AN ABSTRACT OF THE THESIS OF

Andrea J. Humpert for the degree of Master of Science in Botany and Plant

Pathology presented on November 11, 1999. Title: Systematics of the

Genus Ramaria Inferred from Nuclear Large Subunit and Mitochondrial

Small Subunit Ribosomal DNA Sequences.

Abstract approved: Redacted for Privacy

Joseph W. Spatafora

Ramaria is a genus of epigeous fungi common to the coniferous forests of the Pacific Northwest of North America. The extensively branched basidiocarps and the positive chemical reaction of the context in ferric sulfate are distinguishing characteristics of the genus. The genus is estimated to contain between 200-300 species and is divided into four subgenera, i.) R. subgenus Ramaria, ii.) R. subgenus Laeticolora, iii.) R. subgenus

Lentoramaria and iv.) R. subgenus Echinoramaria, according to macroscopic, microscopic and macrochemical characters. The systematics of Ramaria is problematic and confounded by intraspecific and possibly ontogenetic variation in several morphological traits. To test generic and intrageneric

taxonomic classifications, two gene regions were sequenced and subjected

to maximum parsimony analyses. The nuclear large subunit ribosomal DNA

(nuc LSU rDNA) was used to test and refine generic, subgeneric and

selected species concepts of Ramaria and the mitochondrial small subunit ribosomal DNA (mt SSU rDNA) was used as an independent locus to test the monophyly of Ramaria. Cladistic analyses of both loci indicated that Ramaria is paraphyletic due to several non-ramarioid taxa nested within the genus including Clavariadelphus, Gautieria, Gomphus and Kavinia. In the nuc LSU rDNA analyses, R. subgenus Ramaria species formed a monophyletic Glade and were indicated for the first time to be a sister group to Gautieria. Ramaria subgenus Ramaria and Gautieria were derived from R. subgenus

Laeticolora, which formed a paraphyletic grade that included Gomphus.

Ramaria subgenus Lentoramaria species also formed a paraphyletic grade in the nuc LSU rDNA analyses. The Phallales and Clavariadelphus were indicated as sister taxa to the R. stricta complex and Kavinia and R. abietina of R. subgenus Echinoramaria grouped with the basal species, R. pinicola, of

R. subgenus Lentoramaria. In the mt SSU rDNA analyses, Gautieria and

Gomphus again nested within Ramaria; however, the Phallales were indicated as a sister taxon to the Gomphales. A single evolutionary origin of the terrestrial habit was inferred for Ramaria with the terrestrial species, R. rainierensis, bridging the gap between the lignicolous R. subgenus

Lentoramaria and the terrestrial R. subgenus Laeticolora. Species concepts tested included R. amyloidea and R. celerivirescens both of R. subgenus

Laeticolora that differ primarily in the presence of clamp connections. The

results supported these two taxa as distinct, sister species. These analyses were consistent with the ramarioid morphology as ancestral for the

Gomphales with unique derivations of the club, false truffle and gomphoid morphologies. °Copyright by Andrea J. Humpert November 11, 1999 All Rights Reserved Systematics of the Genus Ramaria Inferred from Nuclear Large Subunit and

Mitochondrial Small Subunit Ribosomal DNA Sequences

by

Andrea J. Humpert

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented November 11, 1999 Commencement June, 2000 Master of Science thesis of Andrea J. Humpert presented on November 11, 1999

APPROVED:

Redacted for Privacy

Major ro essor, repres Kg Botany and Plant Pathology

Redacted for Privacy Chair of Department of Botany and Plant Pathology

Redacted for Privacy

Dean of ate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy

An rea J. Humpert, Author ACKNOWLEDGMENT

Many individuals have contributed to the completion of this thesis. I would like to thank Dr. Joseph Spatafora and Dr. Michael Castellano for providing financial support for me and the lab work involved in this project. I would also like to thank Dr. Currie D. Marr and Dr. Ronald H. Petersen for providing the herbarium collections which comprise the bulk of this work.

Without their collections, this study would have taken at least another two years.

I would like to thank my advisor, Dr. Joseph Spatafora, for presenting me with this project and for his enthusiastic support. I would also like to thank my other committee members, Dr. Michael Castellano and Dr. Jeff Stone, for their consultation and support of this project.

The members of the Spatafora lab have been a tremendous source of advice and encouragement, especially when trouble-shooting PCR and

understanding phylogenetic concepts. I would like to thank Dr. Annette

Kretzer who has spent many hours going over concepts and discussing the

peculiarities of this study with me. I would also like to thank Dr. Rekha Meyer

whose laboratory advice helped improve my PCRs and whose personal

advice helped me through some trying times. Dr. Jamie Platt introduced me

to new lab methods and presentation capabilities and provided constant

encouragement. I would also like to thank Dr. Thom O'Dell, Marya Madsen,

Rick Davis, Bryan Fondrick, and Jim Eblin of the Fungal Survey and Manage Team who took me along on several collecting trips in Washington and provided me with additional Ramaria expertise.

My parents, Caroline and Norman Humpert, always believed in me and I thank them for their love and support. My siblings, John, Michael, and

especially Deborah, have encouraged me and pushed me to do my best. I would like to thank all of my friends who have lifted my spirits on numerous

occasions. CONTRIBUTION OF AUTHORS

Dr. Joseph Spatafora participated at all stages of the study including design, data collection and analysis, and editing. Dr. Michael Castellano

provided funding, consultation and information regarding aspects of the

project. Eric Muench contributed the Gautieria nuc LSU rDNA sequences

that were a key component of this study and Admir Giachini provided two

Gomphus nuc LSU rDNA sequences. TABLE OF CONTENTS

Page INTRODUCTION 1

TAXONOMY 2

RAMARIA SUBGENERIC CHARACTERISTICS 7

SYSTEMATICS OF RAMARIA AND THE GOMPHALES ...... 11

OBJECTIVE 13

MOLECULAR PHYLOGENETICS OF RAMARIA (GOMPHALES) AND RELATED GENERA: EVIDENCE FROM NUCLEAR LARGE SUBUNIT AND MITOCHONDRIAL SMALL SUBUNIT rDNA SEQUENCES 16

ABSTRACT 17

INTRODUCTION 18

MATERIALS AND METHODS 21

Sampling 21 DNA extraction 26 DNA amplification of nuc LSU rDNA 27 DNA amplification of mt SSU rDNA 28 DNA purification and sequencing 29 Phylogenetic analysis 29

RESULTS 30

Sequence ambiguities 30 mt SSU rDNA trees 32 nuc LSU rDNA trees 35 Character mapping 40 Kishino-Hasegawa test results 44

DISCUSSION 46

Sequence ambiguities 46 Support for the Gomphales-Phallales relationship 46 TABLE OF CONTENTS (Continued)

Page Rejection of a monophyletic genus Ramaria 47 Testing subgeneric and species concepts within Ramaria using nuc LSU rDNA 48 Character mapping 52 Polarity of basidiocarp morphology 54 Evolution of hypogeous fungi from epigeous Gomphales 56 Gautieria 56 Hysterangium 58

TAXONOMIC CONSIDERATIONS 59

ACKNOWLEDGEMENTS 62

LITERATURE CITED 62

CONCLUSIONS 66

GENERIC, SUBGENERIC AND SPECIES CONCEPTS IN THE GOMPHALES 66

RECOMMENDATIONS FOR FUTURE RESEARCH 68

BIBLIOGRAPHY T

APPENDIX: PAUP NEXUS alignment file of nuclear large subunit and mitochondrial small subunit ribosomal DNA sequences used in maximum parsimony analyses disc LIST OF FIGURES

Figures Page

1.1. Distribution of the Record of Decision (ROD) putative old growth associates 14

2.1. Strict consensus cladogram of 230 equally most parsimonious trees of 1183 steps recovered from maximum parsimony analyses of mt SSU rDNA sequences 33

2.2. Best -In likelihood phylogram of 230 equally most parsimonious trees of 1183 steps recovered from maximum parsimony analyses of mt SSU rDNA sequences 34

2.3. Strict consensus cladogram of 1142 equally most parsimonious trees of 589 steps recovered from maximum parsimony analyses of the conservative nuc LSU rDNA alignment 36

2.4. Best -In likelihood phylogram of 1142 equally most parsimonious trees of 589 steps recovered from maximum parsimony analyses of the conservative nuc LSU rDNA alignment 37

2.5. Strict consensus cladogram of 48 equally most parsimonious trees of 739 steps recovered from maximum parsimony analyses of the less conservative nuc LSU rDNA alignment 39

2.6. Gomphalean morphologies 41

2.7. Substrate habitat 42

2.8. Clamp connections 43 LIST OF TABLES

Table Page

1.1. Ramaria taxonomic terms 4

1.2. Ramaria macrochemical tests 7

1.3. Ramaria subgeneric characters 8

2.1. Taxa included in the phylogenetic analyses 23

2.2. Kishino-Hasegawa likelihood test results 45 DEDICATION

I dedicate this thesis to Patricia Bifferstaff. SYSTEMATICS OF THE GENUS RAMARIA INFERRED FROM NUCLEAR LARGE SUBUNIT AND MITOCHONDRIAL SMALL SUBUNIT RIBOSOMAL DNA SEQUENCES

INTRODUCTION

Ramaria Fr. Ex Bonord. (Basidiomycota, Gomphales) is a genus of epigeous fungi common to the old growth forests of the Pacific Northwest

(PNW) of North America. Ramaria species are informally referred to as coral fungi due to their colorful, extensively branched basidiocarps. Worldwide, the genus Ramaria is estimated to contain 200-300 species, and the Pacific

Northwest of North America is one center of diversity that comprises an estimated sixty-one species (Marr and Stuntz, 1973; Madsen and O'Dell, pers. comm.). The majority of Ramaria species fruit throughout the fall with fewer species fruiting in the spring. Despite the abundance and conspicuous nature of these fungi in forest ecosystems, their phylogenetic relationships are poorly understood.

HolmskjOld (1790) introduced the name Ramaria to include fungi with highly branched basidiocarps, which served to unify the genus in classification schemes. Numerous revisions of Ramaria at the genus and species level have since been proposed based on variation in macroscopic, microscopic and macrochemical characters (Corner, 1950, 1970; Donk,

1961; Marr and Stuntz, 1973; Petersen, 1974, 1975, 1976, 1979, 1981, 1982, 1986, 1987, 1988a, 1988b; Petersen and Scates, 1988). Presently, the genus Ramaria is divided into four subgenera, i.) R. subgenus Ramaria, ii.) R. subgenus Laeticolora, iii.) R. subgenus Lentoramaria and iv.) R. subgenus

Echinoramaria (Corner, 1970; Marr and Stuntz, 1973).

TAXONOMY

Ramaria botrytis (Pers.: Fr.) Ricken is the type species of the genus.

Although Holmskjold (1790) introduced the name Ramaria, his contemporary, Persoon, described R. botrytis and placed it in the genus

Clavaria. In 1821, Fries sanctioned the name Clavaria, treating Ramaria as a section of Clavaria within the Clavariaceae. From 1821 until the first half of the 20th century, this classification scheme was maintained. Donk (1933) elevated the name Ramaria to its current generic nomenclatural status by recognizing Bonorden's (1851) use of the name Ramaria (Corner, 1950,

1970; Donk, 1961; Marr and Stuntz, 1973).

Before 1900, identification of coralloid fungi was primarily limited to macroscopic characters such as branching pattern and spore color (Corner,

1950, 1970; Donk, 1964; Marr and Stuntz, 1973). Patouillard (1900) stressed the use of microscopic characters such as basidial morphology and spore characters. Corner, in his monograph of Clavaria and allied genera (1950), emphasized the taxonomic importance of microscopic characters, specifically hyphal construction, which led to further subdivision of Ramaria species 3

(Marr and Stuntz, 1973; Petersen, 1973). Singer (1962) later described the use of macrochemical tests. Both microscopic characters and macrochemical tests have become an integral component of Ramaria taxonomy (Marr and Stuntz, 1973). See Table 1.1. for a list of terms associated with Ramaria taxonomy.

Early hypotheses originally placed Ramaria in the Clavariaceae of the order Aphyllophorales (Fries, 1821; Corner, 1950, 1970; Donk, 1964; Marr and Stuntz, 1973; Petersen, 1973). Ramaria is now classified in the Family

Ramariaceae (Corner, 1970; Hawksworth et al., 1995) of the order

Gomphales (Basidiomycota, Holobasidiomycetidae); however, some authors maintain Ramaria's conservative placement in the Gomphaceae (Donk,

1961; Petersen, 1988b). Donk (1961, 1964) proposed Gomphaceae to include the resupinate genera Kavinia and Ramaricium, the stalked clavarioid genera Lentaria and Ramaria and the stalked pileate genera

Beenakia, Chloroneuron, Gloeocantharellus and Gomphus. Characters that unite these genera are cyanophilous spore ornamentation, a positive chemical reaction with ferric sulfate, and chiastic basidia. Corner (1970) later proposed Ramariaceae to include Delentaria, Kavinia, Lentaria and Ramaria, excluding the pileate genera because there were no intermediate species linking the gomphoid and ramarioid morphologies. In 1971, Petersen proposed an ancestral gomphoid morphology with multiple derivations 4

Table 1.1. Ramaria taxonomic terms

Ramaria Taxonomic Terms Amphigenous hymenium fertile spore producing layer that lines all sides of a basidiocarp's branches.

Bruise visible color change on the basidiocarp caused by external pressure (i.e., handling).

Chiastobasidia (chiastic basidia) basidia that are clavate in shape with horizontally oriented nuclear spindles in the enlarged upper portion of the basidia.

Clamp connection a hyphal outgrowth that bridges the septum of two adjacent cells to maintain a dikaryotic state in each cell.

Cyanophilous staining blue in aniline blue or cotton blue (spore ornamentation or hyphal structures).

Duff forest litter including leaves, pine needles or pine cones, twigs and other woody debris.

Echinulate spores spiny ornamented spores.

Holobasidia basidia that lack septations.

Lignicolous living on or in wood.

Mycelial cord a discrete filamentous aggregation of hyphae that, unlike rhizomorphs, has no apical meristem (Hawksworth et al., 1995).

Mycelial or hyphal mat a dense subterranean hyphal system that often has a higher microbial biomass and respiration rate than adjacent nonmat soil (Griffiths et al., 1987; Castellano, 1988).

Polychotomous having an apex dividing simultaneously into more than two branches (Corner, 1950).

Ramarioid descriptive term for a highly branched basidiocarp.

Stichobasidia (stichic basidia) basidia that are cylindrical in shape with vertically oriented nuclear spindles in the basidia. 5

Table 1.1. (Continued)

Ramaria Taxonomic Terms Tomentum a covering of soft, matted hairs usually located on the base of the stipe in species belonging to R. subg. Echinoramaria and R. subg. Lentoramaria.

Unilateral Hymenium fertile spore-producing layer that lines only one side of a basidiocarp's branches. 6 of a coralloid morphology and later (Petersen, 1973, 1988b) revised Donk's and Corner's familial classifications to include Gomphus, Ramariopsis,

Ramaria, Kavinia, Beenakia and Ramaricium in the family Gomphaceae.

All Ramaria species possess a highly branched basidiocarp. The

context of the basidiocarp ranges from fleshy-fibrous to cartilaginous to

gelatinous. The hymenium of Ramaria species is generally amphigenous

and composed of layers of fertile hymenial tissue. Ramaria spores are

yellow-brown in mass deposit and range from smooth to warted to echinulate

or striate. There is a range of spore size and the spore ornamentation is

cyanophilous. Basidiocarps can range in color from bright yellow, red, or

orange, to purple, white and shades of tan. Bruises or stains also occur on

some species and often aid in identification. Basidiocarps are either

lignicolous or terrestrial with some species possessing rhizomorphic strands,

a basal mycelial mat or a white tomentum covering the base of the stipe.

Chemical reactions that aid in the identification of Ramaria species are

described in Table 1.2. (Marr and Stuntz, 1973). 7

Table 1.2. Ramaria macrochemical tests

Ramaria Macrochemical Tests CHEMICAL POSITIVE COLOR REACTION Ferric Sulfate some shade of green (delimit Gomphaceae) Pyrogallol orange-yellow, carrot red or orange-brown alpha-Napthol shades of violet and purple

Guaiac Tincture some shade of blue, usually dark blue

Guaiacol pink, red, rust brown or rarely green

Phenol red, red-brown, violet or violet-brown Melzer's Reagent amyloid dull violet (apply to stipe context) nonamyloid no change

RAMARIA SUBGENERIC CHARACTERISTICS

The genus Ramaria is divided into four subgenera: i.) R. subgenus

Ramaria, ii.) R. subgenus Laeticolora, iii.) R. subgenus Lentoramaria and iv.)

R. subgenus Echinoramaria. The subgenera are grouped according to spore ornamentation, context amyloidy, substrate habitat, presence or absence of clamps, basidiocarp size and hyphal construction. Table 1.3. briefly summarizes the characters used to denote the four subgenera as outlined in

Marr and Stuntz's 1973 monograph.

Ramaria subgenus Ramaria includes four species known from the

PNW. Ramaria botrytis (Pers. ex Fr.) Ricken is the type species of this Table 1.3. Ramaria subgeneric characters

Ramaria Subgeneric Characters (Marr and Stuntz, 1973)

Ramaria Ramaria Laeticolora Echinoramaria Lentoramaria subgenus smooth or Spore striate smooth or echinulate ornamentation warted finely warted

amyloid and nonamyloid nonamyloid Context amyloidy usually amyloid nonamyloid lignicolous or Substrate habitat terrestrial terrestrial duff duff present and present present Clamps present absent small to large, small, well- medium, well-well ­ lar g e , Basidiocarp profusely y developed, felty, developed, branched , profusely appearance basal tomentum felty, basal cauliflower branched or mycelia! mat tomentum or appearance mycelia! mat

R. Type species R. botrytis R. formosa cyanocephala R. stricta 9 subgenus. The terrestrial basidiocarps generally possess a stout base and are branched polychotomously. Basidiocarp color ranges from entirely white to alutaceous with some species having upper branches and apices that are orange or red to violet. The spores possess striate ornamentation, which is the primary unifying character of this subgenus. The contextual hyphae have clamp connections and the stipe context is usually amyloid (Marr and Stuntz,

1973).

The majority of terrestrial Ramaria species in the PNW are classified in

R. subgenus Laeticolora which includes species with spore ornamentation ranging from highly warted to smooth. Ramaria formosa (Pers. per Fr.)

Quelet is the type species of this subgenus. Approximately forty-two R. subgenus Laeticolora species are found in the PNW. Species belonging to this subgenus possess large, profusely branched, terrestrial basidiocarps.

The base of the basidiocarp is generally less stout and the branches more elongated in this subgenus than in R. subgenus Ramaria. The spores generally possess either low, flat cyanophilous patches or meandering ridges that give the appearance of irregular warts with a few species having smooth spores. The hyphae are monomitic and clamp connections may be absent. Basidiocarps are spongy to brittle in texture and may range in color from pastel to bright shades of yellow, orange, and red (Marr & Stuntz, 1973).

The nine R. subgenus Lentoramaria species recorded from the PNW are lignicolous except for the terrestrial species, R. rainierensis. Ramaria stricta (Pers. per Fr.) Quelet is the type species of this subgenus. The base of 10 these small to medium-sized basidiocarps often dissipates into a tangle of conspicuous rhizomorphic strands. The rhizomorphs and hyphae of these species may be monomitic or dimitic. Those species that are dimitic possess generative hyphae and skeletal hyphae. The context is stringy to fibrous with the pliability of leather. Basidiocarp color ranges from cream to tan with some yellow and green species. In some species, rapid color changes may occur upon bruising. Clamp connections are present and the cyanophilous spores appear either smooth or finely warted (Marr and Stuntz, 1973).

Several species included in this subgenus and R. subgenus Echinoramaria possess a unilateral hymenium.

Six species belonging to R. subgenus Echinoramaria are recorded from the PNW. The type species of this subgenus is Ramaria cyanocephala

(B. & C.) Corner. The basidiocarps of this subgenus are generally small and possess a well-developed, felty, basal tomentum or mycelial mat that is conspicuous in the duff. Basidiocarps may be cream, yellow, olive, or shades of brown and often change color when bruised. The cyanophilous spores are warted to echinulate with spine lengths ranging from 0.2 to 311m. When viewed with a scanning electron microscope, the spines appear to be

depressed in the center resembling a volcano (Petersen, 1981). The hyphae

are monomitic and clamp connections are present (Marr and Stuntz, 1973).

Ramaria species are typically confined to moist habitats, such as the

mossy, old growth forests of the PNW. The need for a moist habitat is

hypothesized to arise from the extensively branched nature of the 11 basidiocarp, which has a large, exposed surface area that is subject to desiccation (Doty, 1944; Corner, 1950). The branched habit produces a large surface area that allows for the production of the maximum amount of sporogenous tissue from a minimum of flesh (Doty, 1944). Corner (1970) observed in some Ramaria species that the branches grow apically and those cells behind the new growth tend to inflate acropetally thus expanding and creating more surface area for the production of basidia and ultimately spores.

Corner (1950) has suggested that the shape of the basidiocarp may also be due to nutritional factors that the fungus receives from its surroundings. He notes that when the food supply diminishes the basidiocarp continues to grow apically but branching discontinues. The hymenium of many Ramaria species will often produce multiple layers of basidial cells and spores that Corner (1950) suggests may be a result of favorable or unfavorable growth conditions.

SYSTEMATICS OF RAMARIA AND THE GOMPHALES

Although representative gomphalean taxa have been included in molecular studies with larger samplings of holobasidiomycetes, no molecular study has focused strictly on this group. Molecular phylogenies based on

nuclear large subunit, mitochondrial small subunit and mitochondrial large

subunit ribosomal DNA demonstrate strong bootstrap support for a P monophyletic Gomphales Glade including representatives of

Clavariadelphus, Gautieria, Gomphus, Kavinia and Ramaria (Bruns et al.,

1998; Colgan et al., 1997; Hibbett et al., 1997). Molecular analyses of the

nuclear large subunit and mitochondrial small subunit also indicate that the

Phallales, Geastrum and Sphaerobolus are a sister group to the Gomphales

Glade (Colgan et al., 1997; Hibbett et al., 1997).

A recent morphological phylogenetic study of the Gomphaceae sensu

lato used thirty-nine morphological characters from nineteen species in ten

genera including outgroup taxa to infer that the seven genera, Gomphus,

Gloeocantharellus, Kavinia, Beenakia, Ramaricium, Ramaria, and Lentaria,

form a monophyletic group (Villegas et al., 1999). The authors classified this

monophyletic group as the Order Gomphales and divided the above genera

into four families: i.) Gomphaceae, ii.) Beenakiaceae, iii.) Ramariaceae and

iv.) Lentariaceae. In their analyses, the presence of a mycelial cord delimits

the Order Gomphales; however, this character is absent in all members of the

Gomphales except three Ramaria species and one Lentaria species. The

family Lentariaceae is placed basal within the order with subsequent

derivations of the families Ramariaceae, Beenakiaceae and Gomphaceae. 13

OBJECTIVE

This study was designed to test whether the current generic and subgeneric taxonomic classifications of Ramaria reflect natural, monophyletic groups. Nuclear large subunit (approx. 610 bp) and mitochondrial small subunit (approx. 470 bp) ribosomal DNA sequences were used to test the monophyly of the genus Ramaria, the monophyly of three of the four Ramaria subgenera, and selected species concepts within the genus. The analyses included a large taxon sampling of Ramaria species as well as representatives of Clavariadelphus, Gautieria, Gomphus, Kavinia and the

Phallales including Hysterangium, Clathrus and Pseudocolus.

These analyses were partially stimulated by the Record of Decision

(ROD) for Amendments to Forest Service and Bureau of Land Management

Planning Documents within the Range of the Northern Spotted Owl (USDA et al., 1994). Forest Ecosystem Management and Assessment Team (FEMAT) scientists compiled a list of sensitive species that are putative old-growth associates and outlined four survey and management strategies. Fungi represent 57% of the ROD species and Ramaria species account for 10% of the fungi (Figure 1.1.). Twenty-seven Ramaria species are presently listed as survey strategy I and/or III. Strategy I involves managing known collection sites. If rare and endemic fungal species are detected, 160 acres should be withdrawn from ground disturbing activities. Strategy III involves conducting 14

Figure 1.1. Distribution of the Record of Decision (ROD) putative old growth associates.

ROD distribution

Vascular Plants Arthropods Mollusks Mammals Amphibians Bryophytes--/ Other Fungi Lichens

Ramaria 15 extensive surveys during periods when collection conditions are appropriate and identifying those sites that have high priority for protection (USDA et al.,

1994).

Due to the taxonomic difficulties surrounding accurate species identification and the number of Ramaria species on the ROD list, a need exists for improved species classifications. The goal of this work is to develop improved generic, subgeneric and species concepts for the fungi classified in the genus Ramaria and to understand the phylogenetic

relationships of ramarioid taxa. This study will serve as a foundation for

future molecular sampling of Ramaria species in the United States and

abroad. 16

MOLECULAR PHYLOGENETICS OF RAMARIA (GOMPHALES) AND RELATED GENERA: EVIDENCE FROM NUCLEAR LARGE SUBUNIT AND MITOCHONDRIAL SMALL SUBUNIT rDNA SEQUENCES

Andrea J. Humpert, Michael A. Castellano, Eric L. Muench, Admir Giachini

and Joseph W. Spatafora

To be submitted to Mycologia Lawrence, Kansas. 17

ABSTRACT

Phylogenetic relationships of the genus Ramaria and additional

related taxa were examined through maximum parsimony analysis of

ribosomal DNA sequence data. Related genera included Clathrus,

Clavariadelphus, Gautieria, Gomphus, Hysterangium, Kavinia and

Pseudocolus. Outgroup genera included Bondarzewia, Favolus,

Ganoderma, Lactarius and Russula. The nuclear large subunit ribosomal

DNA (nuc LSU rDNA) (approx. 610 bp) from 78 isolates including 34

Ramaria species was used to test generic, subgeneric and selected species

concepts for Ramaria. A representative holobasidiomycete sampling of

mitochondrial small subunit ribosomal DNA (mt SSU rDNA) (approx. 470 bp)

including ten Ramaria species also was used to independently test the

monophyly of the genus Ramaria. Parsimony analyses of both datasets

indicated that the genus Ramaria is paraphyletic and that several

morphologically distinct groups of holobasidiomycetes are derived from a

ramarioid ancestor. In the nuc LSU rDNA analyses, Gautieria was nested

among the terrestrial Ramaria species and was a sister group to R. subgenus

Ramaria. The nuc LSU rDNA results also indicated that R. subgenus

Laeticolora and R. subgenus Lentoramaria form paraphyletic grades.

Ramaria subgenus Laeticolora was paraphyletic due to the terminally

derived R. subgenus Ramaria/Gautieria Glade and a nested Gomphus.

Ramaria subgenus Lentoramaria was paraphyletic due to a nested 18

Clavariadelphus, Kavinia, and R. abietina of R. subgenus Echinoramaria.

Both the nuc LSU rDNA and the mt SSU rDNA analyses indicated a close relationship between the Gomphales and the Phallales, however the exact nature of this relationship was indeterminate. Species concepts tested included R. amyloidea and R. celerivirescens, which were recognized as distinct, sister species in these analyses. R. claviramulata formed a monophyletic Glade with R. celerivirescens with a bootstrap value of 100. R. araiospora and R. stuntzii also were recognized as distinct species, however their relatedness was equivocal. These data reject the monophyly of the genus Ramaria and indicate that the ramarioid morphology is ancestral for the Gomphales.

Key words: Gautieria, Gomphales, Gomphus, mt SSU rDNA, nuc LSU rDNA, Phallales, Ramaria, systematics

INTRODUCTION

Ramaria are commonly described as "coral fungi" due to their extensively branched and colorfully varied basidiocarps. Worldwide, the genus Ramaria is estimated to contain 200-300 species with at least sixty- one species recorded from the Pacific Northwest (PNW) of North America

(Marr and Stuntz, 1973; Madsen and O'Dell, pers. comm.). Identification of

Ramaria species is problematic and involves macrochemical testing and examination of microscopic features (Corner, 1950, 1970; Marr and Stuntz, 19

1973). Species concepts are confounded by intraspecific variability in color and branching patterns that are presumably the result of environmental factors and ontogeny (Corner, 1950). Numerous revisions of Ramaria at the genus and species level have been proposed based on variation in macroscopic, microscopic and macrochemical characters (Corner, 1950,

1970; Donk, 1961; Marr and Stuntz, 1973; Petersen, 1974, 1975, 1976, 1979,

1981, 1982, 1986, 1987, 1988a, 1988b; Petersen and Scates, 1988). The genus is currently divided into four subgenera: i.) R. subgenus Ramaria, ii.)

R. subgenus Laeticolora, iii.) R. subgenus Lentoramaria and iv.) R. subgenus

Echinoramaria, based on basidiospore ornamentation, substrate habitat, context amyloidy and hyphal construction.

are characterized as follows: R. subgenus Ramaria species have striate basidiospores, an amyloid context, a terrestrial habitat, clamp connections and stout basidiocarps with numerous, short, polychotomous branches. R. subgenus Laeticolora species have

basidiospores ranging from smooth to highly warted, a variable amyloid

context, a terrestrial habitat, clamp connections that are absent in some

species and varied basidiocarp branching patterns. R. subgenus

Lentoramaria species have smooth or finely warted basidiospores, a

nonamyloid context, a lignicolous or duff habitat, clamp connections and

small to medium sized basidiocarps that often possess a well-developed,

felty, basal tomentum or mycelial mat. R. subgenus Echinoramaria is

characterized by species possessing echinulate spores, a non-amyloid 20

context, a duff habitat, clamp connections and small basidiocarps that also

possess a well-developed, felty, basal tomentum or mycelial mat (Marr and

Stuntz, 1973).

Marr and Stuntz's 1973 monograph, Ramaria of w estern Washington,

is the most inclusive single publication of Ramaria of the Pacific Northwest.

Additional modern monographs of portions of the genus include those of R.

subgenus Lentoramaria, R. subgenus Echinoramaria and contributions

toward a Ramaria monograph as well as numerous works on tropical and

New Zealand Ramaria species (Corner, 1950, 1970; Petersen, 1974, 1975,

1976, 1979, 1981, 1982, 1987, 1988a, 1988b; Petersen and Scates, 1988).

Despite the amount of taxonomic literature on Ramaria, the phylogenetic

relationships of Ramaria species are unclear.

Representative taxa of the Gomphales have been included with larger

samplings of holobasidiomycetes in previous studies. Cladistic analyses of

nuc LSU rDNA, mt SSU rDNA and mt LSU rDNA have demonstrated strong

support for a monophyletic Gomphales Glade including Clavariadelphus,

Gautieria, Gomphus, Kavinia and Ramaria (Bruns et al., 1998; Hibbett et al.,

1997). Cladistic analyses of nuc LSU rDNA and mt SSU rDNA also have

demonstrated that members of the Phallales are closely related to the

Gomphales (Hibbett et al., 1997; Colgan et al., 1997). To date no molecular

phylogenetic studies have focused strictly on the Gomphales; however, a

recent phylogenetic study of thirty-nine morphological characters provided 21 support for the order Gomphales including the families Lentariaceae,

Ramariaceae, Beenakiaceae and Gomphaceae (Villegas et al., 1999).

Renewed attention was brought to Ramaria with the implementation of

The Northwest Forest Plan which was established to protect the habitat of the northern spotted owl. Putative old growth forest associates were outlined in the Record of Decision (ROD) for survey and management with Ramaria species accounting for 10% of the fungi (U.S.D.A., F.S.: U.S. DOI, BLM,

1994). Due to the inherent taxonomic difficulties of Ramaria, a molecular

investigation of both the nuc LSU rDNA and the mt SSU rDNA was

conducted to test generic, subgeneric and selected species concepts within

Ramaria.

MATERIALS AND METHODS

Sampling

Ramaria specimens from the State University of New York, Oneonta

(SUC) and the University of Tennessee, Knoxville (TENN) were studied

Four Ramaria species from the Fungal Survey and Management Team at the

USDA, Forest Service and one additional species were also included.

These were deposited in the Oregon State University herbarium (OSC). Ten

Ramaria species and one Gautieria species were sampled for the mt SSU

rDNA analyses and combined with an alignment from Hibbett et al. (1997) 22

(Matrix #M176) representing a large sampling of representative holobasidiomycete taxa. Sixty-four Ramaria specimens were sampled for the nuc LSU rDNA analyses representing thirty-four species. When possible, two collections of each species were sampled. This sampling included nineteen Ramaria holotypes and six paratypes. The Ramaria species sampled represent the four Ramaria subgenera as follows: R. subgenus

Ramaria (3 species), R. subgenus Laeticolora (24 species), R. subgenus

Echinoramaria (1 species) and R. subgenus Lentoramaria (6 species).

Seven additional genera were included in the analyses due to their close phylogenetic relationship to Ramaria as inferred from monographic, molecular and morphological analyses (Villegas et al., 1999; Bruns et al.,

1998; Hibbett et al., 1997; Colgan et al., 1997, Petersen, 1971, 1973, 1988b).

Five outgroup taxa were also included to root the ingroup taxa. See Table

2.1. for a list of taxa included in the analyses. 23

Table 2.1. Taxa included in the phylogenetic analyses

Taxon Isolate Gen Bank (nuc LSU, mt SSU) Ingroup taxa Ramaria R. subgenus Echinoramaria R. abeitinat SUC-M3222' R. subgenus Laeticolora R. acrisiccescens SUC-M535* SUC-M897 R. amyloideat SUC-M717* SUC-M670 R. araiosporat var. araiospora SUC-M739* SUC-M108 var. rubella SUC-M741 R. aura ntiisiccescenst SUC-M749* SUC-M738 R. celerivirescenst SUC-M841* SUC-M460 R. claviramulatat SUC-M392* R. conjunctipest var. sparsiramosa TENN-38412 TENN-38272 R. cystidiophora OSC T-5435 R. flavigelatinosa OSC T-5390 R. flavobrunnescens var. aromatica SUC-M7* SUC-M40 R. formosa SUC-M513B4 clone 2 SUC-M95-2 clone 7 SUC-M95-7 R. fumosiavellanea SUC-M730* R. gelatiniaurantiat var. gelatiniaurantia SUC-M479* var. violeitingens SUC-M829*HT R. gelatinosa var. oregonensis SUC-M250* SUC-M721 R. hilarist var. olympiana TENN-45965 24

Table 2.1. (Continued)

Taxon Isolate Gen Bank (nuc LSU, mt SSU) R. subgenus Laeticolora (Continued) R. largentiit SUC-M439* SUC-M441 R. lorithamnust TENN-43382 TENN-43404 R. magnipes OSC AJH-90 R. rubribrunnescenst SUC-M844* SUC-M614 R. spinulosat var. diminutiva TENN-33252 R. stuntziit SUC-M797* SUC-M214 R. thiersiit TENN-47006 TENN-47007 R. velocimutans OSC T-5439 R. versatilis var. violaceibrunnea SUC-M512* SUC-M66 R. vinosimaculans OSC T-5438 R. subgenus Lentoramaria R. concolort f. marrii TENN-34308 TENN-34190 R. gracilist SUC-M1570 R. pinicola SUC-M8V SUC-M752 R. rainierensist SUC-M227* SUC-M231 R. rubellat f. blanda TENN-32598 f. rubella SUC-M4OV SUC-M356 R. stricta var. concolor SUC-M419 var. stricta SUC-M4OV SUC-M261 25

Table 2.1. (Continued)

Taxon Isolate Gen Bank (nuc LSU, mt SSU) R. subgenus Ramaria R. botrytist var. botrytis SUC-M141 var. aurantiiramosa SUC-M803* SUC-M458 R. rubrievanescenst SUC-M584* SUC-M613 R. rubripermanenst SUC-M581* SUC-M582 Related ingroup taxa Clathrus ruber T9354 Clavariadelphus pistillaris OSC-69446 Gautieria G. gautierioides OSC-48547 G. graveolens OSC-41365 G. monticola SNF-115 1333407« G. morchelliformis OSC-41016 G. parksiana OSC-49803 Gomphus clavatust OSC-69447 Gomphus cf. floccosust G-010 G-014 Hysterangium clathroides SZEMORE Hysterangium coriaceum OSC-69448 Kavinia alboviridis SNF-284 1333491« Pseudocolus fusiformis ASM-4705 Outgroup taxa Bondarzewia montanat SARs.n AF042646 Polyporus tessellatulus OSC-54048 Ganoderma lucidum RZ X78776 Lactarius corrugis RV88/61 U11919 Russula mairei RV89/62 U11926 Herbarium source is listed for each isolate: OSC = Oregon State University, USA; SNF = Sierra National Forest; SUC = State University of New York, Oneonta, USA; TENN = University of Tennessee, Knoxville, USA. tSpecies protected by the Record of Decision. *Holotype specimens. Paratype specimens. Accession numbers for the Genome Sequence Database. Bold-faced isolates were included in the mt SSU rDNA dataset. 26

DNA extraction

A 2X CTAB protocol modified from Doyle and Doyle (1987) was used to extract genomic DNA from the five fresh fungal collections. A modified tissue protocol (Winton, pers. comm.) of the QlAamp Tissue Kit (QIAGEN, Inc.,

Valencia, CA) was used to isolate genomic DNA from dried herbarium specimens. The protocol was modified as follows: Dried herbarium material was ground in liquid nitrogen prior to addition of lysis buffer. The ATL Buffer was replaced with a 1% SDS Lysis Buffer (1% SDS, 200mM Tris (pH 7.5),

250 mM NaCI and 25 mM EDTA). For heavily pigmented tissue, polyvinylpolypyrrolidone and f3- mercaptoethanol were added to the lysis buffer prior to use at a final concentration of 1% and 0.1%, respectively.

Ground fungal material was incubated in 700 µL of 1% SDS Buffer for 50 min

@ 60 C followed by one chloroform extraction. The reaction tube was centrifuged for 5 min @ 8000 rpm then 600 µL of liquid lysate was transferred to a 2 mL reaction tube. An equal volume of AL Buffer was added to the lysate and the solution vortexed and incubated for 10 min @ 70 C. To this solution, 630 kL of 100% ethanol was added and vortexed. The lysate was applied to a QIAGEN spin column in 600 kL aliquots and spun 1 min @ 8000 rpm. The flow-through was discarded and more lysate was added and spun down until the lysate was gone. Using a new collection tube 500 kL AW

Buffer was added, centrifuged for 1 min @ 8000 rpm and the flow-through discarded. Again 500 µL AW Buffer was added, centrifuged for 3 min @ 27

8000 rpm and the flow-through discarded. To elute the DNA, 100 ill_ of 70 C ddH2O was added to the spin column, incubated for 5 min @ 70 C, centrifuged for 1 min @ 8000 rpm and collected in a new microcentrifuge tube.

DNA amplification of nuc LSU rDNA

Approximately 650 by of the nuc LSU rDNA was amplified via polymerase chain reactions (PCR) using the primers LROR and LR3 (Vilgalys and Hester, 1990). PCR reactions were performed in 50 viL reaction mixtures containing double distilled H2O, 1 41_ DNA template, 2 !IL of each 10 viM primer, 5µL of Taq 10X buffer, 54 of 1.25 mM dNTP mix, 3 pL of 25 mM

MgCl2 and 0.25 µL of 5 U/4 Taq polymerase. A drop of mineral oil was added to the reaction mixture and the DNA was amplified with a MJ Research

Programmable Thermal Controller (PTC)-100 thermal cycler (Watertown,

Massachusetts). Thermal cycling parameters were as follows: 94 C (1 min),

[94 C (1 min), 50 C (30 s), 72 C (45 s)] x 34 times, 72 C (3 min), and 4 C (15 min).

The primers LROR and LR3 (Vilgalys and Hester, 1990) were unable to amplify the nuc LSU rDNA of several herbarium collections. This inability to amplify appeared to be due to degraded DNA. For these cases, two new primers, RAM1 and RAM3, were designed using the program OligoTech version 1.00 (Oligos Etc. Inc. and Oligos Therapeutics, Inc., 1995) to split the 28

650 by region into two segments. LROR and RAM3 amplified 350 by at the beginning of the nuc LSU rDNA and RAM1 and LR3 amplified the following

400 bp. The RAM1 primer sequence is 5'-GCGAACAAGTACCGTGAG-3' and the RAM3 primer sequence is 5'-CGYGACTGACTTCAARCG-3'. PCR reactions were performed in 50 4 reaction mixtures containing double distilled H2O, 2 4 of DNA template, 3 4 of each 10 t.iM primer, 5 4 of Taq

10X buffer, 5 4 of 2 mM dNTP mix, 3 4 of 25 mM MgCl2 and 0.5 4 of 5

U/4 Taq polymerase. PCR reaction parameters were performed as follows:

94 C (1 min), [94 C (1 min), 45 C (1 min), 72 C (1 min)] x 4 times, [94 C (1 min), 50 C (1 min), 72 C (1 min)] x 29 times, 72 C (5 min), and 4 C (15 min).

DNA amplification of the mt SSU rDNA

Approximately 600 by of the mt SSU rDNA was amplified for eleven gomphalean taxa with the primers MS1 and MS2 (White et al., 1990). PCR reactions were performed in 50 µL reaction mixtures containing double distilled H2O, 2 pt of DNA template, 3 4 of each 10µM primer, 5 4 of Taq

10X buffer, 5 4 of 2 mM dNTP mix, 3 4 of 25 mM MgCl2 and 0.8 4 of 5

U/4 Taq polymerase. Optimal amplification conditions were as follows: 94 C

(3 min), [94 C (30 sec), 48 C (45 sec), 72 C (1 min)] x 4 times, [94 C (30 sec),

50 C (30 sec), 72 C (1 min)] x 35 times, 72 C (5 min) and 4 C (15 min). 29

DNA purification and sequencing

PCR products were electrophoresed on 1% agarose gels (Gibco-BRL ultraPURE, Life Technologies), stained with ethidium bromide and visualized using a transilluminator. Low DNA mass ladder (Gibco-BRL, Life

Technologies) was used to estimate PCR product size. PCR products of both the nuc LSU rDNA and the mt SSU rDNA were purified with the QlAquick

PCR Purification Kit (QIAGEN, Inc., Valencia, CA) following the manufacturer's protocol. Both the coding and template strands of purified

PCR products were sequenced with the primers LROR, LR3, RAM1, and

RAM3 for nuc LSU rDNA and MS1 and MS2 for mt SSU rDNA on an ABI

Model 373A (Perkin-Elmer) automated DNA sequencer at the Central

Services Laboratory of the Center for Gene Research and Biotechnology at

Oregon State University.

Phylogenetic analysis

The rDNA sequence contigs were assembled and edited with SeqEd version 1.0.3 (Applied Biosystems, Inc., 1992) and then aligned by visual examination with Paup* 4.0b2 (Swofford, 1999) and a color font. The mt

SSU rDNA sequence contigs were combined with selected taxa from the

Hibbet et al. (1997) Tree Base matrix. Ambiguous insertions/deletions

(indels) were treated as missing data in both datasets. Phylogenetic analyses were performed in PAUP* 4.0b2. Most parsimonious trees were recovered 30 using the heuristic search option (TBR and MULPARS on) and 100 replicates of random sequence addition. All most parsimonious trees recovered were subsequently compared to each other under the maximum likelihood criterion (Kishino-Hasegawa test); the implemented likelihood model assumed equal rates of substitution and empirical base frequencies. Support for individual branches was tested with bootstrap analysis under the parsimony criterion. Bootstrap analysis was based on 100 bootstrap replicates using the heuristic search option with five random sequence additions (TBR and MULPARS off).

Alternative phylogenetic hypotheses reflecting different classifications and species relationships were constructed in MacClade version 3.03

(Maddison and Maddison, 1992) and were used as constraint topologies in maximum parsimony analyses with the heuristic search option (100 random sequence additions, TBR and MULPARS off). Most parsimonious trees recovered with and without constraints were compared by the Kishino-

Hasegawa test implementing the likelihood model described above.

RESULTS

Sequence ambiguities

Sequence ambiguities were observed in both the forward and reverse nuc LSU rDNA sequence contigs of six Ramaria specimens: R. claviramulata

(SUC-M392), R. formosa (SUC-M513b and SUC-M95), R. hilaris var. 31

olympiana (TENN-45965), R. lorithamnus (TENN-43382) and R. pinicola

(SUC-M752). Of these specimens, the sequence reads obtained from the coding as well as the non-coding strands were completely unambiguous at the beginning of the reads, but at a certain point turned sharply into what appeared to be double sequences with most peaks appearing as double peaks. This phenomenon was interpreted as reflecting the presence of an additional base in some copies of the gene, and has been observed by others (Whittall, 1999; Kretzer, pers. comm.). Unambiguous sequence information from both strands could be assembled in most cases to provide a complete sequence with one-directional confidence. The nuc LSU rDNA of these specimens was amplified and sequenced at least twice with the same results.

All of the ambiguous sequence reads except R. formosa (SUC-M95) appeared to be due to a single base insertion. The nuc LSU rDNA of R. formosa (SUC-M95) was cloned and subsequently amplified because the raw sequences were too ambiguous to allow assembly of one directional sequences. Two cloned PCR products were successfully sequenced and revealed two independent base insertions and one A/G transversion at three different positions. Possible explanations for this phenomenon included either the presence of heterogeneous nuc LSU rDNA, a heterozygous species or an artifact of sequencing. The conserved sequences excluding the dubious base insertion were submitted to Gen Bank with the exception of

R. formosa (SUC-M95) for which both cloned sequences were submitted. 32 mt SSU rDNA trees

The mt SSU rDNA dataset contained 50 representative holobasidiomycete ingroup taxa and one outgroup taxon. The alignment contained 1108 characters of which 705 characters were excluded due to alignment ambiguities and large insertions/deletions (indels). Of the 705 excluded characters, 658 characters were due to three large indels of 142,

262 and 254 characters. Of the remaining characters, 209 were parsimony informative. Maximum parsimony analysis yielded 230 equally most parsimonious trees of 1183 steps. For these trees, the consistency index (CI) was 0.3187 and the retention index (RI) was 0.5179. A strict consensus tree is presented in Figure 2.1. The tree shown in Figure 2.2. was found to have the highest likelihood value of all 230 most parsimonious trees under the

Kishino-Hasegawa test; likelihood values were not significantly different among the trees. Bootstrap values greater than 50 are indicated above the respective internodes. 33

Panus rudis Antrodia carbonica 99 Lentinus tigrinus Trametes suaveolens Laetiporus sulphureus 676 Multiclavula mucida 96 Clavulina cristata Hydnum repandum Bondarzewia berkleyi Gloeophyllum sepiarium Laxitextum bicolor Russula compacta Clavicorona pyxidata Hericium ramosum Stereum hirsutum Panellus stypticus 636 Agaricus bisporus 98 Lepiota procera Tulostoma macrocephala Schizophyllum commune Pleurotus ostreatus Cyathus striatus Typhula phacorhiza 100 Boletus satanas Chamonixia caespitosa Thelephora sp. Phanerochaete chrysosporium Ceriporia purpurea Fomitopsis pinicola 62 Schizopora paradoxa Phellinus igniarius Hyphodontia alutaria 79 Sphaerobolus stellatus 62 Geastrum saccatum Pseudocolus fusiformis Gomphus floccosus 100 Ramaria stricta

Ramaria stricta SUC-M405 a Ramaria stuntzii S UC- M214 80 Ramaria rainierensis SUC-M231 94 Ramaria acris SUC-M404 a Ramaria gelatinosa SUC-M250* Ramaria gelatiniaurantia SUC-M479* Ramaria flavobrunnescens SUC-M7* Gautieria morchelliformis OSC-41016 Ramaria araiospora SUC-M739* Ramaria rubribrunnescens SUC-M844* Ramaria pinicola SUC-M899 Clavariadelphus pistillaris Albatrellus syringae Auricularia auricula.judae

Figure 2.1. Strict consensus cladogram of 230 equally most parsimonious trees of 1183 steps recovered from maximum parsimony analyses of mt SSU rDNA sequences. Bootstrap values greater than 50 are indicated above the respective internode. CI = 0.3187, HI = 0.6813, RI = 0.5179 and RC = 0.1651. 34

Coral Panus rudis Bondarzewia berkleyi

Cantharelloid _ I Gloeophyllum sepiarium _ Laxitextum bicolor El Club Russula compacta O "Stinkhorn" Clavicorona pyxidata Hericium ramosum O False truffle H Stereum hirsutum Panellus stypticus Agaricus bisporus 98 procera Tulostoma macrocephala euagarics

69 1 Schizophyllum commune Typhula phacorhiza II Cyathus striatus Pleurotus ostreatus 100 1-- Boletus satanas Boletales I Chamonixia caespitosa 01 Thelephora sp. Albatrellus syringae Phanerochaete chrysosporium Fomitopsis pinicola Schizopora paradoxa 62 Pheffinus igniarius Hyphodontia alutaria 79 Sphaerobolus stellatus 62 Geastrum saccatum Pseudocolus fusiformis 0 Phallales Gomphus floccosus 100 r Ramaria strict9. 1 Ramaria stricta M405* Ramaria gelatiniaurantia M479*HTII 94 Ramaria stuntzii M214 III Ramaria gelatinosa M250*HT "Gomphales" Ramaria flavobrunnescens M7*HT . Gautieria morcheffiformis OSC41016 0 Ramaria araiospora M739*HT Ramaria rubribrunnescens M844*HT III IQ Ramaria rainierensis M231. I Ramaria acris M404 * Ill .1 Ramaria pinicola M89*. L. Clavariadelphus pistillaris El Antrodia carbonica Lentinus tigrinus 99 L r Trametes suaveolens Laetiporus sulphureus 67 Multiclavula mucidall 96 Clavulina cristatall Hydnum repandum Ceriporia purpurea Auricularia auricula.judae 10 changes

Figure 2.2. Best -In likehood phylogram of 230 equally most parsimonious trees of 1183 steps recovered from maximum parsimony analyses of mt SSU rDNA sequences. Bootstrap values greater than 50 are indicated above the respective internodes. CI = 0.3187, HI = 0.6813, RI = 0.5179 and RC = 0.1651. 35 nuc LSU rDNA trees

The nuc LSU rDNA datasets contained 64 Ramaria sequence isolates,

14 closely related non-ramarioid taxa and five outgroup taxa. Several regions were problematic in the alignment and were characterized by positional homology that was relatively unambiguous among closely related taxa, but ambiguous among more distantly related taxa. Two exclusion sets were constructed to test the alignment, a conservative dataset excluding the ambiguous regions and a less conservative dataset including the ambiguous regions. The conservative nuc LSU rDNA alignment contained 674 characters of which 102 characters were excluded due to alignment ambiguities and indels. Of the remaining characters, 144 were parsimony informative. Maximum parsimony analysis yielded 1142 trees of 589 steps.

For these trees, the CI was 0.3294 and the RI was 0.7134. A strict consensus tree is presented in Figure 2.3. The tree shown in Figure 2.4. was found to have the highest likelihood value of all 1142 most parsimonious trees under the Kishino-Hasegawa test; likelihood values were not significantly different among the trees. Bootstrap values greater than 70 are indicated above the respective internodes.

The less conservative nuc LSU rDNA alignment also contained 674 characters, however only 71 ambiguous characters were excluded, resulting in 166 parsimony informative characters. Maximum parsimony analysis yielded 48 trees of 739 steps. For these trees, the CI was 0.3194 and the RI 36

100 SUC-M227* SUC-M231 R. rainierensis 97 steranitum clathroides steran conaceum ayarta ophus pishilans UU. -m405 R. stricta 100 BUC-M419a 1 ENN-34308 TENN-34190 R. concolor SUC-M261 R. stricta SNF-115 OSC-41365 82 SUC-M803* 96 SUC-M458 R. botrytis 70 SUC-M141 100 SUC--M582M581' SUC -M582 R. rubnpermanens 94 SUC-M584* SUC-M613 R. rubrievanescens QSC -41016 USC -49803 Gautieria OSC-48547 100 TENN-38412 R. conjunctipes 76 TENN-38272 100 TENN-43404 R. lorithamnus TENN-43382 95 SUC-M7' R. flavobrunnescens SUC-M40 QSQ-M1570 a. magnipes Ramaria SUU-M1570 grows and SUC-M404a 100 SUC-M356 R. rubella related taxa TENN-32598 SNF-284 Ilavinta SUC-M322a H. ablettna 100 SUC-M752 SUC-M89a R. pinicola 100 SUC-M797* R. stuntzii 97 SUC-M214 SUC-M479' 92 SUC-M829* R. gelatiniaurantia 83 TENN-45965 a. efts OSC-T5390 H. avigelatinosa 100 lath s ruber ocolus fusiformis 100 uu­ 117' R. amyloidea 82 SUC-M670 SUC-M841' R. celerivirescens 100 SUC-M460 $UQ-M392. R. claviramulata 98 sUu-m844* SUC-M614 R. rubribrunnescens 100 SUC-M513Ba cclloonnee 2 R. formosa

FUCC:MM9955 SUC-M739* 100 SUC-M108 R. araiospora SUC-M741 05Q-T5438 a. vinosimaculans OSC-T5435 H. cystriaropnora clavafus 100 Reirpophus 0014 Gomphus 94 SUC-M512* SUC-M66 R. versatilis SUC-M535* 100 R acrisiccescens SSU-M897UCC-M730* R. fumosiavellanea 99 SUC-M2572­M0* R gelatinosa SUC -M7211 100 TENN-47006 TENN-47007 R.. TENN-33252 R. spinulosa var. diminutiva 100 SUC-M738749M R. aurantiisiccescens 90 SUC SUC-'439 SUC-MM441 R. largentii OSC-T5439 R. velocimutans 96 Bondarzewia montana 100 ifstrti Pas 90 Ganoderma lucidum l-avolus

Figure 2.3. Strict consensus cladogram of 1142 equally most parsimonious trees of 589 steps recovered from maximum parsimony analyses of the conservative nuc LSU rDNA alignment. Bootstrap values greater than 70 are indicated above the respective internode. CI = 0.3294, HI = 0.6706, RI = 0.7134 and RC = 0.2350. 37

100 SUC-M227* R. rainierensisl R. subgenus Lentoramaria SUC-M231 SNF-115 OSC-41365 0-0SC-41016 Gautieria 0SC-49803 -OSC -48547 96 2-{ MAE R. botrytis var. aurantiiramosa 70 SUC-M141 R. bottytis var. botrytis 100 SUC M561 R. subgenus I suc-4582 R. rubnpermanens Ramaria 1 04 I sSuUcC:Mm658413* R. rubrievanescens 94 SUC-M512* R. versatilis var. violaceibrunnea ala var. gelatiniaurantia -2i SUC-M829* 83 e.antiniauranto var vioiettingens TENN-45965 . 1 aps var. olympiana f855AF90 R. flavigelatinosa R. subgenus 99 Laeticolora SUC-M721 R. gelatinosa 100 1.-5NN:4470089 thiersiiR. GomcTa 1 G01 010 I Gomphus I 100 1 G4 100 guUcC-m8M593F R acrisiccescens SUC:M730* R. fumosiavellanea i-iild8:1'474*95 R. flavobrunnescens var. aromatica OSC-AN frl ag11gieS 3 52 -.:pspinulosa var. diminutiva 100 .ilibinE:VA3* R. aurantiisiccescens 90 I §idt:FM R. largentii 100 TENN -38412 R. conjunctipes var. sparsiramosa 100 r TENN-43404 R. lorithamn us . TENN-43382 OSC-T5435 R sutidiophora 100 R. subgenus SUC-M fur R. stuntzii I SUC-M214 Laeticolora EliMloarie 2 I R. formosa Ramaria and 100 I SUC-M95 clone 7 100 related taxa 1 SUC-M717* RA. amyloidea 82 SUCg7An LSUCt640* R. celerivirescens 1°43 SUC-M392* R. claviramulata 98 SUC MR44 C soc:m6i4. R. rubribrunnescens OSC-T5438 R. vinosimaculans SUC-M739* R. araiospora var. araiospora 100 I SUC-M108SUC-MZ41 R. ar,aiospora var. rubella Obl; V39 H verocoutans SUC-M1570 SUCM404 10.100 I suc_10356AR. R rubella f. rubella I R. subgenus Lentoramaria TENN-32598 R. rubella 1. blanda 97 H ysc ia Hyscor 1Hysterangium 100 Clarub Phallales Psetus "stinkhorns" Clapis Clayarjadelphus SUC-M405a H. stncta var. stricta 100 SUC-M419 a R. stricta var. concolor R. subgenus Lentoramaria [-rami ?8 R. concolor f. marrii

SUC-M261 R.R. s,tricta .var. stricta SNF-284 rcavinia I UC-M322 a R. abietina R. subgenus Echinoramaria 100 SUC-M75 R. subgenus Lentoramaria SUC-M89 R. pinicola 96 Bondarzewia montana 100 Lactargssuia 90 ..anpderma lucidum ravolus 5 changes

Figure 2.4. Best -In likelihood phylogram of 1142 equally most parsimonious trees of 589 steps recovered from maximum parsimony analyses of the conservative nuc LSU rDNA alignment. Bootstrap values greater than 70 are indicated above the respective internode. CI = 0.3294, HI = 0.6706, RI = 0.7134 and RC = 0.2350. 38 was 0.7050. A strict consensus tree is presented in Figure 2.5. Bootstrap values greater than 70 are indicated above the respective internodes. 39

100 M22* R. rainierensis UU -M31 _-1,115.366 Gautieria 2 803* 94 458 R. botrytis 74 100 41. 582 R. rubripermanens

98 584* . _ 818 R.N rubrievanescens 49803 Gautieria .8547* 96 vi512 R. versatilis

6 -.U9* vi8299. R. gelatiniaurantia 95 N-45965 a. Naos H. tlavigelatinosa 100 T i9.,Qu R. gelatinosa 721 100 NN-470%06 R. thiersii 100 ff-§74-7 R. stuntzii 100 grus Gomphus floccosus 100 U 1M9* R. acrisiccescens U R. fumosiavellanea 98 76 VI* R. flavobrunnescens -AJI-191 R. magnipes 332528 . ffinulosa var. diminutiva N* R. aurantlistccescens 439* 441 R. largentii UC 739* 100 321 R. araiospora 99 77 -38272-38412 R. conjunctipes 100 -o R. lorithamnus -T5435 R. cystidiophora 100 77* 100 74 670. R. amyloidea 100 46841 R. celerivirescens 392: R. claviramulata 98 vl R. rubribrunnescens 98 N3Tne 2 R. formosa 95 c 0117 Q vi a I n 1g8 .. va?NuFaurg s 94 H clathroides s eran 'um conaceum at rus Der . . us fusitorms rg ppnus pistthans

100 41,9' 4 . R. stricta 73 _34418 R. concolor Uc-M261 stncta F- 4 avihia 4g H. acietina 100 d, R. pinicola -M1570 R. gracilis 100 it\jr\i--R/1_219'18, R. rubella 92 ondarzewia montana 100 cta ussu a 98 ano erma lucidum avo us

Figure 2.5. Strict consensus cladogram of 48 equally most parsimonious trees of 739 steps recovered from maximum parsimony analyses of the less conservative nuc LSU rDNA alignment. Bootstrap values greater than 50 are indicated above the respective internode. CI = 0.3194, HI = 0.6806, RI = 0.7050 and RC = 0.2251. 40

Character mapping

The tree shown in Figure 2.4. from the conservative nuc LSU rDNA alignment was used in Figures 2.6. 2.8. for mapping two morphological characters and the substrate habitat of the taxa sampled. For the taxa included in these analyses, the different gross basidiocarp morphologies are presented in Figure 2.6. A ramarioid morphology is inferred to be ancestral for the Gomphales with multiple derivations of diverse basidiocarp morphologies (i.e. club, false truffle, cantharelloid). The substrate habitat is presented in Figure 2.7. and indicates a lignicolous habitat as ancestral for the Gomphales with a single derivation of the terricolous habitat. The presence or absence of clamp connections is presented in Figure 2.8. The results inferred an ancestral clamped condition with multiple losses of clamps occurring in species of R. subgenus Laeticolora. 41

Gomphalean rainierensis morphologies Gautieria False truffle

botrytis )R. rubnpermanens

mbrievanescens

versatilis

4R. gelatiniaurantia hilatis R. flavigelatinosa gelatinosa

thiersii

Gomphus Cantharelloid \4:R. acrisiccescens R. fumosiavellanea flavobninnescens R. magnipes R. spmulosa R. R aurantiisiccescens

largentii

conjunctipes

lorithamnus R. cystidiophora \/,4,1 R. stuntzii

R. Formosa

) R. amyloidea /41 /2,R. celerivirescens R. claviramulata rubribrunnescens R. vinosimaculans

R. araiospora

R. velocimutans 2 R. gracilis

R. rubella

Hysterangium False truffle "stinkhorns" Phalloid Clavariadelphus Club 1, R. stricta \c: R. concolor R. stricta

A)Pvagetna Resupinate )R. pinicola Bondarzewia montana Poroid $ctartus ussula Gilled anoderma lucidum avolus Poroid

Figure 2.6. Gomphalean morphologies. White branches indicate a ramarioid morphology and shaded branches indicate other Gomphalean morphologies. Specific non-ramarioid morphologies are indicated next to the respective taxa. 42

rainierensis Substrate habitat Gautieria Terrestrial

R. botrytis F Lignicolous rubripermanens Duff or litter Mil . rubrievanescens

versatilis

R. gelatiniaurantia hilans navgelatinosa gelatinosa

. thiersii

Gomphus

R. acrisiccescens R. fumosiavellanea

. flavobrunnescens R. magnipes R. spinufosa aurantiisiccescens

largentii

conjunctipes

lorithamnus cystidiophora stuntzii

formosa

amyloidea

celerivirescens daviramulata rubribrunnescens vinosimaculans

R. araiospora velocimutans R. gracilis

IR rubella

Hysterangium Zchstinkhoms" lavariadelphus stricta concolor stricta Kama R. abet/1'1a

. pinicola Bondarzewia montana ctanus ussule anoderma lucidum )Favolus

Figure 2.7. Substrate habitat. Black branches indicate a terrestrial habitat, white branches indicate a lignicolous habitat and gray branches indicate a duff or litter habitat. 43

R rainierensis Clamp connections autieria Present Absent R. botrytis R. rubripermanens

R. rubrievanescens R. versatilis

4R. gelatiniaurantia hilaris R. flavigelatinosa R. gelatinosa

R. thiersii omphus clavatus )Gomphus floccosus N/4R. acrisiccescens fumosiavellanea R. flavobrunnescens R. ma,gnipes R. spinuiosa ),R. aurantiisiccescens

R. Iargentii z113. conjunctipes

N'1R. lorithamnus

. cystidiophora stuntzii

R. formosa

R. amyloidea 'N/;R. celerivirescens claviramulata rubdbrunnescens N#2R. vinosimaculans

araiospora R. velocimutans R. gracilis R. rubella

ysterangium stinkhorns" Clavariadelphus R. stricta R. concolor stricta avola amettna R. pinicola ondarzewia montana actarius ssula anoaerma lucidum Favolus

Figure 2.8. Clamp connections. Black branches indicate the presence of clamps and white branches indicate the loss of clamps. 44

Kishino-Hasegawa test results

The results of the Kishino-Hasegawa test are presented in Table 2.2.

Hypotheses tested included: i.) The genus Ramaria constrained to monophyly, ii.) R. subgenus Laeticolora constrained to monophyly, iii.) R. subgenus Lentoramaria constrained to monophyly, iv.) Gomphus excluded from the ingroup, v.) Gautieria excluded from the ingroup, vi.) The Phallales excluded from the ingroup, vii.) Kavinia excluded from the ingroup, viii.)

Clavariadelphus excluded from the ingroup, and ix.) R. araiospora and R. stuntzii constrained to monophyly. 45

Table 2.2. Kishino-Hasegawa likelihood test results

Topology # Trees Range -In Range P* Likelihood Unconstrained 1 3056.8826 Best Monophyletic 4 3144.0881 <0.0001 - Ramaria 3151.5676 0.0008 ** Monophyletic R. 5 3079.9483 0.1063 subg. Laeticolora 3089.1307 0.1769 Monophyletic R. 4 3094.2169 0.0091­ subg. Lentoramaria 3098.3134 0.0498**

Exclude Gomphus 1 3070.4077 0.5019

Exclude Gautieria 1 3089.6506 0.1380 Exclude the 3 3071.7337 0.3038 Phallales 3079.0562 0.5854 Exclude Kavinia 4 3070.5099 0.3842 3075.9683 0.4116 Exclude 3 3072.5170 0.2677 Clavariadelphus 3075.7742 0.4315 Monophyletic R. 2 3072.7843 0.3843 araiospora/R. stuntzii 3076.5182 0.5040 * Probability of getting a more extreme T-value under the null hypothesis of no difference between the two trees (two-tailed test) ** Significant at P < 0.05 The best -In likelihood tree of 1142 equally most parsimonious trees 46

DISCUSSION

Sequence ambiguities

Heterogeneous nuc LSU rDNA could result from a hybrid organism, and in such cases, the two cloned sequences of R. formosa would not be expected to group together in phylogenetic analyses. Analyses that included both sequences (Figures 2.3., 2.4. and 2.5.), however, formed a monophyletic Glade and did not show any evidence of a hybrid organism.

Another explanation for this phenomenon is a heterozygous species which possesses two different rDNA alleles due to intraspecific mating. The analyses did not dispute this explanation considering both clones formed a monophyletic group; however, the analyses were not a test of this hypothesis.

The sequencing artifact could result from a constrained loop configuration on one strand of the rDNA that maintains its secondary structure causing the polymerase to skip over a nucleotide as it moves along the template strand.

DMSO was added to the sequencing reaction to reduce the formation of secondary structures; however, the ambiguous sequences remained. This phenomenon was not investigated further and remains an enigma.

Support for the Gomphales-Phallales relationship

The two independent loci examined in these analyses demonstrated strong support for the close relationship of gomphalean taxa and the 47

Phallales with bootstrap values of 94 for the mt SSU rDNA (Figures 2.1. and

2.2.) and 97 (Figures 2.3. and 2.4.) and 100 (Figure 2.5.) for the nuc LSU

rDNA. Both loci demonstrate the close relationship of Clavariadelphus,

2.6.). In the nuc LSU Gautieria , Gomphus and Ramaria (Figures 2.1.

rDNA, members of the Phallales appeared within the Gomphales (Figures

2.4. and 2.5.); however, the one member of the Phallales, Pseudocolus

fusiformis, sampled in the mt SSU rDNA was a sister taxon to the Gomphales

(Figure 2.2.). Conflicts between the two loci were limited to weakly or

unsupported branches. Exclusion of the Phallales from the ingroup taxa in

the nuc LSU rDNA dataset did not produce a significantly worse tree

according to Kishino-Hasegawa p-values (p-values = 0.3038 0.5854). The

possible sister

the mt SSU rDNA analysis was not rejected by the nuc LSU rDNA analyses.

For this discussion, the Gomphales as indicated in the nuc LSU rDNA will

refer to all the ingroup taxa except the Phallales.

Rejection of a monophyletic genus Ramaria

Molecular analyses inferred that the genus Ramaria was paraphyletic

(Figures 2.1. 2.5.). Clavariadelphus, Gautieria and Gomphus were nested

taxa within Ramaria for both the mt SSU rDNA (Figures 2.1. and 2.2.) and

nuc LSU rDNA (Figures 2.3., 2.4. and 2.5.) analyses. Kavinia and the

Phallales including Clathrus, Hysterangium and Pseudocolus were nested 48 within the genus Ramaria for the nuc LSU rDNA analyses (Figure 2.3. and

2.4.). The relationships of related taxa (e.g. Gautieria) to ramarioid fungi were stable throughout taxon sampling but received low bootstrap support.

Kishino-Hasegawa tests indicated a significantly worse tree when the genus

Ramaria was constrained to monophyly (p-value = <0.0001 - 0.0008) (Table

2.2.), but the exclusion of individual non-ramarioid taxa from the ingroup did not result in significantly worse trees according to Kishino-Hasegawa p- values.

Testing subgeneric and species concepts within Ramaria using nuc LSU rDNA

The three species of R. subgenus Ramaria sampled consistently formed a monophyletic group (Figure 2.3., 2.4. and 2.5.). The hypogeous gasteromycete, Gautieria, was a sister group to this Ramaria Glade. Although bootstrap support for this sister group relationship was low, this relationship was consistent throughout the development of taxon sampling. Furthermore, the longitudinally ridged spores of Gautieria provide a strong correlation for its relationship to the only Ramaria subgenus whose species possess striate spores. Kishino-Hasegawa p-values did not indicate a significantly worse tree when Gautieria was excluded from the ingroup (Table 2.2.), however, the p-value (p-value = 0.1380) was lower than for other excluded taxa. The two varieties of R. botrytis formed a biphyletic Glade in these analyses. The two varieties differ macroscopically in the color of their terminal branches and in 49

slight microscopic differences in their spore ornamentation and contextual

hyphae. Although the analyses were not definitive tests of these varieties

representing distinct species, they were consistent with the varieties

representing unique taxa.

Ramaria subgenus Laeticolora species formed a paraphyletic grade

with a nested Gomphus and a terminally derived R. subgenus Ramaria

/Gautieria Glade (Figures 2.4. and 2.5.). Gomphus was a sister taxon to R.

acrisiccescens and R. fumosiavellanea but its placement was not supported

by bootstrap values. The Kishino-Hasegawa p-value (p-value = 0.5019) did

not demonstrate a significantly worse tree when R. subg. Laeticolora was

constrained to monophyly. Variability in R. subgenus Laeticolora species is

reflected in the range of spore ornamentation from warty to smooth seen in

species within the subgenus. Past investigators have suggested further

division of this subgenus but the variability and lack of consistent characters

on which to divide its members has proven this to be a difficult task (Marr and

Stuntz, 1973; Petersen, 1969a). Additional taxon and character sampling of

Gomphus and R. subgenus Laeticolora is needed to clarify the relationships

of this large diverse group.

Molecular analyses indicated several species relationships within the

R. subgenus Laeticolora paraphyletic grade that were supported by high

bootstrap values. R. amyloidea and R. celerivirescens are a species pair that

differ in the presence of clamps, basidiocarp form and spore ornamentation

(Marr and Stuntz, 1973). The analyses supported the hypothesis that these 50 taxa are distinct, closely related species. The species concept for R. celerivirescens was complicated, however, by the placement of R. claviramulata within the R. celerivirescens Glade. R. claviramulata is unique in basidiocarp form with "chubby" blunt branches (Marr and Stuntz, 1973).

The monophyly of R. celerivirescens and R. claviramulata suggests that R. claviramulata may be an aborted or environmental mutant of R. celerivirescens. These three species are united by several characters that are differentially distributed among them. Both R. amyloidea and R. celerivirescens have an amyloid context; whereas, R. claviramulata does not.

Conversely, R. amyloidea and R. claviramulata both possess spores with fine, lobed warts; whereas, R. celerivirescens possesses spores with coarse, irregular warts. turns green with ferric sulfate and regions of dark brown hyphae are apparent on the base of the stipe in all three species.

The gelatinous Ramaria species including R. gelatiniaurantia, R.

hilaris, R. flavigelatinosa, R. gelatinosa and R. thiersii were all closely related

in the nuc LSU rDNA analyses (Figures 2.4. and 2.5). The two varieties of R.

gelatiniaurantia formed a monophyletic Glade with R. hilaris. Both are

gelatinous species that are similar in spore size and ornamentation,

clampless hyphal state and general macroscopic and macrochemical

characters. R. flavigelatinosa was a sister group to this monophyletic Glade

and differs primarily in fruit body color. 51

R. acrisiccescens and R. fumosiavellanea formed a monophyletic

Glade (Figures 2.3 2.5.). These two species differ slightly in most characteristics, however their spores are extremely similar in size and ornamentation. R. aurantiisiccescens and R. largentii, were also supported as sister taxa in these analyses receiving a bootstrap value of 90 (Figure

2.4.). R. aurantiisiccescens does not possess clamps while R. largentii does.

The most striking difference between the two is the grossly enlarged spores of R. largentii. R. conjunctipes and R. lorithamnus were also sister taxa in these analyses, receiving a bootstrap value of 76. Kishino-Hasegawa p- values indicated that constraining R. araiospora and R. stuntzii to monophyly

did not result in a significantly worse tree (p-value = 0.3843 0.5040). These two species differ primarily in their contextual amyloid reaction; however, they

never grouped closely in these phylogenetic analyses.

R. subgenus Lentoramaria species also formed a paraphyletic grade

(Figures 2.4. and 2.5.). Clavariadelphus and the Phallales, including

Hysterangium, Clathrus and Pseudocolus, as well as the one species of R.

subg. Echinoramaria sampled, were nested within R. subgenus

Lentoramaria. Clavariadelphus was a sister taxon to the R. stricta complex

and the Phallales were a sister Glade to the Clavariadelphus-R. stricta Glade.

The relationship between these two clades was not supported by bootstrap

values and exclusion of the Phallales did not produce a significantly worse

tree according to Kishino-Hasegawa p-values (p-value = 0.3038-0.5854).

Excluding Clavariadelphus from the ingroup also did not result in a 52 significantly worse tree (p-value = 0.2677 0.4315). Constraining R. subgenus Lentoramaria to monophyly did, however, produce a significantly worse tree (p-value = 0.0091 0.0498). The R. stricta complex included the paratypes, R. stricta var. stricta and R. stricta var. concolor, two specimens of

R. concolorf. marrii and one R. stricta var. stricta collection (SUC-M261) which grouped outside of the primary R. stricta Glade.

R. abietina, the one member of R. subgenus Echinoramaria sampled, grouped with the resupinate Kavinia alboviridis which were both inferred to share a common ancestor with the smooth-spored R. pinicola in the basal portion of R. subgenus Lentoramaria. Exclusion of Kavinia from the ingroup taxa did not result in a significantly worse tree (p-value = 0.3842-0.4116).

Character mapping

These analyses indicated an ancestral lignicolous habitat for the

Gomphales with a single derivation of the terricolous habitat occurring in the ancestor of R. rainierensis of R. subgenus Lentoramaria (Figure 2.7.). The mycorrhizae of four terrestrial R. subgenus Laeticolora species has been described (Agerer et al., 1996) and it is hypothesized that more terrestrial

Ramaria species are mycorrhizal. Related taxa including Gautieria and

Hysterangium are known mycorrhizal associates (Castellano, 1988; Griffiths et al., 1991; Miller, 1988). 53

The lignicolous nature of the majority of R. subgenus Echinoramaria and R. subgenus Lentoramaria species was inferred to be ancestral for the

Gomphales from these analyses (Figure 2.6.). R. abietina and R. pinicola

both grow on needle or twig litter and possess monomitic rhizomorphs.

These two species appear closely related to the R. stricta complex which has

species that grow on decayed wood and possess dimitic rhizomorphs.

These taxa share a recent common ancestor with R. gracilis and R. rubella

both of which also grow on rotten wood and possess dimitic rhizomorphs.

Members of R. subgenus Laeticolora, R. subgenus Ramaria and R.

rainierensis of R. subgenusLentoramana possess monomitic hyphae and a

terrestrial habitat. From these groupings one could infer a lignicolous nature

as primitive for the Gomphales (Figure 2.7.).

The majority of Ramaria species sampled possess clamps with the

exception of several species in R. subgenus Laeticolora. Marr and Stuntz

(1973) identified species pairs that differ primarily in the possession of

clamps. R. amyloidea and R. celerivirescens are one such species pair. R.

amyloidea possesses clamp connections and R. celerivirescens does not.

These taxa were inferred to be distinct, sister species highlighting the

usefulness of this character to separate the two species. The two Gomphus

species that grouped within R. subgenus Laeticolora were represented by

the species with clamps, G. clavatus, and the clampless species, G.

floccosus. These analyses indicated that the possession of clamp 54 connections was ancestral with multiple losses of this character in members

of R. subgenus Laeticolora (Figure 2.8.).

Polarity of basidiocarp morphology

These analyses suggest a ramarioid or branched coral morphology as

ancestral for the Gomphales with multiple derivations of distinct basidiocarp

morphologies, (i.e. club, false truffle, cantharelloid). Other investigators have

recognized the relatedness of diverse morphologies within this order from

microscopic and macrochemical characters including the cyanophilous

ornamented spores, basidiocarp hyphal construction and positive chemical

reaction in ferric sulfate (Donk, 1961; Petersen, 1971; Villegas et al., 1999).

Eriksson (1954) recognized the cyanophilous nature and similar spore

characteristics of Ramaria and Kavinia. Petersen (1971) proposed an

ancestral gomphoid morphology with multiple derivations of the ramarioid

habit. Two Gomphus species representing two distinct Gomphus subgenera

were included in the analyses. The results of the analyses did not support

Petersen's hypothesis and instead suggested an ancestral ramarioid

morphology for the Gomphales (Figure 2.6.). Petersen (1971) also

hypothesized that the resupinate genera Kavinia and Ramaricium were

derived from the R. stricta complex. These analyses indicated that Kavinia

may be more closely related to R. abietina of R. subgenus Echinoramaria

than to the R. stricta complex (Figures 2.3. 2.5.). 55

Marr and Stuntz (1973) proposed three evolutionary grades within

Ramaria. The first specified a primitive terrestrial Ramaria of medium to large size with coarsely warted spores, clamp connections and either a violaceous fruitbody or a fruitbody that reacted positively with most chemical reactions.

Marr and Stuntz hypothesized that terrestrial Ramarias of medium to large size that were either white or brightly colored were derived from this primitive group. Members of this evolutionary grade possessed either one or more of the following: clampless hyphae, smooth or finely warted narrow spores, an amyloid context and fruitbodies that reacted negatively with most macrochemical tests. The final grade included both lignicolous and terrestrial clamped species of small to large size that were variously colored.

This evolutionary grade was further defined by one or more of the following characters: skeletal hyphae, rhizomorphs and mycelial mats as well as striate, echinulate or smooth spores.

The species complexes outlined by Marr and Stuntz (1973) for each evolutionary grade were consistent with the molecular data; however, the polarity of the evolutionary grades was not supported. The molecular analyses indicated a lignicolous or duff habit as primitive with a progression towards a diverse terrestrial habit (Figure 2.7.). Clamp connections were

inferred to be an ancestral character with multiple losses of the clamped

condition (Figure 2.8.). Corner (1966) noted varying degrees of clamp

possession in the species he studied and assumed that the clampless state

was a derivative of the clamped state with intermediate degrees of clamps. 56

Interestingly, the third evolutionary grade described by Marr and

Stuntz (1973) is characterized by mat-forming species and includes the R. botrytis complex and the R. stricta complex both of which form sister groups to separate genera of hypogeous ectomycorrhizal mat-forming false truffles

(Griffiths et al., 1991) in the nuc LSU rDNA analyses. The R. botrytis complex was sister to Gautieria and the R. stricta complex was sister to the Phallales including Hysterangium.

Evolution of hypogeous fungi from epigeous Gomphales

Gautieria

Both the nuc LSU rDNA and the mt SSU rDNA analyses indicated that

Gautieria Vitt. was closely related to Ramaria. Figure 2.4. from the nuc LSU rDNA analyses inferred Gautieria to be a sister group to R. subgenus

Ramaria. Exclusion of Gautieria from the ingroup did not result in a significantly worse tree; however, the Kishino-Hasegawa p-value was low

(Table 2.2.). Gautieria is a hypogeous fungus in the family Gautieriaceae

(Gautieriales) that forms tuber-shaped sporocarps in the upper layers of the

mineral soil. A peridium is reported in the early stages of Gautieria

development (Zeller and Dodge, 1918); however, with maturity the peridium

of some Gautieria species becomes evanescent and often absent with the

glebal chambers opening to the exterior of the sporocarp. This "false truffle"

possesses a distinct columella that branches into tramal plates that form 57 many glebal chambers that are lined by fertile hymenium. Gautieria sporocarps are defined by their coralloid glebal chambers and striate spores.

Fischer (1933) and Cunningham (1942) both described the development of Gautieria as coralloid with the growth of tramal plates outward from a central sterile base resulting in the formation of pockets or locules. The locules are lined by hymenium which eventually darkens upon spore maturation. Alternatively, the development of Gautieria has been described as (orate by other authors with formation of the branches occurring from inward growth of exterior tissue (Fitzpatrick, 1913; Dring, 1973; Miller and Miller, 1988). The Gautieriales possess a true hymenium with one side of the basidia exposed to an open air space and the other side bound by the trama (Dring, 1973). For this reason, Gautieria was hypothesized to have evolved from a hymenomycete (Dring, 1973).

The relationship of Gautieria to other fungal taxa has been disputed by many authors. Gautieria was hypothesized to be closely related to members of the Boletales through the hypogeous intermediate, Chamonixia, due to

possession by both taxa of longitudinally ribbed spores (Smith, 1973).

Dodge and Zeller (1934), on the other hand, classified Gautieria in the

Hymenogasteraceae while other authors placed the genus in the

Hysterangiaceae due to the distinct columella in the gleba.

These analyses supported the hypothesis that Gautieria evolved from

a hymenomycete, in particular, a ramarioid ancestor and that it is not closely

related to Chamonixia of the Boletales. This finding is consistent with that of 58

Bruns et al. (1998) which showed that Gautieria was closely related to

Gomphus, Kavinia and Ramaria using the mt LSU rDNA gene. The five species of Gautieria sampled form a group that is closely related to the only

Ramaria subgenus with species that possess striate spores, R. subgenus

Ramaria (Figures 2.3, 2.4 and 2.5.). Both taxa possess relatively long striate basidiospores (ie., 10-20um). The spores of Gautieria differ from R. subgenus Ramaria in that they are statismosporic not ballistosporic. The loss of ballistospory is hypothesized to be a consequence of a sequestrate habit (Hibbett et al., 1997; Thiers, 1984). Statismospores generally develop symmetrically on the sterigmata and in the case of Gautieria the spores are longitudinally ribbed.

Hysterangium

In the nuc LSU rDNA tree (Figures 2.4. and 2.5.), the sequestrate

Hysterangium Vitt., was nested along with two other members of the

Phallales within R. subg. Lentoramaria. Bootstrap values and Kishino-

Hasegawa p-values, however, did not support the placement of this taxa in R.

subg. Lentoramaria. The mt SSU rDNA dataset, placed Pseudocolus as a

sister group to the Gomphales Glade thus providing additional support for a

close relationship between the Phallales and the Gomphales. Hysterangium

species produce hypogeous, tuber-shaped sporocarps that possess a

distinct peridium and a gelatinous gleba with a usually distinct columella. 59

The mature gleba is olive or green in color and the spores usually possess a utricle which is a unique character uniting Hysterangium with the "stinkhorns" in the Phallales. The fruitbodies of Hysterangium resemble some "stinkhorn eggs" and the development of sporocarps is described as aulaeate (Miller and Miller, 1988). The spore surface under the utricle may be smooth to finely warted (Castellano, 1988). These findings were consistent with preliminary molecular analyses that support a close relationship between the

Phallales and Hysterangium (Colgan et al., 1997). The relationship of the

Phallales and the Gomphales, however, was indeterminate from these data.

TAXONOMIC CONSIDERATIONS

These phylogenetic analyses indicated that the genus Ramaria is not

monophyletic and therefore raise taxonomic and nomenclatural questions

regarding the division of this potentially paraphyletic genus. In the current

analysis, at least six other fungal genera and the order Gautieriales group

within what is currently recognized as Ramaria. For this reason, further

sampling of gomphalean taxa is needed to make phylogenetically sound

taxonomic revisions within the Gomphales. Two potential taxonomic

proposals are discussed below.

Division of the genus Ramaria into additional monophyletic groups is

one taxonomic approach. R. botrytis is the designated type of the genus and

therefore it is correct for the monophyletic Glade containing this species to 60

retain the generic name, Ramaria. Such a proposition would restrict a generic concept of Ramaria to species of R. subgenus Ramaria. R. subgenus

Laeticolora is not part of the monophyletic Glade including R. subgenus

Ramaria. R. subgenus Laeticolora is a paraphyletic taxon (Figures 2.4. and

2.5.); however, the monophyly of this subgenus is not rejected by the Kishino-

Hasegawa test (Table 2.2.); therefore, additional sampling of R. subgenus

Laeticolora is needed before subdivision of this subgenus can be undertaken. The subgeneric name Laeticolora may be raised to the generic level for the species that form a monophyletic group with the type of this subgenus, R. formosa.

R. subgenus Lentoramaria is the most problematic Ramaria subgenus

in these analyses. This subgenus is paraphyletic and its monophyly is

significantly rejected by these data (Table 2.2.). Revisions within R.

subgenus Lentoramaria will be complicated if further sampling places R.

stricta, the type of the subgenus, sister to Hysterangium and the Phallales.

The remaining species currently recognized in R. subgenus Lentoramaria do

not form a monophyletic group and thus are subject to reclassification.

Additional sampling of R. subgenus Echinoramaria is needed. These

analyses inferred a close relationship between R. abietina and the

resupinate Kavinia.

An alternative taxonomic approach would be the recognition of a

paraphyletic genus Ramaria. Considering that the ramarioid morphology is

ancestral for all the diverse morphological lineages included in this study this 61 approach may seem logical. The difficulty with this proposal, however, lies in the current system of rank and the communication of phylogenetic relationships. If Ramaria were maintained as a paraphyletic genus then the other taxa derived from ramarioid taxa would be treated at a rank lower than genus according to the current system of classification. For example,

Gautieria and Gomphus would be reduced to subgeneric ranks classified within Ramaria. Additional taxon and character sampling, development of more robust phylogenetic hypotheses and further exploration of these taxonomic alternatives is needed before any meaningful and more accurate classification can be developed for the Gomphalean fungi.

Bootstrap support was high for inclusion of all the ingroup taxa in the

order Gomphales. Kishino-Hasegawa p-values indicated a significantly

worse tree when all of the non-Ramaria taxa were excluded from the ingroup.

The monophyly of this order was supported by the mt SSU rDNA dataset

which inferred strong support for a monophyletic Phallales/Gomphales Glade.

Until now investigators have relied on the multitude of characters offered by

the sporocarps of Ramaria to unearth their relation to one another. Additional

sampling and combined morphological and molecular data will contribute to

a better understanding of the evolution of the many diverse morphologies in

the order Gomphales. 62

ACKNOWLEDGEMENTS

We would like to thank Dr. Currie Marr and Dr. Ronald Petersen for loaning Ramaria collections, Dr. Annette Kretzer for critical review of the

manuscript and Dr. David Gernandt for cloning R. formosa. This study was funded by a Joint Venture Agreement with the USDA, Forest Service, PNW

Research Station.

LITERATURE CITED

Agerer, R., R. M. Danielson, S. Egli, K. Ingleby, D. Luoma and R. Treu, eds. 1996. Descriptions of Ectomycorrhizae. 1: 107-130.

Bruns, T. D., T. M. Szaro, M. Gardes, K. W. Cullings, J. J. Pan, D. L. Taylor, T. R. Horton, A. Kretzer, M. Garbelotto, and Y. Li. 1998. A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis. Molecular Ecology 7: 257-272.

Castellano, M. A. 1988. The taxonomy of the genus Hysterangium (Basidiomycotina, Hysterangiaceae) with notes on its ecology. Ph.D. Dissertation, Oregon State University, Corvallis.

Colgan, W., M. A. Castellano, and J. W. Spatafora. 1997. Systematics of the Hysterangiaceae (abstract). lnoculum 48(3): 7.

Corner, E. J. H. 1950. A monograph of Clavaria and allied genera. Oxford University Press, London.

Corner, E. J. H. 1966. Species of Ramaria (Clavariaceae) without clamps. Trans. Br. Mycol. Soc. 49(1): 101-113.

Corner, E. J. H. 1970. Supplement to " A monograph of Clavaria and allied genera. Beihefte zur Nova Hedwigia 33: 1-299.

Cunningham, G. H. 1942. The Gasteromycetes of Australia and New Zealand. John Mclndoe, Dunedin. 63

Dodge, C. W. and S. M. Zeller. 1934. Hymenogaster and related genera. Ann. Mo. Bot. Gard. 21: 625-708.

Donk, M. A. 1961. Four new families of Hymenomycetes. Persoonia 1(4): 405-407.

Doyle, J. J. and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15.

Dring, D. M. 1973. Gasteromycetes. Pp. 451-476. In: The fungi, an advanced treatise. Eds., G. C. Ainsworth and A. S. Sussman. Academic Press, New York.

Eriksson, J. 1954. Ramaricium nov. gen., a corticioid member of the Ramaria group. Svensk Bot. Tidskr. 48: 188-198.

Fischer, Ed. 1933. Gastromyceteae. In: Engler & Prantl. Die Naturl. Pflanzenfam. (II ed.) 7a: 1-122.

Fitzpatrick, H. M. 1913. A comparative study of the development of the fruit body in Phallogaster, Hysterangium, and Gautieria. Ann. Mycologici von Sydow 11: 119-149.

Griffiths, R. P., M. A. Castellano, and B. A. Caldwell. 1991. Hyphal mats formed by two ectomycorrhizal fungi and their association with Douglas-fir seedlings: A case study. Plant and Soil 134: 255-259.

Hibbett, D. S., E. M. Pine, E. Langer, G. Langer, and M. J. Donoghue. 1997. Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences. Proc. Natl. Acad. Sci. 94: 12002-12006.

Maddison, W. P. and D. R. Maddison. 1992. MacClade. Version 3.03. Sinauer Associates, Inc., Sunderland, Massachusetts.

Marr, C. D. and D. E. Stuntz. 1973. Ramaria of Western Washington. Bibliotheca Mycologica 38: 232p.

Miller, 0. K., Jr., and H. H. Miller. 1988. Gasteromycetes: Morphology and Developmental Features. Mad River Press, Eureka, CA.

Petersen, R. H. "1967" (1969a). Type studies in the Clavariaceae. Sydowia 21: 105-122. 64

Petersen, R. H. 1971. Interfamilial relationships in the clavarioid fungi and cantharelloid fungi. Pp. 345-374. In: Evolution in the higher Basidiomycetes. An International Symposium. Ed., R. H. Petersen. University of Tennessee, Knoxville.

Petersen, R. H. 1973. Aphyllophorales II: The clavarioid and cantharelloid Basidiomycetes. Pp. 351-367. In: The fungi, an advanced treatise. Eds., G. C. Ainsworth and A. S. Sussman. Academic Press, New York.

Petersen, R. H. 1974. Contribution toward a monograph of Ramaria. I. Some classic species redescribed. Amer. J. Bot. 61(7): 739-748.

Petersen, R. H. 1975. Ramaria subgenus Lentoramaria, with emphasis on North American taxa. Bibliotheca Mycologica 43.

Petersen, R. H. 1976. Contribution toward a monograph of Ramaria. III. R. sanguinea, R. formosa, and two new species from Europe. Amer. J. Bot. 63(3): 309-316.

Petersen, R. H. 1979. Contribution to a monograph of Ramaria. IV. R. testaceo-flava and R. bataillei. Nova Hedwigia 31: 25-38.

Petersen, R. H. 1981. Ramaria subgenus Echinoramaria. Bibliotheca Mycologica 79.

Petersen, R. H. 1982. Contributions toward a monograph of Ramaria. V. Type specimen studies of taxa described by W. C. Coker. Sydowia Anna les Mycologici 35: 176-205.

Petersen, R. H. 1986. Some Ramaria taxa from Nova Scotia. Can. J. Bot. 64: 1786-1811.

Petersen, R. H. 1987. Contribution toward a monograph of Ramaria. VI. The Ramaria fennica versatilis complex. Sydowia 40: 197-226.

Petersen, R. H. 1988a. Contribution toward a monograph of Ramaria. VII. New taxa and miscellany. Mycologia 80(2): 223-234.

Petersen, R. H. 1988b. The clavarioid fungi of New Zealand. Science Information Publishing Centre, Wellington.

Petersen, R. H. and C. Scates. 1988. Vernally fruiting taxa of Ramaria from the Pacific Northwest. Mycotaxon 33(Oct-Dec): 101-144. 65

Smith, A. H. 1973. Agaricales and related secotioid Gasteromycetes. Pp. 421-450. In: The Fungi IVB, A Taxonomic Review with Keys: Basidiomycetes and Lower Fungi. Academic Press, London.

Swofford, D. L. 1999. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4. Sinauer Associates, Inc., Sunderland, Massachusetts.

Thiers, H. D. 1984. The secotioid syndrome. Mycologia 76(1): 1-8.

U.S.D.A., F.S.: U.S. DOI, BLM. 1994. Record of Decision for amendments to FS and BLM planning documents within the range of the northern spotted owl. 74 p. Plus Attachment A: Standards and Guidelines.

Vilgalys, R. and M. Hester. 1990. Rapid genetic identification and mapping of enzymatically amplified DNA from several Cryptococcus species. J. Bacteriol. 172(8): 4238-4246.

Villegas, M., E. de Luna, J. Cifuentes, and A. E. Torres. 1999. Phylogenetic studies in Gomphaceae sensu lato (Basidiomycetes). Mycotaxon 70(Jan-Mar): 127-147.

White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols: A Guide to Methods and Applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White. Academic Press, New York.

Whittall, J. B. 1999. Molecular phylogeny for the Mimulus moschatus alliance (Scrophulariaceae) and its conservation implications. MS thesis. Oregon State University, Corvallis.

Zeller, S. M. and C. W. Dodge. 1918. Gautieria in North America. Ann. Mo. Bot. Gard. 5: 133-142. 66

CONCLUSIONS

GENERIC, SUBGENERIC AND SPECIES CONCEPTS IN THE GOMPHALES

The current taxonomic classification of the genus Ramaria

(Gomphales) was tested through phylogenetic analyses of nucleotide sequence data from ribosomal DNA. The monophyly of the genus Ramaria was tested as well as the monophyly of three of the four Ramaria subgenera.

Selected species concepts were also examined within the genus.

Phylogenetic analyses inferred that the genus Ramaria was not monophyletic. Genera known to be closely related to Ramaria based on spore characters and a positive macrochemical reaction of the context with ferric sulphate, but with different sporocarp morphologies, were nested within the genus. The analyses demonstrated that several distinct fungal

morphologies were derived from a ramarioid ancestor and inferred a single transition from a lignicolous to a terrestrial Ramaria habitat with the terrestrial

R. subgenus Lentoramaria species, R. rainierensis, bridging the gap between

the lignicolous R. subgenus Lentoramaria species and the terrestrial R.

subgenus Laeticolora species.

R. subgenus Ramaria was monophyletic in this sampling. The two

varieties of R. botrytis were recognized as unique taxa that form a biphyletic

Glade. The sequestrate ectomycorrhizal genus Gautieria formed a sister

group to R. subgenus Ramaria species in the nuc LSU rDNA. The placement 67 of Gautieria in the Gomphales was confirmed by both the mt SSU rDNA and the nuc LSU rDNA. This was the first report of Gautieria being closely related to the striate-spored species of R. subgenus Ramaria.

R. subgenus Laeticolora formed a paraphyletic grade with the cantharelloid genus Gomphus nested within it and R. subgenus Ramaria and

Gautieria derived from its terminal members. The species pair R. amyloidea

and R. celerivirescens were inferred to be two distinct species with R.

claviramulata grouping with R. celerivirescens. R. subgenus Laeticolora is

the only subgenus to contain clamped and clampless species. The

possession of clamps was inferred to be ancestral in these analyses with

eight losses of the clamped condition.

In the nuc LSU rDNA analyses, R. subgenus Lentoramaria also

formed a paraphyletic grade with members of the Phallales, Clavariadelphus,

Kavinia and R. abietina of R. subgenus Echinoramaria nested within R.

subgenus Lentoramaria. The Phallales formed a sister Glade to the Glade

comprising the R. stricta complex and Clavariadelphus in the nuc LSU rDNA.

This sister relationship was not supported by the mt SSU rDNA which

included a smaller sampling of gomphalean taxa and placed the Phallales as

a sister group to the Gomphales.

These analyses redefine the origins of several diverse fungal

morphologies as originating from a ramarioid morphology. The false truffle

(Gautieria), club (Clavariadelphus), cantharelloid (Gomphus) and resupinate

(Kavinia) morphologies were supported by both mt SSU rDNA and nuc LSU 68 rDNA as closely related morphologies within the Gomphales. The nuc LSU rDNA inferred that all of these taxa are derived from a ramarioid ancestor.

Additional taxon sampling of both the mt SSU rDNA and the nuc LSU rDNA is needed, however, to accurately define these relationships.

RECOMMENDATIONS FOR FUTURE RESEARCH

Additional sampling of gomphalean taxa is needed, in particular, those taxa derived from a ramarioid habit including Clavariadelphus, Gautieria,

Gloeocantharellus, Gomphus, Kavinia and the Phallales. In these analyses,

R. subgenus Echinoramaria was represented by only one species collection.

Additional members of this subgenus as well as R. subgenus Lentoramaria should be sampled to clarify the relationship between taxa associated with a duff habitat and a lignicolous habitat. R. subgenus Laeticolora is comprised of the majority of Ramaria species which with additional taxon sampling will likely be subdivided into additional subgenera or possibly even genera.

Extensive sampling of this subgenus will also assist in identifying morphological characters on which to base improved morphological descriptions and keys. R. subgenus Ramaria was represented by only three species in these analyses in part because only four species are recorded from the Pacific Northwest of North America. Additional sampling of striate spored Ramaria species from different geographic regions may provide additional support for the sister relationship of species of Gautieria and R. 69

subgenus Ramaria. A wide geographic sampling of all four Ramaria subgenera particularly from regions of Europe, South America, New Zealand, and the Himalayas would enhance our current understanding of the ramarioid morphology. Ramaria collections are recorded from these regions and herbarium collections should be available.

Sampling of multiple loci would increase confidence in molecular phylogenetic hypotheses of taxonomic relationships. Future phylogenetic studies should employ the internal transcribed spacer regions for clades of closely related gomphalean taxa to improve understanding of interspecific

relationships. Another independent gene region, such as RPB2 which codes for RNA polymerase beta subunit 2, would provide additional characters that can be combined with other molecular data to increase tree resolution. A

detailed morphological study combined with molecular analyses should also

be done for the gomphalean taxa. 70

BIBLIOGRAPHY

Agerer, R., R. M. Danielson, S. Egli, K. Ingleby, D. Luoma and R. Treu, eds. 1996. Descriptions of Ectomycorrhizae. 1: 107-130.

Bonorden, H. F. 1851. Handbuch der allgemeinen Mykologie. E. Schweizerbart. Stuttgart.

Bruns, T. D., T. M. Szaro, M. Gardes, K. W. Cullings, J. J. Pan, D. L. Taylor, T. R. Horton, A. Kretzer, M. Garbelotto, and Y. Li. 1998. A sequence database for the identification of ectomycorrhizal basidiomycetes by phylogenetic analysis. Molecular Ecology 7: 257-272.

Castellano, M. A. 1988. The taxonomy of the genus Hysterangium (Basidiomycotina, Hysterangiaceae) with notes on its ecology. Ph.D. Dissertation, Oregon State University, Corvallis.

Colgan, W., M. A. Castellano, and J. W. Spatafora. 1997. Systematics of the Hysterangiaceae (abstract). lnoculum 48(3): 7.

Corner, E. J. H. 1950. A monograph of Clavaria and allied genera. Oxford University Press, London.

Corner, E. J. H. 1966. Species of Ramaria (Clavariaceae) without clamps. Trans. Br. Mycol. Soc. 49(1): 101-113.

Corner, E. J. H. 1970. Supplement to A monograph of Clavaria and allied genera. Beihefte zur Nova Hedwigia 33: 1-299.

Cunningham, G. H. 1942. The Gasteromycetes of Australia and New Zealand. John Mclndoe, Dunedin.

Dodge, C. W. and S. M. Zeller. 1934. Hymenogaster and related genera. Ann. Mo. Bot. Gard. 21: 625-708.

Donk, M. A. 1933. Revision der Niederlandischen Homobasidiomcetae- Aphyllophoraceae II. Mededeel. Bot. Mus. Rijks-Univ. Utrecht 9: 1­ 278.

Donk, M. A. 1961. Four new families of Hymenomycetes. Persoonia 1(4): 405-407.

Donk, M. A. 1964. A conspectus of the families of Aphyllophorales. Persoonia 3(2): 199-324. 71

Doty, M. S. 1944. Clavaria, the species known from Oregon and the Pacific Northwest. Ph.D. Dissertation, Oregon State University, Corvallis.

Doyle, J. J. and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15.

Dring, D. M. 1973. Gasteromycetes. Pp. 451-476. In: The fungi, an advanced treatise. Eds., G. C. Ainsworth and A. S. Sussman. Academic Press, New York.

Eriksson, J. 1954. Ramaricium nov. gen., a corticioid member of the Ramaria group. Svensk Bot. Tidskr. 48: 188-198.

Fischer, Ed. 1933. Gastromyceteae. In: Engler & Prantl. Die Naturl. Pflanzenfam. (II ed.) 7a: 1-122.

Fitzpatrick, H. M. 1913. A comparative study of the development of the fruit body in Phallogaster, Hysterangium, and Gautieria. Ann. Mycologici von Sydow 11: 119-149.

Fries, E. 1821. Systema Mycologicum. Vol. I. Ex Officina Berlingiana. Lund.

Griffiths, R. P., Caldwell, B. A., Cromack, K., Castellano, M. A., and Morita, R. Y. 1987. A study of chemical and microbial variables in forest soils colonized with Hysterangium setchellii rhizomorphs. p. 96. In: Mycorrhizae in the next decade practical applications and research priorities. 7th N. Am. Conf. Mycorrhizae. Eds., D. M. Sylvia, L. L. Hung, and J. H. Graham. Gainesville, FL.

Griffiths, R. P., M. A. Castellano, and B. A. Caldwell. 1991. Hyphal mats formed by two ectomycorrhizal fungi and their association with Douglas-fir seedlings: A case study. Plant and Soil 134: 255-259.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Dictionary of the Fungi, 8th edition. CAB International, Cambridge, UK.

Hibbett, D. S., E. M. Pine, E. Langer, G. Langer, and M. J. Donoghue. 1997. Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences. Proc. Natl. Acad. Sci. 94: 12002-12006.

Holmskjold, Th. 1790. Beata furls otia fungorum Danicis. Vol. 1. E. Viborg. Copenhagen. 72

Maddison, W. P. and D. R. Maddison. 1992. MacClade. Version 3.03. Sinauer Associates, Inc., Sunderland, Massachusetts.

Marr, C. D. and D. E. Stuntz. 1973. Ramaria of Western Washington. Bibliotheca Mycologica 38: 232p.

Miller, 0. K., Jr., and H. H. Miller. 1988. Gasteromycetes: Morphology and Developmental Features. Mad River Press, Eureka, CA.

Patouillard, N. 1900. Essai taxon. Hymen. These, Paris.

Petersen, R. H. "1967" (1969a). Type studies in the Clavariaceae. Sydowia 21: 105-122.

Petersen, R. H. 1971. Interfamilial relationships in the clavarioid fungi and cantharelloid fungi. Pp. 345-374. In: Evolution in the higher Basidiomycetes. An International Symposium. Ed., R. H. Petersen. University of Tennessee, Knoxville.

Petersen, R. H. 1973. Aphyllophorales II: The clavarioid and cantharelloid Basidiomycetes. Pp. 351-367. In: The fungi, an advanced treatise. Eds., G. C. Ainsworth and A. S. Sussman. Academic Press, New York.

Petersen, R. H. 1974. Contribution toward a monograph of Ramaria. I. Some classic species redescribed. Amer. J. Bot. 61(7): 739-748.

Petersen, R. H. 1975. Ramaria subgenus Lentoramaria, with emphasis on North American taxa. Bibliotheca Mycologica 43.

Petersen, R. H. 1976. Contribution toward a monograph of Ramaria. III. R. sanguinea, R. formosa, and two new species from Europe. Amer. J. Bot. 63(3): 309-316.

Petersen, R. H. 1979. Contribution to a monograph of Ramaria. IV. R. testaceo-flava and R. bataillei. Nova Hedwigia 31: 25-38.

Petersen, R. H. 1981. Ramaria subgenus Echinoramaria. Bibliotheca Mycologica 79.

Petersen, R. H. 1982. Contributions toward a monograph of Ramaria. V. Type specimen studies of taxa described by W. C. Coker. Sydowia Annales Mycologici 35: 176-205.

Petersen, R. H. 1986. Some Ramaria taxa from Nova Scotia. Can. J. Bot. 64: 1786-1811. 73

Petersen, R. H. 1987. Contribution toward a monograph of Ramaria. VI. The Ramaria fennica versatilis complex. Sydowia 40: 197-226.

Petersen, R. H. 1988a. Contribution toward a monograph of Ramaria. VII. New taxa and miscellany. Mycologia 80(2): 223-234.

Petersen, R. H. 1988b. The clavarioid fungi of New Zealand. Science Information Publishing Centre, Wellington.

Petersen, R. H. and C. Scates. 1988. Vernally fruiting taxa of Ramaria from the Pacific Northwest. Mycotaxon 33(Oct-Dec): 101-144.

Singer, R. 1962. The Agaricales in modern taxonomy, 2nd edition. Koeltz Scientific Books, Koenigstein, Germany.

Smith, A. H. 1973. Agaricales and related secotioid Gasteromycetes. Pp. 421-450. In: The Fungi IVB, A Taxonomic Review with Keys: Basidiomycetes and Lower Fungi. Academic Press, London.

Swofford, D. L. 1999. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4. Sinauer Associates, Inc., Sunderland, Massachusetts.

Thiers, H. D. 1984. The secotioid syndrome. Mycologia 76(1): 1-8.

U.S.D.A., F.S.: U.S. DOI, BLM. 1994. Record of Decision for amendments to FS and BLM planning documents within the range of the NSO. 74 p. Plus Attachment A: Standards and Guidelines.

Vilgalys, R. and M. Hester. 1990. Rapid genetic identification and mapping of enzymatically amplified DNA from several Cryptococcus species. J. Bacteriol. 172(8): 4238-4246.

Villegas, M., E. de Luna, J. Cifuentes, and A. E. Torres. 1999. Phylogenetic studies in Gomphaceae sensu lato (Basidiomycetes). Mycotaxon 70(Jan-Mar): 127-147.

White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols: A Guide to Methods and Applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White. Academic Press, New York. 74

Whittall, J. B. 1999. Molecular phylogeny for the Mimulus moschatus alliance (Scrophulariaceae) and its conservation implications. MS thesis. Oregon State University, Corvallis.

Zeller, S. M. and C. W. Dodge. 1918. Gautieria in North America. Ann. Mo. Bot. Gard. 5: 133-142.