Nuclear ribosomal DNA sequence analysis in molecular systematics of Pezizales (Ascomycetes)

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Nuclear ribosomal DNA sequence analysis in molecular systematics of Pezizales (Ascomycetes)
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Momol, Esengul A., 1956-
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Table of Contents
    Table of Contents
        Page i
    Acknowledgement
        Page ii
        Page iii
        Page iv
    Table of Contents
        Page v
    List of Figures
        Page vi
    List of Tables
        Page vii
    Abstract
        Page viii
        Page ix
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
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    Chapter 2. Materials and methods
        Page 19
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    Chapter 3. Results from molecular data
        Page 26
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    Chapter 4. Cladistics: Morphological and ultrastructural data
        Page 47
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    Chapter 5. General discussion
        Page 72
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    Literature cited
        Page 81
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        Page 87
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        Page 89
    Biographical sketch
        Page 90
        Page 91
        Page 92
Full Text












NUCLEAR RIBOSOMAL DNA SEQUENCE ANALYSIS IN MOLECULAR SYSTEMATICS OF PEZIZALES (ASCOMYCETES)
















By

ESENGUL A. MOMOL


















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA 1992















ACKNOWLEDGEMENTS


I would like to express my deepest appreciation and gratitude to Dr. James W. Kimbrough, for his guidance, support, encouragement and his limitless patience throughout this research. I was indeed fortunate to have him as a committee chairman. His attitude toward the students, encouraging their thoughts and creativity, challenging their ideas and most importantly, his constructive criticism make him an excellent example of an educator. I wish to extend my profound appreciation to Jane Kimbrough for her encouragement, support, and thoughtfullness. I also thank Dr. M. M. Miyamoto, who generously provided many helpful discussions, guidance and much encouragement. Although with a very busy schedule, his door was always open for discussion and advice on scientific problems. I would like to extend appreciation to the other members of my committee, Drs. E. Hiebert, G. Moore, and D. Pring who have also greatly assisted in my project and have improved the quality of the dissertation with their comments. Sincere appreciation is also extended to Dr. Hiebert who provided laboratory space and supplies for this research. I also greatly appreciate the willingness of Dr. R. E. Stall to read my dissertation and to participate in my final ii










examination. I have always felt lucky to have such a fine committee. I wish to express my deepest thanks to my department chairman Dr. G. N. Agrios and Dean J. Fry for their generous support and encouragement. The valuable help of Drs. F. Martin, S. Pappu and H. Pappu, E. Almira, B. Zettler, D. Purcifull, and John Taylor is greatly appreciated. I also would like to thank Dr. Gerry and Ulla Benny for their assistance and friendship.

This study could not have been conducted without the support of many others. I would like to acknowledge Colette Jacono, Rick Smith, Gail Wisler, Rose Koenig, Caryle Baker, Gary Marlow, Li-tzu Li, and E. A. Meyer for their sincere friendship. I would like to express my heartfelt thanks to Emine and Hasan Incirlioglu, Isin and Temel Buyuklimanli, Nimet Turel, Sevgi and Dursun Ince, and Hayriye and Turgay Ibrikci for their friendship and support, and for sharing the good and bad moments with me. Very special thanks go to my dearest friend Zekiye Onsan who has put up with me for the last ten years. She was always there when I needed her. Very special thanks are also due to my close friends Nihal and Philip Scarpace for their valuable friendship, support, encouragement, and help.

I would like to express my deepest gratitude to my

parents, Ayten and Mustafa, my brother Bahadir and his wife Zerhan, and my in-laws Sabahat and Rasim for their love,


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support, encouragement, and unlimited understanding.

To my husband Timur and our daughter, Gulengul go my deepest love and gratitude for their love, understanding, continuous encouragement, and support during all phases of this research. They have shared their love, their lives, and their dreams with me. It was through their help and love that I found strength to cope with the difficulties along the way and accomplish my goal. I do not have enough words to thank them.




































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TABLE OF CONTENTS

page

ACKNOWLEGMENTS............................................ ii

LIST OF FIGURES...........................................Vi

LIST OF TABLES........................................... vii

ABSTRACT..... ... ........................................................ viii

CHAPTER I. INTRODUCTION................................. 1

The 5S Ribosomal RNA Genes.................. 10
The 5.8S rRNA Genes......................... 12
The 18S and 28S Genes....................... 13
The Nonconserved Region of rRNA Genes ......15 CHAPTER II. MATERIALS AND METHODS....................... 19

Total DNA Extraction........................ 20
PCR Amplification of rDNA................... 22
Sequencing.................................. 23

CHAPTER III. RESULTS FROM MOLECULAR DATA................ 26

PCR Amplification........................... 26
Sequence Alignment.......................... 33

CHAPTER IV. CLADISTICS: MORPHOLOGICAL AND

ULTRASTRUCTURAL, DATA........................ 47

Explanation for Character States
Used in This Study...................... o... 58
Results of Morphological and
Ultrastructural Analysis.................... 69

CHAPTER V. GENERAL DISCUSSION................... ...... 72

LITERATURE CITED.......................................... 81

BIOGRAPHICAL SKETCH....................................... 90



V















LIST OF FIGURES

Figure Page



1. PCR amplification products of the 5.8S
and ITS1 of rDNA ............................. 27
2. PCR amplification products of the 5.8S
and ITS2 of rDNA ............................. 28

3. Percent G+C composition of rDNA regions ..... 32

4. Multiple sequence alignment of the
5.8S region of rDNA ......................... 34

5. Transversion and transition frequencies
of species against Neurospora crassa ........ 37

6. Multiple sequence alignment of the ITS2
region of rDNA ............................... 44

7. The most parsimonious tree from
the 5.8s sequences .......................... 45

8. The most parsimonious tree from the
ITS2 sequences .............................. 46

9. Septal structures of various families
of Pezizales ................................ 64

10. The most parsimonious tree from
morphological and ultrastructural data ...... 71













vi















LIST OF TABLES


Table page


1. Sizes of rDNA Regions ............................29

2. Percent divergence values in the 5.8S rDNA
Out group= Neurospora crassa ....................40

3. Percent divergence values in the 5.8S rDNA
Out group= Saccharomyces carlbergensis...........41

4. Character and Character States of Epigeous
and Hypogeous Pezizales ..........................54































vii










Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

NUCLEAR RIBOSOMAL DNA SEQUENCE ANALYSIS IN
MOLECULAR SYSTEMATICS OF PEZIZALES (ASCOMYCETES) By

Esengul A. Momol

August 1992


Chairperson: J. W. Kimbrough Major Department: Plant Pathology


The Ascomycete order Pezizales is characterized by

operculate asci borne in an apothecium. Earlier systems of classification were based on morphological features of ascocarp, asci and ascospores. Recently, ultrastructural features of Pezizales have become important in classification. Despite the large number of studies, there is still considerable uncertainty in the group. There are two problems relative to the systematics and phylogeny of Pezizales: 1) the limits of families and the number of families that should be included in the order and 2) the relationship of Pezizales to the other orders of Ascomycetes.

The purpose of this study was to investigate the

relationships of species within the families, to analyze the phylogenetic relationships of families in Pezizales and with other orders of Discomycetes, and to investigate the viii













congruence of molecular findings with ultrastructural and morphological data. Polymerase chain reaction (PCR) amplified 5.8S, ITS1 and ITS2 regions of nuclear ribosomal DNA were used for analyzing the phylogenetic relationships in this group. Although, the 5.8S coding region was 158bp and conserved among the species tested, ITS1 and ITS2 regions were variable in primary size and in sequences. Phylogenetic inferences were made by using parsimony analysis. Three major groups were found by using the sequences from the 5.8S conserved region; however, the positions of eight species which belong to the different families were resolved by using sequences from the ITS region. Parsimony from morphological and ultrastuctural data were congruent with the parsimony analysis from sequence data.



















ix















CHAPTER I

INTRODUCTION



The Pezizales is a large order of Discomycetes, many having cup shaped apothecia and placed in the class Ascomycetes. Apothecial shape, however, may be highly variable but in most, cylindric asci are arranged in a hymenium among sterile paraphyses. Pliny (23-79 A.D.), a Roman scholar, was the first one to describe the Discomycetes as "Pezica", as a kind of mushroom. In 1801, Persoon attempted the first systematic classification of the Discomycetes. His classification was based on the variation of ascocarps. Fries (1822) made a systematic arrangement of the Discomycetes according to hymenial configuration. The Friesian system of classification remained in use for almost 60 years. In 1849, Fries modified his system by including in the Discomycetes six groups, the Helvellaceae, Bulgariaceae, Dermateae, Patellariaceae, Phacidiaceae, and Sticteae, based on the microscopic characters of apothecia. Although a number of mycologists followed the Friesian system, some of them used the different characters of asci and ascocarp for the taxonomy of the Pezizales. Crouan and Crouan (1857) were the first to demonstrate that


1









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Discomycetes had both operculate and inoperculate dehiscence of asci.

As research on Discomycetes advanced, microscopic and cytochemical features were used more extensively. Nylander (1869) used the iodine reaction in asci, Karsten (1869) recognized a number of families based on the cellular nature of sterile elements of the apothecium, and Saccardo (1884), used the size, shape, and septation of ascospores for classification. Boudier (1885) was the first to emphasize in his new natural classification of Discomycetes the presence and absence of an operculum and thus divided the group into operculate and inoperculate Discomycetes. In addition to ascal dehiscence, he included microscopic characteristics such as amyloidity of asci and number of oil drops of spores in his classification. He recognized two families, Morchelles and Helvelles, with stipitate apothecia and alveolate or veined hymenia; two families, Rhizines and Pezizes with cupulate, sessile to short stipitate apothecia, and three families, Ciliaries, Humaries, and Ascoboles, with small, lenticular apothecia. Boudier (1907) modified his earlier system of classification by including 7 operculate and 12 inoperculate families. He used not only external features of apothecia but also cytochemical and microscopic observations for delimiting the families. Boudier's contemporaries such as Saccardo (1889), Gaumann (1926), and










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Seaver (1928), however, did not make the sharp distinction based on ascal dehiscence but placed both inoperculate and operculate taxa together. Nannfeldt (1932) appears to be the first to restrict the Pezizales to those taxa with operculate asci. He recognized the operculate order Pezizales and inoperculate orders Helotiales, Lecanorales, and Ostropales in the Discomycetes based on the characteristics of spore, asci, and sterile structures.

In the 1940s, Discomycetes, including Pezizales, were

reevaluated. Chadefaud (1946) described the internal apical apparatus of operculate and inoperculate asci in detail. Le Gal (1946) proposed the taxon "subopercules' for the groups having a type of ascal dehiscence intermediate to the operculates and inoperculates. She proposed two new names, Homospermales and Heterospermales, to replace the classical orders Pezizales and Helotiales and those with suboperculate asci were placed in the Sarcoscyphaceae. With minor modifications, she continued to recognize the families of Pezizales previously proposed by Boudier (1907). Korf (1954) revised the classification of the operculate Discomycetes and proposed 3 families, the Cyttariaceae, Pezizaceae, and Sarcoscyphaceae.

The decade of the 1960s brought about a number of

changes in the systematics of the Pezizales. Berthet (1964) investigated the nuclear condition of spores and other










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apothecial cells. He found that with a few exceptions spores of Pezizales were uninucleate. Two notable exceptions were the Helvellaceae with tetranucleate and Morchellaceae with multinucleate spores. Brummelen (1967) described apothecial development and anatomy and proposed the term cleistohymenial for those taxa in which the ascogenous system is initially closed within the excipulum, and gymnohymenial for those in which the asci are exposed throughout development. Kimbrough and Korf (1967) examined important characters such as microchemical reactions of asci, the development of asci and ascospores, and the manner of ascal dehiscence in a variety of genera. Rifai (1968), using basically morphological and anatomical features, followed for the most part the system of classification of Le Gal (1953), however, recognizing in addition the family Pyronemataceae. Rifai's most notable proposal was to divide the Pezizales into two suborders, the Sarcoscyphineae with suboperculate asci and the Pezizineae with regular opercula. Kimbrough (1989), using apothecial ontogeny, ascal structure, and septal organelles, proposed to recognize the Pyronemataceae as a third suborder, the Pyronemineae. In 1968, Eckblad divided the order Pezizales into the following families: Thelebolaceae, Ascobolaceae, Rhizinaceae, Pyronemataceae, Helvellaceae, Morchellaceae, Otidiaceae, and Sarcoscyphaceae. The predominant feature of his system was










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placing the genera of Humariaceae into two separate families, Pyronemateceae and Otideaceae. He also pulled the Rhizinaceae from out of the Helvellaceae.

One of the most widely accepted systems of

classification of Discomycetes is in Dennis's (1968) "British Ascomycetes" which includes the Pezizales, Helotiales, Phacidiales, Lecanorales, and Ostropales. In dividing the Pezizales, he followed Le Gal's (1953) classification with the addition of the Thelebolaceae and Aleuriaceae. Arpin (1968) made his classification based on the carotenoid content of the spores. These treatments not only increase in the number of families of Pezizales but also resulted in reclassification of genera within the families.

In the 1970s, ultrastructural studies began to impact

the systematics of Pezizales. The septal pore structures of asci, ascogenous hyphae, and paraphyses have gained lots of attention. The septal structures of some families, such as the Pezizaceae (Curry and Kimbrough, 1983), Ascobolaceae (Kimbrough and Curry, 1985), Ascodesmidaceae (Steffins and Jones, 1983), Helvellaceae (Kimbrough, 1989), and Morchellaceae (Kimbrough, 1990), are unique and consistent at the family level. However, in other families such as Pyronemateceae (Kimbrough and Curry, 1986), and Thelebolaceae (Kimbrough, 1981), there are complex septal









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structures (Kimbrough, 1986). Merkus (1973) did an extensive study on spore ontogeny and she concluded that spore ontogeny may not be an appropriate character for the taxonomy of Pezizales since different types of spore ontogeny may occur in the same genus. However, Dyby and Kimbrough (1987) showed that the type of ascosporogenesis was consistent in the family Pezizaceae and a similiar observation was made by Gibson and Kimbrough (1988) and Kimbrough, Wu, and Gibson (1990) in the Helvellaceae, and Wu and Kimbrough (1992) in Humariaceae.

Phylogenetic relationships of the Pezizales have been very uncertain over the years. The operculate and inoperculate Discomycetes have been considered to be closely related because of apothecial shape and hymenial configurations. Chadefaud (1946) and LeGal (1946) suggested that the Pezizales may have evolved from the Helotiales, inoperculate Discomycetes, by way of the Sarcosyphaceae (suboperculates) with an intermediate ascal type. Their idea was based on the fact that suboperculate asci were also found within some inoperculate genera. Many mycologists proposed that particular Plectomycetes may be directly related to the Pezizales (Malloch, 1979; Benny and Kimbrough, 1980). Because of the size and anatomy of apothecia, mycologists generally agree that Pezizales are closely related to the order Tuberales. The orders have









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usually been separated by the epigeous versus hypogeous habitat and the presence or absence of forcible spore discharge. Trappe (1979) proposed the reorganization of hypogenous Ascomycetes, excluding Elaphomyces, in which the Tuberales were included in the Pezizales. The taxonomic position of Elaphomyces is not stable. Elaphomyces, having a thick peridium and evanescent asci, was thought to be distinct from other Tuberales. Korf (1973) placed Elaphomyces in a separate family within the Tuberales, but at the same time he also noted that it may belong to the Eurotiales.

Controversy also exists relative to the position of

various genera within the families. Eckblad (1968) placed a number of Helvellaceous genera in a new family Rhizinaceae based on spore ornamentation. However, current ultrastructural studies of ascospores by Gibson and Kimbrough (1987) have argued against recognition of the Rhizinaceae. A wide difference of opinion exists as to the limits of the family Ascobolaceae. Ascobolus, Saccobolus, lodophanus, and Thecothus were placed in Ascobolaceae based on morphological studies (Korf, 1973). But ultrastructural data from Kimbrough and Curry (1985) showed that the septal structure of Iodophanus differs greatly from that of other Ascobolaceous genera. They placed lodophanus in the Pezizaceae based on their septal structure similiarity to










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the Pezizaceous genera (Curry and Kimbrough, 1983). Despite these extensive studies, there is disagreement as to the position of certain genera in almost every currently recognized family of Pezizales.

During the last few decades there has been a growing interest among mycologists to reconstruct phylogenetic relationships among the fungi by using molecular methods (Jahnke, 1987). Many reliable molecular methods are available which allow the classification of fungi at different taxonomic levels. Nuclear and mitochondrial DNA analysis, the molar percentage of DNA bases cytosine and guanine, DNA-DNA reassociation experiments, and ribosomal RNA (rRNA) sequencing have been utilized. The usefulness of mitochondrial and nuclear DNA restriction analysis for taxonomic purposes has been repeatedly demonstrated (Kozlowski and Stepien, 1982; Kurtzman, 1985). The smaller genome size and the faster evolutionary rate make mitochondrial DNA more suitable for investigating the phylogenetic relationships among the fungi. The multicopy nature of the mitochondrial genome and difference in base composition from nuclear DNA make it a very valuable taxonomic tool especially in yeast systematics (Clark-Walker et al., 1987). DNA-DNA reassociation techniques are based on the ability of single stranded DNA molecules to recognize complementary single stranded DNA molecules and to form










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duplexes with them in vitro. The data obtained from DNA-DNA reassociation experiments depend very much on the kind of DNA used. Vilgalys and Johnson (1987) found very similiar DNA homology values of repeated DNA and nonrepeated DNA of Collybia species, as did Williams and coworkers (1981) for Neurospora. Therefore, these data suggest that DNA-DNA reasssociation values alone cannot be used as a good taxonomic tool.

Recently, ribosomal RNA genes have proven very informative for phylogenetic studies (Jahnke, 1987). Ribosomal rRNAs as a taxonomic tool show several advantages over other molecular markers, such as being ubiquitous, playing a central role in protein synthesis, being highly expressed and easily purified. The overall organization of rRNA repeat units in higher eukaryotes is in the form of long tandem arrays in a head to tail configuration. This was first visualized by electron microscopic analysis. Synthesis of three RNA molecules found in ribosomes is generally achieved via the production of a single rRNA precursor molecule. In prokaryotes, the precursor contains 16S, 23S, and 5S of rRNA. The prokaryotic pre-rRNA sometimes contains one or more tRNA molecules. In contrast, a eukaryotic pre-rRNA molecule includes a 5.8S rRNA and does not contain 5S rRNA. In prokaryotic systems, rRNA genes exist as few copies, whereas eukaryotic cells contain many










10

copies of them. Especially in yeast and Neurospora more than 100 copies of rRNA genes are repeated in a tandem array (Dutta et al., 1983). The rRNA genes consist of conserved regions also called coding regions which include 5S, 5.8S, 18S, and 28S, and a nonconserved or noncoding regions which consists of nontranscribed spacer (NTS), internal transcribed spacer (ITS), and external transcribed spacer (ETS) regions.

The 5S Ribosomal RNA Genes



Of the different ribosomal RNAs, the 5S rRNA was the first to be studied extensively, since its length of 120 nucleotides and makes it easy to sequence. Selker and his coworkers (1985) reported on a heterogeneity of 5S rRNAs in fungal ribosomes. They showed that six minor kinds of 5s rRNA genes exist in Neurospora crassa.

The rRNA genes function in a folded state by base

pairing within the molecule to form secondary structures. The secondary structure of the 5S rRNA is highly conserved in all organisms and consists of 5 stems and 5 loop regions. In a majority of basidiomyceteous fungi, 5S rRNA sequences fit the general secondary structure model. The 5S ribosomal RNA is widely used in Basidiomycetes for phylogenetic comparisons.











Septal pore structure has been shown to be a valuable

tool in the systematics of Basidiomycetes (Moore, 1978; Khan and Kimbrough, 1982). Walker and Doolitle (1982) using 5S rRNA sequences grouped the Basidiomycetes analyzed so far into five clusters. They observed that all species belonging to clusters 1 and 2 had simple septal pores, whereas those belonging to clusters 3 to 5 (except for Exobasidium vaccini) had dolipores. These results contradict the studies based on morphological characters and do not support the idea that the Hetero- and Homobasidiomycetes are two distinct groups within the Basidiomycetes. According to 5S rRNA sequence similarity, Taphrina deformans is more similiar to Basidiomycetes than Ascomycetes (Blanz and Unseld, 1986). It was hypothesized that Taphrina may have originated from a primitive ancestor of the Ascomycetes or Basidiomycetes.

In the analysis of additional Basidiomycetes,

Gottschalk and Blanz (1984) have shown that the 5S rRNA sequences have a limited value for differentiation within the groups. Mao and his workers (1982) compared the sequences of 5S rRNA from Schizosaccharomyces pombe with the other species of yeasts, S. cerevisiae and Torula utilis, and with the fruitfly Drosophila melanoqaster. They observed that the sequences in 5S rRNA from S. pombe shared more homology to D. melanogaster than to S. cerevisiae and










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T. utilis. All of these results indicate the informative limitation of 5S rRNA sequences in evaluation of phylogenetic relationships.



The 5.8S rRNA Genes



In large subunits of eukaryotic ribosomes, the 5.8S

rRNA is a specifically bound large subunit rRNA (L rRNA) by noncovalent interactions. The 5.8S rRNA-LrRNA complex may be isolated from ribosomes in denaturating conditions. In the 1980s, several researchers (Jacq, 1981 and Nazar, 1980) demonstrated that 5.8S rRNA is homologous with the 5' end of prokaryotic 23S rRNA. Perhaps, in the course of evolution, the 5.8S rRNA originated from the 5' end sequence of prokaryotic LrRNA as a result of incorporation of a noncoding sequence. In the rRNA operon this sequence is represented by the internal transcribed spacer region (ITS)2 between the 3' end of 5.8S rRNA and the 5' end of LrRNA. Comparisons of the nucleotide sequences of 5.8S rRNA from various sources also indicate a stronger conservation of the 5' end of the molecule. The degree of homology between Neurospora and several species of vertebrates is about 50% (Crouch and Bachellerie, 1986). Most of the 5.8S rRNA molecules consist of 156 to 167 nucleotide residues. Variation in length comes from insertions and the presence










13

or absence of additional nucleotide residues at the 5' or 3' ends. In some species, the 5.8S rRNA molecule is found to be heterogeneous at the end. In the yeast Schizosaccharomyces pombe, there are 8 strains in which the

5.8S rRNA is 158 to 165 nucleotide residues long.

The 5.8S rRNA like other RNAs carries out its function in the folded state. There are few models proposed for the secondary structures of 5.8S rRNA but none of them are in full agreement with all the data obtained. The large conservation of 5.8S rRNA can be seen among the taxa. The identity between compared regions of rodents and man is 100% (Nazar et al., 1976). Alignment of the known 5.8S sequences from fungi (S. cerevisiae, N. crassa, and S. pombe), and higher eukaryotes shows a high degree of homology (Schaak et al.,1982). The comparisons of 5.8S rRNA nucleotide sequences may help in determining the degree of affinity between the higher taxa such as classes and divisions.



The 18S and 28S rRNA Genes



Nuclear sequences for the small (18S) and large subunit (28S) ribosomal RNAs are present in the genomes of all eukaryotic organisms and have also been used for phylogenetic comparisons (Sogin, 1991). The small subunit










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is about 1800 base pairs (bp) and the large subunit is about 3000bp.

One major concern when using the structural RNA

sequences is the affect of substitutions on stem regions. The nucleotide change in one strand of the stem region may require the a compensatory change in the other strand. The 18S and 28S rRNAs contain a significant number of independently variable sites or divergent domains which may be subject to different functional constraints. All organisms other than fungi reveal several longer insertions in these regions. Nucleotide sequences of domains II and III of 28S rRNA which consist of about 1000 nucleotides have been determined in species of Saccharomyces, Taphrina, Septobasidium, Ustilaqo, and Exobasidium (Blanz and Unseld, 1987). The 3' ends of 18S and 28S rRNA genes are highly conserved and sequence homology has been found between prokaryotes and eukaryotes in these regions. Two complete sequences of maize and rice 18S genes showed 97% homology to each other (Messing et al., 1984; Takaiwa et al.,1984). Sequence comparisons of the entire 18S genes of Xenopus and Saccharomyces indicate extensive but interrupted areas of homology (Salim and Maden, 1981). Overall, the 18S gene is found to be more conserved than 28S gene.










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The Nonconserved Region of rRNA Genes



The overall structure of the rRNA is described by a spacer region and a region giving rise to a primary precursor transcript. The precursor rRNA consists of an external transcribed spacer (ETS) preceding the 18S RNA gene at the 5' end of the transcript, a 5.8S rRNA gene separated from both the 18S and 28S rRNA by internal transcribed spacers (ITS1 and ITS2), and the 28S rRNA gene at the 3' end of the transcript. The most extensive data base for interspecific relationships at the rDNA locus comes from systematic comparisons of variation in the spacer. The large differences in the length of the rRNA repeat unit among the eukaryotes are mostly accounted for by variations in the size of the nontranscribed spacer regions (NTS). These comparisons have been based primarily on restriction enzyme analyses. Most evolutionary studies have confirmed the idea that the spacer region is the fastest evolving component of the rDNA locus (Crouch and Bachellerie, 1986). The ITSs are nonrepetitive and the two spacers (ITS1 and ITS2) are not related in sequence. Unlike mature rRNAs, no vertebrate ITS sequences show any significant homology when compared to yeast (Michot et al., 1982). Much more significant homologies in ITS sequences are observed when










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comparing closely related species, such as mouse and rat (Michot et al., 1983). Highly conserved sequences are shared between species, and these conserved sequences are interspersed with and laterally displaced by highly divergent sequences. Furlong and Maden (1983) suggested that the displacement is the result of deletions, insertions, and possibly point mutations. The lengths of ITS1 between species are similiar, whereas ITS2 lengths are different. For example, in mouse the ITS2 is 1089 bp, while in rat it is 765 bp. However, in yeast (S. pombe) ITS1 is about 400 bp, and ITS2 is 290 bp (Shaak et al.,1982), whereas in Verticillium ITS1 is about 100 bp and ITS2 is 164 bp (Nazar et al.,1991). The ITS1 of S. carlsbergensis is 363 bp, about two times the 186 nucleotides of ITS1 of N. crassa. The length of ITS2 of S. carlsbergensis is also almost twice that of N. crassa (235 compared to 145). There is little homology between ITS regions of S. carlsbergensis and N. crassa (Crouch and Bachellerie, 1986). ITS1 sequences are more homologous (75%) than ITS2 (30%) between rat and mouse. In contrast, ITS1 (11%) is less homologous than ITS2 (36%) in comparisons of the two Xenopus species.

Base composition of DNA is an important parameter to consider in phylogenetic reconstruction (Larson, 1991). Base compositional biases exist in variable regions of rRNA genes (Bernardi et al., 1988). The coding region of rRNA










17

genes usually are more GC rich than noncoding regions. However, in Drosophila, the 28S gene is more AT rich. The G+C content ranges in the fungal kingdom from about 27% to about 65 mol%. G+C content of Ascomycetes species range from 28 to 52 mol%, whereas Basidiomycetes range from 49 to 68 mol% (Jahnke, 1987). The ITS1 of S. carlsbergensis is 35% G+C, and ITS2 is 39% G+C, whereas in Neurospora, the ITS1 and 2 are 55% G+C and 58% G+C, respectively (Crouch and Bachellerie, 1986). Storck (1972) suggested that fungal evolution was associated with the progressive increase in G+C content of the DNA. Similiar G+C value, by itself does not indicate the a close relationship but dissimiliar G+C value may be a useful characteristic to exclude close relationship or conspecificity.

Because of the absence of a fossil record, evolutionary patterns of fungi have been speculative. In spite of large numbers of morphological, cytochemical, and ultrastructural studies, there is still considerable taxonomic and phylogenetic controversy in the order Pezizales. Among the various ultrastructural studies, septal pore structure of asci and ascogenous hyphae appear to provide the most definitive phylogenetic information (Kimbrough, 1990). There have been two problems relative to the systematics and phylogeny of the Pezizales; first, the limits and the numbers of families that should be included in the order and










18

second, the relationship of Pezizales to other subclasses of Ascomycetes (Kimbrough, personal communication). In this study, molecular methods were used to clarify the taxonomic problems that exist in this group.

The objectives of this research were (1) to investigate the relatedness of species within the families of Pezizales,

(2) to analyze the phylogenetic relationship of families in the Pezizales, (3) to attempt to correlate the ultrastructural data with molecular analyses. These goals were achieved by using molecular techniques such as polymerase chain reaction (PCR) and sequencing of the 5.8S and internal transcribed spacer (ITS) regions of rDNA.














CHAPTER II


MATERIALS AND METHODS



Species used in this study representing different

families of Pezizales were obtained either from cultures or herbarium materials. Fungal cultures (C) and Herbarium specimens (H): FAM: ASCOBOLACEAE

(C) Saccobolus depauperatus (Berk. and Br.)Hans. (FLAS

culture collection 106) (SAC) FAM: ASCODESMIDACEAE

(C) Ascodesmis nigricans Van Tiegh. (FLAS culture

collection 122) (ASN)

(C) Ascodesmis sphaerospora Obrist. (FLAS culture

collection 260) (ASD)

(C) Eleutroascus lectardii (Nicot) Von Arx. (FLAS culture

collection 300) (ELE) FAM: HELVELLACEAE

(H) Gyromitra montana Harmaja. (Cryptogamic collections of

Oregon State University 49452) (GYR)

(H) Helvella lacunosa Afz.:Fr. (Cryptogamic collections of

Oregon State University 49459) (HEL)


19









20

FAM: PEZIZACEAE

(C) lodophanus sp. (FLAS culture collection 327) (IOD)

(H) Peziza vesiculosa Bull.: St.Amans. (University of

Florida Herbarium F53697) (PEV)

(H) Plicaria endocarpoides (Berk.) Rifai. (University of

Florida Herbarium F54729) (PAC) FAM: HUMARIACEAE

(C) Lamprospora sp. I (FLAS culture collection 346 from

Muller) (LAM)

(C) Lamprospora sp. II (FLAS culture collection 343 from

Udagawa) (LAU)

(H) Otidea leporina (Fr.) Fckl. (University of Florida

Herbarium F49001) (OTI) FAM: PYRONEMATECEAE

(C) Pyronema domesticum (Sow.:Fr.) (ATCC 14881) (PYR) FAM: THELEBOLACEAE

(C) Thelebolus sp. (FLAS culture collection IMI 67944)

(THE)

FAM: SARCOSOMATACEAE

(C) Urnula craterium (Schw.) Fr. (ATCC 11067) (URN)


Total DNA Extraction



Total DNA was isolated from the fungal cultures as

described by Lee and Taylor (1989). Cultures were grown in










21

a nutrient rich medium (5 g peptone, 2.5 g yeast extract, 5 g tryptone, and 10 g NaCI in IL water). Lyophilized mycelium (20 to 60 mg dry) was ground with a mortar and pestle and was put in 1.5-ml Eppendorf microcentrifuge tube. Lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 3% SDS, and 1% 2mercaptoethanol) was added, and mixture stirred with a dissecting needle and incubated at 65 C for an hour. An equal amount of phenol-chloroform (1:1) was added, vortexed, and centrifuged at 10,000xg for 15 minutes at room temperature. The aqueous phase containing the DNA was removed and 3 M sodium acetate was added to the aqueous phase followed by 0.54 volumes of isopropanol. The DNA was pelleted by centrifuging in a microcentrifuge for 2 minutes at room temperature and the pellet was washed with 70% ethanol, dried at room temperature for 20-30 minutes, and resuspended in distilled water.

DNA extraction from herbarium materials was done using the procedure described by Bruns, Fogel, and Taylor (1990). To remove any signs of insect damage, external surfaces of herbarium specimens were scraped off. The material (5-10 mg) was ground to a fine powder and placed in 1.5 ml microfuge tubes, suspended in 700 ul of 50 mM EDTA/50 mM Tris pH 7.5/ 3% SDS, incubated for 1 hour at 65 C, extracted once with phenol-chloroform (1:1), and once with chloroform. The phenol was previously equilibrated to pH 7.0. DNA was precipitated from the aqueous phase by addition of 50 ul of









22

3 M ammonium acetate and by 500 ul of isopropanol. After incubation at room temperature, the precipitate was centrifuged for 2 minutes, and the pellet was washed with 70% ethanol and dried at room temperature for 20-30 minutes. The pellet was resuspended in 50 ul sterile water and was diluted 50 or 1000 fold for PCR amplification.



PCR Amplification of rDNA



From 100 to 200 ng of double stranded DNA was amplified by using a Perkin Elmer Cetus thermal cycler (Perkin Elmer Cetus corporation, Emeryville, CA). The primers ITS3 (5'GCATCGATGAAGAACGCAGC-3'), ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), ITS2 (5'-GCTGCGTTCTTCATCGATGC-3'), and ITS5 (5'GGAAGTAAAAGTCGTAACAAGG-3') were used to amplify the 5.8S, ITS1 and ITS2 regions of ribosomal DNA. These primers were provided by John Taylor from the University of California in Berkeley. Total reaction volume was 100 ul and contained the following components: 50 pmoles of each primer, 2.5 units Taq polymerase, reaction buffer containing 50 mM KC1, 10 mM Tris and 2.5 mM MgCl2, and 200 uM of each of the four deoxyribonucleotide triphosphates. These reactions were subject to 30-35 cycles under the following temperatures: 2 minutes at 95 C, 30 seconds at 55 C, 2 minutes at 72 C. Results of amplification were assayed for 2 ul aliquots by










23

gel electrophoresis in a gel composed of 2% NuSieve and 1% normal agarose (FMC, Rocland, Maine). Gels were stained for 15-20 minutes with 0.5 ug/ml ethidium bromide, destained in water for 10-15 minutes, and photographed with polaroid 55 and 57 film.

Sequencing



Products of PCR were extracted once with phenolchloroform and precipitated with 3 M ammonium acetate and 3 volumes of absolute ethanol. After 2 hours at -80 C the precipitate was pelleted by 2-minute centrifugation in a microcentrifuge, washed once with 70% ethanol and dried 2030 minutes at room temperature. The pellet was resuspended in 100 ul sterile water and diluted to 300 ul and excess nucleotides and primers were removed by using the Milipore Ultrafree-MC filter unit (Milipore Corporation, MA). The aliquot was centrifuged for 2 to 4 minutes at 2000 x g in a fixed angle rotor. The filtrate (50-80 ul) was removed to a separate tube and washing steps were repeated twice to ensure efficient separation of DNA from primers and free nucleotides. Sanger's Dideoxy Chain Termination method was used for sequencing (Sanger et al., 1977). Sequencing reactions were carried out on 7 ul aliquots of concentrated samples using each primer, T7 polymerase, and 35S labelled dATP with using the Sequenase kit (U.S Biochemicals,










24

Cleveland, Ohio). Template DNA (0.5-1 ug) was annealed to 50 pmole primer in a 10 ul reaction by heating to 95 C for 4 minutes and allowed to cool at room temperature for 1 minute. Extension was carried out on ice for 5 minutes using 1 ul [a-3SS] dATP (SA>1200ci/mmol), 1 ul of 0.1 M dithiothreitol, 2 ul of diluted (1:5) dNTP mix (USB kit), and 2 ul diluted Sequenase T7 DNA Polymerase (1:5). A 3.5 ul sample of this reaction was added to 2.5 ul of each termination mix (USB kit) and reaction was incubated for a further 5 minutes at 50 C. The reaction was stopped by the addition of 4 ul of formamide dye mix. The samples were electrophoresed in 6% polyacrylamide, 7 M urea sequencing gels at 1700 volts for 1.5-6 hours. Gels were fixed using 10% ethanol, and 10% glacial acetic acid for 20-30 minutes destained with water for 20-30 minutes, dried, and exposed to Kodak X-Omat film.

Also, some of the sequences from a few species were

obtained by using automated sequencers, and these sequences were compared with manual sequences. Automated sequencers utilize electrophoresis but in a different way. They measure the time it takes for a band to traverse a specified distance in the gel. Thus output is presented as detected bands showing peaks on the Y axis and time of electrophoresis on the X axis. Each peak is identified as an A, T, G, or C depending upon the detection sysytem which










25

is based on fluorescent tags to label DNA from different reactions (Feri et al., 1991).















CHAPTER III

RESULTS FROM MOLECULAR DATA


PCR Amplification



Approximately 200-250 ng DNA from cultures was used for PCR amplification. Herbarium materials were diluted 50-to 1000-fold and generally were subjected to 35 cycles of amplification. Primers ITS2 and ITS5 yielded variable sizes of amplification products (Fig. 1) which include part of

5.8S and ITS1 region. Using the primers ITS3 and ITS4, 320 base pair (bp) of amplification products were obtained (Fig. 2). These amplification products included part of the 5.8S coding region and ITS2 region of rDNA.

The sizes of ITS1, 5.8S, and ITS2 regions obtained from sequences of ribosomal DNA are shown in Table 1. Ambiguous sequences from a few species are not included in the Table 1. The size of the 5.8S coding regions for fifteen species tested were 158 bp. However, the ITS1 and ITS2 regions were variable in size. Closely related species such as Ascodesmis nigricans, Ascodesmis sphaerospora, Eleuthroascus lectardii, and Saccobolus depauperatus have a conserved ITS1 region consisting of 170 bp, while the size of this region


26









27



Figure 1. PCR Amplification Products of the 5.8S and ITS1 of
rDNA.






z z (: w U U > 0 w W P .q
0 w w 14 #1:4 4 w 0 4 M 1-4 -4 W E-4 U K4 I< W U) 04 P4 H 0 P P4 0 W !:3 0










492
369
246
123



CON= Control ASN= Ascodesmis nigricans ASD= Ascodesmis sphaerospora ELE= Eleuthroascus lectardii SAC= Saccobolus depauperatus PAC= Plicaria endocarpoides PEV= Peziza vesiculosa IOD= Iodophanus sp. LAM= Lamprospora sp. LAU= Lamprospora sp. THE= Thelobolus sp. PYR= EyEonema domesticum GYR= Gyromitra montana HEL= Helvella lacunosa URN= Urnula craterium OTI= Otidea leporina









28




Figure 2. PCII Amplification Products of the 5.8S and ITS2 of
rDNA.

W4




Z z Q w U U
H 0 U) U) 1-4 4 4. 14 0 >4 >4 w E-1 U) kI U) N P4 0 E-A N 0 M LD 0











492
369
246
123


CON= Control ASN= Ascodesmis nigricans ASD= Ascodesmis spliaerospora ELE= Eleuthroascus lectardli SAC= Saccobolus depauperatus PAC= Plicaria endocarpoides PEV= Peziza vesiculosa IOD= Iodophanus sp. LAM= Lamprospora sp. LAU= Lamprospora sp. THE= Thelobolus sp. PYR= Pyronema domesticum GYR= Gyromitra montana HEL= Helvella lacunosa URN= Urnula craterium OTI= Otidea leporina








29




Table 1. Sizes of rDNA Regions.


SPECIES ITS1(bp) 5.8S(bp) ITS2(bp)


PEV 217 177

PAC 201 158

ELE 170 158 161

ASN 170 158 161

ASD 170 158 161

SAC 170 158 161

LAU 166 158 163

LAM 157 158 160

THE 158 158 149

OTI 201 158 172

PYR 179 158 163

IOD 207 158 185

GYR 363 158

HEL 256

URN 462 172

NEU 185 158 145

VER 110 158 164

SCC 362 157 235


Neu (Neurospora), Ver (Verticillium), and Scc (Saccharomyces) sequences were obtained from the Genbank Database.










30

ranged from 462 bp in Urnula craterium to only 157 bp in Thelebolus. The size variation of ITS2 was considerably less than ITS1. Saccharomyces carlbergensis has a longer ITS2 region than the others and lodophanus sp. with 185 bp is the longest among the other Pezizales tested. Closely related species, such as Saccobolus depauperatus, Eleuthroascus lectardii, Ascodesmis nigricans, and Ascodesmis sphaerospora, showed a very conserved ITS2 region which is 161 bp in size, similiar to observations from ITSl.

The large DNA segments called "isochores," showing biased G+C/A+T compositions, have been reported in the genomes of warm-blooded animals (Bernardi et al., 1988) and plants (Salinas et al., 1988). Figure 3 shows the percent G+C values in the regions of 5.8S, ITS1, and ITS2. The G+C composition values for the 5.8S in the Ascodesmidaceae which includes the species Ascodesmis nigricans, A. sphaerospora, and Eleuthroascus lectardii, Pyronemateceae which includes Pyronema domesticum, Ascobolaceae family which includes Saccobolus depauperatus, Otideaceae family which includes Otidea leporina, Helvellaceae family which includes Gyromitra montana, Humariaceae family which includes Lampspora species, Pyronemateceae family which includes Pyronema domesticum Thelobolaceae family which includes Thelobolaceae, and Pezizaceae family which includes lodophanus species are similar.























ul
P





U) -,j 00 4-4 4
0 a)
41


U) L) V

04
(n
4
ul (a 4-) :
44 0 En
'H z 0 4 0
z co -H 0 > 41

41
.-j 0 >
rn Q)
0 44 04 4 E! Z
0

U U) in
+ 44 (1) 10
0 4j 0
(L) 4 41 U2 U 4-)
F: >4 0 E -H 0
M U 70








tn
.'i
44












32


















[ _
.......










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. . . . . .







'-4


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..........



.........

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o LO 0 0 04










33

Sequence Alignment



The initial crucial step for analyzing nucleotide

sequence data is to align the sequences against one another. Sequence alignment can be done by eye without using any alignment algorithm when DNA homology is conserved (Miyamoto and Cracraft, 1991). Since the 5.8S coding region is very conserved and free of gaps, initial alignment was done by eye. Then different computer assisted alignment programs from GCG such as "pileup" and "pretty" for multiple sequences, and "seqaid" for pairwise comparisons were used to search the best alignment (Figure 4). The 5.8S region of Saccharomyces cerevisiae was 157 bp and one gap was introduced to facilitate the alignment. The 5' end of the

5.8S coding region did not show any substitutions among the species tested. However, the 3' end of the sequences was not as conserved as the 5' sequences.

Because rates of change for different substitution types may vary between sequence regions, greater phylogenetic resolution may be attained by considering transition (TS) to transversion (TV) ratios before assigning weights for phylogenetic analysis. Transition and transversion frequencies of species were calculated against Neurospora for the 5.8S rDNA region (Figure 5). For many species transitions basically represented more than 50% of











34



1 50
Lam58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Pyr58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Asn58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Ele58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Lau58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Asd58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Sac58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Oti58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC The58s AAACTTTCAA CAACGGATCT CTTGGTTCTG GCATCGATGA AGAACGCAGC Gyr58s AAACTTTCAA CAACGGATCT CTTGGTTCCC GCATCGATGA AGAACGCAGC Pac58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Neu58s AAACTTTCAA CAACGGATCT CTTGGTTCTG GCATCGATGA AGAACGCAGC Scc58s AAACTTTCAA CAACGGATCT CTTGGTTCTC GCATCGATGA AGAACGCAGC Ver589 AAACTTTTAA CAACGGATCT CTTGGCTCTA GCATCGATGA AGAACGCAGC Iod58s AAACTTTCAA CAACGGATCT CTAGGCTCTT GCATCGATGA AGAACGCAGC

51 100
Lam58s GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Pyr58B GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Asn58s GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Ele58s GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Lau58s GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Asd58s GAAATGCGAT AAGTAGTGTG AATTGTAGAA TTCAGTGAAT CATCGAATCT Sac58B GAAATGCGAT AAGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Oti58s GAAAGGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT The58s GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Gyr58s GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Pac58s GAAATGCGAT AGGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Neu58s GAAATGCGAT AGGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Scc58s GAAATGCGAT ACGTAATGT. AATTGCAGAA TTCCGTGAAT CATCGAATCT Ver58B GAAACGCGAT ATGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT Iod58s GAAATGCGAT AAGTAATGTG AATTGCAGAA TCTCGTGAAT CATTGAATCT

101 150
Lam58B TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG Pyr58s TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG Asn58s TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG 91e58B TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG Lau58B TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG Asd58B TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG COTGTTCGAG Sac58s TTGAACGCAC ATTGAGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG Oti58B TTGAACGCAC ATTGCGCCTC CTGGTATTCC GGGAGGCATG CCTGTTCGAG The58B TTGAACGCAC ATTGCGCCCT CTGGTATTCC GGGGGGCATG CCTGTTCGAG Gyr58s TTGAACGCAC ATTGCGCCCG CCTGTATTCC GGAGGGCATG CCTGTTCGAG Pac58s TTGAAAGCAC ATTGTGACCT CTGGTATTCC GGAGGGCATG CCTGTTCGAG Neu589 TTGAACGCAC ATTGCGCTCG CCAGTATTCT GGCGAGCATG CCTGTTCGAG Scc58s TTGAACGCAC ATTGCGCCCC TTGGTATTCC AGGGGGCATG CCTGTTTGAG Ver589 TTGAACGCAC ATGGCGCCTT CCAGTATCCT GGGAGGCATG CCTGTCCGAG Iod58s TTGAACGCAC ATTGCGCCCT ATGGTATTCC GTAGGGCATG CCTGTCTGAG


Figure 4. Multiple sequence alignment of the 5.8S region of rDNA from various species of
Ascomycetes. Species abbreviation are in
materials and methods.











35



Figure 4--continued.



151

Lam58s CGTCATTA Pyr58s CGTCATTA Asn58s CGTCATCA Ele58s CGTCATCA Lau58s CGTCATCA Asd58s CGTCATCA Sac58s CGTCATCA Oti58s CGTCATGA The58s CGTCATTA Gyr58B CCTCGTGA Pac58s CGTCAGCT Neu58s CGTCATTT Scc58s CGTCATTT Ver58B CGTCGTTT Iod58s CGTCAGCT



























a,) 0 U


9 N *o
:3P4 (a ta a) 4-4 U) U) H 40 (a~4-4

E4-4 -4" 0 t





0) ) (1)4 E-4 ui (a .HU -4

a)U) HU p -i a )Q E-4 > 4J




ton

r24









37























KNN


F
C
0







C/)
< V) C)


0 co CM 0
T-










38

the substitutions in the 5.85 coding region of rDNA.

Neurospora crassa and Saccharomyces carlbergensis were used as outgroups to determine sequence similarity in pairwise comparisons. The percent divergences were determined by either using the transitions + transversions or transversions only according to the following formula: D= 100(transitions and/or transversions + gaps)/total nucleotides shared. Transitions begin to saturate when they represent about 50% of the substitutions, which is the case for the 5.8S coding region, then transversions may be given additional weight. Table 2 shows the percent divergence values using Neurospora crassa as an outgroup. The percent divergence value for lodophanus sp. was higher than other Pezizales species tested when both transition and transversion values were used. Thelobolus sp. was the least divergent species from Neurospora crassa. However, taking a consideration of transversion values only, smaller divergence values than those with both transitions and transversions resulted. Similar results were obtained when Saccharomyces carlberqensis was used as an outgroup (Table 3). It was found that Pyronema domesticum was 1% and 5% divergent from Saccharomyces, by using the transversion and transition values respectively. The percent divergence value of 10 was found for lodophanus by using the










39

transitions values but only 3% divergence was found by using the transversions.

Since full sequence alignment should be attempted only when it is accepted that the sequences are related from one end to the other, multiple sequence alignments of the ITS1 and ITS2 regions were not possible. Because of similarity in length, Ascodesmis nigricans, Ascodesmis sphaerospora, Eleutheroascus lectardii, Saccobolus pauperatus, Lamprospora sp. I and Lamprospora sp. II, Thelebolus sp. and Pyronema domesticum sequences from ITS2 regions were aligned by using the pileup multiple alignment program (Figure 6). In this study, the comparisons of the 5.8S region from these species are not sufficient for making phylogenetic inferences, because the 5.8S region is too conserved. However, the ITS regions which are not as conserved as coding regions may be used to analyze phylogenetic relationships in closely related species. Since ITS regions were variable in length, gaps were introduced to facilitate the alignment.

The alignment of sequences was formatted for inferring phylogenetic trees using the PAUP (Phylogenetic Analysis Using Parsimony) computer program of D. L. Swofford (1990). Version 3.0 of this program for the Macintosh system was used. All characters were treated as unordered entities. A heuristic approach with the tree-bisection-reconnection (TBR) branch-swapping algorithm was performed. In the first









40








Table 2. Percent Divergence Values in the 5.8S rDNA



SPECIES %DIVERGENCE %DIVERGENCE
(TV+TS+GAP) (TV+GAP)

LAU 8.0 2.0

LAM 6.0 1.8

PYR 8.0 2.0

ASN 8.0 2.0

ASD 9.0 2.0

PAC 8.0 3.0

IOD 12.0 4.0

GYR 7.0 3.0

OTI 7.0 3.0

ELE 8.0 2.0

SAC 8.0 3.0

THE 5.0 1.8




Outgroup = Neurospora crassa
Percent divergence values were calculated by using the formula: D= 100(tv+ts+gap)/total nucleotides shared or D= 100(tv+gap)/total nucleotides shared.









41







Table 3. Percent Divergence Values in the 5.8S rDNA




SPECIES %DIVERGENCE %DIVERGENCE
(TV+TS+GAP) (TV+GAP)


LAU 7.0 2.0

LAM 6.0 2.0

PYR 5.0 1.0

ASN 7.0 2.0

ASD 8.0 3.0

PAC 8.0 3.0

IOD 10.0 3.0

GYR 9.0 5.0

OTI 7.0 3.0

ELE 7.0 2.0

SAC 7.0 3.0

THE 5.0 3.0



Outgroup = Saccharomyces carlbergensis Percent divergence values were calculated by using the formula: D= 100(tv+ts+gap)/total nucleotides shared or D= 100(tv+gap)/total nucleotides shared.










42

analysis transitions and transversions were given equal weights. The undetermined two base pairs of the 5.8S region from Peziza vesiculosa were treated as missing data and included in the analysis. One most parsimonious tree with a consistency index of 0.714 was obtained by analyzing the sequences from the 5.8S region (Figure 7). Three major branches were observed with Neurospora crassa (NEU) as the outgroup. Iodophanus sp. (IOD) with Peziza vesiculosa (PEV) formed a clade with Plicaria endocarpidales. Otidea leporina (OTI) and Gyromitra montana (GYR) were in a separate branch, whereas Pyronema domesticum (PYR), Eleuthoascus lectardii (ELE), Thelebolus sp. (THE), Ascodesmis nigricans (ASN), Ascodesmis sphaerospora (ASD), Saccobolus depauperatus (SAC), Lampospora sp.(LAM), and Lampospora sp.(LAU) formed a single group whose lower-level relationships were not resolved by using the 5.8S coding region sequences. In a second analysis using transversions which were not as subject to multiple hits as transitions, the same relationships were derived for these eight taxa. Since the 5.8S coding region was too conserved to resolve lower-level relationships of these eight taxa, sequences from variable regions such as ITS were used. The ITS2 region was selected for this analysis because it was similiar in length among the species tested. Heuristic approach with TBR algoritm was performed. Iodophanus was










43

used as an outgroup. A single most parsimonious tree with a consistency index value of 0.538 was found (Figure 8). Ascodesmis nigricans, Ascodesmis sphaerospora, and Saccobolus depauperatus were clustered together. Two Lamprospora species with Thelebolus and Eleuthroascus formed a separate clade (Figure 8). Pyronema domesticum formed an independent line in this network.











44






50
Asdit92 AAAACCTCA ACCATAATTT ATTATGAGTT GGTATTGCAT TGGACTTCTT Asnits2 AAAACCTCA ACCATAATTT ATTATGAGTT GGTATTGCAT TGGACTTCTT Sacits2 AAAACCTCA ACCATAATTT ATTATGAGTT GGTATTGCAT TGGCCTTCTT Eleits2 AAAACCTCAA CCCATAATTT ATTATGAGTT GGTTCTGCAT TGGACTTTAT Lamits2 AAGACCACTC AAGCGA.TTT TGCTTGGTAT TGGAAGAAGA G..CGCCTCT Lauit92 AAGACCACTC AAGCGACTCT TGCTTGGTAT TGGAAGAAGA G..AGCTTCA Pyrits2 AAACCTCCT CAAGCTCTTT TGCTTGGTAT TGGAAGAAGA GGCCGCTTGT Theits2 ...... CAAC CCTCAAGCTT TGGTGGGTAT TGGA ...... CATTGCCAGT

51 100
Asdits2 GTGGGTCCTC TGCGAAATTC AATGGCGAAG AGCCACGCAA CCAAAG ....
Asnits2 GTGGGTCCTC TGCGAAATTC AATGGCGAAG AGCCACGCAA CCAAAG ....
SacitS2 GTGGGTCCTC TGCGAAATTC AATGGCGAAG AGCCACGCAA CCAAAG ....
EleitB2 ATGGGTCCTT GGTGAAATTC AATGGCGAAG AGCCACGCAG CCAAAG ....
Lamits2 GGCCCTCCCT TCCGAAATTC AATGGCGGAA AGTCTCACGT GCCCCGGCGT Lauit92 GGCCCTCCCT TCCGAAATTG AATGGCGGAA AGTCTCACGT GCCCACGCGT Pyrits2 CGGTCTCCCT TTCGAAATGC AATGGCAGAT TGCCTCATGT GCCCTGGCGT Theits2 TTCTGGCAGG TCTTAAAATC AGTGGCGG.. TGCCATTTGG CTTCAAGCGT

101 150
Asdits2 CGTAGTATAA CTATTTCGTT ATGGAAGCGT TGGTGCCTCT GCCGTAACCC Asnit92 CGTAGTATAA CTATTTCGTT ATGGAAGCGT TGGTGCCTCT GCCGTAACCC Sacite2 CGTAGTATAA CTATTTCGTT ATGGAAGCGT TGGTGACTCT GCCGTAACCC Eleits2 CGTAGTATAA TAATCTCGTT ATGGATGTGT GGATACCTCT GCCGTAA.CC Lamit92 AGTAAG.TTT ATCTTTCGCT TGGACCCTGA GGCGTTCTCG CCCTCAAATC Lauits2 AGTAAG.TTT ATCTTTCGCT TGGACGCTGA GCCCTTCTCG CCCTCAAATC Pyrits2 AATAAGATTT ATCTTTCGCT TGTGCATTGG GATGATCCCG CCGCAAACCC Theits2 AGTAATTCTT CTCGCTTTGG AGATCCAGGT GGT.TACTTG CCAATAACCC

151 166
Asdits2 CCCAATTTCT TAGTTT AsnitB2 CCCAATTTCT TAGGAT Sacits2 CCCAATTTCT TAGTTT Eleits2 CCCAATTTCT TAGTTT Lamits2 CCCAATACTC TAGG..
Lauits2 CCCAACGCAA CACTGG Pyrits2 CCAATTTTTT CTGG..
Theits2 CCAATTTTTT CAGG..






Figure 6. Multiple sequence alignment of the ITS2 region
from species of different families. Species abbreviations are in materials and methods.







45



NEU PAC IOD

PEV PYR ELE

THE ASN LAU ASP SAC

LAM OTI GYR



Figure 7. The most parsimonious tree for the 5.8S
sequences using Neurospora as an outgroup.
Species abbreviations are in materials and
methods.







46


Asdits2 Asnits2 Sacits2 Eleits2 Pyrits2 Lamits2 Lauits2 Theits2 Iodits2




Figure 8. The most parsimonious tree for the ITS2 sequences
using lodophanus as an outgroup. Abbreviations are
in materials and methods.















CHAPTER IV

CLADISTICS: MORPHOLOGICAL AND ULTRASTRUCTURAL DATA



Among the Ascomycetes, the operculate Discomycetes are unique because the operculum is not found among other Ascomycetes or lichens. Pezizales are also unique in that they all have non-septate ascospores. The size and shape of Pezizales are highly variable, ranging from small globose discoid to large saddle shaped or spongy apothecia (Korf, 1973). The Tuberales and certain Plectomycetes are believed to be derived from the Pezizales (Trappe, 1979; Malloch, 1979). However, without the fossil record or supported hypothesis of geneological relationships within the order, ideas on the origin of Pezizales are speculative. Within the order Pezizales, as many as eighteen families, inclusive of the Tuberales are recognized by different mycologists (Eriksson and Hawksworth, 1991). However, there is considerable controversy as to the limits of genera and families in this order.

The members of Ascobolaceae are small, mostly dunginhabiting, operculate Discomycetes. The concept of the family and the number of genera have changed considerebly over the years with as few as three (Rifai, 1968) and as 47









48

many as seventeen genera (Le Gal, 1947; Kimbrough, 1970) recognized. Boudier (1869) recognized two subgroups," Ascobolei genuini" for the pigmented spored genera, and "Ascobolel spurii" for hyaline spored genera. Later, most of the hyaline-spored species were transferred to Thelebolaceae (Kimbrough and Korf, 1967; Eckblad, 1968) with the exception of Iodophanus. Brummelen (1967) described the two genera, Ascobolus and Saccobolus forming a sharply delimited, natural group in Ascobolaceae. He paid special attention to the development and the microscopic structures of these fungi in connection with the relationships within the genera. Using ultrastructural data, Kimbrough and Curry (1985) placed Ascobolus, Saccobolus, and Thecotheus in this family based on the ascoboloid septal type. Iodophanus was found to have septal structure characteristics of Pezizaceae and was transferred to the Pezizaceae.

Ascodesmidaceae are characterized by small,

gymnohymenial ascocarps. Ascodesmis, on which the family is based, has been placed most often in Ascobolaceae (Brummelen, 1967). The taxonomic revision of the genus Ascodesmis and family Ascodesmidaceae were delimited and defined by Brummelen (1981). The genus Eleuthroascus was included in the Ascodesmidaceae because of its septal, ascal, and ascospore similiarity to Ascodesmis (Brummelen, 1989). Kimbrough (1989) placed Ascodesmidaceae and









49

Pyronemataceae in a new suborder, Pyronemineae, and transferred Amaurascus to Ascodesmidaceae.

There is considerable controversy regarding the natural limits of Helvellaceae (Kimbrough et al., 1990). Nannfeldt (1937) recognized the similarity of sessile taxa such as Discina and stipitate genera such as Gyromitra. Berthet (1964) demonstrated that the tetranucleate condition of ascospores, the habitat, and pigmentation of apothecia were features of Helvella, Gyromitra, Rhizina, and Discina. Eckblad (1968) included Rhizina, Discina, and Gyromitra and some related genera with tetranucleate ascospores in a separate family, the Rhizinaceae, based on spore ornamentation. However, recently Kimbrough and Gibson (1988) and Kimbrough (1991) have shown that there are similarities of septal pore organelles in "discinoid" and "gyromitroid" groups. They include two tribes, Gyromitrae and Discinae in the family Helvellaceae.

Humariaceae is the largest and most confusing family of the order Pezizales. Rifai (1968) demonstrated two types of development, with the first series of genera having pale or brownish apothecia with slender hyphae, and the second series have bright colored apothecium with paraphyses, usually turn green in iodine. He proposed that one part of Humariaceae might have originated from Helvellaceae. He basically recognized four tribes, Otideae, Lachneae,









50

Ciliarieae, and Aleurieae. Many genera of Humariaceae were placed in the large family Pyronemataceae (Korf, 1973), Otideaceae (Eckblad, 1968), and Aleuriaceae (Arpin, 1968). Recently, ultrastructural studies by Wu and Kimbrough (1991) showed that genera such as Aleuria and Leucoschypha (tribe Aleuriae) have an "aleurioid" septal type and produce mostly rough spores, and possessed a "gradual condensation" type of spore ontogeny. They demonstrated that distinct types of spore ontogeny always correlate with specific types of septal structures.

Eckblad (1968) defined a new family Otideacae as having medium-sized sessile to shortly stipitate apothecia. He included within it the humariaceous genera Geopyxis, Otidea, Pustularia, Sowerbyella, and Ascosporassis. Using light microscope data, Rifai (1968) concluded with Eckblad and others that many similiarites existed between "aleurioid" and "otideoid" genera. Korf and Zhuang (1991) placed Aleuria, Antracobia, Octospora, and Lamprospora in the family Otideaceae based on the apothecium and ascospore features. Wu and Kimbrough (1991) determined that Otidea, Anthracobia, and Octospora, produce mostly smooth ascospores, having with an "antracobioid" septal type and "gradual condensation" type of spore ontogeny. They found a direct correlation between spore ontogeny and septal structures for these genera.









51

Pezizaceae is the family of Pezizales with mostly

medium to large-sized, discoid, cupulate apothecia in which the asci turn blue in iodine. Asci that turn blue in iodine are also found in Ascobolaceae, and therefore taxa such as lodophanus and Thecotheus, have been transferred in and out of the two families. Also, the genus Boudiera was placed in Pezizaceae by Korf (1973) and in Ascodesmidaceae by Eckblad (1968). Curry and Kimbrough (1983) have found that septal structures are unique in the Pezizaceae. Dyby and Kimbrough (1987) considered Peziza as a representative genus of the family when they studied spore ontogeny and the chemical nature of spore walls in selected species. Data from these studies and that of earlier work from Curry and Kimbrough (1983) on septa showed that these features were consistent at the family level.

Following Rifai (1968) and Arpin (1968), Kimbrough (1989) restricted the Pyronemateceae to taxa with small gymnohymenial apothecia, hyaline spores, and limited exipular growth. Eckblad (1968) appears to be the first to greatly expand the limits of the Pyronomataceae to include eighteen genera. Among these were genera placed in the Aleuriaceae (Arpin, 1968), Ascobolaceae (Brummelen, 1967), and Humariaceae (Rifai, 1968). Korf (1972) recognized five subfamilies and several tribes which correspond to the Ascobolaceae, Aleuriaceae, and Humariaceae of Le Gal (1947),









52

including the Otideacae and Thelebolaceae of Eckblad (1968), and Aleuriaceae of Arpin (1968). A new suborder Pyronemineae was proposed by Kimbrough (1989) to include the Pyronemataceae (sensu Rifai) with the genera Coprotus and Pyronema.

Most of the taxa placed in the family Thelebolaceae originally represented the "Ascobolei spurii" (Boudier, 1869), and later recognized as Pseudoascobolaceae (Dennis, 1968). Many genera such as Ascozonus, Caccobius, Coprobolus, Coprotus, Lasiobolus, Thelebolus, and Trichobolus were initially placed in this family. Kish (1974) found that Coprotus was related to the Pyronema. Samuelson (1978) concluded that ascal structures in Ascozonus were like those of the Aleuria-Otidea complex. Samuelson and Kimbrough (1978) concluded that the ascal structure of Trichobolus, which was originally placed in Thelebolus, was very different from that of Thelebolus. They suggested a relationship of Trichobolus to Lasiobolus, but a proper family for these genera has not been identified. Kimbrough (1981) and Brummelen (1981) have shown that asci of Thelebolus are bitunicate and should be excluded from the Discomycetes. Subsequent research has shown that the Thelebolaceae family is a very heterogeneous group (Kimbrough, 1981).









53

The family Sarcoscyphaceae was characterized by

suboperculate asci and leathery to corky apothecia with a gelatenous layer in the excipulum. The family was divided into two tribes, Sarcoscyphaceae and Urnuleae, by Le Gal (1947). Korf (1970) proposed placing taxa of Urnuleae in the Sarcosomataceae. However, ultrastructural studies showed that the dehiscence zone of the operculum and ascal wall structures of Sarcosomateceae differed from the taxa of Sarcoscyphaceae (Samuelson et al., 1978; Kimbrough and Li, unpublished).

Six of the families placed in Pezizales by Eriksson and Hawksworth (1991) are hypogeous, the Monoascaceae is a Plectomycetes, and three families are questionable operculate Discomycetes. In this part of the study, the Parsimony analysis based on the morphological and ultrastructural characters of selected families of epigeous Pezizales are used to examine the intra- and inter-specific relationships, and to test congruence between molecular data and morphological observations.

Genera of nine families of Pezizales were selected and subjected to a phylogenetic analysis using PAUP (Phylogenetic Analysis Using Parsimony), a computer program of D. L. Swofford (1990). Version 3 of this program for the Macintosh system was used. These genera correspond to those used in the molecular studies described earlier (Chapter









54

II). Fifty characters were selected and character states were polarized by using Neurospora as the outgroup. The characters used in this study are summarized in Table 4. Table 4. Character and Character States of Epigeous and
Hypogeous Pezizales



I. APOTHECIA

1. Development: 0 = cleistohymenial; 1 = gymnohymenial.

2. Shape: 0 = perithecial; 1 = cupulate; 2 = stalkedcupulate; 3 = saddle-shaped; 4 = morchelloid.

3. Size: 0 = <5mm-5cm; 1 = >5cm.

4. Size: 0 = 2->5cm; 1 = 5mm-0.3mm.

5. Texture: 0 = fleshy; 1 = leathery to corky or

gelatinous.

6. Color: 0 = non-pigmented; 1 = water soluable pigments; 2

= carotenoids; 3 = oxydized carotenoids.

7. Hairs: 0 = absent; 1 = present, nonrooted; 2 = rooted. 8. Substrate: 0 = pyrophilic or soil; 1 = cellulosic; 2 =

coprophilic.

9. Substrate: 0 = cellulosic, coprophilic or soil; 1 =

pyrophilic.

10. Nutrition: 0 = saprobic; 1 = mutualistic symbiosis; 2 =

antagonistic symbiosis.










55

Table 4--continued

II. ASCI

11. Operculum: 0 = not present; 1 = functional; 2 =

nonfunctional.

12. Dehiscence zone: 0 = not present; 1 = differentiation

of endoascal wall; 2 = differentiation of exoascal wall. 13. Operculum: 0 = not present; 1 = apical, thin-walled; 2

= oblique, thickened lid.

14. Subopercular shoulder: 0 = not present; 1 = present. 15. Iodine reaction: 0 = positive; 1 = negative. 16. Number per apothecium: 0 = numerous; 1 = one to ten. 17. Phototropism: 0 = absent; 1 = present. 18. Shape: 0 = narrowly cylindric; 1 = saccate; 2 =

globose.

19. Septal structures: 0 = torus, V-shaped striation,

simple, biconvexed bands; 1 = zonate, hemispherical

dome.

20. Septal structures: 0 = hemispherical dome or simple,

biconvexed bands; 1 = matrix with laminated, translucent

torus; 2 = granular matrix with V-shaped striations.



III. ASCOSPORES

21. Symmetry: 0 = ellipsoid; 1 = globose. 22. Shape: 0 = symmetrical; 1 = asymmetrical. 23. Color: 0 = hyaline; 1 = yellow-green;









56

Table 4--continued

2 = purple-brown; 3 = black

24. Chemistry: 0 = noncyanophilic walls; 1 = cyanophilic. 25. Chemistry: 0 = noncarminophilic nuclei; 1 =

carminophilic.

26. Surface: 0 = smooth; 1 = smooth to slightly ornamented;

2 = highly ornamented.

27. Wall ontogeny: 0 = direct precipitation; 1 = gradual

condensation.

28. Coherence: 0 = free; 1 = fused. 29. Lipids: 0 = absent; 1 = small lipids; 2 = large lipid

droplets.

30. Granular inclusions: 0 = absent; 1 = present. 31. Nuclear condition: 0 = uninucleate; 1 = tetranucleate;

2 = multinucleate.

32. Spores per ascus: 0 = fewer than eight or eight; 1 =

greater than 8.

33. Spores per ascus: 0 = more than eight or eight; 1 =

fewer than eight.

34. Spore liberation: 0 = synchronous; 1 = asynchronous.



IV. PARAPHYSES

35. Presence: 0 = pseudoparahyses; 1 = present; 2 =

absent.

36. Pigmentation: 0 = hyaline; 1 = pigmented.









57

Table 4--continued

37. Branching: 0 = simple; 1 = branched. 38. Shape: 0 = filamentous; 1 = clavate; 2 = setose or

highly modified.

39. Nuclear condition: 0 = uninucleate; 1 = multinucleate. 40. Lipid droplets: 0 = absent; 1 = present. 41. Granular bodies: 0 = absent; 1 = present. 42. Septal occlusions: 0 = lamellate structures absent; 1 =

lamellate structures present.

43. Woronin bodies: 0 = globose; 1 = hexagonal; 2 = long,

rectangular.



V. EXCIPULUM

44. Differentiated: 0 = ectal and medullary areas present;

1 = excipulum of only one tissue type.

45. Tissue types: 0 = textura intricata; 1 = textura

prismatica; 2 = texture angularis; 3 = textura globosa. 46. Gel tissue: 0 = absent ; 1 = present. 47. Nuclear condition: 0 = uninucleate; 1 = coenocytic. 48. Septal structure: 0 = lamellate structures absent; 1 =

lamellate structures present.

49. Woronin bodies: 0 = globose; 1 = hexagonal; 2 = long,

rectangular.









58

VI. ANAMORPHS

50. Anamorphs: 0 = absent; 1 = present.



Explanation for Character-States Used in This Study



I. APOTHECIA:

1(1). Development: All presumed Pyrenomycete and

inoperculate Discomycetes ancestors have a cleistohymenial development. Gymnohymenial development is found in a number of Pezizales.

2(2). Shape: Most Pezizales have sessile, cupulate apothecia (Korf,1972), a character shared by most Helotiales. Stipitate, saddle-shaped, gyrose, and morchelloid apothecial characters are major modifications.

3(3, 4). Size: A large majority of Pezizales apothecia are from 2-5 cm in diameter (Korf, 1972), but several form robust (over 5cm) or with small (less than 5mm) apothecia.

4(5). Texture: Pezizales predominantly have fleshy, succulent apothecia, but some are leathery to corky, or gelatinous.

5(6). Color: The ability to produce pigments, such as carotenoids, is common in many Pezizales. Arpin (1968) considers carotenoids to be good phylogenetic tracers because species more advanced chemically have carotenoids and the most advanced taxa produce oxydized carotenoids.









59

Most families of Pezizales are devoid of carotenoids, but instead have a variety of water soluable pigments.

6(7). Hairs: Excipular hairs are special cellular

modifications for ecological adaptation. Glabrous apothecia are common but others have elaborate and complex types of hairs. Eckblad (1968) notes that when different hair types are present, it is usually correlated with differences in several other characters.

7(8-10). Substrate: An assumption can be made that most Pezizales are saprobic but symbiotic taxa, either antagonistic or matualistic, are present. Also, Pezizales appear basically cellulosic with many pyrophilic, coprophilic, and hypogeous species. Hypogeous Pezizales (Tuberales), appear to have become mutualistic symbionts of epigeous taxa and evolved a hypogeous habitat.



II. ASCI:

8(11-14). Operculum: Although Pezizales are characterized by opeculate asci, there are many modifications of the operculum (Kimbrough, 1972; Korf, 1973). The broad spectrum of epigeous Pezizales has an operculum in which the dehiscence zone results from physical and chemical changes in the inner layer of the ascus wall (Samuelson, 1975-1978). Those with other types of operculum ontogeny, such as the suborder Pyronemineae have been










60

discovered (Kimbrough, 1989). Some have a transition in the inner wall to form the operculum, some have pronounced shoulders below the operculum and the asci may split longitudinally instead of cicumscissily (Kimbrough, 1972), while others form thick, lens-shaped structures within the operculum (suboperculum sensu Le Gal, 1947).

9(15). Iodine Reaction: Samuelson (1978a) determined that the blue reaction in asci was restricted to an outer mucilagenous coat on the outside of the ascus. This state is shared by many inoperculate Discomycetes and lichens.

10(16). Number of Asci per Apothecium: If we derive Pezizales from inoperculate Discomycetes (Le Gal, 1953; Korf, 1958) we must assume that an extensive multiascal hymenium is primitive. Evolution went in two directions, one in which there is a sharp reduction in the number of asci per apothecium. Examples would include special adaptation for the coprophilic habitat (Kimbrough and Korf, 1967) in which there was a selection pressure for organisms with a large projectile. A second direction would be a great proliferation in the production of asci, resulting in extensive gyrose to spongy, undulating hymenium (Korf, 1958); this is treated under ascocarp shape. Most differ from the outgroup in having a larger number of asci.

11(17). Spore Ejection: Most epigeous Pezizales have asynchronous spore production, maturation and ejection.









61

Some asci become phototrophic (i.e. coprophilic taxa, Brummelen, 1967) and protrude strongly above the level of the hymenium at spore liberation. Others mature and eject their spore synchronously (i.e. certain Sarcoscyphaceae, Rifai, 1968). In hypogeous taxa there is for the most part no forceful spore discharge.

12(18). Shape: Eckblad (1968), felt that globose,

saccate, or broadly cylindric asci were primitive. Such conditions are also found in hypogeous taxa in which by growing within the substrate there is a restriction in normal hymenial growth (Korf, 1958; Trappe, 1979). Theoretically, those having evolved the most have lost their expansive hymenium of cylindric asci interspersed with paraphyses and have a restricted growth state with compressed and disarranged asci. Saccate asci in coprophilic taxa is the result of selection pressure to form large projectiles via large, multispored asci (Kimbrough, 1981).

13(19, 20). Septal pore structures: Various types of septal structures were found in epigeous Pezizales. The Peziza type septum shows the greatest simplicity and is somewhat similar to pit plugging in the red algae (Pueschel and Cole, 1982). The ontogeny and structure of pore plugs in the Florideophyceae is similiar to that of the Pezizaceae (Curry and Kimbrough, 1983) in the formation of electron-










62

opaque, biconvex bands. In Pezizales, ascal septa appear to be in two major lines of evolution, one in which there is additional plug deposition onto the convexed or biconvexed band to form a zonate, hemispherical dome and the second in which there is an electron-translucent torus bordering the pore in which there is what appears to be a loose matrix of granular material. This material usually accumulates within the ascus side of the septal pore. The matrix becomes more pronounced and with V-shaped striations in certain Humariaceae (Pulvinula and Geopyxis) and in members of Helvellaceae and Morchellaceae (Kimbrough and Gibson, 1989; Kimbrough, 1991). These two patterns of pore plug development are outlined in Figure 9.



III. ASCOSPORES:

14(21, 22). Shape: Most epigeous and hypogeous

Pezizales have broadly ellipsoid spores (Korf, 1972; Trappe, 1979). When Pezizales evolved opercula, an ellipsoid spore evolved concurrently to optimize spore ejection. There appear to be two lines of evolution, one in which the spores become globose, for example in the hypogeous and few epigeous taxa, and second, those that become cylindric or inequalateral (Sarcoscyphaceous taxa, Rifai, 1968). Several deviations from a broadly ellipsoid shape are present within the order.






























ul ul


.H 41


tH
4J La U) :j
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4 4-) (0 04 > (1)
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ro CY,
m u Q)
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64



F:4 F4 UF:4
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E-1 OW7 0
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65

15(23). Color: With minor exception, spores of Pezizales are hyaline (Korf,1973; Eckblad, 1968). Pigmentation of spores in Ascobolaceae (Brummelen, 1967), Ascodesmidaceae (Brummelen, 1989), and certain Pezizaceae (Korf, 1973) characterize those groups.

16(24, 25). Chemistry: A number of taxa of Pezizales have spores with peculiar staining reactions such as carminophily or cyanophily (Rifai, 1968; Eckblad, 1968; Korf, 1973), while most of the remainder are unreactive in various mounting agents.

17(26). Spore Surface: A majority of Pezizales have a smooth spore surface, a condition shared by most inoperculate Discomycetes (Korf, 1972). A large group has taxa with smooth to ornamented surfaces, while a smaller number have exclusively ornamented spores.

18(29, 30). Inclusions: The primitive ascophyte (Cain, 1972) or ancestral Pezizales probably had merely the essential cellular organelles, i.e., nuclei, mitochondria, and ribosomes. Additional organelles such as large lipid droplets (with or without carotenoids), polysaccaride granules or similiar inclusions occur. DeBary bubble formation (a gaseous interface) appears to be a special reaction to certain mounting media (Kimbrough nad Korf, 1967). It is doubtful that it is of any phylogenetic significance.










66

19(31). Nuclear Condition: With a few exceptions, spores of Pezizales are uninucleate (Berthet, 1964). Tetranucleate spores characterize Helvellaceae, and the multinucleate condition is a characteristic feature of Morchellaceae and is very common in hypogeous taxa. Parguey-Leduc and coworkers (1987) have shown that the multinucleate condition in species of Tuber is the result of atypical spore delimitation by the spore delimiting membranes. Tetranucleate spores of Helvellaceae (Gibson and Kimbrough, 1988) and multinucleate spores of Morchellaceae (Gibson and Kimbrough, 1988) are the result of mitotic divisions within the initial uninucleate spore.

20(32, 3). Number of Spore per Ascus: Eight-spored asci are the rule in Ascomycetes. However, there are some taxa with fewer or more spores per ascus. Multispored asci are most common among coprophilous Discomycetes (Kimbrough and Korf, 1967; Brummelen, 1967). Tetra-spored asci are less common in Pezizales (Eckblad, 1968; Rifai, 1968; Korf, 1972), but some eight-spored taxa produce four or fewer spores when grown in culture. Greatly reduced spore numbers are common in hypogeous Pezizales (Trappe, 1979), and is the result of atypical ascosporogenesis.



IV. PARAPHYSES:

21(35). Presence or Absence: With minor exceptions,










67

asci of Pezizales are interspersed with paraphyses (Korf, 1973). A number of hypogeous Pezizales have asci that are seperated by pseudoparenchyma cells (Gilkey, 1939; Trappe, 1979).

22(36). Pigmentation: A large majority of Pezizales has hyaline paraphyses (Kimbrough, 1970; Korf, 1972). But many taxa have paraphyses containing carotenoids or other pigments.

23(37). Branching: Most epigeous Pezizales have simple, unbranched paraphyses (Korf, 1972). But many have branched and anastomosing paraphyses. Hypogeous taxa are characterized by having highly branched and anastomosing paraphyses (Trappe, 1979).

24(38). Shape: Filamentous paraphyses characterize most Pezizales (Korf, 1972; Trappe, 1979), but broadly clavate or moniliform paraphyses occur.

25(39). Nuclear Condition: Berthet (1964) contends that the uninucleate state is more primitive than the coenocytic condition. The uninucleate condition is found in inoperculate Discomycetes and in a few operculate taxa but most operculates are coenocytic.

26(40, 41). Inclusions: Paraphyses of most Pezizales are without inclusion bodies. The formation of lipid (without or without caretonoids), granules, or other inclusion bodies are also common.










68

27(42, 43). Septal Structures: All Pezizales (including hypogeous taxa) studied thus far have a granular lamellate structure within septal pores of paraphyses (Curry and Kimbrough, 1983; Kimbrough, 1991). They are considered uniquely pezizalean because they are not found in other Ascomycetes. Evidence suggests that Woronin bodies are composed largely of lipoproteins (Wu and Kimbrough, 1991). There appears to be great differences in concentration of proteins in Woronin bodies of various taxa of Pezizales, and perhaps accounts for morphological variations seen in Woronin bodies (Kimbrough, 1991). Simple, globose Woronin bodies are most common, but hexagonal, long, and rectangular types also occur.



V. EXCIPULUM:

28(44). Ectal and Medullary Differentiation: A majority of Pezizales has well differentiated ectal and medullary excipular layers (Korf, 1972; Gilkey, 1939; Trappe, 1979). This trait is not shared with the outgroup, Neurosopora crassa. Reduced and highly modified excipula are common among Pezizales.

29(45). Tissue Types: There are several types of

tissues found within the excipulum of epigeous and hypogeous Pezizales (Korf, 1973). It is difficult to project what types of tissues were present in ancestral Pezizales,










69

although the outgroup, Neurospora, has predominantly a texture prismatica to textura angularis. The simplest type of tissue consists of interwoven hyphae (textura intricata), but other special arrangements or modifications of this type are present.

30(46). Gel Tissues: A number of Pezizales have

specialized gel tissues either in the medullary or ectal excipulum (Moore, 1965).

31(47). Nuclear condition: Berthet (1964) and Eckblad (1968) considered the uninucleate condition of exipular cells to be primitive. Most Pezizales have coenocytic excipular cells.

32(48, 49). Septal Structures: Septal pore structures in excipular cells parallel those of the paraphyses. All are characterized by the presence of a granular matrix with striations, the "lamellate structure" (Curry and Kimbrough, 1983), that varies somewhat in different taxa. Woronin bodies also may be globose, hexagonal, long, or rectangular. Globose Woronin bodies are found consistently throughout Ascomycetes.



Results of Morphological and Ultrastructural
Analysis


Cladistics using parsimony analysis was performed with the PAUP (Phylogenetic Analysis Using Parsimony) software of










70

Swafford (1991). A single most parsimonious tree was found with a consistency index value of 0.511.

The two Ascodesmis species clustered together and appear to be more closely related to Saccobolus and Eleuthroascus than Peziza (Figure 10). Pezizaceous genera, Peziza, Plicaria, and lodophanus, formed a separate branch, suggesting a close relationship to the helvellaceous genera Helvella and Gyromitra. The two Lamprospora species showed a close affinity to Ascodesmidaceae. Otidea (Humariaceae) appears to be more closely related to Urnula (Sarcosomataceae) than to Pezizaceae. Thelebolus (Thelobolaceae) appears not to be closely related to either Ascodesmidaceae, Ascobolaceae, Pezizaceae or Humariaceae.







71




NEU ASSAC
ASN ASD ELE
LAM LAU
IOD
IpPAC IOD

PEV L-f GYR
-- HEL
OTI
-- URN

PYR THE



Figure 10. The most parsimonious tree in the analysis using
the morphological and ultrastructural data.
Abbreviations are in materials and methods.















CHAPTER V


GENERAL DISCUSSION



Fifteen species which belong to different families of the order Pezizales were investigated in this study. For a more complete analysis and identification of inter-specific variation among the species, amplification and direct sequencing of nuclear ribosomal DNA were done. The primers ITS2, ITS3, ITS4, and ITS5 specifically amplified the ITSl, 5.8S, and ITS2 regions of ribosomal DNA. In contrast to the size of the 5.8S region which was conserved among the species, the size of ITSl and ITS2 was variable. The species which were closely related based on morphological characters, such as Ascodesmis nigricans, Ascodesmis sphaerospora, Saccobolus depauperatus, and Eleuthroascus lectardii, showed a conserved ITS1 region of 170 bp in size. Recently, DNA sequence comparisons of the nuclear ITS region showed that genera of the family Sordariaceae (Ascomycetes) are no more divergent in this DNA region than are species of Laccaria or Suillus that belong to different orders of Basidiomycetes. This cannot be easily explained. One thought might be that speciation may


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have occurred more rapidly in Sordariaceae, considering the life cycles of saprobic Ascomycetes, than in mycorrhizal Basidiomycetes. Within Armillaria species, ITS sequences were insufficient to resolve relationships within all species; thus the other variable regions of rDNA such as NTS (nontranscribed spacer) or IGS (intergenic spacer) might be more useful (Bruns et al.,1991). In this study the ITS region was very useful for resolving uncertain positions of closely related species. The cluster of eight species (Figure 8) was resolved by using the variable region ITS2.

In plants and animals, it has been reported that

certain DNA regions called isochores show biases of G+C/A+T compositions (Salinas et al., 1988; Bernardi et al., 1988). Takaiwa and et al. (1985) showed that ITS1 and ITS2 regions of O.sativa have high G+C contents of 72% and 77% respectively. However, Yokota et al. (1989) showed that G+C contents of D. carota and V.faba ITS1 and ITS2 are approximately 50%. The similarity values between these two dicots were 49% in the ITSl and 53% in ITS2 regions, in contrast to the value of 93% observed between their 5.8S rDNA sequences. Both ITS sequences in mouse are very rich in G+C, 70% and 74.3% for ITS1 and ITS2 respectively. Also for each ITS, the number of Gs equals exactly the number of Cs (350:350 in ITS1, 407:407 in ITS2). Both ITS are particularly deficient in As: 7.1% and Ts: 6.2% for ITS1 and










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ITS2 respectively (Furlong and Maden, 1983). In this study, similiar percentage G+C values were found in the ITS and

5.8S regions of Gyromitra, Lamprospora, and Thelebolus, Ascodesmis, Eleuthroascus, Saccobolus, Pyronema, and Iodophanus (Figure 3). These base composition features clearly distinguish ITS sequences from 5.8s rRNA. The data obtained from plants support this argument (Torres et al., 1990). However, discrepancies occur with respect to base composition in protozoans such as between Crithidia fasciculata and Giardia lambia.

When mouse ribosomal ITS sequences are compared with

yeast and Xenopus, a complete lack of homology is observed, except for a short segment (13 bp) located immediately downstream from the 5.8S rRNA which is conserved between the two vertebrates. When Neurospora and Saccharomyces ITS sequences are compared very little homology was found. It has been observed that the sequences of the ITS region from different species of families of Pezizales showed interspecific variation that appears to be more than intraspecific variation. According to Michot (1982) the pattern of distribution of homologies between the species in areas of ITS may have been subject to selective pressure and could have some functional roles, probably in the control of ribosomal biogenesis.

Different parts of the RNA molecule may be subject to different selection pressures. It has been recently









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suggested that unpaired rRNA regions may give more reliable results at distant evolutionary distances than paired regions (Wheeler and Honeycutt, 1988). However, the available data indicated that the nucleotide sequences of

5.8S were highly conserved during evolution, but have changed to such an extent that phylogenetic trees can be constructed among families and orders. By contrast, the nontranscribed and transcribed spacer regions of rDNA were very variable among different organisms and show high sequence heterogeneity.

Based on morphological data, various evolutionary

pathways for Pezizales were proposed by many mycologists. Atkinson (1915) and Gaumann (1926, 1964) viewed Pezizales as evolving from a lower group of Ascomycetes such as yeastlike fungi (Endomycetales). Nannfeldt (1932), LeGal (1953), Berthet (1966), and Arpin (1968) all considered the Pezizales to have evolved from the inoperculate family Sclerotiniaceae. Their ideas were based on the study of LeGal (1946) who visualized the suboperculate Discomycetes as being an intermediate group between the operculate and inoperculate Discomycetes. LeGal (1953) suggested a number of phylogenetic lines in the Pezizales which would suggest a polyphyletic origin of various families. Along one line she projected that members of Otideaceae (=Humariaceae sensu LeGal) gave rise to the Pezizaceae (=Aleuriees) and this









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family in turn gave rise to Ascobolaceae. In another line, members of the Sclerotiniaceae gave rise to other groups of Humariaceae. Gaumann(1964) viewed the evolution of Pezizales from the Plectomycetes, going sequentially from small gymnohymenial taxa such as Pyronema to large complex members of Morchellaceae and Helvellaceae. Using karyotype data, Berthet (1966) suggested an origin of various families of Pezizales from the inoperculate Discomycete order Helotiales. Those with uninucleate ascospores and excipular cells were lower on the evolutionary chain than multinucleate Helvellaceae, Morchellaceae, or Sarcoscyphaceae. Eckblad (1968) viewed Pezizales as evolving from Pyrenomycetes. He felt that highly reduced cleistohymenial members of Thelebolaceae were very similiar in structure and ontogeny to perithecial members of the Pyrenomycetes. Through the elaboration of ascocarps, various other families evolved in which Morchellaceae, and Helvellaceae were at the end of the evolutionary chain. Cytochemical data such as carotenoids or other pigments led Arpin (1968) to agree with LeGal's opinion that suboperculate Sarcoscyphaceae evolved from inoperculate Sclerotiniaceae. He would similarly derive the Otideaceae and Pezizaceae from the inoperculate family Geoglossaceae. Based on septal structures, Wu (1991) demonstrated that there was a strong correlation of septal structures among









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various families of Pezizales. He proposed an evolutionary model linking Humariaceae with other families of Pezizales. Species producing smooth ascospores and simple apothecia such as Pyronema were considered to be primitive, and from this group there seems to be two directions of evolution, one connecting two families, Pezizaceae and Ascodesmideceae then to Ascobolaceae, and the other to both Helvellaceae and Morchellaceae. Ascobolaceae is thought to be an advanced group because of its complex spore ontogeny.

In this study, using the 5.8S and ITS sequences, phylogenetic inferences were made in various species representing different families of Pezizales. The position of Plicaria, Iodophanus, and Peziza in the Pezizaceae appears to be confirmed and tends to support septal ultrastructure data that show these genera to be closely related (Kimbrough and Curry, 1985). Although lodophanus has been placed among the Ascobolaceae (Brummelen, 1967; Dennis, 1978), the 5.8S and ITS data do not support this position. The parsimony analysis of morphological and ultrastructural data would suggest a close affinity of Pezizaceae and Helvellaceae. There appears to a close correlation of results from the parsimony analysis of molecular data and that of morhological and ultrastructural data. Also, the two Lamprospora species which were clustered together in both analyses and appear to be more










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closely related to Ascodesmis than to lodophanus (Figure 8 and 10). This disagrees with the most current systematic arrangements (Eckblad, 1968; Rifai, 1968; Korf, 1973). It has been observed that there seems to be a disagreement regarding the position of Pyronema between morphologicalultrastructural cladistics analysis and molecular parsimony analysis. In the parsimony analysis from molecular data, Pyronema shows a close relationship to species of Lamprospora, whereas, in cladistics analysis from morphological and ultrastructural data Pyronema is more closely related to Otidea than Lamprospora.

For many years the species of Ascodesmis were placed within the Ascobolaceae (Brummelen, 1967; Dennis, 1968). More recently Ascodesmis has been placed in the family Ascodesmidaceae (Brummelen, 1981). Brummelen (1989) and Kimbrough (1989) both concluded that based on morphological and ultrastructural data Eleuthroascus also belongs to the Ascodesmidaceae. All data available from the 5.8S region as well as cladistics of morphological and ultrastructural data support the recognition of this family and a close relationship of Ascodesmis and Eleuthroascus. However, when the ITS2 region was analyzed Eleuthroascus was set apart from Ascodesmis. In fact it appears to be closely related to Pyronema. Also, it appears from these data that the Ascodesmidaceae is more closely related to Ascobolaceae than









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to the other families of Pezizales.

Gyromitra and other Helvellaceae with elaborate ascocarps and unique cytological and ultrastructural features are believed to be highly evolved (Berthet, 1964; Arpin, 1968; Eckblad, 1968; Gibson and Kimbrough, 1987; Kimbrough, 1989). There have always been questions, however, as to from which group of sessile, cupulate Pezizales they may have evolved. Using the 5.8S sequence data, Gyromitra and Otidea appear to be closely related.

The taxonomic position of Thelebolus species in the Pezizales has been controversial for several years (Kimbrough, 1981). The research of Samuelson and Kimbrough (1978) and later Kimbrough (1981) shows that species of Thelebolus belong to the Loculoascomycetes since they have bitunicate asci, with the "jack-in-the-box manner" of ascal dehiscence. However, Eriksson (1984) does not feel that Thelebolus should be removed from Pezizales. But both cladistics analysis using ultrastructural and morphological data, and the parsimony analysis from 5.8S and ITS sequences support the view that Thelebolus is not closely related to the species placed in Pezizales.

Finally, in the comparison of morphological and ultrastructural data with molecular information, the following conclusions were reached:

(1) Thelobolus is not closely related to species of









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Pezizales tested.

(2) A lack of similarity between Ascodesmis

(Ascodesmidaceae) species and Otidea (Humariaceae) was

observed.

(3) Gyromitra (Helvellaceae) is more closely related to

Otidea (Humariaceae) than Ascodesmidaceae.

(4) Ascodesmidaceae is more closely related to the

Ascobolaceae than the other families of Pezizales.

(5) The parsimony analysis from molecular data does not

agree with the cladistic analysis from morphological

and ultrastructural data regarding the position of

Pyronema. In the parsimony analysis, Pyronema

(Pyronemateceae) is closely related to the species of Lamprospora (Aleurieae sensu Korf, 1973), whereas, in

the cladistic analysis Pyronema shows a close

relationship to Otidea (Otideeae sensu Korf, 1973).

(6) The Lamprospora species examined were more closely

related to Ascodesmis species than to lodophanus

(Pezizaceae).

(7) An overall congruence between data from morphologyultrastructural features of Pezizales and data from

5.8S and ITS sequences exit. However, the

quantification of congruence needs to be done.















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BIOGRAPHICAL SKETCH



Esengul Ayse Momol was born May 15, 1956, in Kutahya, Turkey. She received a Bachelor of Science degree from the University of Ege, College of Pharmacy, in 1980. She first started working in Pharmacology department at the University of Florida under the guidance of Dr. E. Meyer, then she continued her work as a director of brain cell culture facility in Dr. M. Raizada's laboratuary located in Physiology Department at the University of Florida. In 1986, she was admitted to graduate school and in 1989, she received her Master of Science degree, specia lizing in the molecular genetics of fungus-plant interaction from Plant Pathology Department of the University of Florida. She has continued her studies toward the completing a Doctor of Philosophy degree in the molecular systematics.















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I certified that I have read this study and that in my opinion it confirms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Jmes W. Kimbroulh, Chairma
(ofessor of Plant Pathol' y



I certified that I have read this study and that in my opinion it confirms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.





Professor of lant Pathology



I certified that I have read this study and that in my opinion it confirms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.





Ernest Hiebert
Professor of Plant Pathology



I certified that I have read this study and that in my opinion it confirms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Gloria Moore
Professor of Horticultural
Science