yang 8 Oberwinkler 1998 Limacella glishra U85301 Drehmel, Moncalvo 8 Vilgalys 1999 Tricholoma flavovirens AD001652 Bruns et al. 1998 The cycling reactions were performed in a DNA thermal cycler (Perkin- Elmer, Norwalk, CT, USA) with an annealing temperature of 55°C following Tank 8 Sang (2001). Alternately, a 60°C to 45°C touchdown protocol was used on some templates that did not amplify with the 55°C annealing temperature. The amplification ended with an additional 10 min extension at 72°C, and storage at 4°C. PCR amplification products were separated, and purified following Hughey et al. (2000). Alternatively, products were gel purified and cloned using TOPO® TA (lnvitrogen, Carlsbad, CA, USA) or pGEM® (Promega, Madison, WI, USA) cloning kits. Sequencing was performed by the Michigan State University Genomics Technology Support Facility, using dye terminator capillary electrophoresis on an ABI Prism® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Phylogenetic analyses Sequences were aligned using Clustal W (Thompson, Higgins 8 Gibson 1994), and were further aligned by eye. Phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford 2002). Maximum parsimony was used with a heuristic search algorithm. A consensus tree was built from all equally most parsimonious trees. Five hundred bootstrap replications (maxtrees=300) were 32 run to attain bootstrap values (Hedges 1992). Maximum likelihood analyses were performed in PAUP* using likelihood settings determined by Modeltest 3.06 (Posada 8 Crandall 1998). Trees that had the shortest length and the greatest In likelihood were displayed using TreeView version 1.6.6 (Page 1996). Results Approximately 100 bp from the 5’ end of ITS 1 were not alignable between sections of Amanita, and were excluded from analyses. The dataset was of 25 taxa and consisted of 571 aligned nucleotides including gaps introduced during alignment (261 informative sites). A heuristic search produced nine equally most parsimonious trees of 894 steps and consistency index (CI) of 0.688, retention index (RI) of 0.726, and rescaled consistency index (RC) of 0.499. Likelihood settings from the best-fit model (HKY+I+G) were selected by hierarchical likelihood rates testing in Modeltest (Posada 8 Crandall 1998). The tree with the greatest likelihood value, In likelihood of -4518.01, is shown in Fig. 3. Toxin- producing members of section Phalloideae form a monophyletic clade with 100% bootstrap support. (Text continues on page 41) 33 ' Amanita pleropus "A. sp. SA 22 A. thiersii A. marmorata ssp. myrtacearum 99 —LA. reidii 9 A. phalloides f. umbrina A. virosa sensu auct. amer. _1_00 A. bisporigera A. virosa France A. ocreata 100 A. bisporigera f. tetraspora A. arochae Phalloideae 32 Amanita sp.T27 80 A. phalloides Norway A. phalloides Australia 88 A. phalloides f. alba France A. phalloides California 97 A. cf. virosa sensu auct. mexic. 100 rA. gilbertii lA. cylindrospora A. muscaria var. guessowii 69 —— A. fulva A. flavoconia A. cf. subphalloides 0.1 A. citrina f. lavendula Fig. 3. Cladogram produced by maximum likelihood analysis of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA operon.Topology corresponds to one of nine equally most parsimonious trees (894 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree. 34 ‘— Amanita pleropus A. thiersii A. fulva A. cf. virosa sensu auct. mexic. _ 6 A. citrina f. lavendula I E A. cf. subphalloides A. flavoconia A. bisporigera 5 A. virosa sensu auct. amer. L— 65\_ A. sp. T27 7 A. magnivelaris A. phalloides Germany -|A. phalloides f. alba France A. phalloides California TIA. virosa China A. virosa France Phalloideae 99 A. arochae A. virosa sensu SUCt. amer. A. ocreata ——1-QQJ A. marmorata ssp. myrtacearum 7 A. phalloides f. umbrina A. reidii 100 A. muscaria var. alba IA. muscaria var. guessowii A. cylindrospora 100 A. gilbertii 0.1 Fig. 4. Cladogram produced by maximum likelihood analysis of the 288 region of the nuclear ribosomal DNA operon.Topology corresponds to one of 132 equally most parsimonious trees (361 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree. 35 Amanita pleropus A. thiersii 99 A. phalloides f. alba France PIA. phalloides California SKLI F—A. virosa sensu auct. amer. \ , A. bisporigera \ I 109 —A. virosa France __A. sp. T27 100 A. ocreata 30L A. arochae A. marmorata ssp. myrtacearum A. reidii 8 A. phalloides r. umbrina Phalloideae A. flavoconia 100 99 — A. cf. subphalloides A. citrina f. lavendula A. muscaria var. guessowii 7 A. fulva A. cf. virosa sensu auct. * mexic. A. gilbertii 100 0.1 A. cylindrospora Fig. 5. Cladogram produced by maximum likelihood analysis of the combined ITS and 288 regions of the nuclear ribosomal DNA operon.Topology corresponds to one of four equally most parsimonious trees (1338 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). Arrow indicates node leading to amatoxin-producing taxa. * indicates branches that collapse on the strict consensus tree. 36 Tricholoma flavovirens Amanita brunnescens A. of. virosa sensu auct. mexic. A. silvicola ___53 100 A. calyptrata I—A. jacksonii Caesareae .. A. magniverrucata _ A. gemmata __ A. pantherina 91 A. muscaria * 66 A. muscaria var. Amanita ‘ guessowii . A. muscaria orange .A. muscaria var. alba I A. excelsa * A. pachycolea - Lilla. fulva I Vagrnatae 96[A- Pha”°"°'°s I Phalloideae A. magnivelaris ____g_1_I———— A. francheti A. flavoconia 77 A. cf. subphalloides I 3%- A. citrina A. citrina f. lavendula 0.1 Fig. 6. Cladogram produced by maximum likelihood analysis of the mitochondrial large ribosomal DNA operon.Topology corresponds to one of 7 equally most parsimonious trees (156 steps). Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). Numbers at nodes are bootstrap indices of support (%). * indicates branches that collapse on the strict consensus tree. 37 Limacella {— Lepidella Limacella Lepidella ISubsection Mappae Validae ISubsection Mappae Phalloidae Amidella Amanita <— Amidella Lepidella 4— Phalloideae* Caesareae Vaginatae <— Lepidella Fig. 7a. Consensus based on 300 equally parsimonious trees (length 1275) of the 28S region of the nuclear ribosomal DNA subunit. Names of sections in genus Amanita are given. Limacella (top) is the other genus in the Amanitaceae and was used as an outgroup. Arrow indicates node leading to amatoxin-producing taxa. * indicates Amanita manginiana and A. pseudoporphyria, traditionally placed in section Phalloideae. 38 ——A. fuliginea _ A. subjunquillea alba "A. ocreata A. phalloides Germany 78 A. phalloides New England A. phalloides California A. phalloides alba France A. bisporigera A. bisporigera 57 A. virosa sensu auct. amer. A. magnivelaris A. virosa sensu auct. amer. A. sp. T27 A. virosa China A. virosa France A. cf. virosa Germany A. virosa Virginia A. arochae A. phalloides umbrina L__ A. reidii A. marmorata myrtacear um Fig. 7b. Amatoxin-producing members of section Phalloideae from the parsimony analysis of the extended 28S dataset. Numbers at nodes are bootstrap indices of support. 39 Limacella glischra A. thiersii Limacella glioderma A. pleropus armillariiformis armillariiformis Fig. 7c. Basal taxa from the parsimony analysis of the extended 288 dataset. Numbers at nodes are bootstrap indices of support. 40 The 28S sequence dataset was of 26 taxa and 526 aligned nucleotides (118 informative sites). A heuristic search produced 132 equally most parsimonious trees of 361 steps, CI = 0.673, RI = 0.722, RC = 0.486. Likelihood settings from the best-fit model (TrN+G) were selected by hierarchical likelihood rate testing in Modeltest (Posada 8 Crandall 1998). One of the three trees with the greatest likelihood value, In likelihood of -2497.75, is shown in Fig. 4. Amatoxin-producing members of section Phalloideae form a monophyletic clade with 99% bootstrap support. The combined ITS and 288 dataset was of 21 taxa and 1089 aligned nucleotides (378 informative sites). A heuristic search produced four equally most parsimonious trees of 1338 steps, CI = 0.661, RI = 0.663 and RC = 0.438. Likelihood settings from the best-fit model (TrN+l+G) were selected by hierarchical likelihood rates testing in Modeltest (Posada 8 Crandall 1998). The tree with the greatest likelihood value, In likelihood of -7418.964, is shown in Fig. 5. Amatoxin-producing members of section Phalloideae form a monophyletic clade with 100% bootstrap support. The mitochondrial large dataset was of 24 taxa: 23 Amanita specimens and Tricholoma flavovirens, which was included as an outgroup. The dataset was of 310 aligned nucleotides (46 informative sites). A heuristic search produced seven equally parsimonious trees of 156 steps, CI = 0.641, RI = 0.726, RC = 0.463. Likelihood settings from the best-fit model (F81+I+G) were selected by hierarchical likelihood rates testing in Modeltest (Posada 8 Crandall 1998). The tree with the greatest likelihood value, In likelihood = - 1142.43, is shown in Fig. 41 6. Sections Amanita, Caesareae, Phalloideae and Vaginata formed monophyletic clades, with bootstrap support of 89% and above. An extended 28S dataset was prepared by combining the sequences of 26 taxa with an additional 86 sequences from GenBank to yield a total sequences. The dataset was of 542 aligned nucleotides (202 informative sites). A heuristic search produced 300 equally most parsimonious trees of 1275 steps, CI = 0.355, RI = 0.761 and RC = 0.271, a strict consensus of which is shown in Fig. 7a. Sections Amide/Ia and Lepidella were both polyphyletic. Amatoxin-producing members of section Phalloideae formed a monophyletic clade with 100% bootstrap support (Fig. 7b). The genus Amanita itself may be polyphyletic, as Limacella glioderma was placed within Amanita with 84% bootstrap support (Fig. 7c). With the exception of the taxon identified as A. cf. virosa sensu auct. mexic. (according to Mexican authors), all purportedly amatoxin-producing members of section Phalloideae formed a monophyletic clade in all analyses. Section Validae formed a monophyletic clade, with a monophyletic subsection Mappae nesting within, in the ITS and 288 analyses, but was found paraphyletic in the mitochondrial large analysis. Discussion Among taxa that produce amatoxins there is little resolution. A. phalloides f. umbrina Ferry, A. reidii Eicker 8 van Greuning and A. marmorata ssp. 42 myrtacearum O.K. Miller, D. Hemmes 8 G. Wong consistently form a clade with high (96% and above) bootstrap support. This supports the belief that these taxa are synonymous (Eicker, van Greuning 8 Reid 1993; Hallen, Adams 8 Eicker in press; RE Tulloss, pers. comm.) The white A. phalloides f. alba Britzelm, to which the name A. vema has sometimes been misapplied, forms a monophyletic clade with A. phalloides f. phalloides specimens from Europe, Australia and North America. The name A. vema (Bull.:Fr.) Lamarck has frequently been misapplied to white morphs of A. phalloides, and is of uncertain validity. Type material would need to be examined to determine whether A. vema is a valid taxon. Amanita virosa in the sense of American authors (sensu auct. amer.) does not cluster with Old World A. virosa Lamarck specimens; nor does it cluster with A. bisporigera or the four-spored A. bisporigera var. tetraspora as morphology would suggest (Tulloss et al. 1995). The amatoxin-producing Phalloideae form a well-supported clade (96- 100% bootstrap support in all analyses, suggesting a single gain of the ability to produce amatoxins in an ancestral member of this section. This affirms the validity of amatoxin presence as a chemotaxonomic character (Beutler 1980). The fact that Phalloideae is a derived clade suggests that amatoxin gain in the genus Amanita occurred separately from amatoxin gain in other genera. There was uncertainty with regard to the identification of the species denoted Amanita cf. virosa sensu auct. mexic. This taxon is placed well apart from other A. virosa specimens and section Phalloideae in all analyses, and the likeliest hypothesis is that the specimen was misidentified. The phylogenetic 43 placement of this taxon is uncertain as it varies depending on the dataset. In all cases it is basal to sections Phalloideae and Validae. Some species that have been placed in section Phalloideae, such as A. manginiana and A. pseudoporphyria, are placed well apart from the amatoxin- producing Phalloideae in the extended 28S dataset. This placement was also evident in the analyses of WeiB, Yang 8 Obenrvinkler (1998), and it is clear that the classification of these species requires some consideration. Sections Amanita, Caesareae, Phalloideae and Vaginata formed monophyletic clades in analyses of the mitochondrial large rDNA. Section Validae and Validae subsection Mappae were polyphyletic. The portion of the mitochondrial large rDNA examined is highly conserved and is not always of utility in distinguishing between species or closely related genera (Bruns et al. 1998). The evident distinctiveness of sections Amanita, Caesareae, Phalloideae and Vaginata on the basis of this data is therefore unexpected. The ability to identify Amanita species to section may prove very useful in mycorrhizal research. However, the relatively low number of informative sites (46 out of 312) for this gene renders it of little utility in formulating evolutionary hypotheses. A. pleropus (Kalchbr. 8 MacOwan) D. Reid and A. thiersii Bas (section Lepidella) have long been considered “primitive” basal taxa on the basis of their morphology (Bas 1969). This is confirmed by the phylogenetic analyses. In the parsimony analysis of the extended 288 dataset, these species, together with A. annilan'ifonnis Trueblood 8 Jenkins, were basal to Limacella glioderma. A. annillan'iformis, A. pleropus and A. thiersii possess dry, membranous universal 44 veils whereas Limacella possesses a gelatinous veil. It can be inferred based on the 28S analysis that the character of a gelatinous veil might have been given inappropriate weight in differentiating genera. The placement of a Limacella species within the basal Amanitas renders both genera polyphyletic. Further Limacella and basal Amanita taxa will need to be evaluated, and additional genes will need to be sequenced in Limacella before we can judge the validity of the generic circumscriptions. Acknowledgements We gratefully acknowledge the help of Rodham Tulloss, who has identified and provided us with many Amanita samples we would not have been otherwise able to obtain. We also thank Greg Mueller and the Field Museum of Natural History, David McLaughlin and the University of Minnesota Herbarium, and Dennis Desjardin and San Francisco State University for providing specimens. This research was supported by a grant from the International Association for Plant Taxonomy. References Bas C. 1969. Morphology and subdivision of Amanita and a monograph of its Section Lepidella. Persoonia 5(4): 285-579. Beutler JA. 1980. Chemotaxonomy of Amanita: qualitative and quantitative evaluation of isoxazoles, tryptamines, and cyclopeptides as chemical traits. Ph.D. thesis, Philadelphia College of Pharmacy and Science. Bruns TD, TM Szaro, M Gardes, KW Cullings, JJ Pan, DL Taylor, TR Horton, A Kretzer, M Garbelotto, Y Li. 1998. A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis. Molecular Ecology 7: 257-272. 45 Corner EJH, C Bas. 1962. The genus Amanita in Singapore and Malaya. Persoonia 2(3): 241-304. Drehmel D, J-M Moncalvo, R Vilgalys. 1999. Molecular phylogeny of Amanita based on large-subunit ribosomal DNA sequences: implications for taxonomy and character evolution. Mycologia 91 (4): 610-618. Eicker A, JV van Greuning, DA Reid. 1993. Amanita reidii — a new species from South Africa. Mycotaxon 47: 433-437 Gardes M, TD Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 1 13-1 18. Glen M, IC Tommerup, NL Bougher, PA O’Brien. 2001. Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations. Mycological Research 105: 138-149. Hallen HE, GC Adams, A Eicker. 2002 in press. Amatoxins and phallotoxins in indigenous and introduced South African Amanita species. South African Journal of Botany 68: 1-5. Hawksworth DL, PM Kirk, BC Sutton, DN Pegler. 1995. Dictionary of the Fungi 8th edn. ,Wallingford, Oxon, UK: CAB International. 616 pp. Hedges SB. 1992. The number of replications needed for accurate estimation of the bootstrap P value in phylogenetic studies. Molecular Biology Evolution 92: 366-369. Hughey BD, GC Adams, TD Bruns, DS Hibbett. 2000. Phylogeny of Calostoma, the gelatinous-stalked puffball, based on nuclear and ribosomal DNA sequences. Mycologia 92: 94-104. Jenkins DT. 1986. Amanita of North America. 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Bioinfonnatics 14: 917-818. Singer R. 1986. The Agarica/es in Modern Taxonomy 4th ed. Koenigstein, Koeltz Scientific Books. 981 pp. Swofford DL. 2002. PAUP*: Phylogenetic Analysis Using Parsimony (and other methods) version 4.0b10. Sunderland, MA, Sinauer Associates. Tank DC, T Sang. 2001. Phylogenetic utility of the glycerol-3-phosphate acyltransferase gene: evolution and implications in Paeonia (Paeoniaceae). Molecular Phylogenetics and Evolution 19: 421 -429. Taylor DL, TD Bruns. 1999. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities. Molecular Ecology 8: 1837-1850. Thompson JD, DG Higgins, TJ Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. Tulloss RE, SL Stephenson, RP Bhatt, A Kumar. 1995. Studies on Amanita (Amanitaceae) in West Virginia and adjacent areas of the mid-appalachians. Preliminary results. Mycotaxon 56: 243-293. Weill. M, Z-L Yang, F Oberwinkler. 1998. Molecular phylogenetic studies in the genus Amanita. Canadian Journal of Botany 76: 1170-1179. White TJ, T Bruns, S Lee, J Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: MA Innis, DH Gelfand, JJ Sninsky 8 TJ White, eds. PCR Protocols: A Guide to Methods and Applications. San Diego, CA, Academic Press, Inc. pp. 315-322. 47 CHAPTER 2 IDENTIFICATION OF AMANITA SPECIES PARASITIZED BY HYPOMYCES H YALINUS Abstract RFLPs and DNA sequencing of PCR products of the ITS-5.88 regions of Amanita specimens infected with the parasite Hypomyces hyalinus were used to identify the host species. Twenty two uninfected Amanita taxa were utilized as reference standards for comparisons of RFLP and sequence homology. Phylogenetic analyses of sequence enhanced identification of several of the hosts. Parasitized Amanitas were in Amanita Section Validae. Several parasitized specimens were identified as taxa allied with Amanita rubescens, supporting earlier reports based on proximity to uninfected fruiting bodies and ecology. The parasitized taxa included A. brunnescens, A. flavoconia, A. novinupta and A. orsonii. Two amorphous basidiocarps parasitized by H. hyalinus and possessing fertile perithecia contained DNA sequence from a Russula and a The/ephora species. Introduction Species of Amanita Pers. are subjected to parasitism by Hypomyces hyalinus (Schw.:Fr.) Tul. (Hypocreales, Pyrenomycetes). The perithecia of H. 48 hyalinus form over the entire host surface, aborting the basidiocarp and rendering it unidentifiable (Fig. 8). Figure 8. Amanita basidocarps parasitized by Hypomyces hyalinus. Infected Amanitas may grow to approximately full height but the pileus does not expand. No recognizable lamellae are present, and basidiospores are not produced. Infected specimens frequently grow intermingled with identifiable, uninfected specimens. The proximity of uninfected fruiting bodies and the tendency of some parasitized specimens to stain reddish upon bruising (rubescent nature) has led to the assumption that the host species is predominantly A. rubescens sensu auct. amer. (Bessette, Bessette 8 Fischer, 1997). Lincoff (1981; quoted by Phillips, 1991) describes the habitat of H. hyalinus as “on Amanita rubescens, Amanita flavorubescens, Amanita frostiana, and possibly Amanita bisporigera." Rogerson and Samuels (1996) include A. 49 muscaria (L.:Fr.) Pers. in the list of parasitized species, but go on to state, “Hypomyces hyalinus is presumed only to occur on Amanita species because the only healthy basidiocarps associated with the Hypomyces are species of Amanita. The parasitism of H. hyalinus is so complete and the host so distorted, that in the absence of nearby healthy basidiocarps it is not possible to determine the host.” A. rubescens and related taxa in Amanita Section Validae are edible, and in some parts of the country parasitized Amanitas are collected and eaten (TJ Volk, pers. comm., 28 August 2001). A. bisporigera Atk. is deadly poisonous and A. muscaria and A. frostiana (Peck) Sacc. contain the central nervous system toxin ibotenic acid. The identity of the species of parasitized Amanita thus becomes of importance. Reports of H. hyalinus on A. frostiana may be based on misidentification; A. flavoconia in section Validae is frequently misidentified as A. frostiana (Jenkins 1977; Tulloss 1998). The type collection of A. frostiana includes material of A. flavoconia (Jenkins 1977). We have undertaken this study to begin to address the question of which species of Amanita are being parasitized. The field of mycorrhizal research has given rise to several molecular techniques for identifying basidiomycete fungi in the absence of a recognizable fruit body. The principle methods used are restriction fragment length polymorphisms (RFLPs) of PCR products (Gardes et al. 1991; Karen et al. 1997) and direct sequencing of PCR products (Glen et al. 2001; Bruns 1996). The use of both techniques is limited by the requirement for data on fungi of known identity. RFLPs are further limited by the existence of sequence variation within a 50 fungal species, which may result in differing restriction patterns (Karen et al. 1997). This, however, may be overcome by subjecting multiple individuals of the same species to restriction digests (Bruns et al. 1998). DNA sequencing is the most reliable method for identifying an unknown fungus unidentifiable by morphological characters (Bruns et al. 1998). Large amounts of sequence data for both the ITS1-5.8S-IT32 region of the nuclear ribosomal RNA operon (ITS region) and the mitochondrial large subunit rRNA gene are available on GenBank, operated by the National Center for Biotechnology Information (). Sequence from the mitochondrial large region can place a hymenomycete at the familial or subfamilial level with a high level of confidence (Bruns et al. 1998), but is inadequate for resolving identity within a genus or between closely related genera. The ITS region is superior at determining relationships between closely related fungi (Bruns 1996). Furthermore, basidiomycete-specific ITS primers exist (Gardes 8 Bruns 1993; Glen et al. 2001) which permit amplification of Amanita without co-amplification of the ascomycete parasite. Our purpose was to use both RFLPs and sequence data from the ITS region to identify the host species in parasitized specimens of Amanita. Materials and Methods Specimens used are outlined in Table 3. RFLP patterns and ITS sequences were obtained from reference species from both subgenera and 51 Table 3. Parasitized and non-parasitized Amanita specimens examined Taxon Sectiona Locale Accession #b Amanita PA 01 Vermont, USA MSC 380560 Amanita PA 03 Vermont, USA MSC 380560 Amanita PA 05 Vermont, USA MSC 380560 Amanita PA 13 Michigan, USA MSC 380561 Amanita PA 17 Michigan, USA MSC 380561 Amanita PA 19 Michigan, USA MSC 380561 Amanita PA 22 Michigan, USA MSC 380561 Amanita PA 33 Michigan, USA MSC 380561 Amanita PA 43 Wisconsin, USA MSC 380562 Amanita PA 8 Michigan, USA MSC 380561 Hypomyces hyalinus Wisconsin, USA MSC 380559 Amanita muscaria orange Amanita Michigan, USA MSC 380556 Amanita muscaria var. alba Amanita Michigan, USA MSC 380555 Amanita muscaria var. guessowii Amanita Micihgan, USA MSC 380549 Amanita cylindrispora Amide/la New Jersey, USA RET S. Tulloss 8- 11-96-B Amanita hemibapha var. hemibapha C Caesarae Japan ABO15699 Amanita thiersii Lepidella Illinois, USA F1127062 Amanita bisporigera Phalloidae Michigan, USA MSC 380551 Amanita fulva ss auct. amer. Vaginatae Indiana, USA MSC 380554 Amanita sinicoflava Vaginatae Minnesota, USA MIN 838924 Amanita vaginata ss auct. amer. Vaginatae Minnesota, USA MIN 839788 Amanita brunnescens Validae Maine, USA MSC 380552 Amanita cf. subphalloides Validae Indiana, USA F1116789 Amanita citrina f. lavendula Validae Michigan, USA MSC 380550 Amanita citrina var. citrina Validae Japan AB015679 Amanita citrina var. grisea Validae Japan ABO15680 Amanita excelsa sensu D. Reid 8 Validae Belfast, Mpumalanga, MSC 375639 Eicker South Africa Amanita flavipes Validae Japan AB015696 Amanita flavoconia Validae Minnesota, USA KH94 Amanita flavoconia Validae Vermont, USA MSC 380548 Amanita flavoconia (white-stiped form) Validae New Jersey, USA RET 9-8-99-J Amanita novinupta Validae Oregon, USA RET 4-14-92-JEL1 Amanita porphyria Validae Minnesota, USA DJM 1148 Amanita porphyria Validae Japan AB015677 . d Validae Japan AB015682 Amanita rubescens Amanita rubescens ss auct. amer. Validae Michigan, USA MSC 380557 Amanita rubescens ss auct. amer. Validae Michigan, USA MSC 380558 Amanita rubescens var. alba Validae South Carolina, USA RET 7-19-86-B Amanita rubescens var. congolensis Validae Zambia RET Arora 00-384 Amanita rubescens var. congolensis Validae Zimbabwe RET Arora 00-443 52 Table 3. Parasitized and non-parasitized Amanita specimens examined. aSection for the parasitized specimens was unknown at the beginning of the study and is not listed. bNumbers beginning “AB” are GenBank accession numbers and refer to sequences from Oda, Taneka 8 Tsuda (1999). “MSC” = Beal Darlington Herbarium, Michigan State University, East Lansing, MI, USA All MSC specimens were collected by H. Hallen. “MIN” = University of Minnesota herbarium, St. Paul, MN, USA. A. sinicoflava and A. vaginata were collected by P. Leacock. “DJM” (collected by DJ. McLaughlin) and “KH” (collected by K. Harris) are unaccessioned collections at MIN. “F” = Field Museum of Natural History, Chicago, IL, USA. A. cf. subphalloides was collected by G. Wesley, det. I. Morrar. A. thiersii was collected by H.L. Monoson. “RET” = herbarium of Rodham Tulloss, Roosevelt, NJ, USA. Both specimens of A. rubescens var. congolensis were collected by D. Arora. A. cylindrispora was collected by S. Tulloss. A. flavoconia (white- stiped variety), A. novinupta and A. rubescens var. alba were collected by R. Tulloss. CThe morphology of Japanese specimens denoted “A. rubescens” suggests that they are most likely A. orsonii (R. E. Tulloss, pers. comm, July 30, 2002) 53 several sections of the genus Amanita, with a special emphasis on section Validae. DNA extraction, amplification and sequencing Parasitized Amanita specimens were collected in the summer of 2000 from Michigan, Vermont and Wisconsin. Herbarium specimens of parasitized Amanita were also procured from the University of Michigan. DNA was extracted using a cetyltrimethyI-ammonium bromide (CTAB) method (Scott and Playford 1996). Dried basidiocarps were rehydrated in the respective extraction buffer before grinding. Amanita DNA was selectively favored by using sections from the interior of the fruiting body, avoiding the outer layer of perithecia. Tissue was ground with a pestle and 0.2 g sand in 4 mL of the extraction buffer. The extract was then filtered through 1 layer of Miracloth (Calbiochem-Novobiochem Corp., La Jolla, California). The filtrate was purified with phenol:chloroform:isoamyl alcohol (24:24:1) extractions and was centrifuged to remove solids. The water- soluble fraction was precipitated with isopropanol and centrifugation. The precipitate was air-dried under vacuum then resuspended in 50 pl water. DNA was extracted from mature, non-parasitized Amanita fruiting bodies and from Hypomyces perithecia following Raeder 8 Broda (1985). Approximately 1-20 ng of the total genomic DNA was used per 25 pl reaction mixture for polymerase chain reaction (PCR) amplification. Various brands of prepackaged buffers and polymerases were used for PCR 54 amplification. The fungal-specific primer ITS 1F (C'I‘I'GGTCATTI’AGAGGAAGTAA; Gardes 8 Bruns 1993) was used in combination with basidiomycete-specific ITS 4B (CAGGAGACTTGTACACGGTCCAG; Gardes 8 Bruns 1993) to selectively amplify the Amanita host and to amplify the reference Amanita species. Additionally, the basidiomycete-specific primer combination of ITS F (CCCTR'ITGCTGAGAAXYTGRTC; Glen et al. 2001) and ITS 4B was tested. ITS 1F was used with the universal primer ITS 4 (TCCTCCGCTTATTGATATGC; White et al. 1990) to amplify the Hypomyces. PCR was performed in a DNA thermal cycler (Perkin-Elmer, Nonrvalk, CT, USA), using the protocol: 4 min at 70°C (1 cycle); 1.5 min at 94°C, 1 min at 52°C, 1.5 min at 72°C (4 cycles); 1 min at 94°C, 1 min at 55°C, 1.5 min at 72°C (29 cycles). The amplification ended with an additional 10 min extension at 72°C, and storage at 4°C. Annealing temperatures of 50°C (4 cycles)/52°C (29 cycles) and 55°C (4 cycles)/57°C (29 cycles) were also tested. PCR amplification products were separated, and purified following Hughey et al. (2000). Alternatively, products were gel purified and cloned using TOPO® TA (lnvitrogen, Carlsbad, CA, USA) or pGEM® (Promega, Madison, WI, USA) cloning kits. Sequencing was performed by the Michigan State University Genomics Technology Support Facility, using dye terminated capillary electrophoresis on an ABI Prism® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). 55 Restriction fragment length polymorphisms MapDraw version 4.05 (DNASTAR, Inc., Madison, WI, USA) was used to evaluate ITS 1F:ITS 4B sequences of 22 reference Amanita taxa in order to determine restriction endonucleases that would allow for easy identification. Alu I and Fnu 4Hl were selected. PCR products of both parasitized and non- parasitized Amanita specimens were separately digested for 2 - 4 h at 37 C with each restriction endonuclease. The reaction mixture consisted of 1.0 pl of the appropriate restriction buffer (supplied by the manufacturer), 4.8 pl water, 0.2 pl restriction endonuclease and 4 pl PCR product. The restriction products were run for 45 min on a 2% agarose gel at 70 V on an electrophoresis system (Fisher Scientific, Pittsburgh, PA, USA), stained with ethidium bromide and visualized using Alphalmager Version 3.2 (Alpha lnnotech Corporation, San Leandro, CA, USA). Phylogenetic analysis Sequences were aligned using Clustal W (Thompson, Higgins 8 Gibson 1994), and were further aligned by eye. The ITS region, covering a portion of the 188, all of the ITS1, 5.8S and ITS2, and the beginning of the 28S nuclear ribosomal operons, was analyzed for the parasitized Amanita specimens and the reference taxa in Amanita section Validae. Amanita bisporigera (section 56 Phalloidae) was used as an outgroup. Alignment difficulties precluded selecting an outgroup from outside the genus. The alignment was 951 positions, including gaps. Additionally, 340 bp (113 bp from ITS 1, the entire 588 region, and 65 bp from ITS 2) were analyzed for parasitized specimens plus representatives from each section of Amanita. As we wished to determine probable identity, and not to formulate evolutionary hypotheses, a distance method was chosen for the analyses. Neighbor joining trees based on Kimura 2-parameter distances were generated using PAUP* 4.0b10 (Swofford 2002). 1000 hundred bootstrap replications were run using Kimura 2-parameter distance as the optimality criterion to attain bootstrap values. Bootstrap values are printed beside the branches; values less than 50 are not shown. Results Annealing temperatures between 52°C and 57°C were tested in the PCR reactions. A 52°C annealing temperature resulted in multiple bands for parasitized Amanita specimens due to lowered stringency, while 57°C resulted in less product (a weaker band) than 55°C. An annealing temperature of 55°C was optimal. Primer ITS F, a basidiomycete-specific substitute for ITS 1F, was evaluated but yielded multiple bands upon amplification, even at 57°C, possibly due to internal primer recognition sites. 57 Out of 46 parasitized Amanita specimens from which DNA was extracted, fifteen yielded discernible PCR products when amplified with ITS 1F:ITS 4B. Herbarium specimens approximately 30 years old did not yield PCR products. RFLP patterns (Figs. 9-12) were able to provide positive identifications of three parasitized Amanita specimens. Specimens PA 13 and PA 33 matched Alu l and Fnu 4HI digests of A. novinupta Tulloss 8 J. Lindgren. Specimen PA 43 matched reference material of A. brunnescens Atk. and A. citrina sensu auct. amer., which differed from A. rubescens sensu auct. amer. by the presence of a faint band of approximately 350 bp in the Fnu 4HI digest. Specimen PA 01 was identified as either A. rubescens sensu auct. amer. or A. flavoconia Atk. Specimens PA 03, PA 08, PA 19 and PA 22 did not match any of the reference material in the Fnu 4Hl digest. 58 PA 03 W " PA 19 g 3f PA 33 3‘ g, " PA 22 ‘ PA 43 9.! PA 13 93, PA 01 ‘3 PA 08 (a; 1 KB+ ladder Ell! Amanita rubescens var. congolensis 2 Amanita rubescens var. congolensis 1 Amanita rubescens var. alba Amanita novinupta Amanita fulva Amanita vaginata Amanita muscaria var. guessowii Amanita muscaria “orange" Amanita muscaria var. alba Amanita porphyria Amanita brunnescens Amanita citrina f. lavendula Amanita citrina Amanita subphalloides Amanita flavoconia Amanita sinicoflava Amanita flavoconia Amanita excelsa Amanita novinupta Amanita rubescens 1 kb+ ladder 59 Figure 10. Alu 1 digest of parasitized Amanita specimens. Shapes indicate matches to the reference gel (Fig. 9) Figure 9. Alu 1 digest of reference Amanita specimens. Shapes indicate reference to the parasitized Amanita gel (Fig. 10). 1 kb+ ladder PA 03 PA 19 PA 33 PA 22 PA 43 PA 13 PA 01 PA 08 Amanita rubescens var. congolensis 2 Amanita rubescens var. congolensis 1 Amanita rubescens var. alba Amanita novinupta 1 kb+ ladder Amanita fulva Amanita vaginata Amanita muscaria var. guessowii Amanita muscaria "orange" Amanita muscaria var. alba Amanita porphyria Amanita brunnescens ._ Amanita citrina f. lavendula Amanita citrina Amanita subphalloides Amanita flavoconia Amanita sinicoflava Amanita flavoconia Amanita excelsa Amanita novinupta Amanita rubescens 60 Figure 12. Fnu 4H1 digest of Figure 11. Fnu 4H1 digest of reference Amanita specimens. Stars indicate reference to the parasitized Amanita gel (Fig. 12). parasitized Amanita specimens. Stars indicate matches to the reference gel. (Fig. 11) We were able to obtain sequence for nine parasitized specimens. Based on the neighbor joining trees (Figs. 13-14) specimens PA 13, PA 22 and PA 33 were identified as Amanita novinupta. PA 43 was identified as A. brunnescens. PA 1 was identified as A. aff. flavoconia. These findings supported the tentative identifications of parasitized Amanita specimens made on the basis of the RFLPs. PA 08 and PA 19 grouped with the A. “rubescens” from the study of Oda, Taneka 8 Tsuda (1999), which is most likely A. orsonii Kumar 8 Lakanpal (R. E. Tulloss, pers. comm., 30 July 2002). We were unable to obtain reference RF LP patterns from A. orsonii. The sequence obtained for PA 17 showed 93% homology with members of the family Thelephoraceae over the entire 657 bp sequence when subjected to a BLAST search. The DNA extraction, PCR reaction, purification, cloning and sequencing of PA 17 were repeated, and the same result was obtained. The sequence obtained from PA 03 showed 87-90% sequence homology with Russula and Lactarius species over the 18S, 5.8S and ITS 2 regions and a portion of the ITS 1 region. No significant homology with species in any Section of the genus Amanita was found for PA 03 or 17, which could not be aligned with the other Amanita sequences. 61 Amanita bisporigera 100 —— PA 43 Amanita brunnescens Amanita cf. subphalloides 100 Amanita citrina f. lavendula Amanita porphyria ABO15677 99 78 Amanita citrina var. citrina ABO15679 54 _— Amanita citrina var. grisea ABO15680 manita rubescens var. congolensis A 100 Amanita rubescens var. alba 99 Amanita rubescens var. congolensis Amanita rubescens A801 5682 9 I £[PA 19 PA8 'n 9 Amanita novinupta ISOTYPE 6r- / PA 22 8,, PA33 81 79/ 100M113 Amanita flavoconia (white-stiped form) PA1 Amanita flavoconia 76 99 98 Amanita flavipes ABO15696 0.1 Fig. 13. Neighbor-joining tree of the ITS region of parasitized Amanita specimens and Amanita Section Validae. Numbers at nodes are bootstrap indices of support (%). Branch lengths correspond to genetic distance (expected number of nucleotide substitutions per site). 62 Amanita thiersii Amanita cylindrospora Amanita hemibapha var. hemibapha A801 5699 la 86 80 67 Amanita bisporigera 100 PA 43 Amanita brunnescens 100 Amanita rubescens congolensis Amanita rubescens var. alba 85 Amanita rubescens congolensis Amanita cf. subphalloides g3 Amanita citrina var. grisea 9 A8015680 Amanita porphyria A8015677 Amanita citrina f. lavendula Amanita citrina var. citrina 624' P A 19 A8015679 PA 8 .. Amanita rubescens A801 5682 85 -Amanita novinupta ISOTYPE ' PA 22 PA 33 8%PA 13 Amanita flavoconia (white-stiped form) Amanita flavipes ABO15696 PA1 7 Amanita flavoconia Amanita muscaria 82 0.1 . var. uessowii Amanita fulva g Fig. 14. Neighbor-joining tree of the 5.88 and partial ITS 1 and ITS 2 regions of parasitized Amanita specimens and representative Amanita species from other sections in the genus. Numbers at nodes are bootstrap indices of support (%). Branch lengths correspond to genetic distance (expected number of nucleotide substitutions per site). 63 Discussion RFLPs were sufficient in three of the nine specimens examined to identify the parasitized Amanita. The cases in which RF LPs did not yield an identification were due to lack of the appropriate reference taxon (PA 08 and 19), amplification of basidiomycete contaminants (PA 03 and 17), and insufficient resolution of reference taxa by the restriction endonucleases used (PA 01 and 22). We based our restriction enzyme selection on the cut sites generated on one member of each species. The inability of restriction digests to match PA 01 conclusively with A. flavoconia and PA 22 with A. novinupta shows that restriction endonuclease choice was suboptimal. Karen and colleagues (1997) observed sufficient intraspecific variation in Mbo I digests of ITS PCR products to enable them to accurately identify 27 species of ectomycorrhizal fungi, including several Amanita species. Expanding the collection of restriction digests of reference taxa, and possibly adding Mbo l digests, would improve the chances of accurately matching a parasitized specimen to a known species. Sequencing of the ITS region is more time-consuming than RFLP analyses, but provides superior resolution. When possible, we would recommend sequencing over RFLPs for identifying unknown fungi, due to the possibility of multiple taxa sharing RFLP banding patterns. A. rubescens (Pers.:Fr.) Pers. is an Old World taxon and is not known from North America (Tulloss 8 Lindgren 1994). A. novinupta and A. orsonii are rubescent taxa phenetically closely allied to A. rubescens (Pers.:F r.) Pers. Both 64 taxa have been called A. rubescens by western North American authors and southern and eastern Asian authors, respectively. The identification of five parasitized Amanita specimens as A. novinupta and A. orsonii is therefore in agreement with North American reports of A. rubescens sensu lato as a primary host for H. hyalinus. A. rubescens is an Old World taxon, and is not known from North America or southern Asia (Tulloss 8 Lindgren 1994; Tulloss et al. 2001). On the other hand, the groupings with A. orsonii and A. novinupta suggest that there is insufficient resolution provided between taxa, since neither taxon is known to occur in Michigan based on current literature. Singer (1986) placed A. brunnescens in Amanita section Mappae, separate from section Validae. However, Singer’s distinction was based on the presence of bufotenine in A. citrina and A. brunnescens. Recent phylogenetic analyses (WeiB, Yang 8 Oberwinkler 1998; Drehmel, Moncalvo 8 Vilgalys 1999; Oda, Tanaka 8 Tsuda 1999) suggest that this chemotaxonomic character is insufficient to merit the separation of Mappae and Validae. Bufotenine is a hallucinogenic compound frequently found on the skin of toads. While bufotenine is not active in humans when ingested (Benjamin 1995), A. brunnescens is considered mildly poisonous and should not be eaten. The fact that A. brunnescens can be parasitized by H. hyalinus provides reason for warning that parasitized Amanitas should not be used as food. Clearly, several species of Amanita are being parasitized by Hypomyces hyalinus. All parasitized species identified to date have been members of section Validae, if they could be identified as Amanita species, but the small sample size 65 does not preclude the possibility of other sections being susceptible. While we did not confirm reports based on morphology and proximity of A. bisporigera as a host (Lincoff 1981), Section Phalloidae is the sister group to section Validae (WeilS, Yang 8 Obenrvinkler 1998; Drehmel, Moncalvo 8 Vilgalys 1999; Oda, Tanaka 8 Tsuda 1999), and, as such, might be susceptible to parasitism. PA 03, from which Russulaceae sequence was obtained, and PA 17, from which Thelephoraceae sequence was obtained, merit discussion. Members of the genera Russula and Lactarius are parasitized by Hypomyces Iactifluorum (Schweinitz:Fr.) Tulasne. The Hypomyces entirely covers the Russula or Lactarius fruiting body, producing a sterile, distorted mushroom. However, the morphology of Russulaceae infected by H. lactiflorum differs significantly from that of Amanita infected by H. hyalinus. An infected Amanita is club-shaped, with minimal expansion of the pileus, while infected Russulaceae show a broad, funnel-shaped pileus with folds visible where the lamellae were initiated. H. hyalinus is pink to white and does not discolor in KOH, while H. lactifluorum is bright orange-red and immediately stains purple in 4% KOH (Rogerson 8 Samuels 1996). H. Iactifluorum produces equally two-celled ascospores with prominent apices, while the ascospores of H. hyalinus are divided into two unequal cells and the apices are not prominent. These characters have enabled us to rule out the possibility of misidentification of the Hypomyces species. The parasite of PA 03 was consistent with Hyopmyces hyalinus on the bases of ascospore characters and KOH reaction, Fruiting bodies of the Russulaceae possess characteristic swollen cells (sphaerocysts), which are visible in infected 66 specimens, while Amanita lacks sphaerocysts. We found no sphaerocysts in PA 03. Therefore, the fruiting body is not one of a Russula and the parasite is not one known to infect the Russulaceae. Members of the Russulaceae and Thelephoraceae are ectomycorrhizal, as are Amanita species. 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DNA extraction technique for PCR in rain forest plant species. BioTechniques 20: 974-978. Simard SW, DA Perry, MD Jones, DD Myrold, DM Durall, R Molina. 1997. Net transfer of carbon between ectomycorrhizal tree species in the field. Nature 338:579-582. Singer R. 1986. The Agaricales in Modern Taxonomy 4th ed. Koenigstein, Koeltz Scientific Books. 981 pp. Swofford DL. 2002. PAUP*: Phylogenetic Analysis Using Parsimony (and other methods) version 4.0b10. Sunderland, MA, Sinauer Associates. Thompson JD, DG Higgins, TJ Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. Tulloss RE. 1998. Seminar on Amanita. 4th edn. San Francisco, CA, USA: North American Mycological Association and Mycological Society of San Francisco. vi + 186 pp. Tulloss RE, JE Lindgren. 1994. Amanita novinupta - a rubescent, white species from the Western United States and Southwestern Canada. Mycotaxon 51: 179- 190. Tulloss RE, SH Iqbal, AN Khalid, RP Bhatt, VK Bhatt. 2001. Studies in Amanita (Amanitaceae) from southern Asia. I. Some species of Pakistan's Northwest Frontier Province. Mycotaxon 77: 455-490. 69 Weill M, Z—L Yang, F Oberwinkler. 1998. Molecular phylogenetic studies in the genus Amanita. Canadian Journal of Botany 76: 1170-1179. White TJ, T Bruns, S Lee, J Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: MA Innis, DH Gelfand, JJ Sninsky & TJ White, eds. PCR Protocols: A Guide to Methods and Applications. San Diego, CA, Academic Press, Inc. pp. 315-322. 70 CHAPTER 3 Hallen HE, GC Adams, A Eicker. 2002. Amatoxins and phallotoxins in indigenous and introduced South African Amanita species. South African Journal of Botany 68: 1-5. 71 AMATOXINS AND PHALLOTOXINS IN INDIGENOUS AND INTRODUCED SOUTH AFRICAN AMANITA SPECIES Heather E. Hallen*, Gerard C. Adams1 and Albert Eicker?- *Michigan State University, Department of Plant Biology, East Lansing, MI 48824-1312 USA 1Michigan State University, Department of Plant Pathology, East Lansing, MI 48824-1312 USA 2University of Pretoria, Department of Botany, Pretoria 0002, South Africa *To whom correspondence should be addressed, (E-mail: hallenhe@msu.edu) 72 Abstract The production of lethal amatoxins and phallotoxins in species of Amanita from South Africa was investigated by HPLC analyses. The indigenous mushrooms Amanita foetidissima and A. pleropus tested negative for production of these toxins. Several introduced species were analyzed; of these, A. phalloides var. phalloides, A. phalloides var. alba and A. reidii contained amatoxins and phallotoxins. Despite reports of rapid degradation of phallotoxins upon drying, phallotoxins and amatoxins were both readily detectable in dried herbarium specimens up to 17 years old. Previous reports of phallotoxins in A. rubescens were not substantiated. 73 Introduction Members of the genus Amanita Pers., with their characteristic white spores, free gills, and the presence of both a universal and a partial veil, are among the most readily recognized fleshy fungi. This genus has been a subject of intensive study over the past century (Corner & Bas 1962; Bas 1969; Jenkins, 1977; Wieland 1986; Reid & Eicker 1991; Yang 1997). Several species of Amanita occur in South Africa (Reid & Eicker 1991; van der Westhuizen & Eicker 1994). Many species, such as A. excelsa (Fr.) Kummer, A. muscaria (L.:Fr.) Pers., A. pantherina (DC.:Fr.) Krombh., A. phalloides (Fr.:Fr.) Link and A. rubescens (Pers.:Fr.) Pers., are believed to have been introduced from Europe on trees as mycorrhizal associates (Reid & Eicker 1991). Amanita reidii Eicker & van Greuning was described from a South African specimen but occurs only in association with Eucalyptus species and may have been introduced from Australia. Amanita pleropus (Kalchbr. & MacOwan) Reid and A. foetidissima Reid & Eicker are believed to be indigenous. Amanita is a large genus, with several hundred species (Hawksworth et al. 1995) divided between two subgenera and several sections. Subgenus Lepidella is characterized by the blackening of the spores in iodine (amyloid reaction), while subgenus Amanita has inamyloid spores. Subgenus Lepidella contains sections Amidella, Lepidella, Phalloideae and Validae. Subgenus Amanita contains sections Amanita and Vaginatae. Sections are sensu Corner & Bas 74 (1962), and are further distinguished on the basis of universal and partial veil characters (Corner & Bas, 1962, Jenkins 1986). The genus has been subject to particular scrutiny due to the production of toxins by several species (Wieland 1986). Toxins produced by Amanita species include the central nervous system toxin ibotenic acid, produced by certain species of Amanita section Amanita (e.g., A. muscaria and A. pantherina) and the hallucinogen bufotenine, produced by A. citn'na (Schaeff.) Pers and A. brunnescens Atk. Most important are the two families of cyclic peptide toxins, amatoxins and phallotoxins, that are produced by several species of Amanita section Phalloideae (e.g., A. phalloides, A. virosa Lamarck, A. vema (Bull.:Fr.) Lamarck, and others). Amanita phalloides is known to produce relatively high quantities of a-, B- and y—amatoxins, the phallotoxins phalloidin and phallacidin, and smaller quantities of related chemicals (Wieland 1986). Amatoxins tend to be localized in the lamellae and annulus, while the area of highest phallotoxin concentration is usually the volva (Enjalbert, Bourrier & Andary 1989; Enjalbert, Cassanas & Andary 1989; Enjalbert et al. 1993). In species that produce amatoxins and phallotoxins, both types of toxins are detectable in Iamellar tissue (Enjalbert et al. 1992; Hallen, unpublished results). Phallotoxins have only been reported in the genus Amanita. Amatoxins are found in three other genera: Conocybe Fayod, Galerina Earle and Lepiota (Pers) Gray (Benjamin 1995). Amatoxins and ibotenic acid have both been implicated in fatal human and animal poisonings (Wieland 1986; Benjamin 1995; Naudé & Berry 1997). The isoxazole toxins, ibotenic acid and its metabolite muscimol, will rarely kill an 75 adult; most fatal outcomes are in child or animal poisonings (Benjamin 1995, Naudé & Berry 1997). Amatoxins are frequently lethal, and are responsible for 90% of fatal human mushroom poisonings worldwide (Benjamin 1995). Amatoxins are potent inhibitors of RNA polymerase II (RNA polymerase B), indirectly halting protein synthesis (Wieland 1986). The human LD50 is 0.1 mg kg'1 body weight. This is approximately 7 mg toxin for an adult male, or approximately 1 cm3 of tissue from A. phalloides. Phallotoxins are structurally similar to amatoxins and are hypothesized to share a common biosynthetic pathway. Phallotoxins have not been implicated in human poisonings because they are not absorbed from the gastrointestinal tract (Benjamin, 1995). In this study, high-performance liquid chromatography (HPLC) has been used to evaluate a number of mushrooms from South Africa for presence of two amatoxins, a- and B-amanitin, and two phallotoxins, phalloidin and phallacidin. We utilized an HPLC protocol that has been proven sensitive enough to detect toxins in nanogram quantities (Enjalbert et al. 1992). We further confirmed the presence of the toxins by mass spectrometry. While detailed toxicological studies of many of the northern hemisphere species of Amanita have been conducted (Malak 1974; Beutler 1980; Wieland 1986), this is the first report of evaluations of endemic and introduced species collected in South Africa. Materials and methods The specimens evaluated are detailed in Table 4. 76 Table 4: Analysis of amatoxins and phallotoxins in Amanita species. Taxon Section Provenance Year collected Accession #a orb [3b Cb Hb A. "capensis"°ve Unknown Mpumalanga I992 PRU 3356 - - - - A. excelsa Validae Belfast, Mpumalanga I998 MSC 375639 - - - - A. excelsad Validae Belfast. Mpumalanga 1999 MSC 375640 - - - - A. foetidissima Lepidella Pretoria I992 PRU 3505 - - - - A. foetidissima Lepidella Pretoria I993 PRU 3498 - - - - A. foetidissima Lepidella Pretoria 1994 PRU 4168 - - - - A. muscaria Amanita Pretoria I998 MSC 377980 - - - - A. nauseosa Lepidella LC deVilIiers sports I989 PRU 2703 - - - - ground A. pantherina Amanita Pretoria I991 PRU 3 I 56 - - - - A. pantherina Amanita Mpumalanga I993 PRU 3667 - - - - A. pantherina Amanita Sabie, Mpumalanga I998 MSC 37564] - - - - A. pantherina Amanita Pretoria I999 MSC 375642 - - - - A. pantherina Amanita Belfast, Mpumalanga I999 MSC 375643 - - - - A. phalloides var. Phalloidae Pretoria I994 PRU 3959 + + + + phalloides A. phalloides var. Phalloidae unknown I994 PRU 4258 + + + + phalloides A. phalloides var. Phalloidae Pretoria I998 MSC 375644 + + - + phalloidesd A. phalloides var. Phalloidae Saasveld, George I983 PRE 47293 + + + + phalloides Cape A. phalloides var. Phalloidae Bergvliet State I985 PRE 48659 + + + + alba Forest, Sabie A. phalloides f. Phalloidae Bergvliet State 1985 PRE 48654 + + + + umbrina Forest, Sabie A. phalloides f. Phalloidae Bergvliet State I984 PRE 48618 + + + + umbrinadae Forest, Sabie A. pleropusd Lepidella Brummesia National I984 PRE 47480 - - - - Research Institute Gardens, Pretoria A. reidiid’e Phalloidae Hide-away, I990 PRU 4306 + + + + Melkrivier, Northern Province A. rubescens Validae Belfast, Mpumalanga I998 MSC 375645 - - - _ A. rubescens Validae. Belfast, Mpumalanga I998 MSC 375646 — - - - A. rubescensd Validae Belfast, Mpumalanga 1999 MSC 375647 - - - _ A. rubescens Validae Pretoria 1999 MSC 375930 - - - - A. speciese Lepidella Lynnwood Glen I993 PRU 36] I - - - - Nature Reserve A. speciese Unknown Darow, Cape I996 PRU 4149 _ _ _ _ Province 77 Table 4. Analysis of amatoxins and phallotoxins in Amanita species. aMSC = Beal-Darlington Herbarium, Michigan State University, East Lansing, MI, USA 48824-1312. PRU = H. G. W. J. Schweickerdt Herbarium, Botany Department, University of Pretoria, Pretoria 0002, Gauteng Province, South Africa. PRE = National Herbarium, National Botanical Institute, Private Bag X101, Pretoria 0001, Gauteng Province, South Africa. Pa = a—amanitin; [3 = B-amanitin; C = phallacidin; H = phalloidin. — indicates no toxin was detectable; + indicates that toxin was detected. cAmanita capensis lacks a type specimen and has never been validly published, so the identification and taxonomic affinities of this taxon are uncertain. Quotation marks are added to indicate its uncertain affinities. dMultiple specimens from this collection were assayed, results were the same for all specimens. eSpecimen is being further examined by DNA sequence analysis for species determination (Hallen, unpublished results). 78 Fungi were evaluated for toxins using a modification of the method of Enjalbert et al. (1992). Dried specimens were rehydrated in KOH, then rinsed thoroughly with distilled water. Excess water was blotted from the specimens and specimens were then diced and weighed. Eight to 200 mg of the tissues were suspended in 1.5 ml extraction medium containing methanolzdistilled water:0.01M HCI (5:421) 9‘1 tissue, Suspended tissues were incubated at 4°C for 12 h. Methanol was HPLC grade (J.T. Baker, Phillipsburg, New Jersey, USA). Samples were then centrifuged at 1000 x g and 4°C for 10 min, and the supernatant was collected. The pellets were resuspended in 0.6 ml extraction medium 9'1 tissue, incubated at 4°C for an additional 12 h and centrifuged. The supernatants from the first and second centrifugation were pooled. Extractions were from Iamellar tissue for all samples except A. rubescens. Both the lamellae and the volva were used in A. rubescens to facilitate testing for phallotoxins which have been reported in this species (Malak, 1974). HPLC analysis of amatoxins and phallotoxins was performed on a Model 114 HPLC apparatus (Beckman Instruments, Inc., Fullerton, California, USA) with detection at 295 nm. Amatoxins and phallotoxins were separated using a 0711-0231 C-18 column (Perkin-Elmer Corporation, Norwalk, Connecticut, USA) and a 30 min gradient of solution A to solution B. Solution A was 0.2 M ammonium acetate, adjusted to pH 5 with glacial acetic acid, and solution B was acetonitrile. Flow rate was 1 ml min-1. Samples were maintained at a temperature of 4°C until injection. Twenty pl of each sample were injected. 79 Standards were purified a-amanitin, B-amanitin, phallacidin and phalloidin (Sigma Chemical, St. Louis, Missouri, USA). Each toxin was at a concentration of 100 pg ml'I, which is comparable to the concentration of toxins naturally occurring in A. phalloides (Enjalbert et al., 1992). Peaks eluted at approximately 70 - 80 °/o acetonitrile (Figure 21). Putative toxin peaks were identified by comparison with the toxin standards, and eluted fractions were manually collected from the HPLC apparatus. Eluted fractions were subjected to fast atom bombardment (FAB) mass spectrometry, at the Mass Spectrometry Facility at Michigan State University, to confirm identity. FAB mass spectra were obtained using a model HX-110 double-focusing mass spectrometer (JEOL USA, Peabody, Massachusetts, USA) operating in the positive ion mode. Ions were produced by bombardment with a beam of xenon atoms (6 kV). The accelerating voltage was 10 kV and the resolution was set at 1000. The instrument scanned from m/z (mass to charge ratio) 50 to 1500. Results and Discussion The results of the analyses are shown in Table 4. It was found that only species in Amanita section Phalloideae showed presence of amatoxins or phallotoxins. These species included A. reidii, A. phalloides var. phalloides, A. phalloides var. alba Gillet (= A. phalloides f. alba Britzelm), and “A. phalloides f. umbrina” (use of quotation marks is explained below). Each of these species showed HPLC peaks that agreed with the standards of a- and B-amanitin, 80 % Acetonltrlle 8 I \ \ 1 30l4 20'— 0 10 15 20 25 Time in Minutes Figure 15. HPLC results for Amanita phalloides f. umbrina (= A. reidii) PREM 48654. The dashed line indicates the percent acetonitrile. Solid line shows absorbance at 295 nm. Peak #1 represents B-amanitin, 3 a-amanitin, 5 phallacidin and 6 phalloidin. Peak 4 is likely y-amanitin; this could not be confirmed due to the lack of a y-amanitin standard. phalloidin and phallacidin. Other amatoxins and phallotoxins, for which standards were not available, may have been present. Peaks identified by HPLC (Figure 15) were confirmed by FAB mass spectroscopy of the eluted fractions in A. reidii and “A. phalloides f. umbn’na”. The duration between collection of the mushrooms and HPLC analysis ranged from less than one month to 17 years. Despite reports of rapid degradation of phallotoxins upon drying (Stijve & Seeger 1979; Klein & Baudisova 1993), both phallotoxins and amatoxins were readily detectable in dried 81 herbarium specimens of “A. phalloides f. umbrina” up to 17 years old. We have detected both toxins in Amanita species up to 21 years old, but there is a diminution in peak strength with increasing sample age (Hallen, unpublished results). Apparently, following a sharp decrease in the concentration of the heat- labile phallotoxins during drying, there is little degradation over time of the remaining phallotoxins. The distribution of amatoxins in mushrooms has long been a subject of controversy. Faulstich and Cochet-Meilhac (1976) reported the presence of trace quantities of amatoxins in all mushroom species tested, including the common edible species Agaricus bisporus (J. E. Lange) Pilat using radioimmunoassay (RIA). Preston et al. (1982) also detected trace quantities of amatoxins in edible mushrooms, using in vitro inhibition of RNA polymerase II activity. Collectively, these findings were taken to indicate that all basidiomycetes produce amatoxins. This was rapidly promulgated through the literature (e.g., Wieland & Faulstich 1978; Horgen et al. 1978) but these findings were later refuted (Enjalbert et al. 1993) because of methodological considerations. The levels of toxin detected by Faulstich & Cochet-Meilhac were at the limits of detection for the RIA procedure. These levels could be accounted for by contamination. When Faulstich repeated the assay in a different laboratory using new glassware, no toxins were detected in edible fungi (Wieland 1986). Similarly, Preston and colleagues based their evaluations solely on inhibition of calf thymus RNA polymerase II, without any further assays. The levels of putative toxin detected in nontoxic species, including Amanita species such as A. brunnescens, 82 were near the limits of detection for this methodology. No toxins have been detected in these species following extensive testing using more sensitive HPLC procedures (Enjalbert et al. 1992, 1993; Hallen, unpublished results). The edible species A. mbescens did not contain detectable toxins in our studies. Neither phallotoxins nor amatoxins were detected in either lamellar or volval preparations of A. rubescens. This contradicts the report of the detection of phallotoxins in A. rubescens using thin layer chromatography (Malak, 1974). The edible A. excelsa, and the poisonous A. pantherina and A. muscaria, contained no detectable amatoxins or phallotoxins using our analytical techniques. However, our methods do not detect other toxins, notably ibotenic acid or muscimol. Neither amatoxins nor phallotoxins were detected in the indigenous species A. foetidissima, A. pleropus, the species of uncertain identity A. “capensis”, or the unidentified species PRU 3611 and PRU 4149. Tests for other fungal toxins need to be performed, and more specimens need to be examined, before these species are considered safe to eat. Despite the fact that A. phalloides f. umbrina was originally used to describe aged specimens of A. phalloides (Ferry, 1911), the name came into colloquial use in South Africa in referring to the streaked, gray-brown mushroom now known as A. reidii (Eicker, van Greuning & Reid 1993). Thus the colloquial usage (which we have denoted by quotation marks: “A. phalloides f. umbrina”) may be considered synonymous with A. reidii, as in van der Westhuizen & Eicker (1994), while the original sense is not taxonomically valid (Eicker, van Greuning & Reid 1993). Amanita reidii may be synonymous with the Australian A. 83 marmorata ssp. marmorata Cleland & Gilbert and the Hawaiian A. marmorata ssp. myrtacearum O.K. Miller, D. Hemmes & G. Wong (R. Tulloss, personal communication). These taxa are all mycorrhizal on Eucalyptus, and appear to have accompanied their host plant from Australia. Amanita reidii was placed in section Phalloideae based on morphological characters, and has been presumed toxic due to its affinities. This study is the first direct analysis of amatoxins and phallotoxins in this taxon, and confirms the presence of the toxins. Acknowledgements The Foundation for Research Development, Pretoria is thanked for financial assistance to G. C. Adams. Dr. Rodham Tulloss and three anonymous referees are thanked for their evaluations and comments. References Bas C. 1969. Morphology and subdivision of Amanita and a monograph of its section Lepidella. Persoonia 5: 285-579. Benjamin DR. 1995. Mushrooms: Poisons and Panaceas. New York, USA: W.H. Freeman and Company. Beutler JA. 1980. Chemotaxonomy of Amanita: qualitative and quantitative evaluation of isoxazoles, tryptamines, and cyclopeptides as chemical traits. Ph.D. thesis. Philadelphia College of Pharmacy and Science, Pennsylvania, USA. Corner EJH, C Bas. 1962. The genus Amanita in Singapore and Malaya. Persoonia 2: 241-304. Eicker A, JV van Greuning, DA Reid. 1993. Amanita reidii — a new species from South Africa. Mycotaxon 47: 433-437. Enjalbert F, MJ Bourrier, C Andary. 1989. Assay for the main phallotoxins in Amanita phalloides Fr. by direct fluorimetry on thin-layer plates. Joumal of Chromatography 462: 442-447. 84 Enjalbert F, G Cassanas, C Andary. 1989. Variation in amounts of main phallotoxins in Amanita phalloides. Mycologia 81: 266-271. Enjalbert F, C Gallion, F Jehl, H Monteil, H Faulstich. 1992. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography. Journal of Chromatography 598: 227-236. Enjalbert F, C Gallion, F Jehl, H Monteil. 1993. Toxin content, phallotoxin and amatoxin composition of Amanita phalloides tissues. Toxicon 31: 803-807. Faulstich H, M Cochet-Meilhac. 1976. Amatoxins in edible mushrooms. FEBS Letters 64: 73-75. Ferry R. 1911. Etudes sur les Amanites. A. phalloides, A. vema, A. virosa. Revue mycologique. Toulouse. Supplement 1. Hawksworth DL, PM Kirk, BC Sutton, DN Pegler. 1995. Ainsworth & Bisby’s Dictionary of the Fungi 8th edn. Oxon, UK: CAB International. Horgen PA, AC Vaisius, JF Ammirati. 1978. The insensitivity of mushroom nuclear RNA polymerase activity to inhibition by amatoxins. Archives of Microbiology 118: 317-319. Jenkins DT. 1977. A Taxonomic and Nomenclatural Study of the Genus Amanita Section Amanita for North America. Bibliotheca Mycologica Band 57. Stuttgart, Germany : J. Cramer. Jenkins DT. 1986. Amanita of North America. Eureka, CA, USA: Mad River Press. Klan J, D Baudisova. 1993. Toxiny muchomi’irky zelené v susenych plodnicich. Casopsi Lékar'u Ceskych 132: 468-469. Malak SHA. 1974. Chemotaxonomic significance of alkaloids and cyclopeptides in Amanita species. Ph.D. thesis. University of Maine, Orono, USA. Naudé TW, WL Berry. 1997. Suspected poisoning of puppies by the mushroom Amanita pantherina. Journal of the South African Veterinary Association 68: 154- 158. Preston JF, BEC Johnson, M Little, T Romeo, JH Stark, JE Mullersman. 1982. Investigations on the function of amatoxins in Amanita species: A case for amatoxins as potential regulators of transcription. In: H Kleinkauf & H von Gohten (eds) Peptide Antibiotics, Biosynthesis and Functions. Berlin, Germany: Gruyter. pp. 399-426. 85 Reid DA, A Eicker. 1991. South African fungi: the genus Amanita. Mycological Research. 95: 80-95. Stijve T, R Seeger. 1979. Determination of or-, B-,,and y-amanitin by high performance thin-layer chromatography in Amanita phalloides (Vaill. ex Fr.) Secr. from various origin. Zeitschrift fu'r Naturforschung 34: 1133-1138. van der Westhuizen GCA, A Eicker. 1994. Mushrooms of Southern Africa. Cape Town, South Africa: Struik Publishers. Wieland T. 1986. Peptides of Poisonous Amanita Mushrooms. New York, USA: Springer-Verlag. Wieland T, H Faulstich H. 1978. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active compounds of poisonous Amanita mushrooms. Critical Reviews in Biochemistry 5: 185-260. Yang Z-L. 1997. Die Amanita-Arten von Stidwestchina. Bibliotheca Mycologica Band 170. Stuttgart, Germany: J. Cramer. 86 CHAPTER 4 TAXONOMY AND TOXICITY OF CONOCYBE LACTEA AND RELATED SPECIES Abstract Conocybe Iactea was examined as part of a larger study on the distribution of amatoxins and phallotoxins in fungi, and the taxonomic relationships between these fungi. Because amatoxins are present in a congener, C. filaris, the locally abundant C. Iactea was examined using HPLC and mass spectroscopy. Amatoxins were not found, but the related phallotoxins were present in small quantities. C. Iactea was the first fungus outside of the genus Amanita in which phallotoxins have been detected. Despite the presence of a related toxin, C. Iactea was found not to be taxonomically close to C. filaris. Phylogenetic analyses using nuclear ribosomal RNA genes indicated that North American specimens of C. Iactea were conspecific with North American specimens of C. crispa in Conocybe Section Candidae. European C. crispa was a distinct taxon. The implications of Hausknecht’s use of the name Conocybe albipes for these taxa are discussed. Nucleotide data confirmed placement of the sequestrate taxon Gastrocybe lateritia in Section Candidae, but as a distinct taxon. It is hypothesized that the unique sequestrate morphology of G. lateritia may be caused by a bacterial infection. 87 Introduction The genus Conocybe Fayod (Bolbitiaceae, Agaricales), with 70 species, is the largest genus in the Bolbitiaceae (Hawksworth et al. 1995). Conocybe species occur worldwide. A variety of secondary metabolites are produced within the genus. Conocybe cyanopus (Atkinson) Kuhner and C. smithii Watling, like many blue-staining agarics, contain the hallucinogenic compound psilocybin (Benedict et al. 1962; Benedict, Tyler & Watling 1967). The cyclic peptide amatoxins are present in North American collections of C. filaris (Brady et al. 1975), but have not been found in European collections (Benjamin 1995). C. Iactea (Lange) Métrod produces an unidentified nematicidal compound in culture (Hutchison, Madzia & Barron 1995). Conocybe Iactea is a common mushroom on cultivated lawns and meadows in North America (Arora 1986; Bessette, Bessette & Fischer 1997), the UK (Watling 1982), Europe, Asia and northern Africa (Breitenbach & Kranzlin 1995). In the central and northern United States and Canada, C. Iactea fruits between early June and early September (Kauffman 1918; Hallen, pers. obs.) The mushrooms are ephemeral, with the buttons first visible around 8:00 pm. Caps expand during the course of the night, but spores do not normally mature until 9:00 am. the following morning. Spore discharge occurs between 9:00 am. and 11:00 am, by which time the fungi normally appear shriveled and 88 deliquescent to desiccated. Collapse of the fruit bodies usually follows (Hallen, pers. obs.). Conocybe Iactea is distinguished by its conical, white to buff pileus. The spores are smooth, ochre-brown and possess a large germ pore. There are four spores per basidium and distinctive lecythiform marginal cystidia are present. The pileus color, spore type, cystidial type, and deliquescent nature of the fungus are the defining features of Conocybe section Candidae. The Section is composed of C. Iactea, C. crispa (Longyear) Singer, C. crispella (Murrill) Singer and C. subcrispa (Murrill) Singer (Singer 1986). C. crispella and C. subcrispa are small, subtropical taxa of limited distribution and are rarely collected. C. crispa differs from C. Iactea in the possession of two-spored basidia and “crisped", or wavy, lamellae. C. crispa shares the distribution of C. Iactea, but is less common. Ecological characteristics of these species are identical. Some authors consider C. crispa to be a form of C. Iactea (Watling 1982; Breitenbach & Kranzlin 1995). The number of spores per basidium can vary considerably within a single fruiting body of either species (Breitenbach & Kranzlin 1995) and intermediate forms, possessing both crisped and straight lamellae, have been observed (D. Malloch, pers. comm, 10 Feb. 2001). Gastrocybe lateritia Watling is known primarily from northeastern North America (Bessette, Bessette & Fischer 1997). Like C. Iactea and C. crispa, it is a grass-inhabiting species forming ephemeral fruiting bodies between dusk and mid-morning the following day, from June to September. Fruiting bodies possess, on average, four spores per basidium. Watling, in the type description, describes 89 the pileus as “ellipsoid-campanulate or conic, hardly or never expanding, greasy to viscid rapidly becoming reduced to a gelatinous mass," (Watling 1968). The pileus is normally ochre-brown upon maturity due to the coloration of the spores showing through the translucent pilear tissue. In immature specimens pileus color resembles that of C. Iactea. Forcible spore discharge is lacking, due to the gelatinous-deliquescent nature of the pileus (Watling 1968; Weber 1989; Hallen, pers. obs.) The stipe is frequently longer in proportion to the pileus size than that of C. Iactea or C. crispa. At maturity, the fragile, elongate stipe can no longer support the weight of the gelatinous pileus and the mushroom collapses (Weber 1989) G. lateritia shares the ecology of C. Iactea and C. crispa. The habitat, season, fruiting time and duration are identical. The similarities between these taxa are noted by Watling: “It is characteristic of members of the C. Iactea group for the pileus to become tacky and soft when mature and this same group parallels [G. lateritia] in that the pileus is often long and slender, cylindrical and only slightly expanding; the stipe is also white or hyaline," (Watling 1968). Despite these similarities, the genus Gastrocybe was created to accommodate this taxon solely on the basis of its unusual sequestrate nature. Intermediate forms occur between G. lateritia and C. Iactea and, rarely, between G. lateritia and C. crispa. In these forms, the surface of the pileus may remain dry or only slightly tacky while the gills deliquesce before completing development. Alternately, the ordinary G. lateritia morphology complete with fully gelatinous 90 pileus may develop, but the coloration remains white to buff as in Gastrocybe buttons or mature C. Iactea and C. crispa specimens. In this paper, we investigate the relationships between C. Iactea, C. crispa and G. lateritia using sequence data from the ITS1, ITS 2, 5.8 S and a portion of the 28S regions of the nuclear ribosomal RNA operon. These regions have been shown to be of good utility in examining relationships between closely related fungi (White et al. 1990). Representative species from each section of Conocybe, as well as Bolbitius and Agrocybe species, have been sequenced to provide resolution. Additionally, several sequestrate taxa with presumed affinities to the Bolbitiaceae have been sequenced. Because certain specimens of C. filaris produce the potentially deadly amatoxins, HPLC was used to evaluate the locally abundant C. Iactea and allied taxa for both amatoxins and the related phallotoxins as part of a larger study of the distribution of these toxins. Mass spectrometry was used as a complementary procedure. Materials and methods The specimens examined are detailed in Table 5. 91 Table 5. Specimens of Conocybe and related genera examined. Taxon c Year Accession # Locale collected A grocybe praecox Ingham Co.. Michigan 2000 MSC 378486 Agrocybe semiorbicularis Ingham Co., Michigan 2000 MSC 378490 Bolbitius lacteus Ingham Co., Michigan 2000 MSC 378485 Bolbitius Iener I945 MICH: W. H. Long I I 121 Bolbitius variicolor Ingham Co., Michigan 2000 MSC 378488 Bolbitius vilellinus Ingham Co., Michigan 2000 MSC 378484 Conocybe coprophi/a Custer Co., Idaho 1962 MICH: A. H. Smith 6564] , a Ingham Co., Michigan 2000 MSC 37849] C onocybe crispa _ b Ingham Co., Michigan 2001 MSC 378493 C onocybe crispa ‘ " Conocybe crispa Yorkshire. United Kingdom 1961 E: G l 37 Conocybe/Harts Marin Co., California 1998 MSC 378482 Conocybe huijsmannii var. Kepong, Kuala Lumpur, 1992 E: Wat 24446 conica Malaysia Conocybe/0019a Yorkshire, United Kingdom 1990 E: Wat.22175 a Ingham Co.. Michigan 1998 MSC 378481 C onocybe Iactea a.b Ingham Co., Michigan 1999 MSC 378483 C onocybe Iactea " a Ingham Co., Michigan 2000 MSC 378487 C onocybe Iactea " Conocybe Iactea a.b Ingham Co., Michigan 2001 MSC 378492 Conocybe Iactea Benton Co., Oregon 2002 MSC 380513 Conocybe Iactea Lane Co., Oregon 2002 MSC 380514 Conocybe Iactea Laramie Co., Wyoming 2002 MSC 380515 Conocybe Iactea Laramie Co., Wyoming 2002 MSC 380516 Conocybe rickem’i Indian Gap, Great Smokies I952 MICH: Hessler 20421 National Park Conocybe subcrispa Alameda Co., California 1933 MICH: E. Morse Conocybe subnuda Multnoma Co., Oregon 1995 L. L. Norvell 1950623-01 Conocybe tenera Portneuf Co., Quebec, 1967 MICH: R. L. Shaffer 5892 Canada Cyttarophyllum besseyr' Santa Fe Co., New Mexico 1967 MICH: K. A. Harrison 6825 Galeropsis deserrorum Moravia 1930 PR 15418] Gastrocybe "yellow" Sierra Co., California 2001 D. E. Desjardin 7326 , c Rooks Co., Kansas 1896 F H: E. Bartholomew 2239 Gastrocybe deceptive , , c Ithaca, Michigan 1947 MICH: V. Potter 3654 Gastrocybe lateritia Gastrocybe Iateritiaa Ingham Co., Michigan I999 MSC 380542 , , a Ingham Co., Michigan 2000 MSC 380543 Gastrocybe lateritia Gastrocybe lateritia Ingham Co.. Michigan 2001 MSC 378494 92 Table 5, cont. d I 'v ., ' ° 3 Intermediate 1 ngham Co Michigan 2000 MSC 380544 d 0h . ' ' 2 Intermediate 2 Inc am Co , Michigan 000 MSC 380545 , e Ingham Co.. Michigan 2001 MSC 380546 lntennediate 3 ” " Lucien-crispa intermediate Ingham Co.. Michigan 2001 MSC 380547 Weraroa cucullata Sierra Co., California 2001 D. E. Desjardin Specimens examined in this study. All specimens were subject to DNA extraction and sequencing except C. Iactea 1998 and 2000, and G. lateritia 2000. aSpecimen(s) subjected to HPLC analysis. bMore than one specimen was examined. cType material. dInten'nediate form between C. Iactea and G. lateritia in which the cap was dry and white but never expanded. elntermediate form between C. Iactea and G. lateritia in which the cap expanded but was gelatinous and ochre—brown at maturity. HPLC and mass spectrometry Fungi were evaluated for the presence of toxins using a modification of the method of Enjalbert et al. (1992). Evaluations were performed only upon fresh specimens, as phallotoxins degrade considerably upon drying (Klan & Baudisova 1993). The toxins were extracted from 0.3 to 1.0 mg of tissue from the lamellae and pileus using 1.5 ml extraction medium containing methanolzdistilled water:0.01M HCI (5:421) 9'1 tissue. Methanol was HPLC grade (J.T. Baker, Phillipsburg, New Jersey, USA). Suspended tissues were incubated at 4°C for 12 h. Samples were then centrifuged at 1000 x g and 4°C for 10 min, and the supernatant was collected. The pellets were resuspended in 0.6 ml extraction 93 medium 9'1 tissue, incubated at 4°C for an additional 12 h and centrifuged. The supernatants from the first and second centrifugation were pooled. HPLC analysis of amatoxins and phallotoxins was performed on a Model 114 HPLC apparatus (Beckman Instruments, Inc., Fullerton, California, USA) with detection at 295 nm. Amatoxins and phallotoxins were separated using a reverse-phase C-18 column (Aquapore OD-300, 7pm, 200x46 mm; Perkin-Elmer Corporation, Norwalk, Connecticut, USA) and a 30 min gradient of solution A to solution B. Solution A was 0.2 M ammonium acetate, adjusted to pH 5 with glacial acetic acid, and solution B was acetonitrile. Flow rate was 1 ml min-1. Samples were maintained at a temperature of 4°C until injection. 20 - 200 pl of each sample were injected. Standards were purified a-amanitin, B-amanitin, phallacidin and phalloidin (Sigma Chemical Company, St. Louis, Missouri, USA), each used at a concentration of 100 pg ml'I. Amanita bisporigera Atk. in Lewis samples of known toxicity were run as additional controls. Peaks eluted at approximately 70 - 80 % acetonitrile. Putative toxin peaks were identified by comparison with the toxin standards, and eluted fractions were manually collected from the HPLC apparatus. Eluted fractions were subjected to fast atom bombardment (FAB) mass spectrometry, at the Mass Spectrometry Facility at Michigan State University, to confirm identity. FAB mass spectra of C. Iactea and a phallotoxin standard were obtained using a model HX-110 double- focusing mass spectrometer (JEOL USA, Peabody, Massachusetts, USA) operating in the positive ion mode. Ions were produced by bombardment with a beam of xenon atoms (6 kV). The accelerating voltage was 10 kV and the 94 resolution was set at 1000. The instrument scanned from m/z (mass to charge ratio) 50 to 1500. DNA extraction, amplification and sequencing DNA was extracted from gill and pileus tissue from dried basidiocarps (10 mg). Fungal tissue was placed in a microfuge tube, frozen in liquid nitrogen and macerated. One ml cetyltrimethylammonium bromide (CTAB) mixture (5% w/v CTAB, 1.4 M NaCl, 20 mM EDTA pH 8.0, 100 mM Tris-HCI pH 8.0, 1% w/v polyvinylpyrrolidone (PVP-360)) and 2 pl B-mercaptoethanol were then added. The tube was incubated at 65°C for 1 h. The mixture was extracted once with phenolzchloroform:isoamyl alcohol (24:24:1) and once with chloroform and centrifuged to remove solids. The water-soluble fraction was precipitated with two volumes absolute ethanol and centrifugation, followed by a rinse with 70% ethanol and a second centrifugation. The precipitate was air-dried under vacuum then resuspended in 50 pl water. Approximately 1-20 ng of the total genomic DNA was used per 25 pl reaction mixture for polymerase chain reaction (PCR) amplification. Various brands of prepackaged buffers and polymerases were used for PCR amplification. Primer combinations used were ITS 1F:ITS 4B (Gardes & Bruns 1993) and ITS F (Glen et al. 2001):ITS 4 (White et al. 1990) for the lTS1/5.BSIITSZ region; and CTB6 (Bruns & Li; cited in Hughey et al. 2000):TW13 (White et al. 1990) for the nuclear large subunit (288) ribosomal DNA. 95 The cycling reactions were performed in a DNA thermal cycler (Perkin- Elmer) following Tank 8. Sang (2001). Alternately, a 60°C to 45°C touchdown protocol was used on some templates that did not amplify with the Tank & Sang protocol. The amplification ended with an additional 10 min extension at 72°C, and storage at 4°C. PCR amplification products were separated, and purified following Hughey et al. (2000). Alternatively, products were purified using TOPO® TA (Invitrogen, Carlsbad, CA, USA) or pGEM® (Promega, Madison, WI, USA) cloning kits. Sequencing was performed by the Michigan State University Genomics Technology Support Facility, using dye terminated capillary electrophoresis on an ABI Prism® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Phylogenetic analysis Sequences were aligned using Clustal W (Thompson, Higgins & Gibson 1994), and were further aligned by eye. The ITS region covered a portion of the 188, all of the lTSt, 5.88 and ITSZ, and the beginning of the 28S nuclear ribosomal operons. The alignment was 930 positions, including gaps. Gaps were coded following Simmons and Ochoterena (2000). The alignment for the 288 region was 537 bases. The ITS and 288 regions were analyzed independently and in combination. A total of 141 positions where alignment was ambiguous were excluded from analyses of the ITS and combined datasets. 96 Phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford 2002). Weraroa cucul/ata (Seaver & Shope) Thiers and Watling was used as an outgroup. Maximum parsimony was used with a heuristic search algorithm. A consensus tree was built from all equally parsimonious trees. Three hundred bootstrap replications (maxtrees=300) were run to attain bootstrap values. Bootstrap values are printed above the branches; values less than 50 are not shown. Modeltest 3.06 (Posada & Crandall 1998) was used to determine likelihood settings, which were used to run maximum likelihood analyses in PAUP*. HKY+G was selected as the optimum likelihood model for all datasets. Parsimony analyses of the combined dataset were run in which 1) C. Iactea was constrained to be monophyletic, 2) C. crispa was constrained to monophyly, and 3) Gastrocybe and Galeropsis were constrained to monophyly. Culture Wedges of lamellar tissue from C. Iactea, C. crispa and G. lateritia were surface sterilized with 70 % ethanol and were placed on PDA with 10 ppm benomyl and 200 ppm streptomycin. G. lateritia samples were additionally grown on PDA with 10 ppm benomyl, 500 ppm streptomycin and 200 ppm tetracycline. A bacterium was present on all G. lateritia samples and was identified by the Michigan State University Plant Diagnostic Clinic using BioLog (BioLog, Hayward, CA, USA). 97 RESULTS HPLC and mass spectrometry Eighteen fresh fruiting bodies of C. Iactea were subjected to HPLC analysis. Putative phalloidin peaks were identified in 11 C. Iactea samples. The identity of these peaks was confirmed by mass spectrometry (Fig. 16). Based on comparison with the toxin standard, the quantity of phallotoxins present in C. Iactea was estimated at approximately 3 ng per 9, less than one tenth the concentration found in Amanita bisporigera. No traces of amatoxins were found in any of the C. Iactea fruiting bodies analyzed, while both amatoxins and phallotoxins were readily detectable in Amanita bisporigera samples used as controls. Three fresh fruiting bodies of C. crispa and five of G. lateritia were also analyzed using HPLC. In none of these cases was any phallotoxin detectable. Phylogenetic analyses North American samples of C. Iactea and C. crispa formed one clade in all phylogenetic analyses (Fig. 17-22), with European C. crispa segregating as a distinct taxon outside of Section Candidae. The European C. Iactea was indistinguishable from North American specimens on the basis of the partial 28 S sequence, but possessed two distinct ITS sequences, both placing the European 98 taxon within Section Candidae, but separate from North American C. Iactea specimens. When North American C. Iactea was constrained to monophyly the C. Iactea clade nested within a paraphyletic C. crispa. Likewise, a monophyletic C. crispa nested within a paraphyletic C. Iactea. Inclusion of the European C. crispa specimen in a monophyletic C. crispa clade resulted in a tree 71 steps longer than the most parsimonious trees. Gastrocybe lateritia formed a sister group to the C. Iactea/C. crispa clade and clearly belongs in Conocybe Section Candidae. G. deceptive and an unidentified Gastrocybe-like fungus from the Sierra Nevadas (Gastrocybe “yellow”) are placed in the same clade as Bolbitius species. Constraining G. lateritia to monophyly with Galeropsis desertorum, in keeping with Moreno et al.’s (1989) placement of Gastrocybe in Galeropsis, resulted in a tree 40 steps longer than the most parsimonious trees. Bacterial identification Bacteria were isolated from G. lateritia whenever attempts were made to culture the latter. One type that was consistently present proved to be resistant to 500 ppm streptomycin and 200 ppm tetracycline. Metabolic testing (BioLog) was used to identify this type as a member of the Chryseobacterium gleum/rndologenes group. (Text resumes on page 107) 99 .6663 6980200 m .2357. c_o_o__m;n_ < 2026032 .8368 + 522223 95 SSE + £22.93 u «.212 ncm +1.2 .xEmE _ocoo_m 32099:: u 5.65. .86QO mmmE mam .2. 0.59". N\E mzm .mzm mam gum mma am mm“ 8mm mum on“ man am“ F.»i H» _iiLLFL eriL r_.~ .EL.»»—>»cr.i. »Hitiir : Eggs, . mm mm“ , w m.® . \ \ .V mm ®_m mm.mmm _k mm + 7L <4 +;L<4 rm%_ km.mmm 5222 m N\E mam ®_m mam saw mm“ 8mm mmm 8mm man an“ mam ....—I..»i-.._t..._...t»t.r.e...E. t_ L I_.r._. 822/? 225 £32, . . ,m®.emm . r m.® 7 m®._zm q®.mmm em.mmm ..®.z mz+_>_ +I_>_ x522 < 100 Weraroa cucullata 100l— Agrocybe praecox Cyttarophyllum besseyi 9 Agrocybe semiorbicularie Galeropsis desertorum J’EConocybe coprophila Con. filaris Con. subnuda A astrocybe “yellow” Gastrocybe deceptive TYI 1008 Bolbitius variicolor Bolbitius vitellinus 9 Bolbitius Iacteus 100 10°J'Con. rickenii Con. crispa UK Con. Iactea UK clone 2 Con. Iactea 2 Con. Iactea 1 74 Con. Iactea 4 ‘ Con. crispa 5 Con. Iactea 5 Con. crispa 2 10° Lactea-crispa intermediat 3 Con. crispa 1 53 Con. Iactea Corvallis Con. huijsmannii conica 37 Con. crispa 4 {Con crispa 3 6 Con. Iactea 3 Con. Iactea Eugene IQI gar—Con. subcrispa Con. Iactea UK clone 5 3%; Intermediate 3 5 Intermediate 2 Gastrocybe lateritia 4 51 Gastrocybe lateritia 3 1 00 Gastrocybe lateritia 5 Gastrocybe lateritia 2 L31]: lnterrnediate 1 Gastrocybe lateritia 1 Fig. 17. Consensus based on 300 equally parsimonious trees (length 1090) of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA subunit. Numbers at nodes are bootstrap indices of support (%). 101 Weraroa cucullata '—Agrocybe praecox LE:- Cyttarophyllum besseyi Agrocybe semiorbicularis Galeropsis desertorum Conocybe filaris Conocybe coprophila Conocybe subnuda Bolbitius variicolor FE-Bolbitius vitellinus Bolbitius lacteus maybe deceptiva astrocybe “yellow” I: Conocybe rickenii Conocybe crispa UK Conocybe Iactea UK clone 2 Conocybe Iactea 4 Conocybe crispa 2 Lactea-crr'spa intermediate Conocybe crispa 1 Conocybe Iactea 2 Conocybe huijsmannii conica Conocybe Iactea 1 Conocybe crispa 5 Conocybe Iactea 5 Conocybe crispa 4 Conocybe crispa 3 Conocybe Iactea Corvallis Conocybe Iactea 3 Conocybe Iactea Eugene Conocybe subcrispa lnterrnediate 2 Intermediate 3 Conocybe Iactea UK clone 5 Gastrocybe lateritia 3 Gastrocybe lateritia 4 Gastrocybe lateritia 5 Gastrocybe lateritia 2 Intermediate 1 0.1 Gastrocybe lateritia 1 Fig. 18. Cladogram of the maximum likelihood analysis of the ITS 1, 5.88 and ITS 2 regions of the nuclear ribosomal DNA subunit. One of three trees with In likelihood = -5938.207. Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). 102 Weraroa cucullata Galeropsis desertorum Conocybe subnuda Conocybe filaris Bolbitius vitellinus Bolbitius lacteus Gastrocybe “yellow” Bolbitius variicolor Gastrocybe lateritia TYPE Intermediate 1 Gastrocybe lateritia 5 Gastrocybe lateritia 3 Gastrocybe lateritia 2 Gastrocybe lateritia 4 Bolbitius tener Conocybe Iactea UK Conocybe Iactea 5 Conocybe Iactea 3 Conocybe Iactea 2 Conocybe Iactea Cheyenne Conocybe Iactea Corvallis Conocybe Iactea Lactea-crispa intermediate Conocybe crispa 1 Conocybe crispa 2 Conocybe crispa 3 Conocybe crispa 4 Conocybe crispa 5 Conocybe subcrispa Intermediate 3 lntennediate 2 Conocybe Iactea Eugene Conocybe huijsmanii conica Conocybe rickenii Conocybe crispa UK Agrocybe praecox A grocybe seimorbicularis Cyttarophyllum besseyii Fig. 19. Consensus based on 70 equally parsimonious trees (length 171) of the partial 288 ribosomal DNA subunit. Numbers at nodes are bootstrap indices of support (%). 103 I . Weraroa cucullata __J A -r, Galeropsis desertorum Conocybe subnuda Conocybe filaris Bolbitius vitellinus Bolbitius lacteus Gastrocybe “yellow” olbitius variicolor Gastrocybe lateritia TYPE Intermediate 1 Gastrocybe lateritia 5 Gastrocybe lateritia 3 Gastrocybe lateritia 2 Gastrocybe lateritia 4 Bolbitius tener Conocybe Iactea UK Conocybe Iactea Cheyenne lnterrnediate 3 'i Intermediate 2 r-Conocybe Iactea 5 Conocybe Iactea 3 Conocybe Iactea 2 _ Conocybe Iactea Corvallis Lactea-crispa intermediate Conocybe crispa 1 H Conocybe crispa 2 _Conocybe crispa 3 Conocybe crispa 4 - Conocybe crispa 5 _‘—— Conocybe Iactea Eugene Conocybe _LC_onocybe Iactea 1 hurjsmanrr conrt Conocybe subcrispa Conocybe rickenii Conocybe crispa UK grocybe praecox A grocybe seimorbicularis Cyttarophyllum besseyii Fig. 20. Cladogram of the maximum likelihood analysis of the partial 288 ribosomal DNA subunit. One of two trees with In likelihood = -1833.145. Branch lengths correspond to genetic distance (expected nucleotide substitutions per 104 Weraroa cucullata 1 Agrocybe praecox Cyttarophyllum besseyi 9 A grocybe semiorbicularis 100 68 Galeropsis desertorum 100 Conocybe filaris Conocybe subnuda 99I Gastrocybe “yellow” 100 Bolbitius variicolor 95 Bolbitius vitellinus 9 Bolbitius lacteus 100 l..Conocybe rickenii '— Conocybe crispa UK Conocybe Iactea 2 Conocybe Iactea 1 Conocybe crispa 5 Conocybe Iactea 5 Conocybe crispa 2 100 {actea-crispa intermediate Conocybe crispa 1 Conocybe Iactea 3 Conocybe Iactea Corvallis Conocybe Iactea Eugene 75 Conocybe huijsmannii conica 88 rConocybe crispa 4 iConocybe crispa 3 99 Conocybe subcrispa Intermediate 3 90 Intermediate 2 Gastrocybe lateritia 4 Gastrocybe lateritia 3 19.0. Gastrocybe lateritia 5 lnterrnediate 1 Gastrocybe lateritia 2 Fig. 21. Consensus based on 300 equally parsimonious trees (length 1122) of the combined ITS and partial 288 regions. Numbers at nodes are bootstrap indices of support (%). 105 Weraroa cucullata — Agrocybe praecox _E: Cyttarophyllum besseyii A grocybe semiorbicularis Galeropsis desertorum Conocybe filaris Conocybe subnuda olbitius variicolor astrocybe “yellow” [I Bolbitius vitellinus Bolbitius lacteus I: Conocybe rickenii Conocybe crispa UK ”Conocybe Iactea 5 ’Conocybe Iactea 1 Conocybe crispa 5 Conocybe crispa 2 Conocybe crispa 1 Lactea-crispa intermediate Conocybe Iactea 2 Conocybe Iactea Eugene l:— Conocybe huijsmannii conica l Conocybe crispa 4 Conocybe crispa 3 I:— Conocybe Iactea 3 Conocybe Iactea Corvallis "_ _|:|Conocybe subcrispa Intermediate 3 Intermediate 2 Intermediate 1 Gastrocybe lateritia 3 Gastrocybe lateritia 4 Gastrocybe lateritia 5 0,1 Gastrocybe lateritia 2 Fig. 22. Cladogram of the maximum likelihood analysis of the combined ITS and partial 288 regions. In likelihood = -7263.045. Branch lengths correspond to genetic distance (expected nucleotide substitutions per site). 106 Discussion HPLC and mass spectrometry This is the first report of phallotoxins outside of the genus Amanita, and the first report of phallotoxins consistently being present in the absence of amatoxins. Phallotoxins are destroyed by heat and are not absorbed by the mammalian digestive system (Benjamin 1995), and thus have not been implicated in any human poisonings. However, the L050 of 0.2 mg/kg body weight in mice for phallotoxins given in interperitoneal injection, and the possibility of a shared biosynthetic pathway for phallotoxins and amatoxins, suggest that C. Iactea should not be considered edible, despite reports to the contrary (Bessette, Bessette & Fischer 1997). The inability of our methods to detect phallotoxins in 39% of fresh specimens examined may mean either that phallotoxins were present below the level of detection, or that the toxins were not present in all specimens. Several fungal cyclic peptides are known to occur sporadically. Yocum and Simons (1977) reported no evidence of either amatoxins or phallotoxins in three out of four specimens of Amanita vema examined, a mushroom capable of producing both. Sporadic phallotoxin distribution in Amanita has also been reported by Beutler (1980). No phallotoxins were found in C. crispa or in G. lateritia. The latter is not particularly surprising, as the phylogenetic evidence suggest that G. lateritia is 107 indeed a separate taxon from C. Iactea and C. crispa. The lack of phallotoxins in C. crispa is less readily explained; however, C. crispa is less common than C. Iactea and few samples were examined. Systematics Despite the presence of cyclic peptide toxins in both species, phylogenetic analyses suggest that C. Iactea is not closely related to the amatoxin-producer, C. filaris. No phallotoxin has been detected in C. filaris, and it is probable that the biosynthetic pathways for the different toxins arose separately in the C. filaris and C. Iactea lineages. Hausknecht (1998), working with European material, renames Conocybe Iactea as Conocybe albipes (Otth) Hausknecht, because the basionym Bolbitius albipes Otth 1871 predates Galera Iactea J. E. Lange 1940, on which C. Iactea is based. His analyses are morphological, and are based primarily upon European specimens, although one specimen apiece from Egypt and Mexico, and three from New Zealand, are examined as well. Two differing ITS sequences were obtained from one fruiting body of the European C. Iactea, rendering proper phylogenetic placement of the organism difficult. However, the European taxon was clearly closely related to the North American C. Iactea. Divergent ITS sequences have been reported in other fungi (O’Donnell 1992). C. huijsmanii var. conica Watling, which Hausknecht treats also as C. albipes, fell within the North American C. Iactea-C. crispa clade. We 108 will need to examine further European specimens before a definitive placement is possible. Whether the North American taxon belongs, with the European specimens, in C. albipes var. albipes, or whether it would be better treated as a subspecies, remains unresolved. Molecular phylogenetic analyses were consistently unable to distinguish between C. Iactea and C. crispa, suggesting a very recent time of divergence. Alternatively, the wavy pileus and crisped gills of C. crispa may be an environmentally-determined variant of the more common C. Iactea form. This hypothesis receives support from the ecology of the fungi, and the plasticity of such features as gill morphology and number of spores per basidium. The placement of the European C. crispa specimen in a clade separate from North American C. Iactea and C. crispa specimens, and the nomenclatural implications thereof, is discussed below. Conocybe crispa was described from North American material (Longyear 1899). The holotype of C. crispa was at MSC, but was misplaced and appears to have been accidentally destroyed in 1973 by someone who did not know its value (Watling & Gregory 1981). The European C. crispa is rare, and is separated from the North American taxon by Hausknecht (1998), who reduces it to C. albipes var. pseudocrispa Hausknecht, while the North American C. crispa is transferred to C. albipes var. crispa Hausknecht. Given the evident differences in DNA sequence (see Figs. 17-22), separating the two taxa at the species level would appear to be warranted. This raises potential nomenclatural problems as 109 the European C. crispa is the taxon that is clearly distinct from the C. albipes group. Until further molecular comparisons can be made between European and North American specimens, we encourage the adoption of C. albipes to refer to North American specimens that have been referred to C. Iactea, and C. albipes var. crispa for North American C. crispa specimens. The name “Conocybe Iactea" is problematic (Hausknecht 1998; Watling, pers. comm., 3 April 2002) and ought not be used. Our analyses clearly distinguished the sequestrate taxon G. lateritia from C. Iactea and C. crispa, but placed G. lateritia as sister to these taxa within Conocybe Section Candidae. G. deceptiva Baroni and the Gastrocybe-like specimen denoted Gastrocybe “yellow” both formed a clade with Bolbitius vitellinus and allies. While Gastrocybe thus was not monophyletic, Watling’s original placement of Gastrocybe within the Bolbitiaceae (Watling 1968) was supported. Moreno et al. (1989) synonymized Gastrocybe Watling with Galeropsis Velenovsky & Dvorak on the basis of morphological features of the type collections, while retaining both taxa in the Bolbitiaceae. Gastrocybe “yellow” is morphologically similar to Bolbitius elegans E. Horak, G. Moreno, A. Ortega & Esteve-Rav., a gasteroid-like fungus that Horak and colleagues (2002) have nevertheless placed in Bolbitius. Some authorities (e.g. Hawksworth et al. 1995) recognize the Galeropsidaceae Singer as the appropriate place for sequestrate forms of Bolbitiaceae and Strophariaceae. 110 The development of secotioid, or sequestrate, forms has been observed repeatedly in the Agaricales, Boletales and Cortinariales (Singer 1958; Thiers 1984). Secotioid fungi appear intermediate between agarics and gastromycetes: a differentiated pileus and stipe are present, but the pileus never expands and forcible spore discharge is lost. The lamellae are usually recognizable when the fungus is sectioned, but are contorted and frequently anastomosed. A close and direct relationship is frequently seen between secotioid and agaricoid taxa. After decades of debate, the consensus is that the direction of evolution is from the agaricoid morphology to the secotioid (Thiers 1984; Baura, Szaro & Bruns 1992; Hibbett et al. 1997). G. lateritia, with its moist, gelatinous nature, is unusual among secotioids, the majority of which are dry and frequently appear in arid habitats. While certain other taxa (Bolbitius Fr., Coprinus Pers., Coprinellus P. Karst., and Coprinopsis P. Karst.) deliquesce at maturity, these do so following spore discharge, not prior to discharge as in G. lateritia. Combined with the elongation of the stipe, the deliquescence of G. lateritia produces a fungus which looks sick, not secotioid. The consistent presence of the bacterium, coupled with the sick morphology and the existence of intermediate forms, has led us to hypothesize that G. lateritia may in fact represent a diseased Conocybe species from Section Candidae, but of unknown identity. Ill Acknowledgements Roy Watling was of considerable assistance, supplying European Conocybe specimens, sharing his notes and slides, and informing us of hard-to-find references. Robert Fogel and the University of Michigan Herbarium were very kind in permitting us to use their extensive fungal collections. This research was funded in part by the A. L. Rogers Medical Mycology Scholarship at Michigan State University. References Arora D. 1986. Mushrooms Demystified 2nd edn. Berkeley, CA, Ten Speed Press. 959 pp. Baura G, TM Szaro, TD Bruns. 1992. Gastrosuillus laricinus is a recent derivative of Sui/lus grevillei: molecular evidence. Mycologia 84: 592-597. Benedict RG, LR Brady, AH Smith, VE Tyler, Jr. 1962. Occurrence of psilocybin and psilocin in certain Conocybe and Psilocybe species. Lloydia 25: 156-159. Benedict RG, VE Tyler, R Watling. 1967. Blueing in Conocybe, Psilocybe and Stropharia species and the detection of psilocybin. Lloydia 30: 150-157. Benjamin DR. 1995. Mushrooms: Poisons and Panaceas. New York, W.H. Freeman and Company. 422 pp. Bessette AE, AR Bessette, DW Fischer. 1997. Mushrooms of Northeastem North America. Syracuse, NY, Syracuse University Press. 582 pp. Beutler JA. 1980. Chemotaxonomy of Amanita: Qualitative and quantitative evaluation of isoxazoles, tryptamines, and cyclopeptides as chemical traits. Ph.D. dissertation, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania, USA. Brady LR, RG Benedict, VE Tyler, DEJ Stuntz, MH Malone. 1975. Identification of Conocybe filaris as a toxic basidiomycete. Lloydia 38: 172-173. Breitenbach J, F Kranzlin. 1995. Fungi of Switzerland 4. Lucerne, Switzerland, Edition Mykologia Lucerne. 368 pp. Enjalbert F, C Gallion, F Jehl, H Monteil, H Faulstich. 1992. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography. Journal of Chromatography 598: 227-236 112 Gardes M, TD Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 1 13—1 18. Glen M, IC Tommerup, NL Bougher, PA O’Brien. 2001. Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations. Mycological Research 105: 138-149. Hausknecht A. 1998. Beitrage zur Kenntnis der Bolbitiaceae 4. Die Sektion Candidae und andere hellht’rtige Arlen der Gattung Conocybe. Osterreich Zeitschrift fL’ir Pilzkunde 7: 91 -1 21. Hawksworth DL, PM Kirk, BC Sutton, DN Pegler. 1995. Dictionary of the Fungi 8th edn. ,Wallingford, Oxon, UK, CAB International. 616 pp. Hibbett DS, EM Pine, E Langer, G Langer, MJ Donoghue. 1997. Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences. Proceedings of the National Academy of Sciences, USA 94: 12002-12006. Horak E, G Moreno, A Ortega, F Esteve-Raventés. 2002. Bolbitius elegans, a striking new species from southern Spain. Persoonia 17: 615-623. Hughey BD, GC Adams, TD Bruns, D8 Hibbett. 2000. Phylogeny of Calostoma, the gelatinous-stalked puffball, based on nuclear and ribosomal DNA sequences. Mycologia 92: 94-104. Hutchison LJ, SE Madzia, GL Barron. 1995. The presence and antifeedant function of toxin-producing secretory cells on hyphae of the lawn-inhabiting agaric Conocybe Iactea. Canadian Journal of Botany 74: 431-434. Kauffman CH. 1918. The Agaricaceae of Michigan 1. Lansing, MI, Michigan Biological and Geological Survey. 924 pp. Klan J, D Baudisova. 1993. Toxiny muchomurky zelené v susenych plodnicich. Cas Lek Ces 132: 468-469. Longyear BO. 1899. Two new Michigan fungi. Botanical Gazette 28: 272-273. Moreno G, M Heykoop, C Illana. 1989. Studies on Galeropsis and Gastrocybe (Bolbitiaceae, Agaricales). Mycotaxon 36: 63-72. O’Donnell K. 1992. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Current Genetics 2: 213-220. 113 Posada D, KA Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinfonnatics 14: 917-818. Simmons MP, H Ochoterena. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 396-381. Singer R. 1958. The meaning of the affinity of the Secotiaceae with the Agaricales. Sydowia 12: 1-43. Singer R. 1986. The Agaricales in Modern Taxonomy 4th edn. Konigstein, Germany, Koeltz Scientific Books. pp. 540-556. Swofford DL. 2002. PA UP*: Phylogenetic Analysis Using Parsimony (and other methods) version 4.0b10. Sunderland, MA, Sinauer Associates. Tank DC, T Sang. 2001. Phylogenetic utility of the glycerol-3-phosphate acyltransferase gene: evolution and implications in Paeonia (Paeoniaceae). Molecular Phylogenetics and Evolution 19: 421-429. Thiers HD. 1984. The secotioid syndrome. Mycologia 76: 1-8. Thompson JD, DG Higgins, TJ Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. Watling R. 1968. Observations on the Bolbitiaceae. IV. A new genus of gastromycetoid fungi. The Michigan Botanist 7: 19-24 Watling R. 1982. Bolbitiaceae: Agrocybe, Bolbitius and Conocybe. British Fungus Flora 3. Edinburgh, Scotland, Royal Botanic Garden. Watling R, NM Gregory. 1981. Census Catalogue of Worid Members of the Bolbitiaceae. Bibliotheca Mycologica 82. Vaduz, Germany, J. Kramer. Weber NS. 1989. Mushrooms in a mycologist's yard: Gastrocybe lateritia. Mclllvainea 9(1): 7-14. White TJ, T Bruns, S Lee, J Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: MA Innis, DH Gelfand, JJ Sninsky & TJ White, eds. PCR Protocols: A Guide to Methods and Applications. San Diego, CA, Academic Press, Inc. pp. 315-322. Yocum RR, DM Simons. 1977. Amatoxins and phallotoxins in Amanita species of the northeastern United States. Lloydia 40: 178-190. 114 CHAPTER 5 NON-RIBOSOMAL PEPTIDE SYNTHETASES AND GALERINA MARGINA TA Abstract Amatoxins are cyclic peptides, and are believed to be biosynthesized by an enzymatic (non-ribosomal) system. The putative enzyme, amatoxin synthetase, is expected to be encoded by a ca. 30 kb gene. The gene is expected to share conserved sequence motifs found in other fungal cyclic peptide synthetases. The wood decay mushroom Galerina marginata has been selected as a model organism for elucidating the pathway of amatoxin biosynthesis because it produces amatoxins in culture and grows more readily in culture than Amanita species. Fourteen primers, six of which were newly designed and eight taken from the literature, were used in various combinations to amplify amatoxin synthetase gene fragments. These attempts were unsuccessful, due to low primer specificity and possibly to flaws in the primer design approach. Therefore, attempts to use pyrophosphate exchange assay to isolate the synthetase enzyme were initiated. Placing amatoxin synthetase in an evolutionary framework using future sequence data has the potential to resolve many questions about the nature and ecological role of amatoxin synthetase, and cyclic peptide synthetases in general. 115 Introduction Cyclic peptides are biosynthesized in a unique process. While these peptides are composed of amino acids, an enzymatic mode of synthesis is employed, as opposed to the ribosomal synthesis employed in the synthesis of typical proteins. The enzymes involved, cyclic peptide synthetases (CPSs), are large proteins encoded by some of the longest known open reading frames (Weber et al. 1994), and much remains unknown about their synthesis, ecological function and evolutionary history. The enzyme obtains, activates, bonds and cyclizes the cyclic peptide end product (Kleinkauf & von Dohren 1996). Each cyclic peptide amino acid requires a separate domain of several hundred amino acids in the synthetase enzyme (Panaccione, 1996). Each domain contains certain conserved regions, which allow for the development of degenerate primers for use in the polymerase chain reaction (PCR) (Panaccione, 1996; see Figure 23 and Tables 6-8). Amatoxins are a family of cyclic octapeptides that are responsible for more than 90 % of fatal mushroom poisonings in humans. Amatoxins are produced in four genera of mushrooms, Amanita, Galerina, Lepiota and Conocybe. a- and B- amanitin are the prevalent amatoxins in the death cap mushroom, Amanita phalloides, while or- and y-amanitin prevail in Galerina and Lepiota (Benedict & Brady 1967; Haines, Lichstein & Glickerman 1985; Faulstich & Zilker 1994). Conocybe contains only very low levels of any given amatoxin (Brady et al. 1975) 116 m>ZWqum m>Zwomqm m>Ewmmgm m>zwmmq> m>zaqmq> m>zwmmqm mszqaqm mszmmqm m>zwmqu m ZququmHUZHx mHmmqumoozax mHmmqumooqu mHmoqumoom>x mHmmoume mHmoqumooqu m>moqmgmoozzm m>ooqm>mowm >zooemaHo >qooemy>m wqawequm >aowequm maawmmw>m >qooexwam >qowexymm >qooemm>m >qowexyqe >qooemwmm amawwama >Haoequm >qoowmem a mow>aqmoHamo>qmem monaqmoHome mwoqaqmo>ooem eaonamoemowm mmmHoqmo>mon momHquo>m moquqmw>mon «oquqmu>wme Hom>>qm@>z <¢muz mumdmwwwz m>Q Om>d4>OHm>UmQ¢HmH mddEDmOAmHUmAHmOH <¢2qmmOAmHUmN>DmH Q¢>QQmOEmHUmm>QmH QmZQUmOEmHUmw>Qm> AmQQOMOAmHUmW>QmH Adddwmothodw>QmH m dmmKUHmDmHm wxmxoemomhw OMQAUHHUmHZ >U>Uoozmd Ummxwwwmmmm QHUAUUUUQA Uxmeobeomhm m>UAH¢U>QQ DXmmUBmwm9> m>U>H¢Ude DXmMUHmUmHm mHUAUUOM>A UMmKOHmUmHm mHmduooqu onxDmHUmb mHUQUUU>09mwmem mHUAHOUVmA UXm>DHmUmBm mHUHUUOmZA UXmmwemDmHm aHU>UwU$ZQ OXmAUHmUmBm m>UHHUOLHA OMmeWmDmHm U demQAOAOmA demQHqumb >quQAU¢EHA demQQD>mHm HmHmDOUAmwb ddeQUUHUAm demmqumbm QBHmawommHm aeHmoooasz mmHmoooqmeg DUOU¢xAH< ”um: Huma ”um: ”um: ”meoaue “>eoape "Hmem names ”Hmem ”swam “24 ”24 ”24 HumD ”um: "umD ”N >mHU "H >mHU ”mcoflub ”>LUAMB Hamem ”Hmkm "HmBI "Hmem HZ< ”Ed HZ¢ wmmflmficfim on: o 2.0 o .m :2 E 22 9 comecoo .m min... 117 Ig_ble 6. Conserved regions in cyclic peptide synthetases Conserved regions in fungal cyclic peptide synthetases. Cyclic peptide sequences were obtained from NCBI GenBank. AM = AM-toxin synthetase from Altemaria altemata AF184074, Clav1 and Clav2 are ergopeptine synthetases from Claviceps purpurea AF022911 and U30621, HT81 = HC-toxin synthetase from Cochliobolus carbonum M98024, TrichH = peptide synthetase from Trichodenna harzianum AF304355, TrichV = peptide synthetase from Trichodenna virens AF351825, UST = ferrochrome siderophore from Ustilago maydis AAB93493. Headings C, D, E, F, G, H, J and K refer to conserved motifs as outlined in Kleinkauf and von Doehren (1996) and shown in Figure 1. 118 Figure 23. A- IIIIIIIIITHIIT ABC DEFGHIJKLMNO Structure of a typical cyclic peptide synthetase. A The entire synthetase for an eight amino acid cyclic peptide, as predicted for amatoxin synthetase. Shaded areas represent domains for the separate amino acids. Each domain shares conserved sequence motifs. B Enlargement of one domain, showing roughly where various activities are encoded (shaded areas). A-l encode adenylation functions, J encodes the acyl carrier, and K-O function in condensation. Some CPSs contain additional sequences P-Q, which function in epimerization, N- methylation and other modifications. After Kleinkauf & von Dohren (1996) and Panaccione (1996). The ecology of amatoxins is unknown. Most known enzymatically- produced peptides are toxic; examples include the antibiotics gramicidin S and penicillin, the host-specific phytotoxin HC-toxin, and the eukaryotic RNA polymerase II inhibitor amanitin. However, the role of amatoxins in the organism and in nature is unclear. Unlike penicillin, which is secreted by Penicillium species into the substrate and can thus discourage potential competitors, amatoxins are not secreted (Murayoka & Shinozawa, 2000). While plants have been experimentally shown to be sensitive to amatoxins (Faulstich, 1980), the lack of secretion by the fungus means that plants are not exposed to these toxins in nature. Indeed, Amanita species are obligate mycorrhizal associates of trees, and cannot afford to poison their hosts. Galerina species are decomposers of dead wood, while Conocybe and Lepiota species are grass saprophytes; the lack 119 of any toxin secretion would eliminate any potential use of the toxin in substrate colonization. Due to the mode of action, amatoxin poisoning exhibits an inevitable delay of several hours. An animal is not going to learn to avoid a particular mushroom if sickness does not occur until several hours later. Prokaryotic RNA polymerases are not affected by amatoxins (Wieland 8 Faulstich, 1991). The genera of mushrooms that produce amatoxins are distributed through four families in two orders of fungi. The lack of any close relationship between amatoxin-producing genera raises questions about the evolutionary history of amatoxins. Why are amatoxins produced by Amanita and Lepiota, but not by Limacella, which falls between the two in phylogenetic analyses? Why by some Amanita species in Section Phalloidae and not others? Why by some A. virosa individuals and not others? Are amatoxins produced in many additional basidiomycetes at levels below detection? Is a nonfunctional amatoxin synthetase gene present in non-producers? Has amatoxin synthesis arisen multiple times independently, or is it a case of horizontal transfer? The hypothesis that amatoxins are produced routinely in basidiomycetes was put forward by Faulstich and Cochet-Meilhac (1976) and elaborated by Preston et al. (1982), who posited a regulatory role. The support for this hypothesis was the low levels of amatoxin the authors found in analyses of several mushroom species, including edible ones. However, in both studies, the toxins in nontoxic species were near the limits of detection for the protocols 120 employed, and more sensitive HPLC analyses have consistently failed to detect amatoxins in these species (Enjalbert et al. 1992; Hallen, unpublished results). Cyclic peptide synthetases are encoded by large genes. A 15.7-kb open reading frame (ORF) encodes HC-toxin synthetase, which produces a four amino acid peptide (Scott-Craig et al. 1992). Cyclosporin synthetase is encoded by a 45.8-kb ORF and synthesizes an eleven amino acid product (Weber et al. 1994). The synthetase for the octapeptide amatoxins, by extrapolation, might be expected to require a 30 kb ORF. The likelihood of a gene this size evolving independently on four occasions is presumably low. The hypothesis that the gene arose once, in an ancestral agaric, and that multiple independent point mutations have rendered the gene nonfunctional in most species cannot be so readily dismissed. A fourth possibility is horizontal transfer (Walton 2000). Peptide synthetases are widely distributed in prokaryotes. Eukaryotic peptide synthetase genes possess certain prokaryotic gene features, notably the lack of introns. Additionally, transposon-like sequences have been observed in HC-toxin regulatory loci (Panaccione et al. 1996). Horizontal transfer of a mobile gene could explain the disjunct distribution at both the inter- and intraspecific levels observed in the case of amatoxins. None of the potential explanations of amatoxin distribution can be satisfactorily addressed by the traditional methods of evaluating fungi for amatoxins. A molecular genetic approach is needed. Once the amatoxin synthetase gene has been isolated and sequenced from one organism, it can be 121 used in evaluating other organisms. Is a mutated gene present in the nontoxic morph of Amanita vema? Is the gene wholly absent, as is the case in Cochliobolus carbonum mutants that lack HC-toxin (Panaccione et al. 1992)? What is the degree of relationship between amatoxin synthetase genes in the different genera of amatoxin producers? These questions could be addressed by the use of DNA-DNA hybridization or the development of amatoxin synthetase- specific PCR primers. Finally, a gene tree could be constructed and compared with existing species evolution data for these fungi. Extensive work has been done to develop molecular phylogenies of Amanita (WeiB, Yang & Oberwinkler 1998; Drehmel, Moncalvo & Vilgalys 1999; Hallen, unpublished) and Galerina (Gulden, Dunham & Stockman 2001). Despite the fame (or infamy) of poisonous Amanita species, we have chosen to begin our search for amatoxin synthetase in Galerina marginata. G. marginata contains 0.1 - 0.8 mg amatoxin per g dry weight (Bresinsky & Besl 1990). These are lower toxin levels than in Amanita but still sufficient to cause harm. Occasional poisonings occur among people seeking hallucinogenic mushrooms, many of which are superficially similar to Galerina species. Galerina is the only amatoxin-producing genus that will produce the toxins while growing in culture (Benedict & Brady, 1967). In the other three genera, toxin is only produced in the mature mushroom, which usually cannot be produced in the lab. The woodrotting Galerina marginata is a relatively more tractable organism than mycorrhizal Amanita species. Amanita species grow very slowly in culture and contain a number of inhibitory compounds that interfere with DNA extraction and 122 the use of molecular biology techniques (Hallen, pers. obs.) Additionally, Amanita species produce several other cyclic peptides that would interfere with the isolation and identification of the targeted toxin. Materials and Methods Culture and HPLC Galerina cultures were obtained from the USDA Forest Products Laboratory, Madison, WI. Cultures were Galerina autumnalis HHB-11959-sp (dikaryon), G. autumnalis HHB-11959 55-1 (monokaryon), G. heterocystis CBS (Wy-4228) (dikaryon), G. marginata RLG-8365-sp (dikaryon) and G. stylifera HHB-12845-sp (dikaryon). Thirty ml liquid shake cultures were grown in HSV for biomass growth, and transferred to 30 ml carbon-starved GFV liquid medium (Muraoka & Shinozawa 2000). Cultures were grown for an additional 15 - 20 days, filtered through miracloth and blotted to remove excess liquid. Additionally, cultures were grown on HSV broth only. Cultures grown on HSV broth only were transferred every 15 - 20 days. Cultures were evaluated for toxins using a modification of the method of Enjalbert et al. (1992). Additionally, dried fruiting bodies of G. marginata were rehydrated and subjected to HPLC. Eight to 200 mg of the tissues were suspended in 1.5 ml extraction medium containing methanolzdistilled water:0.01M HCI (524:1) 9'1 tissue. Methanol was HPLC grade (J.T. Baker, 123 Phillipsburg, New Jersey, USA). Suspended tissues were incubated at 4°C for 12 h. Samples were then centrifuged at 1000 x g and 4°C for 10 min, and the supernatant was collected. The pellets were resuspended in 0.6 ml extraction medium 9'1 tissue, incubated at 4°C for an additional 12 h and centrifuged. The supernatants from the first and second centrifugation were pooled. HPLC analysis of amatoxins was performed on a Model 114 HPLC apparatus (Beckman Instruments, Inc., Fullerton, California, USA) with detection at 295 nm. Amatoxins were separated using a reverse-phase C-18 column (Aquapore OD-300, 7pm, 200x46 mm; Perkin-Elmer Corporation, Norwalk, Connecticut, USA) and a 30 min gradient of solution A to solution B. Solution A was 0.2 M ammonium acetate, adjusted to pH 5 with glacial acetic acid, and solution B was acetonitrile. Flow rate was 1 ml min'1. Samples were maintained at a temperature of 4°C until injection. Twenty pl of each sample were injected. Standards were purified a-amanitin and B-amanitin (Sigma Chemical Company, St. Louis, Missouri, USA). Twenty pl of a toxin standard solution, containing each toxin at a concentration of 100 pg mI‘I, were injected Peaks eluted at approximately 70 - 80 % acetonitrile. Putative toxin peaks were identified by comparison with the toxin standards. In the case of y—amanitin, for which a standard was not commercially available, peaks could be tentatively identified by comparison with published HPLC data (Enjalbert et al. 1992). 124 Primer development and PCR Primers were taken from the literature or designed based on the conserved sequence motifs given in Table 6. Primers and the combinations in which they were used are listed in Tables 7 and 8. lane 7. Primers used in this study. Primer Secmgmge (5' - 3') Amglifies 80m TGD AWIGARKSICCICCIRRSIMRAARAA YRTGD UR R (f) Turgay 8 Marahiel 1994 G TCTAGAGGNAARCCNAARGG RGKPKG (f) Panaccione 1996 JA 1 CARGARGGIYTIATGGC QEGLMA (f) Wrest (pers. comm.) JA 4F TTYACITCIGGITCIACIGG FTSGSTG (f) Wiest (pers. comm.) ELGEIE GARYTNGSNGARATHGA EL G/A EIE (f) Hallen YGP TAYGGNCCNACNGA YGPTE (f) Hallen YRT TAYMGIACIGGIGAYYTIGT YRTGDLV (f) Hallen LGG TWYCGIACIGGIGAYYKIGKICG LLXLGGXS (r) Turgay 8 Marahiel 199‘ Y ARRTCNCCNGTYTTRTATCTAGA YKTGDL (r) Panaccione 1996 JA2 CCIGAIAYIGTIGYICCRAA FG T/A T W SG (r) Wiest (pers. comm.) JA 5 GGIACYTGITGRTCYT‘I' KDTQVK (r) Wrest (pers. comm.) DW GTKCANGSRWANACRTCYTC EDV Y/F AlP CT (r) Hallen GGDS GCNGYDATNSWRTCNCCNCC GGDSI AlT A (r) Hallen PCTPLQ TGIARIGGIGTRCAIGG PCTPLQ (r) Hallen 3(f) = forward primer, (r) = reverse primer. Table 8. Estimated product size (bp) for each primer combination Primer LGG Y M JA5 DVY GGD PC_T TGD 630 3500* 1690 50 780 630 780 G 1290 750 2360 820 1510 1290 1510 JA 1 3060 2430 800 2510 3500* 3060 3500* JA 4F 1290 750 2360 820 1510 1290 1510 ELGEIE 520 3450 1570 3500* 780 520 780 YGP 740 210 1950 270 1130 730 1130 YRT 630 3500* 1690 50 780 630 780 Estimates are derived from sequence of Cochliobolus carbonum. * Primers are partially complementary. An approximately 3500 bp product could result from primers binding to analogous sites in different domains. DNA was extracted from 250 mg of a fruiting body collected in 1996 that had tested positive for amatoxins. Approximately 10 mg of gill tissue apiece was 125 placed in each of 20 microfuge tubes, frozen in liquid nitrogen and macerated. Then, 1 ml cetyltrimethyammonium bromide (CTAB) mixture (5% w/v CTAB, 1.4 M NaCl, 20 mM EDTA pH 8.0, 100 mM Tris-HCI pH 8.0, 1% w/v polyvinylpyrrolidone (PVP-360)) and 2 pl B-mercaptoethanol were added to each tube. The tube was incubated at 65°C for 1 h. The mixture was purified with phenol:chloroform:isoamyl alcohol (24:24:1) and chloroform extractions, then centrifuged to remove solids. The water soluble fraction was precipitated with absolute ethanol and centrifugation, followed by a rinse with 70% ethanol and a second centrifugation. The precipitate was air-dried under vacuum then resuspended in 50 pl water. Approximately 1-20 ng of the total genomic DNA was used per 25 pl reaction mixture for polymerase chain reaction (PCR) amplification. Various brands of prepackaged buffers and polymerases were used for PCR amplification. Primers were used in the combinations given in Table 2. Additionally, primers were used individually as controls. An HC-toxin producing strain of Cochliobolus carbonum was used as a positive control. The cycling reactions were performed in a DNA thermal cycler, model PTC-100 (MJ Research, Inc., Waltham, MA, USA). A 60°C to 45°C touchdown protocol was used. The amplification ended with an additional 10 min extension at 72°C, and storage at 4°C. PCR products were visualized on 1.5% agarose with uv light. A band was considered to represent a putative cyclic peptide synthetase gene product if the band was unique to a combination of two primers and did not appear in either of the individual-primer control reactions. Additionally I26 the band size could be compared with the predicted size, based on known CPS gene sequences. Any such band was gel purified and cloned using a TOPO® TA cloning kit (lnvitrogen, Carlsbad, CA, USA). As CPS genes contain multiple domains, each of which may be expected to amplify with a given primer pair, clones were subject to a restriction digest with Hae III, which has a four base pair recognition site (four-cutter). A clone of each restriction pattern was sequenced. Sequencing was performed by the Michigan State University Genomics Technology Support Facility, using dye terminator capillary electrophoresis on an ABI Prism® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were submitted to a BLAST search (Altschul et al. 1997) through NCBI GenBank for comparison with known CPS gene sequences. Pyrophosphate exchange assay Pyrophosphate exchange assays followed Walton (1987). Results All Galerina cultures including the G. autumnalis monokaryon tested positive with HPLC for a-amanitin. The monokaryon had approximately one tenth the amatoxin of the dikaryon. No peaks consistent with y-amanitin were observed. It should be noted that Gulden and colleagues (2001) have recently synonymized G. autumnalis and G. marginata (as G. marginata), based on 127 molecular phylogenetic evidence. Cultures that had been transferred to the carbon-stewed GFV broth consistently showed a two— to threefold increase in toxin levels than cultures that had been grown in HSV only. PCR products were readily obtained. However, most products were consistent with single primer reactions (Figure 24). Seven products that did appear unique to a primer combination were gel purified, cloned and sequenced. Without exception, the sequences matched bacterial DNA, but not cyclic peptide synthetases of bacterial origin. Therefore the sequences were considered to originate from probable contaminants. No matches to peptide synthetases were obtained. +abcdefghijklmnopqrs Figure 24. Typical gel showing PCR products from G. marginata amplified by CPS primers. + = 1 kb+ ladder, a - s are lanes loaded with PCR products. Lane “d” is G. marginata amplified by primers JA1 and LGG; it contains no bands that are not also shown in lane ‘5”, amplified by JA1 alone. Lane "n” was amplified by primer G alone. 128 Discussion Attempts to amplify amatoxin synthetase gene fragments using PCR presented many problems. The primers were degenerate, requiring low annealing temperatures that lowered primer specificity. The profusion of bands produced caused difficulty in reading the gels. This difficulty could be overcome by using a higher percentage of agarose in the gel which would interfere with gel purification attempts. Finally, we were using primers based on sequences from four ascomycetes and one heterobasidiomycete in attempts to amplify sequence from a homobasidiomycete. It is possible that homobasidiomycete CPS gene sequences differ sufficiently from those of other groups of fungi to prevent amplification of the desired products. Attempts to BLAST the Phanerochaete chrysosporium genome database () with conserved sequence from HC-toxin synthetase yielded no matches. This may mean that P. chrysosporium, a white-rotting homobasidiomycete, contains no cyclic peptide synthetases. Alternately, it may indicate divergence. Summary and future directions The molecular phylogenetics of Amanita suggest one acquisition of amatoxin synthesis within the genus (see Chapter one). Amatoxins are found only in one monophyletic, derived clade. The structurally-similar phallotoxins do 129 not occur in any Amanita species that does not also produce amatoxins (Wieland 1986). Amatoxins are produced by one species of Conocybe, C. filaris, while phallotoxins are produced by Conocybe Iactea, which is not closely related to C. filaris (Chapter four), suggesting a separate acquisition event for each cyclic peptide in Conocybe. In Galerina and Lepiota, 288 rDNA data places amatoxin- producing species in monophyletic, derived clades (Moncalvo et al. 2002). Amanita, Conocybe, Galerina and Lepiota are all well separated from each other in molecular phylogenetic analyses (Moncalvo et al. 2002). Together, these data support the hypothesis of multiple independent gains of amatoxin synthesis. We have begun to search for the amatoxin synthetase enzyme in Galerina marginata from culture and dried fruiting bodies using pyrophosphate exchange assay (Walton 1987), but this work is still in the initial stages. We plan to continue efforts to isolate and, ultimately, sequence amatoxin synthetase. Amatoxin synthetase gene sequence would enable us to determine whether amatoxins share a biosynthetic pathway with the heptapeptide phallotoxins. We also plan to sequence the portion of the RNA polymerase 11 large subunit (RPB1) responsible for amatoxin binding from toxin producers and nonproducers. The complete RPB1 sequence from Amanita phalloides has recently become available (Liu, pers. comm., 11 July 2002), enabling us to design specific primers. The amatoxin binding region of pol II is encoded by regions D - F of RPBl, and amatoxin-resistant mice are known to have mutations in this region (Bartolomei 8 Corden 1995). Toxin producing fungi are known to possess a modified pol ll (Wieland 1986). With the data already available on the 130 evolutionary history of amatoxin-producing mushrooms, RPB1 and amatoxin synthetase sequence data should be a powerful tool in examining the origins and evolutionary history of amatoxin synthetase. References Altschul SF, TL Madden, AA Schaffer, J Zhang, Z Zhang, W Miller, DJ Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402. Bartolomei M8, Corden JL. 1995. Clustered alpha-amanitin resistance mutations in mouse. Mol Gen Genet 246:778-782. Benedict RG, LR Brady. 1967. Further studies on fermentative production of toxic cyclopeptides by Galerina marginata (F r.) Kiihn. Lloydia 30:372-378. Brady LR, RG Benedict, VE Tyler, DE Stuntz, MH Malone. 1975. Identification of Conocybe filaris as a toxic basidiomycete. Lloydia 38:172-173. Bresinsky A, H Besl. 1990. A Colour Atlas of Poisonous Fungi - A Handbook for Pharmacists, Doctors, and Biologists. London, Wolfe Publishing Ltd. 295 pp. Drehmel D, J-M Moncalvo, R Vilgalys. 1999. Molecular phylogeny of Amanita based on large-subunit ribosomal DNA sequences: implications for taxonomy and character evolution. Mycologia 91 :610-618. Enjalbert F, Gallion C, Jehl F, Monteil H, Faulstich H. 1992. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography. Journal of Chromatography 598:227-236. Faulstich H. 1980. The amatoxins. Progress in Molecular Subcellular Biology 7:88—1 22. Faulstich H, M Cochet-Meilhac. 1976. Amatoxins in edible mushrooms. FEBS Letters 64(1): 73-75. Faulstich H, TR Zilker. 1994. Amatoxins in Handbook of Mushroom Poisoning - Diagnosis and Treatment. DG Spoerke, BH Rumack, eds. Boca Raton, FL, CRC Press, Inc. pp. 233-248. 131 Gulden G, S Dunham, J Stockman. 2001. DNA studies in the Galerina marginata complex. Mycological Research 105:432-440. Haines JH, E Lichtstein, D Glickerman. 1985. A fatal poisoning from an amatoxin containing Lepiota. Mycopathologia 93: 15-17. Kleinkauf H, H von Dbhren. 1982. A survey of enzymatic peptide formation. In: Peptide Antibiotics Biosynthesis and Functions. H Kleinkauf, H von Dohren, eds. Berlin, Walter de Gruyter. pp. 1-21. Kleinkauf H, H von Ddhren. 1996. A nonribosomal system of peptide biosynthesis. European Joumal of Biochemistry 236:335—351. Moncalvo J-M, R Vilgalys, SA Redhead, JE Johnson, TY James, MC Aime, V Hofstetter, SJW Verduin, E Larsson, TJ Baroni, RG Thorn, S Jacobsson, H Clemencon, OK Miller, Jr. 2002. One hundred and seventeen clades of euagarics. Molecular Phylogenetic Evolution 23: 357-400. Murayoka S, T Shinozawa. 2000. Effective production of amanitins by two-step cultivation of the basidiomycete, Galerina fasciculata GF-060. Joumal of Bioscience and Bioengineering 89:73-76. Panaccione DG. 1996. Multiple families of peptide synthetase genes from ergopeptine-producing fungi. Mycological Research 100(4):429—436. Panaccione DG, JW Pitkin, JD Walton, 8L Annis. 1996. Transposon-like sequences at the TOX2 locus of the plant pathogenic fungus Cochliobolus carbonum. Gene 176: 1 03-1 09. Panaccione DG, JS Scott-Craig, J-A Pocard, JD Walton. 1992. A cyclic peptide synthetase gene required for pathogenicity of the fungus Cochliobolus carbonum on maize. Proceedings of the National Academy of Science, USA 89:6590-6594. Preston JF, BEC Johnson, M Little, T Romeo, HJ Stark, JE Mullersman. 1982. Investigations on the function of amatoxins in Amanita species: a case for amatoxins as potential regulators of transcription. In: Peptide Antibiotics - Biosynthesis and Functions. H Kleinkauf 8 H von Ddhren, eds. Berlin, Walter de Gruyter. pp. 399-426. Scott-Craig JS, DG Panaccione, J-A Pocard, JD Walton. 1992. The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame. Joumal of Biological Chemistry 267:26044-26049. Turgay K, MA Marahiel. 1994. A general approach for identifying and cloning peptide synthetase genes. Peptide Research 7(5): 238-241. 132 Walton JD. 1987. Two enzymes involved in biosynthesis of the host-selective phytotoxin HC-toxin. . Proceedings of the National Academy of Science, USA 84: 8444-8447. Walton JD. 2000. Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: an hypothesis. Fungal Genetics and Biology 30(3):167-171. Weber G, K Schorgendorfer, E Schneider-Scherzer, E Leitner. 1994. The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Current Genetics 26:120- 125. WeiB, M., Z.-L. Yang, and F. Oberwinkler. 1998. Molecular phylogenetic studies in the genus Amanita. Canadian Journal of Botany 76:1170-1179. Wieland T. 1986. Peptides of Poisonous Amanita Mushrooms. New York, Springer-Verlag. Wieland T, H Faulstich. 1991. Fifty years of amanitin. 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