AN ABSTRACT OF THE THESIS OF

David S. Gernandt for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on May 7, 1998. Title: Phylogeny, Cospeciation, and Host Switching in the Evolution of the Ascomycete Genus Rhabdocline on Pseudotsuga and Larix (Pinaceae).

Abstract Approved: Redacted for Privacy Jeffrey K. Stone

The relative role of cospeciation and host switching in the phylogenetic history of ascomycete foliar symbionts is addressed in the orders Leotiales and Rhytismatales, fungi associated predominantly with Pinaceae (Coniferales). Emphasis is placed on comparing the evolution of the sister genera Pseudotsuga and Larix (Pinaceae) with that of the pathogenic and endophytic fungi in the genus Rhabdocline. Pinaceae evolved during the Mesozoic and divergence of all extant genera and several infrageneric lineages (esp. in Pinus) occurred prior to the Tertiary, with subsequent species radiations following climatic changes of the Eocene. The youngest generic pair to evolve from Pinaceae, Larix and Pseudotsuga, diverged near the Cretaceous-Tertiary boundary in East Asia or western North America. Rhabdocline is comprised of seven species and subspecies, six known from two species of Pseudotsuga and one, the asexual species Meria laricis, from three species of Larix. Evidence from host distributions and from nuclear ribosomal DNA suggests that Rhabdocline speciated in western North America and has been involved in several host switches. The ancestor ofMeria laricis appears to have switched from P. menziesii to its current western North American hosts, L. occidentalis, L. lyallii, and very recently may have extended its host range to the European species, L. decidua. The occurrence of two lineages of R. weirii ssp. weirii on both North American species of Pseudotsuga is also probably the result of a recent host switch. Evidence of host- mediated divergence is seen in R. parkeri, which has different internal transcribed spacer types in the geographically isolated coastal and interior forms of P. menziesii. The wide host ranges of fungal genera closely related to Rhabdocline indicates that host switching is a prevalent pattern in the evolution of foliar symbionts in Leotiales and Rhytismatales. The prevalence of host switching in this group relative to other endosymbiotic organisms can probably be attributed to differences in dispersal mechanisms. Spores of foliar fungi are dispersed horizontally by wind and rain, rather than vertically from parent to offspring. Over evolutionary time, this provides more opportunities to shift to new hosts, particularly when the hosts are closely related and have overlapping distributions. ©Copyright by David S. Gernandt May 7, 1998 All Rights Reserved Phylogeny, Cospeciation, and Host Switching in the Evolution of the

Ascomycete Genus Rhabdocline on Pseudotsuga and Larix (Pinaceae)

by

David S. Gernandt

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Completed May 7, 1998 Commencement June 1998 Doctor of Philosophy thesis of David S. Gernandt presented on May 7, 1998

APPROVED:

Redacted for Privacy Major Professor, repiesenting Botany and Plant Pathology

Redacted for Privacy Chair of Department of Bot ny and Plant Pathol.:41

Redacted for Privacy Dean of Graduate, chool

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

Redacted for Privacy David S. Gernandt, Author Acknowledgments

It is with great pleasure that I extend my thanks to the many people who assisted and sometimes participated in the creation of this work. First and foremost, I thank my major professor, Jeffrey K. Stone for his countless hours of training that started while I was an undergraduate at the University of Oregon, continued throughout graduate school and which I hope will continue with future mycological collaborations. Jeffs confidence in me and willingness to guide my education have been critical in the positive graduate experience I had at Oregon State University. I thank the members of my graduate studies committee, Aaron Liston, Joseph Spatafora, Steven Strauss, and Patrick Breen. It was Aaron Liston who first introduced me to both the theoretical and practical aspects of molecular systematics. I have thoroughly enjoyed participating in the several collaborations that we have pursued together. Since his arrival at Oregon State University, Joseph Spatafora has been an excellent mentor to me in both molecular systematics and fungal evolution. I thank Patrick Breen for being my graduate school representative and for letting me off the hook towards the end of my preliminary oral exams. Steven Strauss kindly agreed to sit on my committee as a replacement member for the last hurdle of my graduate school career. I would also like to extend my thanks to former committee member Everett Hansen, who had to be replaced when he took a sabbatical in France. Everett served as a co-advisor with Jeff in the early stages of my graduate studies. Everett was very patient with me and was indispensible in planning my graduate program. Everett gave me room to roam in the laboratory, and in what was certainly a selfless desire to broaden my mycological education, provided me with ample opportunities to assist in his research on Basidiomycetes and Oomycetes. George Carroll and Howie Bonnett served as my undergraduate thesis advisors at University of Oregon. I thank them for first introducing me to the life of a research

scientist. Along with Aaron Liston, fellow student Ankie Camacho gave me my first training in DNA isolation, PCR, sequence analysis, and several other molecular methods that have proved invaluable. Over the years I have studied specimens generously provided from researchers around the world. Thanks are due to Ernest Ash III, Brenda Callan, George Carroll, Greg DeNito, John Dennis, Martin Gardner, Everett Hansen, Ottmar Holdenrieder, Dan Jones, Alan Kanaskie, Rolf Kehr, Blakey Lockman, Bill Merrill, Jim Oliphant, Catherine Parks, Miguel Soto, Yoshihiko Tsumura, Craig Schmitt, and Gerard Verkley. I also extend my gratitude to the American Type Culture Collection and Centraalbureau voor Schimmelcultures. Over the years my labmates have given me useful advice on everything from biological theory to technical support to lab courtesy. My sincerest thanks (approximately in order of appearance), to Tina Dreisbach, Marion Murray, Jerry Hefner, Mike McWilliams, Brennan Ferguson, Deborah Clark, Ankie Camacho, Wes Messinger, John Wheeler, Jim Oliphant, Susan Massey, Pablo Rosso, Koren Dennis, Aleta Groenig, Lori Trummer, Lori Winton, Kimberly Davis, and Bryan Capitano. Although I have not worked side-by-side with members of the Spatafora lab, they too have been availed themselves to me on countless occassions. Thanks to Jamie Platt, Lisa Grubisha, and Eric Peterson in that regard. Funding for this project has come from discretionary funds of Jeff Stone, and from personal contributions by David Miller Sr. and David Miller Jr. at Moore Mill and Lumber Company in Bandon, Oregon and by my grandmother, Frances Cavoretto. The Mycological Society of America awarded me several travel grants to their annual meetings as well as the Backus award. Sigma Xi contributed a grant in aid of research. The last several months of writing and analyses for this thesis were done in Mexico

City. I thank my future wife Sol Ortiz Garcia for her love and support during that time. I also thank her brother Salvador Ortiz Garcia for providing critical computer support. Finally, I would like to thank my parents, and once again, my grandmother, for all the extra little things they did to help me reach this point in my life. This thesis is dedicated to the loving memory of my grandmother, Frances Cavoretto. CONTRIBUTION OF AUTHORS

Chapter three was coauthored with Aaron Liston, who advised in the design and analysis, provided laboratory resources, and reviewed several drafts. Jeffrey K. Stone advised in the sampling, participated in acquisition of fungal isolates, provided laboratory resources, and edited several drafts for chapters one, four, five, six, and seven. Despite its placement in this thesis, chapter five was the first chapter written. Francisco J. Camacho kindly trained me in the molecular techniques and sequence analysis that were used in this and several other chapters. He also participated in the benchwork necessary for sequencing Meria laricis and Rhabdocline parker!. TABLE OF CONTENTS

Page

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1

1.1 Abstract 1 1.2 Introduction 2 1.3 Strengthening hypotheses regarding the evolution of Pseudotsuga and Larix 4 1.4 Phylogenetic reconstruction of inoperculate discomycetes 6 1.5 Speciation of Rhabdocline on Douglas-Fir and Larch 9 1.6 Literature Cited 12

CHAPTER TWO: A PHYLOGENETIC RECONSTRUCTION OF PINACEAE BASED ON RBCL AND THE FOSSIL RECORD 16

2.1 Abstract 16 2.2 Introduction 17 2.3 Materials and Methods 22 2.4 Results 25 2.5 Discussion 34 2.6 Literature Cited 35

CHAPTER THREE: INTERNAL TRANSCRIBED SPACER REGION EVOLUTION IN LARIX AND PSEUDOTSUGA (PINACEAE) 39

3.1 Abstract 39 3.2 Introduction 40 3.3 Materials and Methods 42 3.4 Results 49 3.5 Discussion 63 3.6 Acknowledgements 70 3.7 Literature Cited 70

CHAPTER FOUR: A PHYLOGENETIC RECONSTRUCTION OF INOPERCULATE DISCOMYCETES INFERRED FROM PARTIAL SMALL SUBUNIT SEQUENCES OF NUCLEAR RIBOSOMAL DNA 76

4.1 Abstract 76 4.2 Introduction 77 TABLE OF CONTENTS (continued)

Page

4.3 Materials and Methods 79 4.4 Results 86 4.5 Discussion 100 4.6 Acknowledgements 108 4.7 Literature Cited 109

CHAPTER FIVE: MERIA LARICIS, AN ANAMORPH OF RHABDOCLINE 115

5.1 Abstract 115 5.2 Introduction 116 5.3 Materials and Methods 117 5.4 Results 121 5.5 Discussion 126 5.6 Acknowledgements 131 5.7 Literature Cited 131

CHAPTER SIX: ANALYSIS OF NUCLEAR RIBOSOMAL DNA PLACES THE NEMATODE PARASITE, DRECHMERIA CONIOSPORA, IN CLAVICIPITACEAE (HYPOCREALES) 135

6.1 Abstract 135 6.2 Introduction 136 6.3 Materials and Methods 137 6.4 Results 138 6.5 Discussion 146 6.6 Acknowledgements 148 6.7 Literature Cited 148

CHAPTER SEVEN: THE RELATIVE ROLES OF COSPECIATION, DUPLICATION, HOST SWITCHING, AND SORTING IN THE EVOLUTION OF RHABDOCLINE (HEMIPHACIDIACEAE) 152

7.1 Abstract 152 7.2 Introduction 153 7.3 Materials and Methods 156 7.4 Results 157 TABLE OF CONTENTS (continued)

Page

7.5 Discussion 163 7.6 Acknowledgements 169 7.7 Literature Cited 170

BIBLIOGRAPHY 174 LIST OF FIGURES

Figure Page

2.1 Observed rbcL nucleotide substitutions for 17 members of Pinaceae 26

2.2 Strict consensus from a weighted parsimony analysis of conifer rbcL 27

2.3 Phylogenetic tree from parsimony analysis of Pinaceae rbcL 28

2.4 Distance tree from neighbor joining analysis of Pinaceae rbcL 29

2.5 Maximum likelihood phylogeny of Pinaceae rbcL 30

2.6 Maximum likelihood with molecular clock 31

3.1 Diagram for the ITS region of Larix 47

3.2 Diagram for the ITS region of Pseudotsuga 47

3.3 Secondary structure of ITS1 for Pseudotsuga menziesii 55

3.4 Phylogeny of ITS1 subrepeats 56

3.5 Nucleotide substitutions in the ITS region of Larix and Pseudotsuga 59

3.6 Strict consensus of two most parsimonious trees from the 5.8S and ITS2 60

3.7 Single most parsimonious tree from the full ITS region 62

4.1 Parsimony analysis of small subunit nrDNA including non-euascomycetes 87

4.2 Neighbor joining tree of small subunit nrDNA including non-euascomycetes 91

4.3 Parsimony analysis excluding non-euascomycetes and Orbiliaceae 93

4.4 Nucleotide substitutions in the small subunit for 36 inoperculate discomycetes 96

5.1 ITS region phylogeny of inoperculate discomycetes in analysis I 122

5.2 Alternative rooting approaches for Rhabdocline 123 LIST OF FIGURES (continued)

Figure Page

6.1 Phylogenetic tree of perithecial ascomycetes inferred from the small subunit 144

6.2 Phylogenetic tree of perithecial ascomycetes inferred from the ITS region 145

7.1 Single most parsimonious tree from the full ITS region of Rhabdocline 158

7.2 Comparison of the phylogenies of Rhabdocline and hosts 159

7.3 Comparison of host and parasite phylogenies 159 LIST OF TABLES

Table Page

1.1 Summary of the families and genera of Rhytismatales and Leotiales 7

2.1 Conifer taxa included in the rbcL study 23

3.1 Synopsis of Larix and Pseudotsuga species 43

3.2 Phylogenetic hypotheses proposed for extant Larix and Pseudotsuga 45

3.3 Nuclear ribosomal DNA ITS region sequence statistics for Larix and Pseudotsuga 53

4.1 Taxa included in the small subunit analyses 80

4.2 Intron distribution for inoperculate discomycetes sequenced 84

5.1 Taxa included in the ITS region analyses of Rhabdocline 119

6.1 Accessions included in the partial SSU analysis of perithecial ascomycetes 139

6.2 Accessions included in the ITS region analysis 142 Phylogeny, Cospeciation, and Host Switching in the Evolution of the Ascomycete Genus Rhabdocline on Pseudotsuga and Larix (Pinaceae)

CHAPTER 1

Introduction and Literature Review

1.1. Abstract

Several evolutionary lines of inoperculate discomycete fungi in the orders Rhytismatales and Leotiales have longstanding, intimate associations with coniferous hosts, particularly with members of Pinaceae. Leotiales is divided into approximately 13 families comprising 392 genera and Rhytismatales is divided into three families comprising 71 genera. While many of the genera are found mainly associated with members of Pinaceae, the exceptions of limited numbers of species on angiosperms and other coniferous families suggest that host switching has occurred. The objective of this study is to reconstruct nucleotide-based phylogenies for the inoperculate discomycetes and for Pinaceae. Particular attention is focused on the foliar fungus Rhabdocline and its evolutionary history on Pseudotsuga and Larix. Instances of host-mediated speciation and host switches are traced for several lineages of foliar fungi in the genus. Cladistic analyses are performed on nucleic acid sequence data from the internal transcribed spacer (ITS) region and the 5' end of the small subunit of nuclear ribosomal DNA. Besides allowing for the documentation of cospeciation and host switching, the nucleotide sequence-based phylogenies reported herein for Rhytismatales and Leotiales are useful in settling confusion regarding systematic relationships in these two understudied fungal orders. 2

1.2. Introduction

Symbiotic associations between fungi and vascular plants date to the earliest colonization of land by plants over 400 Ma (million years ago) during the Silurian Epoch. Pirozynski and Malloch (1975) hypothesized that during this epoch a symbiotic association between an autotrophic green alga and fungal heterotroph enabled the successful colonization of the terrestrial environment. Subsequent radiations in the Carboniferous resulted in a diversity of plant-fungus symbioses and hence a diversity of ecological interactions. Conifers probably arose at an early point during the Carboniferous period (360-286 Ma)(Florin, 1951; Miller, 1977). Over 50 genera of extant conifers in eight families include such well-known trees as redwood, yew, cedar, pine, fir, and spruce. Much is known about the fossil record and present distribution of conifers as well as their evolutionary relationships relative to other plant families (reviewed in Miller, 1988). Receiving considerably less attention are numerous ascomycetous fungi that are abundant and widespread as pathogens, neutral symbionts, and commensal saprobes of bark, twigs, and leaves of conifers. The inoperculate discomycete orders Rhytismatales and Leotiales are comprised of many such pathogens and inconspicuous, asymptomatic endophytes. The diversity and widespread distribution of foliar fungi with their hosts make them an excellent model system for studying cospeciation between plants and fungi. Before meaningful studies of cospeciation and host switching can be addressed for inoperculate discomycete fungi and their conifer hosts, it is imperative to obtain a more rigorous hypothesis of the systematic relationships among the organisms involved in the symbiosis. My first objective in this study is to reevaluate phylogenetic hypotheses for the organisms involved. In chapters 2 and 3, I address the evolutionary histories of the conifer hosts. In chapter 2, the chloroplast-encoded large subunit of ribulose 1,5-bisphosphate carboxylase (r b cL) is used to reconstruct a phylogeny of Pinaceae. Using an assumption of clocklike behavior, the phylogeny is evaluated in light of the fossil record of Pinaceae. Chapter 3 uses the ITS region of nuclear ribosomal DNA (nrDNA) to reconstruct the phylogeny of Larix and Pseudotsuga, the hosts of Mena laricis and Rhabdocline spp. respectively. Chapters 4-6 address the phylogenetic relationships among Rhabdocline and 3 its relatives. In chapter 4, partial sequences from the small subunit are used to reconstruct a phylogeny for the ascomycetes. The closest relatives of Rhabdocline are identified as needle cast fungi in the family, Hemiphacidiaceae. In chapter 5 the ITS region of nrDNA is used to tease apart the relationships among the species and subspecies ofRhabdocline and Meria laricis. The nematode endoparasite, Drechmeria coniospora, was formerly conspecific with Meria laricis. In chapter 6, sequence data from the ITS region and the small subunit of nrDNA are used to demonstrate that D. coniospora is phylogenetically related to perithecial fungi in the family, Clavicipitaceae. The disassociation ofMeria and Drechmeria removes an element of doubt as to whether Hemiphacidiaceae is a family comprised solely of conifer parasites. In the seventh and final chapter, the relative importance of cospeciation and host shifting of Rhabdocline and closely related genera parasitic on Pinaceae is examined. Factors favoring either of the two processes, include life history differences in hosts and parasites, recent range overlaps, and allopatric speciation of hosts. The characters used for formal analysis in this dissertation will be nucleic acid sequences. Except for Chapter 2, the source ofthe sequences will be from the nuclear- encoded ribosomal DNA. Partial sequences from the relatively conserved small subunit will be used to address higher level relationships among the ascomycetes, and the more variable ITS region (5.8S cistron, ITS1 and ITS2) will be used to describe relationships among closely related groups in both the inoperculate discomycetes and in Larix and Pseudotsuga. The use of nucleotides as characters has the advantage over using morphological characters because it can often result in the generation of significantly more raw data. This is of special advantage with foliar fungi because they often yield few informative morphological characters with which to work and many are subject to convergence, making systematic interpretations difficult. For hosts and parasites, morphological, ecological, and biogeographical characters will not be analyzed formally, but have been used to interpret molecular analyses where alternative explanations are presented. Morphological and ecological diversity among species and subspecies of Rhabdocline make the genus an ideal group for studying host-mediated speciation, shifts 4 in host, shifts in symbiotic interactions (e.g. from antagonistic to mutualistic), and establishing relationships between asexual (anamorphic) species and genera and families of ascomycetes. Recent nucleotide-based phylogenies have been used to demonstrate congruent evolution in eubacterial symbionts of aphids (Munson et al., 1991), eubacteria and whiteflies (Campbell, 1993), and fungus cultivating ants associated with basidiomycetes (Chapela et al., 1994). In these studies, vertical transmission of symbionts yielded highly congruent trees for both partners (see Page, 1994). In contrast, foliar fungi of conifers are dispersed horizontally by means of sexual and asexual spores, and the occurrence of several genera of foliar fungi on phylogenetically distant hosts suggests that there are numerous instances of host shifting between inoperculate discomycetes and plants.

1.3. Strengthening hypotheses regarding the evolution of Pseudotsuga and Larix.

An accurate picture of the evolutionary history of foliar fungi requires due consideration of the evolution and biogeography of their hosts. Morphological, immunological, and nucleotide based data have been used by others to reconstruct phylogenies of Pinaceae (Farjon, 1990; Prager et al., 1976; Price et al., 1987, Chase et al., 1993; Chaw, 1997). Pinaceae sequences from rbeL have recently been made available in Genbank. These sequences are analyzed in chapter 2. By using Podocarpus as an outgroup, and by sampling more genera and species from Pinaceae, several proposed phylogenetic hypotheses for the family are reevaluated. Then a previously published estimate of the substitution rate of rbcL is used to estimate the divergence time for main lineages of Pinaceae. In the second part of my investigations into host phylogenies, nucleotide characters are used to address several longstanding hypotheses regarding the evolution of Pseudotsuga and Larix. Pseudotsuga and Larix are recently derived genera, considered on the basis of fossil evidence and molecular clocks to have shared a common ancestor only 45-60 Ma (Axelrod, 1990; Strauss et al., 1990; Schorn 1994). The sister relationship 5 between Pseudotsuga and Larix has been established on the basis of fertilization and pollen development (Christiansen, 1972), karyotype analysis (El-Kassaby et al., 1983), immunoassays (Prager et al., 1976: Price et al., 1987) chloroplast DNA RFLPs (Tsumura et al., 1995), and rbcL phylogenies (Chase et al., 1993). Farjon (1990) conservatively recognizes ten species of Larix and four species of Pseudotsuga. The genus Pseudotsuga is found today in western North America and East Asia. Native Pseudotsuga species can be found in the Canada, the United States, Mexico, Japan, Taiwan, and mainland China. Although Farjon recognizes only one, others have traditionally recognized five species in Taiwan and mainland China (El-Kassaby et al., 1983; Hermann, 1985). Furthermore, some botanists have historically segregated Mexican P. menziesii as three or four separate species (Martinez, 1949). Bigcone Douglas-fir (P. macrocarpa) grows in the mountains of southern California. Pseudotsuga macrocarpa has been hypothesized to be the most plesiomorphic species of Pseudotsuga as well as the closest extant relative of P. menziesii. Pseudotsuga menziesii is the most abundant and widely distributed species in the genus, ranging from British Columbia south to California, and extending inland both along the Sierra Nevada Mountains, and from British Columbia to central Mexico along the Rockies. In an earlier investigation of the genus, Strauss et al. (1990) hypothesized that a common ancestor of P. macrocarpa and P. menziesii originated in western North America and migrated around the Bering Land Bridge to give rise to species of Pseudotsuga in Asia. Larix has a cooler, more circumboreal distribution than Pseudotsuga. Fossil records suggest that Larix probably originated in western North America before extending its range to Asia and Europe (Schorn, 1994). Three species are recognized in North America: western larch (L. occidentalis), subalpine larch (L. and tamarack (L. laricina). Based on the assumption that long, exserted bract scales on the ovulate cone are the primitive state of Larix and Pseudotsuga, L. occidentalis and L. lyallii have been proposed as the oldest extant lineages of the genus (LePage and Basinger, 1991; Schorn, 1994). Japanese larch (L. kaempferi) shares several intermediate characters of the female cone with a common ancestor that gave rise to the short-bracted species L. decidua (European larch), L. .sibirica (Siberian larch), L. gmelinii (Korean larch) and L. laricina. 6

Cone morphology, L. griffithiana (Himalayan larch), L. mastersiana (Master's larch), and L. potaninii (Chinese larch) have been hypothesized to be an intermediate lineage, perhaps representing an early migration by a primitive North American species to Asia (Schorn,

1994). In chapter 3, molecular data are used to reanalyze the evolutionary history of Pseudotsuga and Larix. The outcome suggests that exserted bracts have been overemphasized in reconstructing phylogenies for the group. Basal lineages with exserted bracts do not exist in the ITS phylogeny. It is more likely that the first split within both genera occurs by continent, with reduction occurring in a single lineage of Pseudotsuga and two separate lineages of Larix.

1.4. Phylogenetic reconstruction of inoperculate discomycetes

The diversity and abundance of inoperculate discomycete foliar fungi on conifers suggests that the group originated in the Mesozoic simultaneously with their hosts. The infrequency of their associations with angiosperms suggests that host shifts played an important role only after their cospeciation with conifers. The current taxonomic framework for the foliar fungi in this group has obscured this pattern of associations with conifers. The classification of foliar fungi at all taxonomic ranks is tenuous and based on few reliable morphological characters. Currently, the majority of genera of inoperculate discomycetes associated with conifer foliage are disposed in two orders, Leotiales and Rhytismatales (Table 1.1). Leotiales is a large and ecologically diverse order that includes, in addition to foliar fungi, ground-inhabiting, fleshy members (Geoglossaceae) and lichenized forms placed in Baeomycetaceae and Icmadophilaceae. Rhytismataceae is comprised of parasites and saprophytes primarily on foliage but also on bark and wood. Many members of these orders are endophytes-- generally asymptomatic colonists of live tissue. The two orders are defined by characters that overlap in all respects. Most notably, fungi in these two orders possess sheathed or unsheathed ascospores, their asci 7 either possess or lack an amyloid apical pore, and they include both pathogens and innocuous endophytes. The delimitation between these two orders is ill-defined, resulting in difficulties in controversial ordinal and familial placements of many problematic genera (DiCosmo et al., 1983a; 1983b). Rhytismatales is comprised primarily of foliar and bark fungi typically with immersed erumpent ascomata which are typically surrounded by black fungal tissue. Leotiales, although a more heterogeneous group, includes some foliar fungi with these same morphological features. Two features that overlap with Rhytismatales, ecological habits as foliar fungi and variability with respect to the presence or absence of an apical pore on the ascus, recently have been used as ajustification for broadening the concept of Rhytismatales (Livsey and Minter, 1993).

Table 1.1. Summary of the families and genera of Rhytismatales and Leotiales. Key: a specimens available in collections at Oregon State University, b cultures available at CBS.

Leotiales (13 families) Rhytismatales (3 families) genera genera Baeomycetaceaea 2 Ascodichaenaceaea b 3 Dermateaceaea b 72 Rhytismataceaea b 43 Geoglossaceaea 16 ?Cryptomycetaceae b 4 Hemiphacidiaceaea b 5 Incertae sedisa b 21 Hyaloscyphaceaea b 59 Icmadophilaceaea 5 Leotiaceaea b 104 Loramycetaceaeb 1 Orbiliaceae b 1 Phacidiaceaea b 4 Sclerotiniaceaea b 33 Vibrisseaceae b 3 ?Ascocorticiaceae b 3 Incertae sedis 89 total 392 71 8

Families within Leotiales show some interesting trends with regard to host affinity. For instance, genera within Dermataceae are commonly associated with angiosperm hosts. In some cases, as with Dermea, Durandiella, Mollisia, and Pezicula, the majority of species occur on angiosperms with the exception of a small subset on gymnosperms. However, genera within Geoglossaceae (Leotiales) have rarely if ever been described in close association with plant tissue. Instead, Geoglossum, Microglossum, Spathulariopsis, and Mitrula are ground-inhabiting fungi found in forests, grasslands, and bogs. In contrast to the Dermateaceae, many genera within Rhytismataceae (Rhytismatales) are found primarily on conifers and less often on angiosperms and genera in Hemiphacidiaceae (Leotiales) are known exclusively from conifers. Hemiphacidiaceae was erected in 1962 by Korf to include plant parasites and saprophytes living superficially or internally in host tissue. Although all members of Hemiphacidiaceae are known exclusively from conifers, Korf recently stated that the Hemiphacidiaceae is more of an ecological than a phylogenetic group (Korf, 1994). This interpretation is controversial, yet ecological role and variability with respect to the ascus tip are characters cited as justification for the transfer of genera from Hemiphacidiaceae to Rhytismatales (Minter, 1994). Genera of Hemiphacidiaceae as originally circumscribed that have been transferred to either the Rhytismataceae or Rhytismatales incertae sedis are: Rhabdocline, Cyclaneusma, Darkera, and Naemacyclus (Hawksworth et al., 1995). Members retained in the Hemiphacidiaceae include Didymascella, Fabrella Hemiphacidium, Korfia, and Sarcotrochila. The tenuous systematic framework for Leotiales and Rhytismatales makes it difficult to identify the sister group to Rhabdocline, and offers further justification for a broader study. A nucleotide based phylogeny would make a significant contribution towards understanding the relationships in Rhytismatales and Leotiales. Characters of the ascomata (presence or absence of a darkened excipulum and a clypeus covering the hymenium), ascus (presence or absence of an amyloid pore), and ascospores (septate or aseptate, sheathed or unsheathed) suggest that Rhytismataceae could be more closely related to Hemiphacidiaceae and Phacidiaceae than its current ordinal placement suggests. Are Rhytismataceae and Hemiphacidiaceae monophyletic groups? Evolutionary 9

reconstructions based on molecular characters are used herein to address whether the disposition of genera in the Rhytismataceae or Hemiphacidiaceae is justified. A revised phylogenetic framework would help to resolve whether the foliar fungi associated with Pinaceae are monophyletic (or paraphyletic). If this group is polyphyletic, cladistic patterns may be helpful in proposing hypotheses for coevolutionary patterns between disparate fungal groups and their hosts. The ITS region of nrDNA evolves quickly relative to other regions of nrDNA, and is most appropriate for phylogenetic reconstruction within a genus and among closely related genera. In chapter 4, A conservative region of nrDNA, the small subunit, has been used to analyze familial and ordinal relationships in Leotiales and Rhytismatales. The small subunit has been used in favor of the large subunit (which is intermediate in variation between the small subunit and the ITS region) because there are numeraous inoperculate discomycete small subunit sequences available in Genbank, and because the small subunit is conservative enough to reconstruct a phylogeny that includes all the main lineages of ascomycetes. The 1000 by at the 5' end will be analyzed for genera placed in the Hemiphacidiaceae (as originally and currently comprised) and from most families within Leotiales and Rhytismatales (Table 1.1). A more robust phylogeny for fungi in these two orders will be useful in providing a framework for a wide range of taxonomic and evolutionary investigations.

1.5. Speciation of Rhabdocline on Douglas-Fir and Larch

Rhabdocline is characterized as having immersed fruit bodies with little or no stromatic or excipular tissue, capitate paraphyses, and ascospores that become one-septate with one brown cell and one hyaline cell upon germination. Asci either possess an apical pore that turns blue in Meltzer's iodine reagent (R. weirii and R. parkeri), or lack it (R. pseudotsugae). The genus as currently circumscribed contains five taxa (five subspecies of two species) pathogenic on a single species of Pseudotsva (Parker and Reid 1969) and a single endophytic species on the same host (Sherwood-Pike et al. 1986). Although all 10

these fungi are found on the same host, niche separation is achieved through differences in geographic distribution, anatomical location and by symbiotic role. Morphological similarities suggest a connection between Rhabdocline and Meria laricis, an asexual species (fungi without known sexual states are form-taxa) that has been described on three species of Larix. The anamorphic (asexual) state of Rhabdocline parkeri, given the form- name Meria parkeri, bears a strong morphological similarity to the Larix needle cast pathogen, Meria laricis. Rhabdocline parkeri, a nonpathogenic species, is ubiquitous on Douglas-fir foliage in Washington, Oregon, and northern California (Carroll and Carroll, 1978; Sherwood- Pike et al., 1986). Infections of R. parkeri are confined to a single epidermal cell, and remain inactive until immediately prior to leaf abscision (Stone, 1986; 1988). It is also found in the interior western states, where its abundance is unknown. This species has an iodine-positive apical ascus pore, and while the two pathogenic species of Rhabdocline have sheathed ascospores, the ascospores of R. parkeri have evanescent polar appendages. The distribution of pathogenic Rhabdocline throughout the range of P. menziesii varies by species and subspecies. The distribution of R. pseudotsugae ssp. pseudotsugae has been documented most thoroughly, and unsurprisingly it is the widest ranging. It is present on every variety of P. menziesii and is also found in the eastern United States and Europe, where P. menziesii has been introduced. Rhabdocline weirii ssp. weirii has a similarly widespread range. Other pathogenic Rhabdocline subspecies appear to have a more restricted range. Rhabdocline pseudotsugae ssp. epiphylla has only been described in the coastal region of North America, R. weirii ssp. obovata has mostly been reported from northern interior hosts, and R. weirii ssp. oblonga has been described from the southern interior hosts (Ellis and Gill, 1945; Parker and Reid, 1969, unpublished observations). Rhabdocline pseudotsugae and R. weirii are difficult to grow in pure culture, as are a number of other foliar ascomycetes examined in this study. To obtain ITS region PCR products for unculturable species, the primers ITS5 and ITS4 (White et al., 1990) were used to specifically amplify fungal ITS region from genomic DNA isolated from host 11 needles bearing Rhabdocline apothecia. This circumvents the need for a pure culture and makes it possible to perform PCR directly from host tissue. A limitation of this method is a greater hazard of nontarget DNA contamination. This method requires cautious comparison with sequences from closely related species grown in pure culture because conifer needles contain small quantities of a diverse assemblage of endophytic and epiphytic fungi (Carroll and Carroll, 1978; Camacho et al., 1997). PCR conditions in this procedure favor genomic DNA that is abundant relative to the amount that would be expected from other fungi living within or on the surface of needles. Rhabdocline is a particularly interesting group for a molecular systematic study because there is evidence of both geographical and host-mediated speciation, and of a recent host switching event from one host genus (Pseudotsuga) to its sister genus (Larix). Species and subspecies of Rhabdocline with sexual states appear to have diversified on one species of North American Douglas-fir, P. menziesii. One species of Rhabdocline has also been reported from the other North American species, P. macrocarpa. Limited searching on other species of Pseudotsuga from East Asia has not revealed any more fungal species. In contrast to the association of Rhabdocline predominantly with one species of Pseudotsuga, Meria laricis has been reported on two species of Larix in western North America, L. occidentahs and L. lyallii, and on L. decidua in Europe. It is not known whether the occurrence of M laricis on these widely disjunct species is a result of ancient cospeciation on Larix or a more recent host shift. Phylogenetic reconstructions based on anatomical characters of the ovulate cone suggest that L. decidua and L. occidentahs belong to different lineages within the genus (LePage and Basinger, 1991; Schorn, 1994). Furthermore, the symbiosis between Meria laricis and Larix spp. could date back to the divergence of the common ancestor of Pseudotsuga and Larix or could be the result of one or more recent host shifts between genera. Phylogenetic information on the hosts, as well as a molecular comparison of M laricis from each continent, make it possible to evaluate which of the two possibilities is more likely. The biogeography of the Rhabdocline species and subspecies associated with Douglas-fir suggests that the fungal parasites are undergoing host mediated isolation and speciation. Pseudotsuga menziesii is the most abundant and widely distributed 12

Pseudotsuga species. The pollen of Pseudotsuga (and Larix) is nonsaccate and disperses poorly by wind. This morphological feature has been used to explain the restricted gene flow between and among populations (Li and Adams, 1989) relative to that observed in other genera of Pinaceae. The range of the coastal variety extends from British Columbia south to California, and the interior variety is further divided into northern and southern forms that extend inland along the Sierra Nevada Mountains, and from British Columbia to central Mexico along the Rocky Mountains. This population fragmentation is important because levels of resistance to the disease "Rhabdocline needle cast" are different in the three host varieties. When progeny from the three host varieties are grown in humid environments favorable to infection by the pathogenic R. pseudotsugae, the southern form is most susceptible, the northern form is slightly less susceptible, and the coastal form has a high level of resistance (Stephan, 1980; Hoff, 1988). Within each region, susceptibility among siblings varies, and seems to increase in drier or colder high elevation locations unfavorable for ascospore infection of flushing buds. A combination of host escape and the evolution of resistance within isolated host populations may be driving cospeciation of Rhabdocline on P. menziesii. Host shifting and subsequent host-mediated isolation may also be driving the evolution of Rhabdocline and Mena species on other hosts. The goal of this dissertation is to gather molecular sequence data and to advance the state of systematics for hosts, their parasites, and the nearest relatives of each of these groups. Ultimately, this information can be used to evaluate the relative importance of cospeciation and host shifting in the evolution of inoperculate foliar fungi with their hosts.

1.6. Literature Cited

Axelrod, D. I. 1990. Environment of the Middle Eocene (45 Ma) Thunder Mountain flora, central Idaho. National Geographic Research 6: 355-361. 13

Camacho, F. J., D. S. Gernandt, A. Liston, J. K. Stone, and A. S. Klein. 1997. Endophytic fungal DNA, the source of contamination in spruce needle DNA. Molecular Ecology 6: 983-987.

Campbell, B. C. 1993. Congruent evolution between whiteflies (Homoptera: Aleyrodidae) and their bacterial endosymbionts based on respective 18S and 16S rDNAs. Current Microbiology 26: 129-132.

Carroll, G. C., and F. E. Carroll. 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56: 3034-3043.

Chapela, I. H., S. A. Rehner, T. R. Schultz, and U. G. Mueller. 1994. Evolutionary history of the symbiosis between fungus-growing ants and their fungi. Science 266: 1691-1694.

Chase, M. W., D. E. Soltis, R. G. Olmstead et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Annals of the Missouri Botanical Garden 80: 528-580.

Christiansen, H. 1972. On the development of pollen and the fertilization mechanisms of Larix and Pseudotsuga menziesii. Silva Genetica 21: 166-174.

DiCosmo, F., T. R. Nag-Raj, and B. Kendrick. 1983a. Prodromus for a revision of the Phacidiaceae and related anamorphs. Canadian Journal of Botany 61: 31-44.

H. Peredo, and D. W. Minter 1983b. Cyclaneusma gen. nov., Naemacyclus and Lasiostictis, a nomenclatural problem resolved. European Journal of Forest Pathology 13: 206-212.

El-Kassaby, Y. A., A. M. Colangeli, and 0. Sziklai. 1983. A numerical analysis of karyotyes in the genus Pseudotsuga. Canadian Journal of Botany 61: 536-544.

Ellis, D. E., and L. S. Gill. 1945. A new Rhabdogloeum associated with Rhabdocline pseudotsugae in the southwest. Mycologia 37: 326-332.

Farjon, A. 1990. Pinaceae: drawings and descriptions of the genera Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Koeltz Scientific Books, Konigstein, Federal Republic of Germany.

Florin, R. 1951. Evolution in cordaites and conifers. Acta Horti Bergiani 15: 285-388.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp. 14

Hermann R. K. 1985. The genus Pseudotsuga: ancestral history and past distribution. Forest Research Laboratory, College of Forestry, Oregon State University. sp. pub. 2b.

Hoff, R. J. 1988. Susceptibility of inland Douglas-fir to Rhabdocline needle cast. USDA Forest Service Research Note, INT-375, pp. 3.

Korf, R. P. 1962. A synopsis of the Hemiphacideaceae, a family of Helotiales (discomycetes) causing needle blights of conifers. Mycologia 54: 12-33.

. 1994. Quoted from Ascomycete systematics: problems and perspectives in the nineties. (David L. Hawksworth, Ed). New York:Plenum Press, pp. 453.

LePage, B. A., and J.F. Basinger. 1991. A new species of Larix (Pinaceae) from the early Tertiary of Axel Heiberg Island, Arctic Canada. Review of Palaeobotany and Palynology 70: 89-111.

Li, P., and W. T. Adams. 1989. Range-wide patterns of allozyme variation in Douglas fir (Pseudotsuga menziesii). Canadian Journal of Forest Research 19: 149-161.

Livsey, S., and D. W. Minter. 1993. The taxonomy and biology of Tryblidiopsis pinastri. Canadian Journal of Botany 72:549-557.

Martinez, M. 1949. Las Pseudotsugas de Mexico. Anales del Instituto de Biologia 20: 129-184.

Miller, C. N. 1977. Mesozoic conifers. Botanical Review 43: 217-280.

. 1988. The origin of modern conifer families. in C. B. Beck (editor), Origin and Evolution of Gymnosperms. New York, Columbia Univ. Press, pp. 448-487.

Minter, D. W. 1994. Quoted from Ascomycete systematics: problems and perspectives in the nineties. (David L. Hawksworth, Ed). New York: Plenum Press, pp. 453.

Munson, M. A., P. Baumann, M. A. Clark, L. Baumann, N. Moran, D. J. Voegtlin and B. C. Campbell. 1991. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. Journal of Bacteriology 173: 6321-6324.

Parker, A. K., and J. Reid. 1969. The genus Rhabdocline Syd. Canadian Journal of Botany 47:1533-1545.

Petrini, 0. 1984. Cryptocline arctostaphyli sp. nov., an endophyte of Arcostaphylos uva­ ursi and other Ericaceae. Sydowia Annales Mycologici 37: 238-241. 15

Pirozynski, K. A, and D. Mal loch. 1975. The origin of land plants: a matter of mycotrophism. Biosystems 6: 153-164.

Prager, E. M., D. P. Fowler, and A. C. Wilson. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

Price, R. A., J. Olsen-Stojkovich, and J. M. Lowenstein. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Systematic Botany 12: 91-97.

Schorn, H. E. 1994. A preliminary discussion of fossil larches (Larix, Pinaceae) from the arctic. Quaternary International 22/23: 173-183.

Sherwood-Pike, M., Stone, J. K., and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canadian Journal of Botany 64: 1849­ 1855.

Stephan, Von B. R. 1980. Testing of Douglas-fir provenances for resistance against Rhabdocline pseudotsugae by infection trials. European Journal of Forest Pathology 10: 152-161.

Stone, J. K. 1986. Initiation and development of latent infections by Rhabdocline parkeri on Douglas-fir. Canadian Journal of Botany 65: 2614-2621.

. 1988. Fine structure of latent infectins by Rhabdocline parkeri on Douglas-fir, with observations on uninfected epidermal cells. Canadian Journal of Botany 66: 45-54.

Strauss, S. H., A. H. Doerksen, and J. R. Byrne. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canadian Journal of Botany 68:1502-1510.

Tsumura, Y., K., Yoshimura, N. Tomaru, and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theoretical and Applied Genetics 91: 1222-1236.

White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. (M.A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White eds). San Diego: Academic Press, Inc. pp. 315-322. 16

Chapter 2

Using the rbcL Gene to Estimate the Order and Timing of Evolution in Pinaceae (Coniferales)

David S. Gernandt Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331-2902

2.1. Abstract

Estimated rates of substitution in rbcL were evaluated by comparing an rbcL phylogeny of Pinaceae to its fossil record. The fossil record of Pinaceae extends to the lower Cretaceous and the family is thought to have evolved in the early to middle Mesozoic. Fifteen species representing nine of the approximately 11 genera of Pinaceae were evaluated using parsimony, neighbor joining, and maximum likelihood. Analyses including Podocarpus gracilior as an outgroup failed to identify an unambiguous root to Pinaceae. Maximum likelihood analyses were performed with and without a constant molecular clock assumption (ML and MLK, respectively). Significant rate heterogeneity was observed by comparing log likelihoods of the resulting trees. The MLK tree was calibrated using a reported rbcL divergence rate of 2.05 X10-1° substitutions/site/year. Mapping fossil ages onto the MLK tree revealed that taxon divergence dates estimated under the assumption of a molecular clock were consistent with minimum ages of lineages inferred from a patchy fossil record. 17

2.2. Introduction

Zuckerkandl and Pau ling (1965) were the first to suggest that the clocklike accumulation of changes in proteins and DNA could be used to estimate the time since divergence among lineages of organisms. This clocklike behavior has been used frequently to estimate the timing of speciation among a wide variety of organisms, yet the molecular clock hypothesis has been criticized on both theoretical and empirical grounds. Empirically, numerous cases have been documented where the rate of nucleotide substitution differs across lineages of organisms. As a general example, the rate of mitochondria] DNA substitution in most plants is estimated to be 40-100-fold slower than its rate in animals (Palmer, 1992). Lineage differences in genome complexity, generation time, rate of mutation, intensity of selection, and historical bottlenecks are all potential scenarios where a molecular clock could fail, or at the very least provide but a crude estimation of the timing of evolutionary events. Nevertheless, the allure of calibrating a molecular clock and dating historical events has been strong, and a number of advances have been made in the evaluation of substitution rates. Such advances include calibrations based on fossil data (absolute rate comparisons), the assessment of rate similarity in parallel lineages relative to an outgroup (relative rate comparisons) (Wilson et al., 1987; Springer, 1995), and the assessment of maximum likelihood trees with and without the assumption of clocklike behavior in DNA (ML vs. MLK, see Felsenstein, 1993). Pinaceae (the pine family) is commonly a dominant component of north temperate vegetation, often having a competitive advantage over angiosperm tree species in regions with dry, seasonal climates. Except for one species of Pinus in Sumatra, the 11 genera of Pinaceae have a distribution confined to the Northern hemisphere (Critchfield and Little, 1966). Pinaceae can be distinguished from the other seven families of conifers based on characteristics of the female cone, which has numerous, flattened scales, each accompanied by a flattened bract and arranged in a compact helix (Miller, 1988). Biogeographic, phylogenetic, and paleobotanical concepts in Pinaceae are still incomplete. However, researchers in these disciplines have made extensive contributions toward the understanding of this group, and integration of these disciplines has been 18 addressed for the evolution of Pinus (Axelrod, 1986; Millar and Kin lock, 1991; Millar, 1993) and for the divergence of Pseudotsuga and Larix (Strauss et al., 1990). Currently, the most rapid advances are being made in the field of molecular phylogenetics, where several new sources of data are being exploited (discussed below). A relatively recent and important new source of data made available is the nucleotide sequence of the chloroplast-encoded, large subunit of ribulose 1,5-bisphosphate carboxylase (r b cL) which catalyzes the fixation of CO2 during photosynthesis. The enzyme is encoded by a 1428 base pair open reading frame. Comparative studies of its nucleotide sequence have been used widely for reconstructing phylogenies in the plant kingdom (see Chase et al., 1993). The molecule evolves slowly enough that the majority of substitutions in closely related groups, for instance, the pine family (see below), occur at neutral (synonymous) positions. Assignment of positional homology involves less ambiguity than do comparisons among non-protein coding regions such as nuclear ribosomal DNA (nrDNA). This is expected because nrDNA does not require an in-frame translation, and therefore can possess regions that are more prone to insertion-deletion events. Albert et al. (1994) argued that rbcL behaves in a more or less clocklike manner, describing it as quasi-ultrametric. They used patristic distance between rbcL sequences for 19 woody taxon pairs together with fossil and vicariant disjunction dates to estimate a mean rate of rbcL substitution of 2.05 X 104° substitutions/site/taxon pair (SD 0.75 X 10" 10). Their estimate included only two conifer families, Cupressaceae and Taxodiaceae. These two families had the highest divergence rates of all the pairs tested, 3.85 X 10-10 and 2.80 X 10-10, respectively. These rates are likely to be too high because of conservative estimates of the minimum time of divergence for the genera examined. Both divergence ages were based on vicariance episodes. The Cupressaceae value was determined by comparison of the Australian species, Callitris rhomboidea with the African species, Widdringtonia cedarbergensis, which were calculated to have diverged at least 100 Ma based on the breakup of Gondwana. The more precise episode of its breakup is estimated to have occurred 130 Ma, but the authors rounded to the nearest 100 Ma. Assuming a divergence time of 130 Ma, the divergence rate falls from 3.85 X 1010 to 19

2.96 X 100 subst./site/taxon pair. The Taxodiaceae value was determined by comparison of the Asian species, Metasequoia glyptostroboides, with the North American species, Sequoiadendron giganteum, which were calculated to have diverged at least 40 Ma based on the "disruption of the boreotropical interchange between North America and Eurasia." These rate inconsistencies relative to those from other woody taxon pairs can be partly explained by noting that our knowledge of evolutionary history based on the fossil record is imperfect, and that timing of vicariance disjunctions provides minimum, not precise divergence estimates. The utility of rbcL as a molecular clock has been questioned by Goremykin et al. (1996). They argued that the disparity between observed substitutions at synonymous and nonsynonymous sites caused a systematic error in the calculation of the total substitution rates in rbc1, calculated by Albert et al. (1994). This source of error will probably be of less concern for taxon-divergences that occurred recently but does highlight the preliminary nature of the rate estimate. These rate estimates can be improved by differentially weighting synonymous and nonsynonymous substitutions and transitions and transversions. Distance methods that use a distance estimate that takes "multiple hits" at a single nucleotide position into account, such as the Jukes and Cantor (1969) or Kimura 2­ parameter correction (Kimura, 1980) would be more useful than using patristic distances to calculating divergence values. Such methods have also been recommended for more accurate phylogenetic reconstruction of protein coding sequences (Albert et al., 1993, Swofford et al., 1996). It should be pointed out that the major impediments to rate estimates are the dates of divergence, which are based on an imperfect fossil record and conservative minimum dates based on vicariance events.

The fossil history of conifers-- Eight extant families comprise the conifers. These include Araucariaceae, Cephalotaxaceae, Cupressaceae, Pinaceae, Podocarpaceae, Sciadopityaceae, Taxaceae (Chaw et al., 1993), and Taxodiaceae. The fossil record of conifers may begin with Cordaitales, an extinct order with fossils known from the Carboniferous (286-360 Ma)(Florin, 1951). The fossil record offers few clues regarding the order of origin for these modern families (Miller, 1988). With the exception of 20

Pinaceae, (Sciadopityaceae uncertain) there are fossil representatives of each family from the Jurassic (190-135 ma), and fossil evidence has indicated the presence of Podocarpaceae and Araucariaceae during the Triassic (225-190 ma). While reconstructing the order of branching that occurred among conifer families is of considerable interest, even in the absence of a solid understanding of that order, it can be generalized that modern conifers originated near the Paleozoic/Mesozoic boundary, were dominant components of world vegetation during the Mesozoic, and their importance was diminished as the changing climate (in Northern hemisphere became hotter and wetter) toward the end of the Mesozoic favored success of the angiosperms, which originated in the early Cretaceous and became the dominant component of the vegetation in the later part of that Epoch (Florin, 1951; Millar and Kinloch, 1991).

Familial relationships and generic pairs--Identification of the most closely related family to the Pinaceae should provide optimum information regarding the polarity of evolution in the family (Smith, 1994). Of the seven other families of conifers, three have been plausibly suggested as sharing a most recent common ancestor with Pinaceae. Cladistic studies of morphological characters by Hart (1987) placed Pinaceae sister to the Podocarpaceae but concluded that placement of Podocarpaceae and Araucariaceae was problematic. While Miller (1982) used a phenetic analysis of fossil data to support the relationship between Pinaceae and Podocarpaceae, his subsequent cladistic analysis (Miller, 1988) suggested that Araucariaceae was a slightly better sister group than Podocarpaceae. A cladistic analysis of morphological characters by Nixon et al. (1994) also supported the Pinaceae/Araucariaceae sister relationship, and unlike previous reports, Podocarpaceae was removed from Pinaceae by three to four of the other conifer families. A study of chloroplast RFLP's by Tsumura (1995) demonstrated a sister relationship between Pinaceae and Sciadopityaceae, but the analysis did not include either Podocarpaceae or Araucariaceae. Chaw et al. (1997) used the small subunit of nrDNA to construct a phylogeny in which Pinaceae was the basal member of Coniferales (all eight families were in the Glade as well as Phyllocladaceae), which in turn was derived from an Ephedraceae, Gnetaceae, Welwitschiaceae Glade. The phylogenetic grade leading from Pinaceae to the 21

other families of conifers had poor bootstrap support, but the next lineage to be derived was an Araucariaceae, Phyllocladaceae, and Podocarpaceae Glade. Several molecular studies of the Pinaceae have been reported over the last 20 years. While these studies have not resulted in a robust understanding of the deeper branches in the phylogeny of the pine family, a consensus regarding the relationships

between pairs of genera can be identified. Praeger et al. (1976) used comparative immunological studies to generate a Fitch-Margoliash tree support pairings between Pinus subgenus Pinus (hard pines) and subgenus Strobes (soft pines). The other strong relationship inferred from their analysis was the close relationship between Larix and Pseudotsuga. Picea was basal to the Pinus/Pseudotsuga/Larix Glade, and Abies, Cedrus, and Tsuga, were in another deeply branching Glade, but these relationships were tenuous, and four genera were not included in the analysis.

Price et al. (1987) reported results from a radioimmunoassay on nine genera. Their UPGMA and Fitch-Margoliash analyses corroborated that of Praeger et al. in supporting the pairing of the hard and soft pine and Larix with Pseudotsuga. Instead of Picea being basal to Pinus, Pseudotsuga, and Larix, a sister relationship between it and Pinus was supported, with a deeper branch connecting this genus pair to Pseudotsuga and Larix. These four genera formed the Pinoid group, and a second group, comprised of Keteleeria, Abies, Cedrus, Tsuga, and Pseudolarix formed the Abetoid group. In the latter, Keteleeria was paired with Abies, and sister to this grouping was Cedrus. The final taxon pair identified was between Tsuga and Pseudolarix. The enzymatic evidence of Price et al. was in complete agreement with earlier morphological classifications. These early classifications relied on differences in root anatomy, resin canals, and resin vesicles in

seed coats that had been addressed by several investigators since 1891, while it refuted other classifications based on dimorphism in the length of needle clusters (shoots). The first published rbcL tree of nine genera of Pinaceae was by Chase et al.

(1993). Pinaceae was included in a larger analysis that including other families of conifers as well as flowering plants. Their analysis supported pairings for Pinus-Picea, Pseudotsuga-Larix, and Abies-Keteleeria. The Abietoid taxa sampled were along a paraphyletic grade at the basal portion of the Pinaceae Glade. Their analysis most probably 22 did not reflect the true branching order of older lineages because Pinaceae was a sister group to the angiosperms, while other members of the conifers were basal to this Pinaceae-angiosperm Glade. Tsumura et al. (1995) reported results from an RFLP analysis of chloroplast genes from six conifer families (their analysis did not include representatives of Podocarpaceae or of Araucariaceae, see above). Their analysis included nine members of the pine family, and the most basal lineage within the Pinaceae was Pinus (both hard and soft subgenera were sampled). Pseudotsuga and Larix were a sister group, and Abies, Tsuga, and Keteleeria formed a generic triplet. However, their parsimony and neighbor-joining tree conflicted with respect to relationships in the abietoid group. The Wagner maximum parsimony tree supported a pairing between Abies and Tsuga, with Keteleeria, and Pseudolarix successively basal on a paraphyletic grade, while the neighbor-joining tree supported a pairing of Abies and Keteleeria, with Tsuga, a Picea-Cedrus pair, and then Pseudolarix successively basal on a grade. The root to Pinaceae reported in the 18S nrDNA study of Chaw et al. (1997) split the Pinaceae into a Larix-Pseudotsuga Glade, and an Abies-Pines Glade (only four genera and seven species of Pinaceae were represented). The consensus of these studies is that Pinaceae consists of the following subgroups: a tentative pairing between Picea and Pines, and more certain groupings of Pseudotsuga/Larix, Keteleeria/Abies/Cedrus, and Tsuga/Pseudolarix. The interrelationships of these natural groups has not been consistent in past studies, nor has the identification of a sister family.

2.3. Materials and Methods

All rbcL sequences were downloaded from Genbank (Table 2.1). The conserved nature of the rbcL open reading frame rendered alignment straightforward. Manual alignment was performed using Genetic Data Environment (Smith et al., 1994). A gap was inserted at only one position near the 3' end of the reading frame for 16 species. The gap was necessary because there was a 19 by direct repeat in two of the three spruce 23

species, Picea abies and Picea pungens (Relle et al., 1995). The two repeats differed at two nucleotide positions. An optimal assignment of homology was achieved by inserting a gap near the end of the open reading frame for the 15 species that lacked the repeat, thus using the second direct repeat in P. abies and P. pungens for the phylogenetic analysis.

Table 2.1. Conifer taxa included in the rbc1, study. Sequences for Pinaceae have been deposited in Genbank by S. Strauss and other collaborators except for P. abies (see Relle et al., 1995). Genera not sampled include Cathaya and Nothotsuga. Podocarpus gracilior was selected as the outgroup for the Pinaceae analysis based on maximum parsimony analysis with a broader sampling of six conifer families (Fig. 2.2).

Taxon Genbank accession no. Coniferales Araucariaceae Araucaria augustifolia (Bertol.) Kuntze U87750 Cupressaceae Callitris rhomboidea R. Br. ex L.C. Rich L12537 Cupressus sempervirens L. L12571 Diselma archeri Hook. f. L12572 Juniperus conferta Parl. L12573 Libocedrus plumosa (D. Don) Sarg. L12574 Microbiota decussata Komar. L12575 Tetraclinis articulata (Vahl) Mast. L12576 Thuja occidentalis L. L12578 Thuja plicata Donn ex D. Don L25758 Thujopsis dolobrata (L.f) Sieb. et Zucc. L12577 Widdringtonia cedarbergensi Marsh L12538 Pinaceae Abies magnifica Andr. Murray X58391 Cedrus deodara (D. Don) G. Don X63662 Keteleera davidiana Beissn. X63664 Larix occidentalis Nutt. X63663 Picea abies (L.) H. Karst. X75478 Picea pungens Engelm. X58136 Picea sitchensis (Bong.) Carriere X63660 Pinus balfouriana Balf X63661 Pinus edulis Engelm. X58137 Pinus griffithii M'Clell.(=P. wallichiana) X58131 Pinus krempfii Lecomte X63665 Pinus longaeva D.K. Bailey X58132 24

Table 2.1. (continued)

Taxon Genbank accession no. Pinus pinea L. X58133 Pinus radiata D. Don X58134 Pseudolarix kaempferi Gordon X58782 Pseudotsuga menziesii (Mirb.) Franco X52937 Tsuga heterophylla (Raf.) Sarg. X63659 Podocarpaceae Podocarpus gracilior Pilg. X58315 Sciadopityaceae Sciadopitys verticillata (Thunb.) Sieb. et Zucc. L25753 Taxaceae Amentotaxus argotaenia Pilger L12580 Taxodiaceae Athrotaxus laxifolia Gord. L25754 Cryptomeria japonica (L. fil.) D. Don L25751 Cunninghamia lanceolata (Lamb.) Hook. L25757 Glyptostrobus lineatus Druce L25750 Sequoia sempervirens (D. Don) Endl. L25755 Taiwania cryptomerioides Hayata L25756 Other Gymnosperms (used for outgroups) Ephedraceae Ephedra tweediana C.A. Mey L12677 Gnetaceae Gnetum gnemon Linn. L12680 Wilwitschiaceae Welwitschia mirabilis Hook. f. D10735

Analysis of the aligned open reading frame was performed using the neighbor joining algorithm (Saitou and Nei, 1987) with the Kimura 2-parameter correction (Kimura, 1980) and the maximum likelihood algorithm in the PHYLIP computer package (Felsenstein, 1993) as well as weighted and unweighted parsimony (Swofford, 1993). All parsimony analyses (including bootstraps) were performed using heuristic searches with 10 random sequence additions, tree-bisection-reconnection branch swapping, mulpars in effect, 100 bootstrap replicates, and maxtrees set at 500. Maxtrees was never exceeded in these analyses. 25

2.4. Results

In the alignment of 17 members of Pinaceae and Podocarpus, 179 out of 228 variable sites were at third codon positions (78.5%). Substitutions occurred at a minimum of 37.6% of the possible 476 third codon positions. When Podocarpus gracilior was excluded, the total number of variable characters fell to 154 (90 were parsimony informative). The number of variable positions is 144 when P. gracilior and the two Picea species with direct repeats (P. abies and P. pungens) are excluded. Substitutions occurred at 23% of the third codon positions across all fifteen taxa (108 substitutions). The Pinaceae data set (including all 17 taxa) showed a high transition:transversion (ts:tv) bias (FIG. 2.1). Of the 154 variable positions, 113 were transitions, 28 were transversions, and 13 were a combination of both. Excluding the thirteen positions that were a combination of both, the ts:tv ratio was 4.03. This transitional bias suggested that weighted parsimony could be used to increase the accuracy of phylogenetic reconstruction. A parsimony analysis of Coniferales, as well as outgroup taxa from Gnetaceae, Ephedraceae, and Welwistchiaceae (Table 2.1) was performed in which transversions were weighted four times more than transitions. Weighting in this manner increases the influence of the more conservative transversions in choosing among different trees (Swofford et al., 1996). Weighted parsimony analysis of rhcL sequences from eight conifer families, failed to identify a single conifer family most closely related to Pinaceae (FIG. 2.2). Pinaceae was the basal member of the Coniferales Glade. Araucariaceae, Podocarpaceae, and Sciadopytis formed a polytomy basal to a Taxaceae, Taxodiaceae, Cupressaceae paraphyletic grade. Within Pinaceae, the strict consensus tree supported generic pairings between Larix and Pseudotsuga, and Ahies and Keteleeria. Pinus and Picea both included multiple species, and both genera were monophyletic. There was no single most parsimonious placement of the root between Pinaceae and the other families sampled. 26

8

7

6

5 4 3

2

1 io 0 1,1 ill 0 250 500 750 1000 Position transitions El transversions both

FIG. 2.1. Observed rbcL nucleotide substitutions for 17 members of Pinaceae. Substitutions are inferred from the alignment. Number of substitutions are displayed at 25 by intervals.

Several aspects of the tree topologies obtained from distance, maximum likelihood, and parsimony analyses were consistent with each other and with the topologies reported by Prager et al. (1976), Price et al. (1987), and Tsumura et al. (1995). The hard and soft pines were sister groups, Pseudotsuga and Larix were sister groups, and there was support for an Abietoid Glade that included Pseudolarix and Tsuga as generic pairs and Abies and Keteleeria as pairs (FIGS. 2.3-2.4). Several of the deeper branches were equivocal. Outgroup rooting with Podocarpus consistently resulted in Picea branching from the Pinaceae before Pinus or any other genera. However, this branch was very short (FIG 2.4) and it was not supported by bootstraps above the 50% level. The placement of the Pseudotsuga/Larix branch conflicted in the two trees. Parsimony analysis revealed multiple topologies with the same minimal number of steps that differed in its position relative to ('edrus and the remaining members of the Abietoid Glade (FIG. 2.3). Neighbor 27

CCupressus sempervirens Microbiota decussata Calocedrus decurrens Juniperus conferta Thujaoccidentalis Thuj a ph cata Cupressaceae Thujopsis dolobrata Tetraclinus articulata _r- Diselma archeri IWiddringtonia cedarbergensi Callitris rhomboidea Libocedrus plumosa Cryptomeria japonica Glyptostrobus lineatus Sequoia sempervirens Taxodiaceae Cunninghamia lanceolata Athrotaxus laxifolia Taiwania cryptomerioides Amentotaxus argotaenia Taxaceae Arauc aria augustifolia Araucariaceae Podocarpus gracili or Podocarpaceae Sc iadopitys verticillata Sciadopityaceae Pinus edulis Pinus krempfi Pinus balfouriana Pi nus griffithi i Pinus longaeva Pinus pinea Pinus radiata Picea abies Picea pungens Picea sitchensis Pinaceae Abies magnifica Keteleeria davidiana Larix occidentalis P seudotsuga menziesi Pseudolarix kaempferi Cedrus deodara Tsuga heterophylla Gnehtm gnemon Gnetaccae Welwitschia mirabilis Welwitschiaceae Ephedra tweediana Ephedraceae

Fig. 2.2. Strict consensus from a weighted parsimony analysis of conifer rhcL. 28

Pinus edulis 71 Pinus krempfii 56 Pinus balfouriana 91 Pinus griffithii

100 Pinus longaeva Pinus pines 96 Pinus radiata Pseudolariz kaempferi

Tsuga heterophylla 64 Abies magnifica

Keteleeria davidiana Larix occidentalis 97 Pseudotsuga menziesii Cedrus deodara Picea ahies s4

99 Picea pungens

Picea sitchensis Podocarpus gracilior

FIG. 2.3. Phylogenetic tree from parsimony analysis of Pinaceae rbcL. Strict consensus of eight equally most parsimonious trees (360 steps, CI=0.543, RI=0.660, RC=0.475). Bootstrap values (above branches) were generated using 100 replications. Values lower than 50% are not shown.

joining analysis placed it basal to the Abietoid group. These alternate interpretations conflict with the UPGMA analysis of Price et al. (1987), which placed Pseudotsuga, Larix, Pinus, and Picea in a cluster separate from the Abietoid group. Two features of the data set may be identified as causing misleading topologies. First, although Podocarpus was chosen from Coniferales as an outgroup, the branch attaching it to the Pinaceae ingroup was extremely long (FIG. 2.4), and it did not appear to provide a robust rooting with respect to Pinaceae or three closely related families in a 29

Pseudolarix kaempferi 5 Tsuga heterophylla 69 Abies magnifica 94 Keteleeria davidiana Cedrus deodara Larix occidentalis 98 Pseudotsuga menziesii Pinus balfouriana Pinus griffithii

50 Pinus edulis 89 Pinus longaeva

100 Pinus krempfii Pinus pinea 96L Pinus radiata Picea abies

100 Picea pungens Picea sitchensis Podocarpus gracilior 0.01

FIG. 2.4. Distance tree from neighbor joining analysis of Pinaceae rbc-L. The Kimura 2­ parameter correction was used at a ratio of 2:1 tr:tv. Bootstrap values (at branches) were generating with 100 replications. Values lower than 50% are not shown.

broader analysis (FIG. 2.2). Second, the presence of the 19 by repeat in two of the three Picea species confounds assumptions of positional homology. Therefore, these three taxa were removed and the analyses were repeated. Two most parsimonious trees were identified. The two trees differed only in the placement of two of the soft pines, P. balfouriana and P. griffithii. The most parsimonious trees resolved the Pinoid and Abietoid clades as monophyletic, and generic pairings were consistent with the 18 species analysis. Maximum likelihood analysis was also performed (FIG. 2.5). The tree generated using the data set with Podocarpus and the two species of Picea with repeats removed 30

agreed with the Abietoid/Pinoid grouping when the root is placed on the internal branch separating the Cedrus from the Pseudotsuga/Larix branches. To assess whether the molecular clock assumption could apply to the rbcL sequences in this data set, analyses was performed both with and without the assumption of clocklike behavior (FIGS. 2.5 and 2.6, respectively). For the clock assumption to be met, the resulting pair of trees must have an identical topology and the log likelihood of the tree assuming a molecular clock (MLK) must not be significantly different.

Pinus edulis

Pinus krempfii

Pinus griffithii

_Pinus balfouriana

Pines longaeva

Pinus pinea

Pinus radiata

Pseudolarix kaempferi

Tsuga heterophylla

Abies magnifica

Keteleeria davidiana

Cedrus deodara

Larix occidentalis

Pseudotsuga menziesii

Picea sitchensis

FIG. 2.5. Maximum likelihood phylogeny of Pinaceae rbcL. Tree generated using the fastDNAml algorithm (15 taxa, Ln likelihood=-3406.9). 31

225 190 136 65 26 Mesozoic Cenozoic Triassic Jurassic Cretaceous Paleogene Neogene /Quaternary a2-5 Pinus pinea d5 c5 Pinus railiata

c2 Pinus grii. thii bl c 1 al b3 b2 Pinus kreinPfii c3

Pinus edi /is

Pinus Ion aeva c4 Pinus bal. ouriana

d4 Picea site tensis

dl d2 . Pseudotsuga menziesii

d3 Larix oco dentalis

a6 Cedrus de odara

a9 Keteleeria davidiana a7 Abies mm nffica

a8 Pseudolatix kaenytferi

Tsuga he erophylla a 1 1 a 1 0 0.01

FIG 2.6. Maximum likelihood with molecular clock (15 taxa, Ln likelihood = -.3423.8). The topology of the tree shown here is not identical to the tree in FIG. 2.5, so the log likelihoods are not directly comparable. The geological time scale has been overlayed by dividing branch lengths by the estimated substitution rate per taxon of 1.025 X 10-10 (this figure is one-half the divergence rate per taxon pair). See below for key to fossil records and other estimates of ages within Pinaceae. 32

Key to fossil records and age estimates of Pinaceae genera made by paleontologists. a. Miller 1977

1 "subgenus Pinus is first known in the lowermost Cretaceous" P. belgica (Alvin, 1960) 2. subgenus Pinus. P. triphylla, a 3-needled pine 3. subgenus Pinus. P. tetraphylla, a 4-needled pine 4. subgenus Pinus. P. quinquefolia, a 5-needled pine 5. subgenus Films. P. flabellifolia, a 3-needled pine, and P. pseudostrobifolia, a 5-needle form 6. Cedrus alaskensis from early Cretaceous of Alaska. This petrified wood is the only well founded record of the genus from the Mesozoic. 7. Abiocaulis. Structurally preserved twigs and stems from Belgium and Japan, respectively. Resembles an ancestor of Abies and Keteleeria. 8. two reports of Pseudolarix from the Cretaceous 9. Keteleerioxylon. Petrified wood similar to Keteleeria from early Cretaceous. 10. Pitytes erinensis. Late Jurassic or early Cretaceous, has anatomical features of pine needles 11 a. Jurassic and Triassic seed cones of Compsostrobus 1 lb. vegetative twigs of Pityocladus, same genus widespread in Jurassic 11c. associated seed cones of Schizolepis b. Millar 1993 1. "Origin of the genus [Finns] thought to be early-middle Mesozoic" 2. Pine pollen in Alaskan amber deposits 3. Pinus/Strobus and five subsections represented in Cretaceous c. Axelrod 1986 1. Pinus probably evolved from a Pityostrobus complex by late Jurassic 2. Strobi at 50 Ma, Del Mar, Princeton 3. Cembroides at 25 Ma., Creede (date from 47 Ma, Copper Basin, revised by Wolf and Schorn, 1990). 4. Balfourianae, Copper Basin d. Miscellaneous 1. Pseudotsuga miocena-Porcupine Creek Paleocene wood. Hermann 1985 citing Penhallow 1908 2. 32 Ma estimated from Pseudotsuga macrocarpa-like cone. Axelrod 1990 3. 45 Ma, Larix cone. Schorn 1994 citing Axelrod 1990 4. Picea magna from the latest Eocene Wolf and Schorn 1990 5. Films mutoi (subsect. Sylvestres) from Upper Cretaceous in Japan. (Saiki, 1996) 33

Analyses using over 20 combinations of taxon sampling, including the full dataset and removal of combinations of Picea abies, Picea pungens, one of the soft pines, and Podocarpus gracilior, were performed. All except one combination failed to generate congruent ML and MLK trees (the topologies in FIGS. 2.5 and 2.6 are not identical). The trees from the various taxon samplings conflicted in the same, deep branching positions for Cedrus, Picea, and Pseudotsuga/Larix as they had in the parsimony analysis and as reflected in phylogenies based on other molecular analyses (Prager et al., 1976; Price et al., 1987; Chase et al., 1993; Tsumura et al., 1994). The only analysis found where the topologies matched was a fourteen taxon analysis that excluded Podocarpus gracilior, Picea abies, Picea pungens, and Pinus griffithii (results not shown). The log likelihoods for ML and MLK were -3350.3 and -3369.8, respectively. Chi-squared analysis revealed that the MLK tree was a significantly poorer (p < .005, 12 degrees of freedom) interpretation of the phylogeny and therefore failed the ML vs. MLK test. Inspection of branch lengths for the Abies/Keteleeria and Tsuga/Pseudolarix groups in the ML tree in FIG 2.3) reveals two instances where rates of substitution differed greatly. The molecular clock assumption fails in the MLK analysis by the comparison of log likelihoods. It is still useful to combine the best estimate of the data under the molecular clock assumption using the Albert et al. (1994) mean value of rbcL divergence time (2.05 X 10-10 substitutions/site/taxon pair) with the fossil record of Pinaceae. Using the divergence rate and the segment lengths estimated by the MLK analysis, the geological time scale has been superimposed over the tree generated using MILK (FIG. 2.6). The timing of the origin of major groups of Pinaceae based on molecular clocks and fossil evidence are consistent with one anotherclock-based estimates consistently report longer divergence times than the oldest known fossils for each lineage yet the clock estimates do not exceed conventional estimates by paleontologists. 34

2.5. Discussion

Savard et al. (1994) showed that rbcL and four other genes from either nuclear or chloroplast DNA are evolving at relative rates that are consistent among the main lineages of plants (excluding annual angiosperms, see Bousquet et al., 1992). Synonymous sites of substitution were excluded from their rbcL analysis, which compared much older lineages. Ideally, an rbcL-based phylogeny could be generated that meets the assumptions of clocklike behavior for Pinaceae. Such a phylogeny could be compared with an extensive fossil record that consistently supports the relative branch lengths in the phylogeny. This concordance between the fossil record and the topology and relative branch lengths could in turn be used to estimate the average substitution rate for rbcL. Such criteria have not been met here. However, I would argue that even in the absence of an ideal agreement between nucleotide sequence analysis and the fossil record, preliminary attempts to reconstruct the order and timing of evolution have succeeded in demonstrated congruence between an rbcL based molecular clock and the fossil record. In probably all cases except Picea (which has no fossil record from the Mesozoic), the fossil record only slightly underestimates the age of genera based on MILK and the estimated rate of substitution in rbcL (FIG. 2.6). The analyses presented here suggest several overlooked gaps in our knowledge about evolution of the Pinaceae. Did all extant genera of the Pinaceae share a most recent common ancestor in the Triassic? Molecular evidence suggests that the Pinus-Picea split is very old relative to other genera of Pinaceae, but the fossil record of Picea does not even extend to the Cretaceous. Is Picea an ancient genus that branched off from all other extant members of the Pinaceae sometime early in the Mesozoic? Did Pseudotsuga and Larix diverge at the very end of the Cretaceous, corresponding to the Mesozoic-Cenozoic boundary? These questions, and many more of the same nature could be raised to justify further paleobotanical research on Pinaceae. Furthermore, it emphasizes that several avenues in molecular evolution of Pinaceae need to be explored. A better estimate of the order of branching in the Pinaceae could be made by the inclusion of the remaining extant 35

genera, Cathaya and Nothotsuga. Sampling more species within Pinaceae genera may also help. A more stable placement of Cedrus and Picea in an rbcL analysis might be achieved by using a weighting scheme that makes adjustments for multiple substitution events, synonymous versus nonsynonymous substitutions, and transitions versus transversions (Albert et al., 1993). Finally, with time we will know more about the practicality of using rbcL as a molecular clock as it is applied to an increasingly broad range of plant families. The average rate used here is based on estimated divergence dates that may beimproved in the future through further sampling in other families. They will also be improved with more accurate estimates of divergence times based on vicariance disjunctions and fossil data. A better estimate of the average substitution rate, and its departures in the various plant lineages should be a fruitful direction for future research.

2.6. Literature Cited

Albert, V. A., Chase, M. W., and B. D. Mishler. 1993. Character-state weighting for cladistic analysis of protein-coding DNA sequences. Ann. Missouri Bot. Gard. 80: 752-766.

Albert, V. A., Backlund, A., Bremer, K., Chase M. W., Manhart, J. R., Mishler, B. D., and K. C. Nixon. 1994. Functional contraints and rbcL evidence for land plant phylogeny. Ann. Missouri Bot. Gard. 81: 534-567.

Alvin, K. L. 1960. Further conifers of the Pinaceae from the Wealden Formation of Belgium. Mem. Inst. Roy. Sci. Nat. Belgique. 146: 1-39.

Axelrod, D. I. 1990. Environment of the Middle Eocene (45 Ma) Thunder Mountain flora, central Idaho. National Geographic Res. 6: 355-361.

Bousquet, J., S. H. Strauss, A. H. Doerksen, and R. A. Price. 1992. Extensive variation in evolutionary rate of rbcL gene sequences among seed plants. Proc. Natl. Acad. Sci. USA 89: 7844-7848. 36

Chase, M. W., Soltis, D. E., Olmstead, R. G. et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Missouri Bot. Gard. 80: 528-580.

Chaw, S.-M., H. Long, B. -S. Wang, A. Zharkikh, and W. -H. Li. 1993. The phylogenetic position of Taxaceae based on 18S rRNA sequences. J. MoL Evol. 37: 624-630.

, Zharkikh, A., H.-M.Sung, T.-C. Lau, and W.-H. Li. 1997. Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences. Mol. Biol. Evol. 14: 56-68.

Critchfield, W. B., and E. L. Little. 1966. Geographic distribution of the pines of the world. U.S. Department of Agriculture, Forest Service. Miscellaneous Publication 991. Washington D.C.

Felsenstein, J. 1993. DNAMLK--DNA maximum likelihood program with molecular clock. http://utmmg.med.uth.tm.../phylip_info/dnamlk.doc, version 3.5c.

Florin, R. 1951. Evolution in cordaites and conifers. Acta Horti Bergiani 15: 285-388.

Goremykin, V., Bobrova, V., Pahnke, J., Troitsky, A., Antonov, A., and W. Martin. Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support the Gnetalean affinities of angiosperms. MoL Biol. Evol. 13: 383-396.

Hart, J. A. 1987. A cladistic analysis of conifers: preliminary results. Journal of the Arnold Arboretum 68: 269-307.

Hermann, R. K. 1985. The genus Pseudotsuga: ancestral history and past distribution. Spec. PubL No. 2b, Forest Research Laboratory, Oregon State University, Corvallis, Oregon.

Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules. pp. 21-123. In: Munro, H. N. (ed.) Mammalian Protein Metabolism. Academic Press, New York.

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. MoL Evol. 16: 111-120.

Millar, C.I. and B.B. Kinloch. 1991. Taxonomy, phylogeny, and coevolution of pines and their stem rusts. In: Rusts of pine. Proceedings of the HIER° rusts of pine working party conference, September 18-22, 1989, Banff, Alberta, Canada. (Hiratsuka, Y., Samoil, J.K., Blenis, P.V., Crane, P.E., and B.L. Laishley eds.) 37

Information report NOR-X-317, Forestry Canada Northwest Region Northern Forestry Centre, pp. 1-38.

Miller, C. N. 1977. Mesozoic conifers. Bot. Rev. (Lancaster) 43: 217-280.

. 1982. Current status of Paleozoic and Mesozoic conifers. Rev. Paleobot. Palynol. 37: 99-114.

. 1988. The origin of modern conifer families. Pp. 448-487 in: Origin and Evolution of Gymnosperms. C. B. Beck, (ed.), Columbia Univ. Press, New York.

Nixon, K. C., Crepet, W. L., Stevenson, D., and E. M. Friis. 1994. A reevaluation of seed plant phylogeny. Ann. Missouri Bot. Gard. 81: 484-533.

Palmer, J. D. 1992. Mitochondrial DNA in plant systematics: Applications and limitations. In: Molecular Systematics of Plants, P. S. Soltis, D. E. Soltis, and J. J. Doyle, (eds.) Chapman and Hall, New York.

Prager, E. M., Fowler, D. P., and A. C. Wilson. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

Price, R. A., Olsen-Stojkovich, J., and J. M. Lowenstein. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Syst. Bot. 12: 91-97.

Relle, M., Furst, and A. Wild. 1995. Short duplication in a cDNA clone of the rhcL gene from Picea abies. Plant Physiol. 107: 1037-1038.

Saiki, K.1. 1996. Pinus mutoi (Pinaceae), a new species of permineralized seed cone from the Upper Cretaceous of Hokkaido, Japan. Amer. J. Bot. 83: 1630-1636.

Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method reconstructing phylogenetic trees. Mol. Biol. Eva 4: 406-425.

Savard, L. P. Li, S. H. Strauss, M. W. Chase, M. Michaud, and J. Bousquet. 1994. Chloroplast and nuclear gene sequences indicate Late Pennsylvanian time for the last common ancestor of extant seed plants. Proc. Natl. Acad Sci. USA 91: 5163­ 5167.

Schorn, H. E. 1994. A preliminary discussion of fossil larches (Larix, Pinaceae) from the arctic. Quaternary International 22/23: 173-183.

Smith, A. B. 1994. Rooting molecular trees: problems and strategies. Biological Journal of the Linnean Society 51: 279-292. 38

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gil levet. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Springer, M. S. 1995. Molecular clocks and the incompleteness of the fossil record. J. Mol. Evol. 41: 531-538.

Strauss, S. H., A. H. Doerksen, and J. R. Byrne. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canad. I Bot. 68: 1502-1510.

Swofford, D. L. 1993. PA UP: Phylogenetic analysis using parsimony, Version 3.0s +4. Illinois Natural History Survey, Champaign, Illinois.

G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. Pp. 407-514. In: Molecular systematics. Eds., D. M. Hillis, C. Moritz, and B. K. Mable. Sinauer Associates, Inc., Sunderland, Massachusetts.

Tsumura, Y., Yoshimura, K., Tomaru, N., and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theor. Appl. Genet. 91: 1222-1236.

Wilson, A. C., Ochman H., and E. M. Prager. 1987. Molecular time scale for evolution. Trends Genet. 3:241-247.

Wolf, J. A., and H. E. Schorn. 1990. Taxonomic revision of the Spermatopsida of the Oligocene Creede flora, Southern Colorado. US Geological Survey Bulletin 1923.

Zuckerkandl, E., and L. Pauling. 1965. Evolutionary divergence and convergence in proteins. Pp. 97-166 in: Evolving Genes and Proteins, V. Brysra and H. J. Vogel (eds.) Academic Press, New York. 39

Chapter 3

Internal Transcribed Spacer Region Evolution in Larix and Pseudotsuga (Pinaceae)

David S. Gernandt and Aaron Liston Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331-2902

submitted to American Journal of Botany, February 1998. In review.

3.1. Abstract

The nuclear ribosomal DNA internal transcribed spacer (ITS) region has been characterized in the sister genera Larix and Pseudotsuga (Pinaceae). Complete sequences were obtained for seven species of Larix from North America and Eurasia and five species representing the geographic range of Pseudotsuga in western North America and eastern Asia. The ITS region lengths ranged from 1759 to 1770 by in Larix and from 1564 to 1571 by in Pseudotsuga. In both genera, the ITS1 is three times as long as the 5.8S plus ITS2, and contains subrepeats as observed in other members of Pinaceae. Secondary structure models predicted that the subrepeats folded into terminal stem and loop domains. Evidence of ITS polymorphism within species of Larix and Pseudotsuga was detected and may be attributable to a slow rate of concerted evolution among ribosomal DNA loci. Except for the placement of L. sibirica, phylogenetic analyses of the ITS region data set were in agreement with previously reported restriction site analyses of Larix and Pseudotsuga. The data were not consistent with phylogenetic hypotheses for Larix based primarily upon ovulate cone characters. A stepping stone model of evolution for Pseudotsuga has been proposed by others, but cladistic approaches offer limited power to identify the center of origin for the genus. 40

3.2. Introduction

Morphological and molecular systematic treatments of Pinaceae (Coniferales) have been extensive at the generic level (Prager, Fowler, and Wilson, 1976; Miller, 1977; Hart, 1987; Price, Olsen-Stojkovich, and Lowenstein, 1987; Chase et al., 1993; Tsumura et al., 1995; Chaw et al., 1997). The main source of nucleotide sequence data for the family has been the plastid-encoded ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) gene, but its slow rate of substitution limits its utility for studies among recently derived species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) appears to have fewer functional constraints on substitution and has been used widely in reconstructing generic and infrageneric eukaryote phylogenies (Baldwin et al., 1995). Although the ITS region is approximately 565-700 by in angiosperms, it is longer in non­ flowering seed plants, particularly in Podocarpaceae and Pinaceae (Liston et al., 1996). In Pinaceae, lengths reportedly vary from 1550 by in Pseudotsuga menziesii (Mirb.) Franco to 3 000 by in Pinus pinea L. This substantial length variation suggests that alignment of the ITS1 may be difficult across genera of Pinaceae, and therefore unsuitable for phylogenetic studies above the genus level. Complete nucleotide sequences for the ITS region have been reported for Pinus pinea (Marrocco, Gelati, and Maggini, 1996). To date, detailed comparisons of full length ITS region sequences between species or genera in Pinaceae have not been published. To determine the utility of the ITS region for reconstructing phylogenies in Pinaceae we undertook an ITS region analysis of two genera, Larix Miller (larch) and Pseudotsuga Corriere (Douglas-fir). The sister relationship between these two genera has been well-established on the basis of fertilization and pollen development (Christiansen, 1972; Takaso, 1996), immunoassays (Prager et al., 1976; Price et al., 1987), cladistic analysis of anatomical and chemical characters (Hart, 1987), sequence comparison of rbcL (Chase et al., 1993) chloroplast DNA RFLPs (Tsumura et al., 1995; Xiao-Quan et al., 1997), and 18S rRNA sequences (Chaw et al., 1997). Larix is comprised of ten species (Table 3.1). The genus is more widely distributed than Pseudotsuga and it occurs in cooler environments at higher altitudes and 41

more northerly latitudes throughout the Northern Hemisphere (Farjon, 1990). Larix systematics has been the subject of morphological (Farjon, 1990; Schorn, 1994; LePage and Basinger, 1991, 1995) and molecular examination (Qian, Ennos, and Helgason, 1995). Patschke (1913) was the first to divide the genus into two sections based upon the morphology of the female cone. Section Larix (or Pauciserialis) included species with bracts on the ovulate cone that did not extend well beyond the cone scales and section Multiserialis included species with bracts extending far beyond the cone scales. Several classifications based on ovulate cone morphology have been proposed. Schorn (1994) placed the two western North America species, L. occidentalis and L. lyallii, both of which have bracts on the ovulate cone that are extended well beyond the cone scales (exserted), into section Aristatus. Larix griffithiana, L. mastersiana, L. potaninii, and L. kaempferi, all of which have bract scales that extend only slightly beyond the cone scale, were placed in section Laminatus group Ha. Larix decidua, L. siberica, L. gmelinii, and L. laricina all possess bracts that are shorter than the cone scales and were placed by Schorn in section Laminatus group Hb. Using RFLP analysis of chloroplast DNA (cpDNA), Qian, et al. (1995) contributed the first molecular evidence that classification based on exserted or inserted bract scales was inconsistent with the evolutionary history of Larix. The recent systematic relationships proposed for Larix are summarized in Table 3.2. Recent classification of Pseudotsuga have recognized from four (Farjon, 1990) to eight (Hermann, 1982) species (Table 3.1). Pseudotsuga menziesii has been studied extensively by population geneticists (Li and Adams, 1988; Moran and Adams, 1989, Schnabel, Hamrick, and Wells, 1993; Ponoy, 1994; Aagaard et al., 1995) and the systematics of the genus has been the subject of both morphological (Farjon, 1990) and molecular examination (Doerksen and Ching, 1972; El-Kassaby, Colangeli, and Sziklai, 1982; Strauss, Doerksen, and Byrne, 1990). Pseudotsuga menziesii sensu lato (s.l.) is distributed across a wide range of western North America, P. macrocarpa in montane regions of southern California, P. japonica in southern Japan and P. sinensis s.l. in both mainland China and Taiwan. Pseudotsuga menziesii s.l. includes var. glauca, an interior form occurring from Canada to Mexico. Four Mexican species (Table 3.1) have been 42 accepted by Martinez (1949) but they are generally considered synonyms of P. menziesii var. glauca (Farjon, 1990). At least eight species of Pseudotsuga have been described from as many regions of mainland China and Taiwan, but they are generally synonymized with the mainland P. sinensis and the Taiwanese P. wilsoniana (Hermann, 1982). Farjon (1990) recognized a single species, P. sinensis, and designated only two additional varieties, var. brevifolia (Cheng et Fu) Farjon and var. gaussenii (Flous) Farjon. The evolution of Pseudotsuga has been discussed by Strauss et al. (1990), who used evidence based on restriction fragment analysis to hypothesize that a North American lineage migrated around the Pacific Rim to Asia, giving rise to P. japonica which in turn gave rise to P. sinensis and P. wilsoniana (a stepping stone model of evolution, Table 3.2). The current study provides structural information about the ITS region for Larix and Pseudotsuga, and reexamines phylogenetic hypotheses for these genera. Complete ITS region sequences for seven species of Larix, and five species of Pseudotsuga provide an opportunity to reevaluate conflicting phylogenies for Larix based either on fossil evidence, features of the ovulate cone, or cpDNA restriction sites. The phylogenetic analysis of the ITS region also permits a reevaluation of the stepping stone hypothesis for Pseudotsuga.

3.3. Materials and Methods

Taxa included--A total of twelve species of Larix and Pseudotsuga were selected for sequencing of the ITS region (Table 3.1). Sampling of Larix included members of all three bract length types of Larix and a member from each cpDNA type identified by Qian et al. (1995). The Asian species L. gmelinii in Asia was included in the restriction site study. The four species of Pseudotsuga recognized by Farjon (1990) and P. wilsoniana (treated by Farjon as a synonym of P. sinensis) were also sequenced. Restriction site comparisons included P. flahaultii, P. guinieri, and P. rehderi, three of four named Table 3.1. Synopsis of Larix and Pseudotsuga species. Unless indicated otherwise, the ITS region was sequenced directly from PCR products.

Taxon Source Voucher no./GENBANK no.

Eurasian Larix

L. deciduaMill.1 Hoyt Arboretum, Portland, Oregon, USA DSG001/AF041343

L. gmelinii (Rupr.) Rupr.2 Hoyt Arboretum, Portland, Oregon, USA DSG002/none

L. griffithiana (Lindl. et Gord.) Lachung, Sikkim, India DSG003/AF041349

Carr.

L. kaempferi (Lamb.) Carr. Mt. Yatsugatake, Nagano Prefecture, Japan DSG004/AF041344

L. mastersiana Rehder et Wilson not sampled

L. potaninii Bata lin not sampled

L. sibirica Ledeb. Forestfarm Nursery, Williams, Oregon, USA DSG016/AF041345

North American Larix

L. laricina (Du Roi) K. Koch 19951411, Edinburgh, United Kingdom DSG005/AF041348

L. lyallii Parl. Anthony Lakes, Malheur Co., Oregon, USA DSG006/AF041346

L. occidentalis Nutt. J. H. Stone Nursery, Jackson Co., Oregon, USA DSG007/AF041347 Table 3.1. (Continued)

Taxon Source Voucher no./GENBANK no. Asian Pseudotsuga

P. sinensis Dode Ps871, USDA FS Arboretum, Benton Co., Oregon, USA DSG014/AF041350

P. wilsoniana Hayata 19934044, Edinburgh, United Kingdom DSG015/AF041351 North American Pseudotsuga

P. flahaultii Flous2' 3 Edinburgh, United Kingdom DSG008/none

CC Sierra de Arteaga, Cerro El Coahuilon, Coahuila, Mexico DSG017/none

P. guinieri Flous2'3 19922183, Edinburgh, United Kingdom DSG009/none

P. japonica (Shiras.) Beissn. Botanical Garden, Kukizaki, Ibaraki, Japan DSG010/AF041352

P. macrocarpa (Vasey) Mayr' Palomar Mt., California, USA DSG011/AF041354

P. menziesii (Mirb.) Franco' McDonald Forest, Benton Co., Oregon, USA DSG012/AF041353

P. rehderi Flous2'3 19870763 Edinburgh, United Kingdom DSG013/none

The ITS region was cloned and partially sequenced, in addition to direct sequencing. 2 The ITS region was only restriction site mapped. 3 Mexican species generally synonymized with P. menziesii var. glauca (Beissn. ) Franco. A fourth species, P. macrolepis Flous, has also been described from Mexico. Table 3.2. Phylogenetic hypotheses proposed for extant Larix and Pseudotsuga. Hybrids, varieties, and fossil taxa have been excluded. Key to abbreviations: anc = hypothetical ancestor, Lde = L. decidua, Lgm = L. gmelinii, Lgr = L. griffithiana, Lka = L. kaempferi, Lla = L. laricina, Lly = L. lyallii, Lma = L. mastersiana, Loc = L. occidentalis, Lpo = L. potaninii, Lsi = L. sibirica, Pja = P. japonica, Pma = P. macrocarpa, Pme = P. menziesii, Psi = P. sinensis, Pwi = P. wilsoniana.

Source Phylogenetic hypothesis data source analysis

LePage and Basinger, 1991 (((( Lgm, Lsi ),Lde),Lla)(((((Lma,Lgr),Lpo) cones, geographical unspecified

,Lka),(Loc,Lya)),anc)) distribution, fossil record

Schorn, 1994 (Lgm,(Lla,((Lsi,Lde),((((Lma,Lpo), Lgr),Lka) female cone anatomy unspecified

((Loc,Lya),anc)))))

Qian et al., 1995 ((((Lla, Loc), Lsi), Lgr),(Lka, Lpo, Lde, Lgm)) cpDNA RFLPs phenetic

Strauss et al , 1990 1. ((((Psi, Pwi), Pja), Pme), Pma, Loc) nuclear, cpDNA, mtDNA parsimony

2. (((Psi, Pwi), Pja)(Pma, Pme), Loc) 46

Mexican taxa generally synonymized with P. menziesii var. giauca-(Farjon, 1990; Hermann, 1982).

DNA isolation- -DNA was extracted using the method of Doyle and Doyle (1987). Samples were ground in 65° C 2X CTAB (United States Biochemical, Cleveland, OH) buffer supplemented with 2% NaBisulfite and 2% PVP, extracted twice in chloroform:isoamyl alcohol 24:1, precipitated in isopropanol and 7.5 M ammonium acetate for 60 min at -20° C, spun, washed in 70% EtOH, and resuspended in Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.0).

PCR amplification and DNA sequencing- -PCR reactions were performed in 100 [IL volumes with 2.5 U ReplithermTm DNA polymerase (Epicentre Technologies, Madison, WI), ReplithermTM buffer, 1.5 mM MgC12, 10 pmol dNTP (10 mM, pH 7.0, Epicentre Technologies, Madison, WI), 16.7 pmol ITS5* (Liston et al., 1996), 16.7 pmol 26S-25R (Nickrent, Schuette, and Starr, 1990), 5% DMSO, 1% BSA, and approximately 100 ng sample DNA. PCR amplification reaction conditions were 35 cycles of 60 s at 94° C denaturing, 60 s at 55° C annealing, and 3 min at 72° C extension. Reactions were terminated following a final extension for 7 min at 72° C. Products were prepared for sequencing by gel purification (Qiagen, Chatsworth, CA). Additional primers used for sequencing were 5.8GYM (Liston et al., 1996), ITS-3 (White et al., 1990) and six genus specific primers designed for sites along ITS1 of Larix and Pseudotsuga (FIGS. 3.1-3.2). Cycle sequencing with dye-terminator chemistry was performed using an ABI model 377 fluorescent sequencer. Products were sequenced in both 5' to 3' and 3' to 5' directions. Full length PCR products were digested with the restriction enzymes HaeIII, Hhal, and Hinfl according to manufacturer's instructions. Digested products and a 100 by ladder size standard (Life Technologies, Gaithersburg, MD), were electrophoresed on a 2% agarose gel in Tris-Borate-EDTA buffer (pH 8.6). Gels were visualized with ethidium bromide under UV light. Bands greater than 100 by were compared against restriction 47

LAITS1R LAITSICR 5.8GYM 26S-25R -4 M -1M - -4 SR1 SR2 ITS5* LAITS1B ITS3

SSU ITS1 5.8S ITS2 LSU

2

DFITS1R DFITS1CR 5.8GYM 26S-25r MI SR1 SR2-3 ITS5* DFITS1B ITS3

SSU ITS1 5.8S ITS2 LSU

FIGS. 3.1-3.2. Diagram for the ITS region of representative members of Larix and Pseudotsuga. 1. Larix occidentalis. 2. Pseudotsuga macrocarpa. Arrows above and below Figures represent the reverse and forward primers respectively. Primers designed for this study are LAITS1R (5' -CATCCGAGTTGGTACACGC-3' ), LAITS1B (5' ­ CCAAGGGCCTTGCATCAT-3 ' ), LAITS 1CR (5' -AGCGACAACAAGCAATGC-3 ' ), DFITS1R (5' -GTATGCAAAGGCAGGCGG-3 ' ), DFITS1B (5' ­ CCGCCTGCCTTTGCATAC-3' ), and DFITS1CR (5' -GGCCAACAGCAAACAATGC­ 3' ). 48

site maps generated from the available full ITS region sequences. Restriction site maps indicated that some restriction enzymes would produce multiple similar sized bands (within 10 bp) in the 50-200 by range. For this reason, several digestion products smaller than 200 by were not interpreted. Bands smaller than 50 by were not resolved with agarose electrophoresis. Direct sequencing of PCR products revealed several species with potential site and length polymorphisms at the 5' end of ITS1. PCR products from four of these species (P. macrocarpa, P. menziesii, L. decidua, and L. sibirica) were cloned into a pCR®2.1 vector using a TOPO TA Cloning Kit (Invitrogen, San Diego, CA). Seven to eight cloned inserts per species were reamplified with ITS5* and 26S-25R and digested with HaeIII (GGCC), HhaI (GCGC), and Hinfl (GANTC). Restriction profiles were compared to that of the original PCR product. For each of the four species, a single clone matching the restriction profile of the original PCR product was sequenced with ITS5*. In addition, 400 by was sequenced of the P. menziesii clone using the forward primer DFITS1B. A single clone from L. sibirica with a restriction site loss relative to the original PCR product was also sequenced with ITS5*.

Sequence analysis -- Sequence contigs were assembled and edited using Genetic Data Environment (GDE) (Smith et al., 1994). Ambiguous sites determined from multiple peaks at a single position were designated by IUBMB symbols. Sequence data for the ITS region were aligned using the pileup option in Genetics Computer Group (GCG, 1996) (gapweight = 1.0, gaplengthweight = 0.2). The alignment is available from the authors upon request. Secondary structures were generated using mfold version 2.3 (Jaeger, Turner, and Zuker, 1990). BLAST (Altschul et al., 1990) and Gapped BLAST (Altschul et al., 1997) were used to search DNA sequence databases for high similarity with other accessions. Parsimony and distance analyses were performed using PAUP* 4.0.0d60 for UNIX (Swofford, 1998). Gaps were treated as missing characters. Parsimony and distance trees for the full ITS region were found using the branch-and-bound and neighbor joining methods respectively. Bootstrap values for the parsimony analysis were generated with 500 replicates (100 for subrepeat analysis) each using 50 random addition sequences and a maxtrees limit of 1 500. Neighbor joining analyses were performed with a Kimura 49

two-parameter correction. Molecular and morphological phylogenetic analyses of Pinaceae suggest that Pinus is an appropriate outgroup for Pseudotsuga and Larix. Sequences from Pinus pinea were used to root the phylogenetic analyses of ITS1 subrepeats and the 5.8S plus ITS2. The full length ITS region analysis was midpoint rooted based on the assumption of a sister group relationship between Pseudotsuga and Larix.

3.4. Results

Sequence heterogeneity--Direct sequencing of gel-purified PCR products from Larix and Pseudotsuga resulted in sequence reads of fair to poor quality at the 5' end of ITS1. A prominent feature of the signal degradation was the presence of multiple peaks at several positions that could be observed in both the 5' to 3' and 3' to 5' direction. In most cases where there was a multiple peak, one peak was stronger than the other and this peak was scored by the automated sequencer or by manual editing. When peaks were estimated to be of equal strength, an IUBMB symbol was used to represent all nucleotides present. Signal degradation was observed in L. decidua, L. sihirica, and L. kaempferi and in all five Pseudotsuga species. It was not observed in L. lyallii, L. occidentalis, L. laricina, L. kaempferi, or L. griffithiana. In two species of Larix (L. decidua and L. ,sibirica) and two species of Pseudotsuga (P. menziesii and P. macrocarpa), sequence reads had ca. 400 by stretches of degraded signal characterized by multiple peaks at most positions. Each of these sequence reads was interpreted as having peaks from a sequencing template with small length polymorphisms. Restriction digests of cloned PCR products from these four species made it possible to confirm the presence of restriction site variation. RFLP patterns differing from those of the original PCR product were found in six of eight L. sibirica clones, three of eight L. decidua clones, two of eight P. menziesii clones, and three of seven P. macrocarpa clones. In L. sihirica, the Hhal RFLP pattern of the 50

original PCR product included bands at 209 and 150 by (precise sizes determined from sequence). Clones with RFLPs that lacked one of these bands had apparently gained Hhal sites. The 209 by and 150 by bands in the original PCR product from L. sibirica were of low intensity relative to larger and smaller bands, suggesting that only a fraction of the ITS region products possessed these patterns and therefore polymorphisms were also present in the original PCR product. In clones from the other three species, restriction site differences resulted in large bands that were not visible in restriction digests of the original PCR product. The polymorphic 209 by product in L. sibirica mapped to the ITS2, although there was no evidence of polymorphism in the direct sequence reads from that region. One clone had another Hhal site in ITS2 that gave two bands of ca. 97 and 112 bp. Two sites differing by one by from an Hhal site (GGGC and GCGG) were identified 90-115 by downstream of the invariant ITS2 site. Sequencing reactions using the ITS5* primer were obtained for each of the four species that were cloned. The ITS5* read from a L. decidua clone matching the RFLP pattern of the original PCR product differed at eight of the first 565 sites in ITS1. Four of the site differences could be seen as weak or multiple peaks in the direct sequence reads and the remaining four were not. This ITS type was interpreted as not matching the main type present in the L. decidua exemplar. An ITS5* sequence was determined for two RFLP profiles of L. sibirica. Discrepancies among the two clones and the direct sequence read were traced to eight polymorphic positions. Five of those positions were also polymorphic in either L. kaempferi or L. decidua. In the P. menziesil clone, the ITS5* sequence differed at only one of the first 600 by of the direct sequence determined from the original PCR product. The ITS5* read for P. macrocarpa differed at seven of the first 600 bp. In L. sibirica, L. decidua, and P. macrocarpa, the first discrepancy between the sequence of the clone and the sequence of the original PCR product occurred at the onset of signal degradation. Sequences from clones lacked a nucleotide present in the original PCR products at ITS1 position 4 of L. sibirica and L. decidua, and ITS1 position 62 of P. macrocarpa. In L. sibirica, an additional gap relative to the direct sequence read occurred at position 422, and this gap corresponded to the onset of signal degradation in the reverse read from the LAITS1R primer site. Additional ambiguity symbols were 51

placed into polymorphic positions in these four species to account for the discrepancies between sequences from the clones and the direct PCR products. The final alignment had a total of 20 ITS1 positions that were ambiguous in at least one species. This represents 5.1% of all variable positions. Fifteen ambiguity symbols were placed at ten positions in one or more species of Larix, and five ambiguity symbols were placed at five positions, each in one species of Pseudotsuga.

Conserved sequence motifs, subrepeats, and secondary structure--The 16 by conserved motif observed in the ITS1 of angiosperms (Liu and Schardl, 1994) is not present in the ITS of Larix, Pseudotsuga, or Pinus pinea. Yet subrepeats with sequence homology to those found for Pinus pinea (Marrocco et al., 1995) were observed along the 3' 620 by of ITS1 in both Larix and Pseudotsuga (FIGS. 3.1-3.2). Although the ITS1 length of Larix was observed to exceed that of Pseudotsuga by approximately 200 bp, Pseudotsuga had three copies of the subrepeat and Larix only two. All subrepeats had a highly conserved, central region with the sequence GGCCACCCTAGTC. By searching for the occurrence of this region, a sixth, unreported subrepeat was found in Pinus pinea. The sixth subrepeat (referred to as SRO in FIG. 3.4), was more divergent than the five previously reported. The similarities among the first five subrepeats ranged from 81.9­ 95.5%, compared to a similarity range of 48.3-57.5% with the sixth subrepeat. The conserved sequence region occurred at position 572, which was 883 by upstream of the conserved region of the first of the five tandem subrepeats in Pinus pinea. The length of the Larix and Pseudotsuga subrepeats on either side of the conserved region was judged by visual examination of an alignment. The first subrepeat in Larix and Pseudotsuga was interpreted as ranging in size from 68-72 by (compared to a range of 215-237 by reported in P. pinea). The second subrepeat in Larix and the second and third subrepeats in Pseudotsuga were interpreted as ranging in size from 77-78 bp. Secondary structure analysis of ITS1 revealed that each of the three subrepeats consistently folded into a terminal stem and loop structure (FIG. 3.3). The stem loops were predicted at 20, 25, and 37 °C and were also observed in less optimal folds. Other portions of ITS1 were sensitive to the inclusion or exclusion of flanking small subunit and 5.8S nucleotides (data 52

not shown). Structures reported here included the final eight by of the small subunit and the first 32 by of the 5.8S. These regions have been shown to interact with ITS1 using yeast as an experimental system (Yeh, Thweatt, and Lee, 1990; van Neus et al., 1994). In preliminary folds, portions of the 5' 100 by and 3' 100 by of ITS1 interacted with one another. This interaction was prohibited in the final folds by constraining nucleotides to base pair only with neighbors within 400 positions, which is more consistent with studies on yeast. The free energy of the unconstrained fold (not shown) is -590.8 kcal/mol, compared to -580.7 kcal/mol in the constrained fold. A 230 by region separated the first subrepeat from the second subrepeat in both Larix and Pseudotsuga (FIGS. 3.1-3.2). The final two subrepeats in Pseudotsuga were in tandem (FIG. 2). The Larix and Pseudotsuga subrepeats were more similar to each other regardless of position than they were to those reported in Pinus pinea. A heuristic search was performed in PAUP* to formally test the relationships among the subrepeats (FIG. 3.4). The topology of Larix and Pseudotsuga subrepeats in the resulting strict consensus of 87 most parsimonious trees (not shown) was not consistent with the hypothesis that each subrepeat of Pseudotsuga and Larix is evolving in concert. A Glade comprised of the first Larix subrepeat from each species of Larix was a sister group to a Glade comprised of the first subrepeat from each species of Pseudotsuga, and a second Glade comprised of the second Larix subrepeats was a sister group to a Glade comprised of the third Pseudotsuga subrepeats. The six Pinus pinea subrepeats are monophyletic, a pattern contradictory to that seen in Larix and Pseudotsuga but consistent with the hypothesis that concerted evolution is homogenizing subrepeats in this species. BLASTn searches with the two subrepeats in L. occidentalis and L. griffithiana, and the three subrepeats in P. macrocarpa and P. wilsoniana, found high similarity to the Pinus pinea ITS region sequence (accession X87936). BLASTn also found matches in nonhomologous molecules. These matches had a low degree of similarity that could have been explained by chance alone based upon the size of the nucleotide databases at the time the search was performed. These hits included a match between the first P. macrocarpa subrepeat and an mRNA for unconventional myosin from Sus scrota (p = 1.00, accession TABLE 3.3. Nuclear ribosomal DNA ITS region sequence statistics for Larix and Pseudotsuga.

Parameter ITS1 5.8S ITS2 Total Aligned length 1481 162 233 1876 Length range Larix 1365-1375 162 232-233 1759-1770 Pseudotsuga 1170-1177 162 232 1564-1571 Mean length and s.d. (bp) Larix 1369.7 ± 5.1 162 ± 0.0 232.4 ± 0.5 1764.1 ± 5.6 Pseudotsuga 1171.8 ± 2.9 162 ± 0.0 232 ± 0.0 1565.8 ± 2.9 G + C content mean & s.d. Larix 0.575 ± 0.004 0.512 ± 0.0 0.600 ± 0.004 0.572 ± 0.004 Pseudotsuga 0.589 ± 0.003 0.525 ± 0.0 0.644 ± 0.002 0.590 ± 0.002 Number of variable sites 348 2 45 395 Larix 73 0 5 78 Pseudotsuga 77 0 8 85 Number of informative sites (%) 285 (19.2) 2 (1.2) 42 (18.0) 329 (17.5) Larix 35 (2.4) 0 (0.0) 3 (1.2) 38 (2.0) Pseudotsuga 31 (2.1) 0 (0.0) 5 (2.1) 36 (1.9) Sequence divergence mean & s.d. 0.136 ± 0.107 .0066 ± 0.094 ± 0.078 0.115 ± 0.009 .006 Larix 0.021 ± 0.011 0 0.009 ± 0.006 0.018 ± 0.009 Pseudotsuga 0.031 ± 0.018 0 0.02 ± 0.01 0.026 ± 0.016 Transitions (minimum) 197 2 27 226 Transversions (minimum) 159 0 20 179 Transitions/Transversions 1.24 undefined 1.35 1.26 54

Z35331), between the third P. macrocarpa subrepeat and a Danio rerio (zebrafish) complement factor B mRNA product (p = 0.73, accession U34662), and a match between the first L. occidentalis subrepeat and a Rattus norvegicus ash mRNA (p = 0.59, accession X62853). While most of the matches found using BLASTn exceeded 40 by in length, searches using gapped BLAST found matches ranging from 17-22 bp. Positions 141-153 of Macaca mulatta (rhesus monkey) cytokine gene, interleukin-12 beta p35 subunit (accession U19842), were identical in sequence to the conserved, GGCCACCCTAGTC region in the Larix and Pseudotsuga subrepeats. Additional similarity upstream and downstream of this region resulted in gapped BLAST hits between M mulatta and all subrepeat searches, with similarities of 100% along 18-19 bp. The number of expected hits of this length ranged from 0.24-1.6 at the time the searches were performed. A 100% match occurred between subrepeat 1 of L. occidentalis and Homo sapiens ITS2 (p = 1.00, accession X17626). The match included 2 by at the 3' end of the conserved region plus the next 15 by in L. occidentalis. The expected number of hits for this length was 3.6. In comparison, the score of the Pinus pinea gapped BLAST searches were among the lowest retrieved, with 17 by of 100% identity (including the conserved region) and an expect score of 4.0. There was a high degree of phylogenetic diversity represented in the matching accessions, all of which had scores equal to or lower than that for M mulatta. Organisms subject to intensive sequencing comprised the majority of the matches. These included Bacillus subtilis, Oryza saliva, Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Rattus norvegicus, and Homo sapiens. Except for the hits to Pinus pinea subrepeats and H. sapiens ITS2, the matches were to protein coding genes with no apparent relationship to rDNA.

Phylogenetic analyses -- Lengths of the ITS region for the species sampled were close to those reported based on restriction site mapping (Liston et al., 1996). Excluding the flanking small and large subunits, the length of the region in Larix ranged from 1759 to 1770 by and in Pseudotsuga from 1564 to 1571 by (Table 3.3). Most of the site variation among species (88%) occurred in ITS1. Large indels in ITS1 explained most of 55

:GX.11sGS.: UK. PA

ENO 614-I

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C. A C ,-0G.. UGLY0 Lroco e ` A-:.I "G.ue C Gc.,x "t.r. ',4-"' u c G. c---f., . c c" Su ue uu izo-st u-c u4 cc " uu u c.c- .. BEGIN u sas cue ju u.c Vo, "c. u ct-u.,,a00 uo.e0,0 .40 c'ufcu u, E. Si. ucr. 00 ,.1 u, uo.. ..b:-u- G G A U G-C '. 4. OG A U U at uo..,c u-­ A woo4,c, L,A A ocec' c c," ,'c 4 GUU G, UGC %0%. U GP MO GU C C .G.CAC 4U: CbCrhj CtiA., .0 4,qt urtltib' CG -, u" cuu , u ..­ GA cifja-ce 9.. u , c,-,:sI fql S4 oc 't-',0,C 0 GA U ' ' 0 1 ' °C VC. t.tc ' UA A ' G 0 CC P U CC OUA

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'1GA04, Gc cq '' GcA Gc Gc Gc G. °G.Gc UG tIC

GGtL%A

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ALt "u u

FIG. 3.3. Secondary structure of ITS1 for Pseudotsuga menziesii (energy = -580.7 kcal/mol). Nucleotides are constrained to pair only within 400 bp. Pseudotsuga menziesii has a 3 by autapomorphic insertion in SR-1. The interaction between SR-2 and SR-3 is predicted in folds for all species of Pseudotsuga. 56

FIG. 3.4. Phylogeny of ITS1 subrepeats. One of 87 most parsimonious trees found in a heuristic search of ITS1 subrepeats (116 steps, CIe = 0.667, RI = 0.902 and RC = 0.630). Trees were found using 10 random sequence addition replicates. The maxtrees limit of 1 500 trees was reached 40 times. Bootstrap values (100 replicates) equal to or greater than 50% are displayed on the branches. The scale bar represents the number of steps. 57

L. decidua SR2 56 L. griffithiana SR2 L. sibirica SR2 96 L. kaempferi SR2 L. lyaffii SR2 L. laricina SR2 56 L. occidentalis SR2 P. sinensis SR3 P. japonica SR3 3 TP. wilsoniana SR3 P. menziesii SR3 54 P. macrocarpa SR3 P. sinensis SR2 P. wilsoniana SR2 P. japonica SR2 P. menziesii SR2 P. macrocarpa SR2 L. lyaffii SR1 L. occidentalis SR1 L. laricina SR1 L. kaempferi SR1 6d L. decidua SR1 94 L. sibirica SR1 74 L. griffithiana SR1 _r- P. menziesii SR1 P. macrocarpa SR1 66 P. sinensis SR1 P. japonica SR1 P. wilsoniana SR1 P. pinea SR1 P. pinea SR2 P. pinea SR3 99 P. pinea SR5 83 P.. pinea SR4 P. pinea SRO 58

the size differences between the two genera. Most of the ITS1 variability occurred at the 5' end (FIG. 3.5). The alignment of the 5.8S and ITS2 of all Larix and Pseudotsuga species with Pinus pinea was 426 positions in length. The P. pinea 5.8S sequence is 161 nucleotides in length, one nucleotide short of the number found in Larix, Pseudotsuga, and other species of Pinus (Liston et al., submitted). The shorter length is attributable to a missing guanine residue 62 by from the 5'end that is invariant in all other species sequenced. After insertion of the guanine residue into the P. pinea sequence prior to the analysis, there were no gaps in the 5.8S portion of the alignment. The alignment included seven variable sites in the 5.8S, only two of which were parsimony informative. The ITS2 alignment included 11 gaps. The ITS2 had 89 informative sites, 42 of which were parsimony informative. Maximum parsimony analysis of the ITS2 and 5.8S using branch and bound searching found two most parsimonious trees that differed only in the placement of L. decidua relative to L. griffithiana (FIG. 3.6). Both Larix and Pseudotsuga were monophyletic, and there was limited resolution of some species. The North American species of Larix formed an unresolved trichotomy separate from the Eurasian species. Of the Eurasian species of Larix, L. kaempferi and L. sibirica formed a monophyletic group while L. decidua and L. griffithiana occupied a basal position in the same Glade. The North American species of Pseudotsuga were sister taxa, forming a separate lineage from the three East Asian species sampled. The Larix and Pseudotsuga alignment of the full length ITS region was 1876 characters in length. A total of 63 gaps were placed in ITS1, 51 of which were exclusively for alignment between the two genera. The mean maximum length of the gaps was 6.7 by (range 1-43 bp). A single nucleotide gap was inserted near the 3' end of the ITS2 to account for a nucleotide position present in L. decidua, L. kaempferi, L. sibirica, but absent in the other nine species. Along the entire ITS region, a total of 329 out of 395 variable sites were parsimony informative. Maximum parsimony analysis of the entire ITS region for Pseudotsuga and Larix gave a single, fully resolved tree of high consistency with bootstrap values exceeding 70% at all nodes (FIG. 3.7). The neighbor joining topology was identical (result not shown). 59

ITS1 s 5.8S ITS2 14

12

10

8

a) () 6

4

0 250 500 750 1000 1250 1500 1750 Nucleotide position

FIG. 3.5. Nucleotide substitutions in the ITS region of Larix and Pseudotsuga. Minimum number of nucleotide substitutions inferred from the alignment of the twelve full ITS region sequences of Larix and Pseudotsuga. Class size is 25 bp. SR = subrepeat.

For both genera, the deepest branch separated species according to continent. The Pseudotsuga species were divided into a North American and an Asian Glade, while the Larix species were divided into a North American and Eurasian Glade. Pseudotsuga sinensis and P. wilsoniana, the species from mainland China and Taiwan, were identical in sequence (although P. sinensis had one ambiguous site). The Eurasian species L. kaempferi and L. sibirica were the most closely related pair sampled in Larix, while L. griffithiana was the most divergent. Pseudotsuga macrocarpa and P. menziesii each had a high number of autapomorphies (unique nucleotide substitutions) relative to other taxon pairs. Maximum parsimony analysis of ITS 1 alone found a single most parsimonious tree identical in topology to that of the full data set, while analysis of the ITS2 alone failed to resolve the three species of Pseudotsuga from Asia or the three species of Larix from North America (results not shown). 60

L. kaempferi 64 L. sibirica 69 L. decidua

100 L. griffithiana

L. laricina

64 L. occidentalis

L. lyallii

P. wilsoniana

8 2 P. sinensis

100 P. japonica

P. menziesii 83 P. macrocarpa

P. pinea

FIG. 3.6. Strict consensus of two most parsimonious trees generated with a branch and bound search of the 5.8S and ITS2 only (112 steps, CIe = 0.967, RI = 0.988, and RC = 0.971). Pinus pinea is included as an outgroup. Bootstrap values (500 replicates) equal to or greater than 50% are displayed on the branches. 61

Several additional branch and bound analyses of the full ITS region were run in PAUP* in which the topology was constrained to conform with previously proposed phylogenetic hypotheses for Larix and Pseudotsuga (Table 3.1). Constraining P. macrocarpa to occupy a basal position in the Pseudotsuga Glade (tree 1 in Strauss et al., 1990) required 433 steps, six steps more than the most parsimonious tree. Enforcing the phylogenetic hypotheses proposed for Larix by either Schorn (1994) or LePage and Basinger (1991 and 1995) both required an additional 30 steps. Constraining L. sibirica to a position outside of L. decidua, L. kaempferi, and L. griffithiana required an additional 13 steps. Only a single additional step was required to force L. decidua and L. sibirica to be sister groups instead of L. kaempferi and L. sibirica. Restriction site analysis of PCR products allowed for the examination of four additional taxa whose ITS region sequences were not determined. The digests yielded a total of 27 scoreable sites, eleven HaeIII sites, eight Hhal sites, and eight Hinfl sites. Larix gmelinii had a restriction profiles identical to those of the other three Eurasian larches, L. decidua, L. kaempferi, and L. sibirica. No restriction sites were found to be shared by the Eurasian larches and absent in the North American larches, but all Eurasian larches lacked an HaeIII site in the ITS2 and two Hhal sites in ITS1. These sites were shared by the three North American larches. Larix griffithiana has a unique HaeIII site in ITS1 absent in all other Larix examined, and a unique 11 by insertion in ITS1 that gives it a larger band when digested with Hhal. Restriction digests of the Pseudotsuga species allowed for the separation of three groups: P. macrocarpa, P. menziesii, and the Asian species of Pseudotsuga. The endemic Mexican taxa sampled (both accessions of P. flahaultii, P. guinieri and P. rehderi) had a potential ITS2 length polymorphism visible as a double banding pattern in the fragment mapping to the ITS2 for all three restriction enzymes. The second band was ca. 5-10 by longer than the fragment present in the other species of Pseudotsuga. Otherwise, the Mexican taxa had restriction site profiles identical to P. menziesii. 62

kaempferi

00 L. sibirica Eurasia 78 L. decidua

L. griffithiana

laricina 95 North L. occidentalis America 98 L. lyallii

P. wilsoniana 100 East P. sinensis 100 Asia

100 P. japonica

P. menziesii North P. macrocarpa America 10

FIG. 3.7. Single most parsimonious tree from the full ITS region. Generated using a branch and bound search (427 steps, CIe = 0.9478, RI = 0.9835, and RC = 0.9397). Bootstrap values (500 replicates) are displayed on the branches. The scale bar represents the number of steps. 63

3.5. Discussion

Evaluating systematic and evolutionary hypotheses for Pseudotsuga and Larix--Although the sampling of Larix species in this study does not correspond completely with the cpDNA study of Qian et al. (1995), it corroborates their conclusion that previous systematic arrangements of Larix based on ovulate cone characters or fossil evidence (LePage and Basinger, 1991, 1995; Schorn, 1994) do not correspond with the evolutionary history of the group as inferred from two independent sources of molecular data (Table 3.2, FIG. 3.7). Instead, phylogenies constructed from both cpDNA and the ITS region suggest that if exserted bracts are a symplesiomorphic character of Larix and Pseudotsuga, then reduction in bract length occurred independently in the North American and Eurasian lineages of Larix. The phylogenetic position of L. sibirica as inferred from cpDNA restriction site analysis differs from its position inferred from ITS region sequences. Considering the morphological similarities, geographic proximity, and ITS region similarity among L. sibirica, L. decidua, L. gmelinii, and L. kaempferi, we suggest that the cpDNA placement of L. sibirica as a species removed from this seemingly natural group is inconsistent with the phylogeny of Larix. Furthermore, recent allozyme studies have documented a close genetic relationship between L. decidua and L. sibirica (Lewandowski, 1997). The unique cpDNA restriction sites scored for L. sibirica could be the result of a plastid rearrangement, hybridization and subsequent chloroplast capture (Rieseberg and Soltis, 1991, Liston and Kadereit, 1995), or an artifact of that study. In the ITS region study, the position of L. sibirica within the northern Eurasian Glade is more consistent with morphological and geographical evidence. However, its sister relationship with L. kaempferi, inferred using the ITS region, is only one step less parsimonious than a sister relationship with L. decidua. Considering the high ITS region and cpDNA sequence similarity of the north Eurasian species of Larix, as well as the evidence of ITS region polymorphism in the group, further investigation is necessary before strong conclusions can be made regarding the relationships among members of the northern Eurasian Glade. 64

The sampling in this study resulted in a phylogeny that first divides Larix into a North American and a Eurasian lineage. The North American lineage includes two long­ bracted species (L. occidentalis and L. and a short-bracted species (L. laricina). The confinement of all North American species to a single Glade eliminates the need to postulate a recent North Atlantic/Polar migration for the establishment of short-bracted species in both North America (L. laricina) and Eurasia (L. gmelinii and L. decidua) (Schorn, 1994) or allopatric speciation as a consequence of Pleistocene glaciation (LePage and Basinger, 1995). The Eurasian Glade is comprised of two lineages, one leading to the Himalayan species L. griffithiana, and the other to the more widely and northerly distributed L. decidua, L. sibirica, and L. kaempferi. Although L. gmelinii was not sequenced, its ITS region restriction site profiles (reported here), as well as cpDNA restriction site profile, cone anatomy, and geographical distribution, all support L. gmelinii as a member of the north Eurasian Glade. Two species of Larix were not sampled. Based on other studies (Qian et al., 1995), Larix potaninii seems best placed with the north Eurasian Glade, while L. mastersiana, a rare species of Larix reported from high altitudes in the West Sichuan Province of China, awaits inclusion in a molecular phylogenetic analysis. Strauss et al. (1990) addressed the systematics of Pseudotsuga using thirty parsimony informative characters from DNA restriction fragment analysis of nuclear, mitochondrial, and chloroplast-encoded genes. The study included five species representing all the main lineages of the genus, as well as L. occidentalis as an outgroup. Only six L. occidentalis character states were shared with a subset of the Pseudotsuga species (symplesiomorphic), and as a result, the authors were unable to discern between two equally most parsimonious roots connecting Pseudotsuga to Larix. One topology placed P. macrocarpa in the basal position of Pseudotsuga, with a paraphyletic grade branching to give P. menziesii and then a Glade that included P. japonica in a sister relationship between P. sinensis and P. wilsoniana (Table 3.1). In the second topology, P. macrocarpa and P. menziesii were sister species. The ITS region tree (FIG. 3.7) agrees in topology to the second tree found by Strauss et al., favoring the hypothesis that P. macrocarpa is a sister taxon to P. menziesii rather than the basal lineage of the genus. 65

Constraining the ITS region tree to agree with the paraphyletic tree from Strauss et al. requires an additional six steps. Based on the high number of autapomorphies in P. menziesii and P. macrocarpa relative to the branch lengths among P. japonica, P. sinensis, and P. wilsoniana, divergence of extant North American species preceded the divergence of extant Asian species. Based on the two alternate topologies found in their study, Strauss et al. (1990), hypothesized that the basal extant lineage of Pseudotsuga was North American, probably P. macrocarpa. The remaining species evolved in a pattern conforming to a stepping stone model of evolution. The ancestral form migrated north, probably giving rise to a P. menziesii lineage before extending its range across the Bering land bridge, giving rise to the lineage in Japan and then the taxa in China. This hypothesis was only supported by one of their most parsimonious trees and required an additional six steps in the ITS region tree. The alternate topology from the restriction fragment study, which agrees with the topology of the single most parsimonious ITS region tree (FIG. 3.7), does not allow a continent of origin to be inferred for the genus. This is because the most recent common ancestor of the extant species of Pseudotsuga is at a node that bifurcates to give rise to a North American and an Asian Glade. On this particular topology, a North American origin or an East Asian origin are equally parsimonious. Furthermore, although phylogenetic examination of extant taxa can potentially identify an extant basal lineage within a Glade, the present range of that lineage does not necessarily correspond to its centers of origin. While phylogenetic analysis alone cannot identify the centers of origin for a related group of organisms, combining knowledge of phylogeny with paleontological evidence can strengthen hypotheses about centers of origins. The concentration of Tertiary fossils near the present ranges of several extant species suggests a western North American/eastern Asian origin for Pseudotsuga and Larix (Schorn, 1994). Pseudotsuga has a deeper fossil record in North America than in East Asia. Fossil evidence of North American Pseudotsuga dates to the Early Oligocene for a macrocarpa-like form occupying the lowland Willamette Valley Oregon and west Central Oregon (ca. 32 Ma.) (Schorn, 1994). The fossil history of Pseudotsuga in Asia extends only to the Pliocene (Hermann, 1985). The oldest fossil evidence of Larix dates back to a long-bracted lyallii­ 66

like form from the Thunder Mountain and Coal Creek, Idaho floras (45 Ma.) (Axelrod, 1990; Schorn, 1994), and a short-bracted form from the Canadian High Arctic found in sediments dating to the Middle to Late Eocene (LePage and Basinger, 1991). Fossil vegetation and cones dating to the Oligocene have also been collected from sites in Russia (LePage and Basinger 1995). While the observed high number of unique derived ITS region nucleotide substitutions in the North American species of Pseudotsuga could be cited as support for the hypothesis that the extant North American species of Pseudotsuga are older than the extant East Asian species, it is possible that there were older lineages from Asia that went extinct and whose fossil records remain undiscovered. Including multiple outgroup taxa has been shown to improve the polarization of ingroup character states (Smith, 1994). The single most parsimonious root between the North American and Asian representatives of Pseudotsuga in this study resulted from inclusion of a greater number of informative characters together with a greater number of Larix species than in the study of Strauss et al. (1990). Midpoint rooting between Pseudotsuga and Larix also allowed the inference of a basal North American Eurasian dichotomy in Larix. An additional analysis that included a Pinus pinea partial sequence (5.8S and ITS2) permitted the construction of the hypothetical common ancestor for Pseudotsuga and Larix and resulted in a topology that is consistent with the hypothesized monophyly of both genera (FIG. 3.6).

Use of the ITS region in systematic studies of PinaceaeNuclear ribosomal DNA occurs in tandem repeats at one to several chromosomal locations. The presence of nrDNA in tandem repeats comprised of hundreds to thousands of copies (paralogues) has facilitated its study by making it easier to amplify using PCR. The use of nrDNA for constructing phylogenetic estimates of organisms is possible because concerted evolution homogenizes the paralogues within populations. In most systematic studies using the ITS region, sequences determined directly from PCR products of paralogues are assumed to represent a homogenized functional unit. However, there is a growing awareness of ITS region polymorphism in plants. Buckler, Ippolito, and Holtsford (1997) review evidence for recombination among divergent paralogues and the presence of pseudogenes and 67

functional ITS regions in several studies. These authors have also demonstrated that including DMSO in PCR reactions as a denaturing agent increased the proportion of putatively functional paralogues amplified relative to nonfunctional ones (Buckler and Holtsford, 1996. Restriction site mapping and Southern hybridization have been used to document nrDNA variability in Picea and Films (Karvonen and Savolainen, 1993; Karvonen, Szmidt, and Savolainen, 1994). Multiple IGS (intergenic spacer) and ITS types were found among and sometimes within individuals of Pinus sylvestris from populations in Finland (Karvonen and Savolainen, 1993) and within individuals of Pinus rzedowskii from populations in Mexico (Quijada et al., 1998). The Mendelian inheritance pattern of one ITS region size variant of P. sylvestris was consistent with the hypothesis that rare variants are found at a single locus and that concerted evolution is occurring more rapidly within chromosomes than among sister chromatids or nonhomologous chromosomes. Restriction mapping and DNA sequencing of direct and cloned PCR products was used to verify ITS region polymorphism in two species of Larix and two species of Pseudotsuga. Length variations and substitutions among ITS region types within an individual were observed in direct sequence reads of PCR products. Analysis of clones showed that several of the divergent ITS types had RFLP fragment sizes that were not visible in digests of the original PCR product. Comparison of sequences from the direct PCR product to those of the clones also revealed that some discrepancies in the clones could be traced back to weak, subordinate peaks in the sequence reads from the original PCR product. These observations suggest that even when using DMSO in PCR reactions, divergent ITS region paralogues present in very low abundance relative to a dominant paralogue are preferentially cloned. Although the polymorphism was interpreted as being present at low levels, it is probably present to a varying extent in all species of Larix and Pseudotsuga. In angiosperms, ITS region polymorphism has been attributed to hybridization, polyploidy, slow rates of concerted evolution across nrDNA loci on nonhomologous chromosomes, and agamospermy (see Campbell et al., 1997; Buckler et al., 1997). The polymorphism in Pinaceae may be a result of a slow rate of concerted evolution among nrDNA loci. Species in Pinaceae, which typically have a diploid chromosome number of 68

2n = 24, have more nrDNA loci than angiosperms. The number of chromosomes pairs with 18s-5.8S-26s loci has been shown to range from 5-8 in Pinus and Picea (Doudrick et al., 1995; Brown and Carlson, 1997). Recent studies on nrDNA organization of Pseudotsuga found 18S-5.8S-26s loci on three or four chromosome pairs of Pseudotsuga (J. E. Carlson, Department of Forest Science and Biotechnology Laboratory, University of British Columbia, pers. comm.). ITS region lengths are atypically long throughout conifers (Liston et al., 1996). The number of nrDNA loci and extent of ITS region polymorphism in other families of conifers remains to be explored. Because of the great effort it would have required to sequence several full ITS region paralogues for one or more species of Larix or Pseudotsuga, the nature, the distribution, and the extent of the polymorphism were not examined thoroughly. Most importantly from a phylogenetic perspective, strict orthology of the sequences used in the cladistic analyses could not be demonstrated, and synapomorphic character states could not be distinguished from biased detection of ancestral polymorphism. Despite these limitations, the result of the phylogenetic analysis was a single most parsimonious tree with high bootstrap values (FIG. 3.7). Furthermore, the topology was mostly congruent with other molecular studies performed on the genera. We attribute this success to the long length of the ITS region in Larix and Pseudotsuga, the accompanying large number of informative sites available, and the small percentage of ambiguous sites detected. The presence of polymorphism should most seriously affect the placement of closely related species with low sequence divergence, and this may have occurred in the northern Eurasian Larix Glade. This study documents the presence of two subrepeats in the ITS1 of Larix and three in Pseudotsuga. In both genera, the subrepeats consistently form a terminal stem and loop in secondary folding models (FIG. 3.3). In Pseudotsuga, the second and third subrepeats are in tandem and they fold together. Subrepeats have also been found in the ITS1 for two other members of Pinaceae, P. pinea (Marrocco et al.,1996), and species of Tsuga (Vining and Campbell, 1997). Parasitic flukes also have ITS1 subrepeats (Luton, Walker, and Blair, 1992; Kane and Rollinson, 1994). As in Pinaceae, the number of subrepeats varies among taxa in the fluke genus Schistosoma (Kane and Rollinson, 1994). 69

The length of the Schistosoma subrepeats ranges from 72-80 bp, which is comparable to the 69-74 by range in Pseudotsuga and Larix. Although the function of the subrepeats in Pinaceae is unknown, their conserved nature in Pinaceae and robust behavior in secondary folding models suggests that they may be involved in rRNA processing. No conclusive homologous matches were detected using BLASTn or gapped BLAST, except to P. pinea. With the exception of a low scoring match to Homo sapiens ITS2, the assemblage of sequences retrieved using BLAST and gapped BLAST did not conform to any obvious phylogenetic or functional pattern, and the small size of the conserved region made it difficult to determine whether several low-scoring BLAST hits were homologous or coincidental. If subrepeats in the ITS1 of Pinaceae are evolving in concert, they could violate assumptions of character independence. We observed that within the recently derived sister genera, Pseudotsuga and Larix, there is no evidence of concerted evolution homogenizing repeats within a species. The phylogenetic pattern of P. pinea subrepeats relative to those in Larix and Pseudotsuga is consistent with a process of concerted evolution that is too slow to be detected in recently diverged lineages, but becomes apparent over longer periods of time. This study demonstrates the utility of nucleotide sequence data from the ITS region for reconstructing a phylogeny for two genera in Pinaceae. Using the full region made it possible to identify a highly consistent, completely resolved tree. The ITS2 alone, though easy to align and consistent with the full ITS region phylogeny, was inadequate for resolving relationships among closely related species of Pseudotsuga and Larix. The ITS1 in these genera is longer, and the number of substitutions per site within a genus is roughly twice that for the ITS2. These observations are consistent with recent analyses demonstrating the potential for obtaining deep level phylogenetic signal from the ITS2 of plants (Hershkovitz and Lewis, 1996). If rates of substitution are comparable across other genera of Pinaceae the ITS region could be expected to resolve generic and infrageneric relationships, but alignment difficulties may diminish the utility of the ITS1 for a family- wide phylogeny. 70

3.7. Acknowledgements

The authors thank John Carlson, Aljos Farjon, Ben LePage, James Oliphant, Jeffi-ey Stone, and Don Zobel for their thoughtful comments and suggestions and William Robinson for his technical assistance in the laboratory. Dave Swofford kindly provided access to test versions of PAUP* for UNIX and permission to publish results. All sequencing was performed by the Central Services Laboratory within the Center for Gene Research and Biotechnology at Oregon State University. We are indebted to Martin Gardner, James Oliphant, Miguel Soto, Yoshihiko Tsumura, and Forestfarm Nursery for providing specimens. DSG would like to especially thank Frances Cavoretto, David Miller Sr., and David Miller Jr. for making this project possible. The work was completed with the help of a Sigma-Xi Research Grant.

3.8. Literature Cited

AAGAARD, J. E., S. S. VOLLMER, F. C. SORENSEN, AND S. H. STRAUSS. 1995. Mitochondrial DNA products among RAPD profiles are frequent and strongly differentiated between races of Douglas-fir. Molecular Ecology 4: 441-447.

ALTSCHUL, S. F., W. GISH, W. MILLER, E. W. MYERS, AND D. J. LIPMAN. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.

, T. L. MADDEN, A. A. SCHAFFER, J. ZHANG, Z. ZHANG, W. MILLER, AND D. J. LIPMAN. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.

AXELROD, D. I. 1990. Environment of the Middle Eocene (45 Ma) Thunder Mountain flora, central Idaho. National Geographic Research 6: 355-361.

BALDWIN, B. G., M. J. SANDERSON, J. M. PORTER, M. F. WOJCIECHOWSKI, C. S. CAMPBELL, AND M. J. DONOGHUE. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247-277. 72

FARJON, A. 1990. Pinaceae: drawings and descriptions of the genera Abies, Cedrus, PseudoLarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Koeltz Scientific Books, Konigstein, Federal Republic of Germany.

HART, J. A. 1987. A cladistic analysis of conifers: preliminary results. Journal of the Arnold Arboretum 68: 269-307.

HERMANN, R. K. 1982. The genus Pseudotsuga: historical records and nomenclature. Forest Research Laboratory, Oregon State University, Corvallis, OR, Special Publication 2a.

. 1985. The genus Pseudotsuga: ancestral history and past distribution. Forest Research Laboratory, Oregon State University, Corvallis, OR, Special Publication 2b.

HERSHKOVITZ, M. A., AND L. A. LEWIS. 1996. Deep-level diagnostic value of the rDNA-ITS region. Molecular Biology and Evolution 13: 1276-1295.

JAEGER, J. A., TURNER, D. H., AND M. ZUKER. 1990. Predicting optimal and suboptimal structures for RNA. Methods in Enzymology 183: 281-306.

KANE, R. A., AND D. ROLLINSON. 1994. Repetitive sequences in the ribosomal DNA internal transcribed spacer of Schistosoma haematobium, Schistosoma intercalatum, and Schistosoma mattheei. Molecular Biochemical Parasitology 63: 153-156.

KARVONEN, P. AND 0. SAVOLAINEN. 1993. Variation and inheritance of ribosomal DNA in Pinus sylvestris L. (Scots pine). Heredity 71: 614-622.

, A. E. SZMIDT, AND 0. SAVOLAINEN. 1994. Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species. Theoretical and Applied Genetics 89: 969-974.

LEPAGE, B. A., AND J. F. BASINGER. 1991. A new species of Larix (Pinaceae) from the early Tertiary of Axel Heiberg Island, Arctic Canada. Review of Palaeobotany and Palynology 70: 89-111.

AND 1995. The evolutionary history of the genus Larix (Pinaceae). In W. C. Schmidt and K. J. McDonald, [compilers], Ecology and management of Larix forests: a look ahead, pp. 19-29. United States Department of Agriculture, Forest Service, Intermountain Research Station, Ogden, UT, General Technical Report GTR-INT-319. 73

LEWANDOWSKI, A. 1997. Genetic relationships between European and Siberian larch, Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species? Plant Systematics and Evolution 204: 65-73.

LI, P., AND W. T. ADAMS. 1989. Range-wide patterns of allozyme variation in Douglas-fir (Pseudotsuga menziesii). Canadian Journal of Forest Research 19: 149-161.

LISTON, A. AND J. W. KADEREIT. 1995. Chloroplast DNA evidence for introgression and long distance dispersal in the desert annual Senecio flavus (Asteraceae). Plant Systematics and Evolution 197: 33-41.

, W. A. ROBINSON, J. M. OLIPHANT, AND E. R. ALVAREZ-BUYLLA. 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering seed plants. Systematic Botany 21: 109-120.

LIU, J.-S., AND C. L. SCHARDL. 1994. A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Molecular Biology 26: 775-778.

LUTON, K., D. WALKER, AND D. BLAIR. 1992. Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes: Trematoda: Digenea). Molecular Biochemical Parasitology. 56: 323-328.

MARROCCO, R., M. T. GELATI, AND F. MAGGINI. 1996. Nucleotide sequence of the internal transcribed spacers and 5.8S region of ribosomal DNA in Pinus pinea L. DNA Sequence 6: 175-177.

MARTINEZ, M. 1949. Las Pseudotsugas de Mexico. Anales del Instituto de Biologia 20: 129-184.

MILLER, C. N. 1977. Mesozoic conifers. Botanical Review 43: 217-280.

MORAN, G. F., AND W. T. ADAMS. 1989. Microgeographical patterns of allozyme differentiation in Douglas-fir from Southwest Oregon. Forest Science 35: 3-15.

NICKRENT, D. L., K. P. SCHUETTE, AND E. M. STARR. 1994. A molecular phylogeny of Arceuthobium (Viscaceae) based on nuclear ribosomal DNA internal transcribed spacer sequences. American Journal of Botany 81: 1149-1160.

PATSCHKE, W. 1913. Ober die extra tropischen ostasiatischen Coniferun und ihre Bedeutung fur die pflanzengeographische Gliederung Ostasiens. Botanische Jahrbucher fiir Systematik, Pflanzengeschichte und Pflanzengeographie. 48: 626­ 776. 74

PONOY, B., Y.-P. HONG, J. WOODS, AND J. E. CARLSON. 1994. Chloroplast DNA diversity of Douglas-fir in British Columbia. Canadian Journal of Forest Research 24: 1824-1834.

PRAGER, E. M., D. P. FOWLER, AND A. C. WILSON. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

PRICE, R. A., J. OLSEN-STOJKOVICH, AND J. M. LOWENSTEIN. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Systematic Botany 12: 91-97.

QIAN, T., R. A. ENNOS, AND T. HELGASON. 1995. Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA. Canadian Journal of Forest Research 25: 1197-1202.

QUIJADA, A., A. LISTON, P. DELGADO, A. VAZQUEZ-LOBO, AND E. R. ALVAREZ-BUYLLA. 1998. Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP. Theoretical and Applied Genetics 96: in press.

RIESEBERG, L. H., AND D. E. SOLTIS. 1991. Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants 5: 65-84.

SCHNABEL, A., J. L. HAMRICK, AND P. V. WELLS. 1993. Influence of Quaternary history on the population genetic structure of Douglas-fir (Pseudotsuga menziesii) in the Great Basin. Canadian Journal of Forest Research 23: 1900-1906.

SCHORN, H. E. 1994. A preliminary discussion of fossil larches, (Larix, Pinaceae) from the Arctic. Quaternary International 22/23: 173-183.

SMITH, A. B. 1994. Rooting molecular trees: problems and strategies. Biological Journal of the Linnean Society 51: 279-292.

SMITH, S. W., R. OVERBEEK, C. R. WOESE, W. GILBERT, AND P. M. GILLEVET. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

STRAUSS, S. H., A. H. DOERKSEN, AND J. R. BYRNE. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canadian Journal of Botany 68: 1502-1510.

SWOFFORD, D. L. 1998. PAUP* version 4.0. Sinauer Assoc., Inc., Sunderland, MA (in press). 75

TAKASO, T. 1996. Pollination mechanisms in conifers. Acta Phytotaxonomica et Geobotanica 47: 253-269.

TSUMURA, Y., K. YOSHIMURA, N. TOMARU, AND K. OHBA. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theoretical and Applied Genetics 91: 1222-1236.

VAN NUESS, R. W., J. M. J. RIENTJES, C. A. F. M. VAN DER SANDE, S. F. ZERP, C. SLUITER, J. VENEMA, R. J. PLANTA, AND H. A. RAUE. 1994. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Research 22: 912-919.

VINING, T. F., AND C. S. CAMPBELL. 1997. Phylogenetic signal in sequence repeats within nuclear ribosomal DNA internal transcribed spacer 1 in Tsuga. American Journal of Botany 84: 702 (Abstract).

WHITE, T. J., T. BRUNS, S. LEE, AND J. TAYLOR. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White [eds.], PCR Protocols, a guide to methods and applications, chapt. 38, 315-322. Academic Press, San Diego, CA.

WISCONSIN PACKAGE VERSION 9.0. 1996. Genetics Computer Group (GCG), Madison, WI.

XIAO-QUAN, W., H. YING, D. ZHENG-RONG, AND H. DE-YUAN. 1997. Phylogeny of the Pinaceae evidenced by molecular biology. Acta Phytotaxonomica Sinica 35: 97-106.

YEH, L.-C. C., R. THWEATT, AND J. C. LEE. 1990. Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common motifs. Biochemistry 29: 5911-5918. 76

Chapter 4 A Phylogenetic Reconstruction of Inoperculate Discomycetes Inferred from Partial Small Subunit Sequences of Nuclear Ribosomal DNA

David S. Gernandt Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331-2902

4.1. Abstract

Partial sequences from the nuclear encoded small subunit of ribosomal DNA were obtained from collections, cultures, or from Genbank for 11 families of inoperculate discomycetes, and representative species from other ascomycete orders. The discomycete orders Caliciales, Lecanorales, Leotiales, Peltigerales, Pezizales, and Rhytismatales were represented by multiple genera. Distance and parsimony analyses indicate that representatives from eight families of inoperculate discomycetes occur in either a monophyletic or paraphyletic lineage. These include Cyttariaceae, Hemiphacidiaceae, Hymenoscyphaceae, Leotiaceae, Loramycetaceae, Phacidiaceae, Rhytismataceae, and Sclerotiniaceae. Thelebolaceae, usually classified in the operculate order, Pezizales, also occurs in this Glade. Representative species from Orbiliaceae are placed closer to Pezizales than Rhytismatales and Leotiales. Lichenization and plant parasitic lifestyles predominate in derived clades. Several genera historically having problematic ordinal or familial placement from a morphological perspective and from developmental features remain equivocal in the small subunit analysis. These genera include Cudonia, Cyclaneusma, Darkera, Gremmeniella, Mitrula, Naemacyclus, Phacidium, Piceomphale, and Spathularia. The relative positions of Hemiphacidiaceae, Sclerotiniaceae, and Rhytismataceae clades among the inoperculate discomycetes, and the delimitation between Leotiales and Rhytismatales are discussed. 77

4.2. Introduction

The current lack of consensus in higher order classificaton of ascomycetes appears to stem from their rapid radiation along with the paucity of reliable developmental or morphological features in many groups. The result is that the taxonomic categories of class, subclass, and sometimes order, are ambiguous, often failing to have predictive value with respect to systematic relationships. Early classifications based on the arrangement of ascogenous structures inspired the categories of Hemiascomycetes, Plectomycetes, Pyrenomycetes, Discomycetes, Laboulbeniomycetes, and Loculoascomycetes (see Hawksworth et al., 1995 for a recent review). Contemporary views of systematic relationships are being refined by results from studies on developmental characteristics, modes of ascospore discharge, and phylogenetic analysis of DNA sequences. A recent development has been the abandonment of the use of formal ranks between order and phylum and the relegation of these class and subclass level categories to descriptive names of ascomatal types by some investigators (Eriksson and Hawksworth, 1993; Hawksworth et al., 1995). This abandonment of class and subclass categories was based on the grounds that current delineation of higher level categories is poorly understood and should await further investigation. Subsequent molecular systematic treatments have upheld classes (sometimes in a more restricted sense) for Hemiascomycetes, Archiascomycetes, Plectomycetes and Pyrenomycetes (Berbee and Taylor, 1992, Spatafora and Blackwell, 1993; 1994; Ogawa et al., 1997). Discomycetes have been treated by Boudier (1885; 1907), Nannfeldt (1932), Seaver (1951) and Dennis (1978). Today, the term discomycete is commonly used to describe unitunicate ascomycetes with open, saucer- or cup- shaped apothecia exhibiting ascohymenial development (Hawksworth et al., 1995). Several orders of lichenized apothecial ascomycetes do not fit into this definition. Patellariales and some members of Dothideales possess apothecial ascocarps, but exhibit ascolocular development and have bitunicate asci that split at discharge (fissituicate). Other lichenized apothecial orders with 78

bitunicate asci include Arthoniales, Lecanorales, and Pertusariales. Apothecial ascomycetes lacking two separable ascus wall layers include the orders Caliciales, Cyttariales, Gyalectales, Peltigerales, and Teloschistales, which are comprised predominately of lichenized members. Apothecial ascomycetes with a large number of nonlichenized members include Pezizales, all of which discharge ascospores through an operculum, and Leotiales (which includes two lichenized families, Baeomycetaceae and Icmadophilaceae), Neolectales, Ostropales and Rhytismatales, with inoperculate asci. DNA sequence data have been of particular importance in studying groups such as fungi in which morphological features are simple and subject to evolutionary convergence, and where sexual states may be lacking. Recent systematic treatments of apothecial ascomycetes have relied on sequence data generated from the highly conserved small subunit of nuclear ribosomal DNA (nrDNA). The slow rate of sequence evolution of the small subunit has resulted in its widespread use for phylogenetic studies at and above the family level in fungi (reviewed in Bruns et al., 1991; Hibbett, 1992). Small subunit studies of lichenized orders have identified a monophyletic group comprised of members from Caliciaceae, Lecanorales, and Peltigerales (Gargas and Taylor, 1995; Gargas et al., 1995; Wedin and Tibell, 1997). Caliciales s.l. has been shown to be polyphyletic in these analyses. Curiously, analysis of nrDNA places species of Neolecta, apothecial fungi that lack paraphyses, in a basal position in the Archiascomycetes, a group of fungi that otherwise do not form an ascocarp (Landvik et. al., 1993; Landvik, 1996). LoBuglio et al. (1996) and Landvik et al. (1996) demonstrated that Elaphomycetales are allied with the cleistothecial ascomycete orders Eurotiales and Onygenales. Based on a weakly supported sister relationship between Sclerotinia sclerotiorum and Blumeria graminis, the cleistothecial order Erysiphales has also been reported as a possible member of the discomycetes (Saenz et al., 1994). The placement of Erysiphales with the cleistothecial ascomycetes is controversial because members of this order produce asci from a basal hymenium and forcibly discharge their ascospores, both characters of Pyrenomycetes. O'Donnell et al. (1997) and Landvik et al. (1997) have used nrDNA to study relationships within Pezizales, but since it was not their objective to test the monophyly or the placement of the group, few other ascomycetes were represented in those studies. The 79

discomycetes have occupied a basal position among the ascomycetes in several molecular systematic treatments of the ascomycetes (Gargas et al., 1995; Gargas and Taylor, 1995; Wedin and Tibell, 1997; but see Ogawa et al., 1997 for an alternative placement using neighbor joining analysis). The basal position of the discomycetes relative to the pyrenomycetes and cleistothecial ascomycetes in nrDNA analyses has been cited as supporting the hypothesis of Nannfeldt (1932) that the perithecium and cleistothecium were derived from enclosure of the apothecium (Gargas and Taylor, 1992). To date, published molecular systematic treatments of the ascomycetes as a whole have sampled very lightly from among the inoperculate discomycetes, particularly in the orders Rhytismatales and Leotiales. This state of affairs is unfortunate considering the uncertainty regarding the relationships among the members of these two orders, as well as their position among the euascomycetes. This study reconstructs a phylogeny of the discomycetes using sequence data from approximately 1000 by of the nuclear-encoded small subunit ribosomal DNA. Our aims are to evaluate 1) the monophyly of inoperculate discomycetes, 2) the phylogenetic position of inoperculate discomycetes within the ascomycetes, and 3) the ordinal and familial relationships of the group. To this end, we have included partial small subunit sequences from 66 discomycete taxa representing 33 families in 11 orders (Table 3.1). Of the 100 sequences used in this study 36 are from taxa classified in the inoperculate orders Leotiales or Rhytismatales.

4.3. Materials and Methods

Sequence determination For the discomycete sequences determined for this study, DNA was extracted from either pure fungal cultures, apothecia, lichen thalli or host tissue bearing diagnostic fructifications using a variety of procedures described elsewhere (Gernandt et al., 1997; Hamelin and Rail, 1996; Holst-Jensen et al., 1997; Platt and Spatafora, in prep). A single, uniform procedure was not used for PCR amplification. In general, primers used for amplification of the small subunit were either NS1, NS17, or NS3 and NS4, NS17, or NS24 (White et al., 1990). In addition, NS2, SR7R, and SR11R 80

Table 4.1. Taxa included in the small subunit analyses. Family placements are given for the apothecial ascomycetes.

Classification Taxon Gen Bank no. Apothecial ascomata Caliciales Caliciaceae Cyphelium inquinans (Sm.) Trevis. U86695 Texosporium sancti-jacobi (Tuck.) Nadv. U86696 Thelomma mammosum (Hepp) A. Mass. U86697 Mycocaliciaceae Chaenothecopsis savonica (Ras.) Tibell U86691 Mycocalicium albonigrum (Nylander) Tibell L37538 Stenocyhe pullatula (Ach.) Stein. U86692 Sphinctrinaceae Sphinctrina turbinata (Pers.) de Not. U86693 Cyttariales Cyttariaceae Cyttaria darwinii Berk. em Espinosa U53369 Elaphomycetales Elaphomycetaceae Elaphomyces maculans U45440 Lecanorales Cladoniaceae Cladia aggregata U72713 Porpidiaceae Porpidia crustulata (Ach.) Hertel & Knoph L37540 Lecanoraceae Lecanora dispera Pers. ex Sommerfelt L37535 Sphaerophoraceae Sphaerophorus globosus (Hudson) Vainio L37532 Stereocaulaceae Pilophorus acicularis (Ach.) Th. Fr. U70960 Stereocaulon ramulosum U70961 inc. sedis Siphula ceratites U72712 Leotiales Geoglossaceae Microglossum viride (Pers. ex Fr.) Gill. U46031 Hemiphacidiaceae Fabrella tsugae (Farl.) Kirsch (F256) Hemiphacidium longisporum Ziller & Funk Meria laricis Vuill. (CBS 298.52) Rhabdocline parkeri Sherwood-Pike, Stone, & Carroll Sarcotrochila macrospora Ziller & Funk Hymenoscyphaceae Hymenoscyphus ,fructigenus (Bull ex. Merat) S. F. Gray U67430 Leotiaceae Chloroscypha chloromela (W. Phillips & Harkn.) Seaver Chloroscypha enterochroma (Peck) Petrini Gremmeniella ahietina (Lagerberg) Morelet var. abietina Leotia lubrica Pers. ex Fr. Neobulgaria premnofila U45445 81

Table 4.1. (continued)

Classification Taxon GenBank no. Loramycetaceae Loramyces juncicola W. Weston Orbiliaceae Orbilia auricolor (Bloxam ex Berk.) Sacc. Orbilia delicatula Phacidiaceae Phacidium coniferarum (Hahn) DiCosmo, Nag Raj, & Kendrick Phacidium infestans Karst.( P431) Rutstroemiaceae Rutstroemia firma (Pers.) P. Karst Z81395 Sclerotinia homoeocarpa F.T. Bennett Z81397 Sclerotiniaceae Ciborinia foliicola (E.K. Cash & R. W. Davidson) L. R. Batra Z81376 Mitrula elegans (Berk.) Fr. U92310 Monilinia laxa (Aderh. & Ruhl.) Honey Y14210 Monilinia oxycocci (Woronin) Honey Z73727 Sclerotinia sclerotiorum (Lib.) de Bary X69850 incertae sedis Cudonia confusa Bres. Z30240 Piceomphale bulgarioides (Rabenh.) Svrcek Spathularia flavida Pers. ex Fr. Z30239 Neolectales Neolectaceae Neolecta vitellina (Bresadola) Korf & J. K. Rogers Z27293 Ostropales Stictidaceae Stictis radiata Pers. U20610 Patellariales Patellariaceae Rhytidhysteron rufulum (Spreng.: Fr.) U20506 Petrak Peltigerales Peltigeraceae Peltigera neopolyclactyla (Gyelnik) Gyelnik X89218 Solorina crocea (L.) Crocea X89220

Pezizales Ascobolaceae Ascobolus lineolatus Brumm. L37533 Helvellaceae Gyromytra montana Harmaja U42652 Morchellaceae Morchella esculenta Pers. ex St. Amans L36998 Verpa conica Swartz ex Pers. U42644 Otideaceae Otidia leporina (Batch) Fuckel U53381 Trichophaeopsis bicuspis (Boud.) Korf & Erb. U53391 Pezizaceae lodaphanus carneus (Pers.) Korf U53380 Peziza vesiculosa Bull. U53384 Sarcosomataceae Sarcosoma globosum (Schmidel) Rehm. U53386 82

Table 4.1. (continued)

Classification Taxon GenBank no. Thelebolaceae Ascozonus woolhopensis (Berk. & Br.) Boud. AF010590 Thelebolus crustaceus Tode U53394 Tuberaceae Tuber rufus Rhytismatales Rhytismataceae Colpoma quercinum (Pers.ex St.-Am.) Wallr. (C313) Cyclaneusma minus (Butin) DiCosmo, Peredo, Minter (93197) Elytroderma deformans (Weir) Darker Lirula macrospora (R. Hartig) Darker Lophodermium pinastri (Schrad. ex. Fr.) Chev. (L230) Meloderma desmazierii (Duby) Darker Naemacyclus .fimbriatus (Schwein.) DiCosmo, Peredo & Minter Rhytisma salicinum (Pers.:Fr.) Fr. U53370 Tryblidiopsis pinastri (Pers.: Fr.) P. Karst. (CBS 234.83)

incertae sedis Darkera parca (Berk. & Broome) H. S. Whitney, J. Reid & Pirozynski

Cleistothecial ascomata Erysiphales Blumeria graminis (de Candolle) Speer f. sp. hordei L26253 Eurotiales Aspergillus fiimigatus Fresenius Eremascus albus Eidam M83258 Eurotium rubrum Konig, Spieckermann & Bremer U00970 Monascus purpureus Went M83260 Onygenales Ajellomyces capsulatus (Kwon-Chung) McGinnis & Katz X58572 Coccidioides iminitis Rixford et Gilchrist Perithecial ascomata Diaporthales Diaporthe phaseolorum (Cook & Ellis) Sacc. L36985 Hypocreales Balansia sckrotica (Pat.) Han. U32399 Claviceps paspali F. Stevens & J. G. Hall U32401 Hypocrea lutea (Tode) Petch D14407 Nectria cinnabarina (Tode: Fr.) Fr. U32412 83

Table 4.1. (continued)

Classification Taxon GenBank no. Microascales Microascus trigonosporus C. W. Emmons & B. 0. Dodge L36987 Ceratocystis fimbriata Ellis & Halst. U32418 Phyllacorales Colletotrichum gloeosporioides Penzig M55640 Ophiostomatales Ophiostoma ulmi (Buisman) Nannf M83261

Sordariales Chaetomium elatum Kunze & Schmidt: Fr. M83257 Neurospora crassa Shear & Dodge X04971 Xylariales Daldinia concentrica (Bull.:Fr.) Ces. & de Not. U32402 Xylaria carpophila (Pers.: Fr.) Fr. Z49785 Pseudothecial ascomata Chaetothyriales Capronia pilosella U42473 (Karsten) Muller, Petrini Fisher, Samuels & Rossman Coniosporium perforans Y11714 Dothideales Botryosphaeria rhodina (Berk. & Curtis) von Arx U42476 Dothidea hippophaeos U42475 Phaeosclera dematioides Y11716 Pleosporales Pleospora rudis Berl. U00975 Lophiostoma crenatum Fr. U42485 conidial fungi of unknown placement Chalara fusidioides (Corda) Rabenh. Phialocephala .fortinii Wang & Wilcox L76626 ascomata lacking Saccharomycetales Candida albicans (Robin) Berkhout M60302 Dipodascopsis uninucleata (Biggs) L. R. Batra & Millner U00969 Kluyveromyces lactis (Drombrowski) van der Walt X51830 Pichia anomala (Hansen) Kurtman X58054 Saccharomyces cerevisiae Meyer ex Hansen V01335 Zygosaccharomyces rouxii (Boutroux) Yarrow X58057 Taphrinales Taphrina deformans (Berk.) Tul. U20376

Basidiomycetes Sporidiales Rhodosporidium toruloides Banno D12806 Stereales Athelia bombacina Pers. M55638 a CBS = Centraalbureau voor Schimmelcultures, Baarn, Netherlands 84

Table 4.2. Intron distribution for inoperculate discomycetes sequenced. Only introns at the 5' end of the small subunit for Leotiales and Rhytismatales sequenced for this study are included.

Taxon flanking seqs (position of insertion) length (bp)

Leotiales

Rutstroemia firma ACGGGGGATT/AGGGTTCTAT (369) 218

Chloroscypha seaveri CAAGTCTGGT/GCCAGCAGCC (563) 292

Hemiphacidium longisporum AAGTCTGGTG/CCAGCAGCCG (564) 386

Sarcotrochila macrospora AAGTCTGGTG/CAGCAGCCGC (564) 384

Rhytismatales

Colpoma quercinum ACGGGGGATT/AGGGTTCTAT (369) 217

AAGTCTGGTG/CCAGCAGCCG (564) 214

Elytroderma deformans GTAACGGGGT/ATTAGGGTAC (366) 194

AAGTCTGGTG/CCAGACTCTC (564) 198

Lirula macrospora AAGTCTGGTG/CCAGCAGCCG (564) 212

CGAAGACGAT/CAGATACGGT (998) 211

Meloderma desmazierei GTAACGGGGT/TTAGGGTACT (366) 197

AAGTCTGGTG/CCAGCAGCCG (564) 215

Tryblidiopsis pinastri AAGTCTGGTG/CCAGCAGCCG (564) 205

Cyclaneusma minus CGAAGACGAT/CAGATACCGT (998) 218

Naemacyclus fimbriatus AATGGCTCAT/TATATCAGTTA (101) 198

AAGTCTGGTG/CCAGCAGCCG (564) 264 85

(White et al., 1990; Spatafora et al., 1995; R. Vilgalys, unpublished) were used as internal sequencing primers. PCR products were amplified, purified, and sequenced according to procedures described in Ho lst-Jensen et al. (1997) or Spatafora et al. (1995). Sequence reads were determined in both the 5' to 3' and 3' to 5' direction, except that the presence of introns in some species (Table 3.2) prevented the use of one internal primer, resulting in 5-10 percent of these sequences lacking a complementary read.

Sampling--The fungi sampled are alphabetized by ordinal classification in Table 3.1. The classification system outlined by Hawskworth et al. (1995) is used except where otherwise specified. Eighty-two of the 103 taxa used here have been included in previous studies and were downloaded from Genbank. PCR products from five isolates representing as many genera of Sclerotiniaceae, some of which have been recently proposed for transfer to Rutstroemiaceae (Holst-Jensen et al., 1997), were provided by A. Hoist- Jensen so that sequence reads could be obtained for the 5' end. Gapped-BLAST (Altschul et al., 1997) was used to identify accessions with high sequence similarity to inoperculate discomycetes. Sequences having greater than 15% of sites ambiguous along the 1000 by included in this study were not used. To test the monophyly and to determine the placement of the apothecial ascomycetes, species representing 23 of the 45 ascomycete orders recognized by Eriksson and Hawksworth (1993) were sampled. Among the non­ apothecial ascomycetes, an attempt was made to select species that did not have problematic placements in prior analyses. An attempt was made to include at least two different families per order. This included 33 species from 16 orders of non-apothecial ascomycetes representing plectomycetes, pyrenomycetes, loculoascomycetes, hemiascomycetes, and archiascomycetes. Discomycetes sampled included 77 species from ten orders of apothecial ascomycetes: Caliciales, Cyttariales, Elaphomycetales, Lecanorales, Patellariales, Peltigerales Leotiales, Rhytismatales, Ostropales, and Pezizales. Taxa from the apothecial orders Gyalectales, Pertusariales, and Teloschistales were not available, and those sequences available from Arthoniales were not long enough for inclusion. A single exemplar (Blumeria graminis) was available from Erysiphales, which 86

grouped with Sclerotinia sclerotiorum (Leotiales) in a previous small subunit analysis (Saenz et al., 1994).

Sequence analysis -- Sequences were aligned manually using Genetic Data Environment version 2.2 (Smith et al., 1994). Sequence positions 75-1143 with respect to S. cerevisiae were included in the analysis. Introns (Gargas et al., 1995) were removed from the phylogenetic analyses, otherwise, all sequence positions were included. Sequences were analyzed using PAUP v. 4.0.0d63 for UNIX (Swofford, 1998). Heuristic searches were performed under the following conditions: gaps treated as missing characters, tree- bisection reconnection, and random sequence addition with five replicates. The maximum number of trees saved for each replicate varied among analyses. Bootstrap values were generated using 100 replicates, with the mulpars option deactivated. Because a transitional bias in was observed in the data set (see results), additional searches were performed in which transversions were weighted 1.6:1 over transitions. Weighting in this manner gives preference to phylogenetically informative transversions over transitions and is especially useful when unweighted parsimony gives very high numbers of conflicting equally most parsimonious trees (Swofford et al., 1996). Neighbor joining analyses were performed using the Kimura 2-parameter correction.

4.4. Results

Outgroup selection- -Smith (1994) demonstrated that it is more desirable to choose a single closely related sister group than a nested succession of taxa when choosing an outgroup. Previous studies have demonstrated that Saccharomycetales occupies a sister position to the euascomycetes (Berbee and Taylor, 1992a; Spatafora, 1995; Gargas et al., 1995; Ogawa et al., 1997). Placement of the root to the euascomycetes is problematic. Spatafora (1995) showed that several different rooting schemes are not significantly less parsimonious. In most analyses, the most parsimonious root is placed either 1) between the euascomycetes and a Glade that includes both hemiascomycetes and archiascomycetes 87

FIG 4.1. Parsimony analysis of small subunit nrDNA including non-euascomycetes. Majority-rule consensus of 1000 equally most parsimonious trees (364 parsimony informative characters, 2175 steps, CI = 0.3, RI = .5, and RC = 0.2). The search was stopped prematurely with the first 1000 trees recovered. Apothecial orders are indicated on the left (Ma = anamorphic, Cal = Caliciales, Cyt = Cyttariales, Ela = Elaphomycetales, Ery = Erysiphales, Lec = Lecanorales, Leo = Leotiales, Neo = Neolectales, Ost = Ostropales, Pat = Pattelariales, Pet = Peltigerales, Pez = Pezizales, Rhy = Rhytismatales). 88

.6Meloderma desmazierii Rhytisma salicinum Lophodermium pinastri FY Elytroderma deformans Tryblidiopsis pinastri 6Spathularia flavida Cudonia confusa IlLeo Colpoma quercinum Lirula macrospora ,Rh)/ Mitrula elegans Leo Siphula ceratites ELec Gremmeniella abietina Leo Naemacyclus fimbriatus L Cyclaneusma minus IRhY Thelebolus crustaceous ElPez L Ascozonus woolhopensis iRhabdocline parkeri Meria laricis Fabrella tsugae Hemiphacidium longisporum L Sarcotrochila macrospora Piceomphak bulgarioides oxycocci Leo Ciborinia foliicola - S'clerotinia sclerotiorum Monilinia laxa Sclerotinia homoeocarpa Darkera parca EiRhy Phacidium conijerarum IMILeo _Claviceps paspali Nectria cinnabarina Hypocrea lutea trigonosporus j-MicroascusCeratocystis fimbriata Colletotrichum gloeosporioides rNeurospora crassa Chaetomium datum Ophiostoma ulmi Diaporthe phaseolorum Xylaria carpophila L Daldinia concentrica Neobulgaria premnofila MILec Blumeria graminis Loramyces juncicola liaLeo Phialocephala fortinii Phacidium infestans Chloroscypha chloromela Chloroscypha seaveri Chalara fusidioides I eo Hymenoscyphus fructigenus Rutstroemia firma LLeotia lubrica Microglossum viride Cyttaria darwinii MCYt 89

FIG 4.1. (continued)

1Texosporium sancti-jacobi Thelomma mammosum Cal Cyphelium inquinans _r- Solorina crocea Pel L Peltigera neopolydactyla Porpidia crustulata ICIadia aggregata Pilophorus acicularis Lee S'tereocaulon ramulosum Lecanora dispersa L Stictis radiata 1110st Sphaerophorus glohosus Lec Sphinctrina turbinata Chaenothecopsis savonica Cal Mycocalicium albonigrum Aspergillus fumigates Eurotium rubrum Ajellomyces capulatus _ Coccidioides iminitis Elaphomyces maculans III Ela r Capronia pilosella L Coniosporium peyforans Ascobolus lineatus E3Pez Pleospora rudis Lophiosioma crenatum 6 Rhytidhysteron rufulum IMIPat Botryosphaeria rhodina r Phaeosclera dematioides L Dothidea hippophaeos fMorchella esculenta Verpa conica Gyromytra montana Tuber rufus Trichophaeopsis bicuspis Pez Otidia leporina Sarcosoma globosum i Peziza vesiculosa L lodaphanus carneus [Orbilia auricolor ,Leo Orhilia delicatula S'accharomyces cerevisiae ..6Zygosaccharomyces rouxii Kluyveromyces lactis Pichia anomala Candida albicans Dipodascopsis uninucleata Taphrina deformans Neolecta vitellina El Neo Athelia hombacina Rhodosporidium toruloides 90

(Berbee and Taylor, 1992a; Spatafora et al., 1995a), and 2) between archiascomycetes and a Glade that includes euascomycetes and hemiascomycetes (Gargas et al., 1995; Ogawa et al., 1997). Sampling of the three groups of ascomycetes varied greatly among these studies. Parsimony and distance analyses were performed under two different sampling schemes. The resulting trees were displayed with an outgroup designated. Analyses la (neighbor joining), lb (maximum parsimony), and lc (weighted parsimony) were performed not only to examine the position of all discomycete orders within the ascomycetes, but also to identify an outgroup suitably close in relationship to Leotiales and Rhytismatales. This more closely related outgroup was used in analysis 2. Analysis 1 used 103 taxa including two archiascomycetes (T. deformans and N vitellina) and two basidiomycetes (A. barnbacina and R. toruloides) and six genera from Sacharomycetales. The parsimony analyses indicated that Saccharomycetales was a sister group to the euascomycetes (FIG. 4.1), but the distance analysis placed the Saccharomycetales and Archiascomycetes as sister groups (FIG. 4.2). Pezizales and Orbiliaceae (Leotiales) were in a basal position within the euascomycetes. Pezizales was selected as the outgroup in analysis 2. All non-euascomycetes as well as Orbiliaceae were removed from the second analysis.

Supraordinal and ordinal relationships among the ascomycetes sampledIn the taxon sampling presented here, cleistothecial ascomycetes (s.s.), perithecial ascomycetes, hemiascomycetes, and archiascomycetes each formed monophyletic clades in all analyses in which they were included (FIGS. 4.1-4.3). These clades all received moderate to high bootstrap support. Bitunicate loculoascomycetes were paraphyletic in the majority-rule consensus trees from unweighted parsimony analysis 1 (FIG. 4.1), monophyletic in the neighbor joining tree in analysis 1 (FIG. 4.2), but polyphyletic in the strict consensus trees for weighted analysis 2 (FIG. 4.3). Loculoascomycetes sampled included representative members from Dothideales, Pleosporales, and Patellariales (an apothecial order of bitunicate ascomycetes). In analysis 2, species from Pleosporales grouped with Rhytidhysteron rufulum (Patellariales), separate from three genera from Dothideales. Bootstrap values were lower than 50% in all topologies. 91

Figure 4.2. Neighbor joining tree of small subunit nrDNA including non-euascomycetes (analysis 1). The Kimura 2-parameter correction was used. Transversions were weighted 1.6:1 over transitions. Bootstrap values equal to or greater than 50% are displayed above the branches. 92

100 , Cudonia cpnfusa L Spathulana flawda 60 Colpoma duercinum Lim' le madrospora LopiggidiogrstrirrsIntri Ilrodermrdeformans hyttsma salicinum Me °Warr:7a desmazied

100 1 Ft' habdocIthe parken 2 L Mena lancts Fabrella 100 liemiphaci aim Ion ;spot= Sardotroc spora Piceomphale bu panoi es Thelebolusrcrus aceous 4 L Ascozonus woofhopensis Gremmentella abtebna Leotiales, Rhytismatales, 98 r- Chlorescypha chloromela hlabscypha seaven Erysiphales, Cyttariales 00 Naemadyclus fimbriatus Cyclaneusma minus Lorarnyces tuncicola Phialocephela fort= Chalara fustdtoides Phacidium infestans 8498 Monilinja taxa Sclerobrlla sclerotiorum Cibonnta_foltipola 74 Monthrna oxycocci Sclerotirda homoeocarpe Microplossum Vinde 3 Leotia labnca Rutstroemta firma Darkera pared Phacidium cornferarum Hymenoscyphus fructigenus Blumena graMirns Cyttalia darwinii Clavipeps paspali 9i5 Nectna ophabanna Hypoc .ea lutea 99 9 icroascus tngonosporus eratocystis 17mbnata Colletotrichum ploposponordes Pyrenomycetes 100 83 Ophlostoma aim 65 DiapOrthe phaseolorum 100 Neurospora crassa elatum 57 94 Xylaria carpopglaaebmium Daldinta concentnca Neobulgaria premnofila Pleospora herbarum 4 1 crenatum Rhytidh steron rufulum Botryosp aena rhopfina Loculoascomycetes Phaeosclera demaboides Dothidea ea 98 I 9' i Op 0 S ocularis 62 Stereocau on ramuiosum I Lecanorales I Lecanora dispersa Snobs raaiata Ostrop ales 97 Texosporium sancti-jacobi 100 i neiomma mammosum Cyphefi um inqunans i Caliciales s.s. SphaerOphorus giabosus 1 Porp4a clustulata I Sphinctnna turbinata Lecanorales II Chaenothecopsis ,sa vonica Mycocaliciumcium arbonigrumru Mycocaliciaceae/Sphinctrinaceae 100 .Splonna crocea Pelbgera neopolydaclaty I Peltigerales Siphula ceratites .Morchella esculenta 57 Verpa conica Gyromitra montana Tuber tvfus 96 54 Trichophaeopsis bicuspis 5 Otidea leponna Sarcosoma _geloboSum Operculate discomycetes 99 P ziza vesiculosa 1 rodaphanus cameus Ascobolus lineatus 100 Aspergillys fumigatus rubrum 100 ep maculatus 94 worn co4 capsulatus Cleistothecial ascomycetes C cadiotdes /m/nibs 100 Ottilia.auncolor Orbtha delicatula I Orbiliaceae 100 aproniia pilosella I ( ConiosponuM pertorans Cleistothecial ascomycetes 77 S.accharomyces perevisiae 86 Kluyveromyces lactis 94 ZygpsacCharomyces roux!! 94 PI ichia anomala Hemiascomycetes 77 a/b/cans 56 ipodascopsts uninucleata 531 aphnna deformans Neolecra wtelina I Archiascomycetes 99 Athelia bombacina Basidiomycetes Rhodosporium toruloides 0.1 93

FIG. 4.3. Parsimony analysis of small subunit nrDNA excluding non-euascomycetes and Orbiliaceae. Weighted analysis outgroup rooted with Pezizales. Strict consensus of 360 equally most parsimonious trees (296 parsimony informative characters, 1982.8 steps, CIe = 0.347, RI = 0.584, and RC = 0.251). Transversions: transitions weighted 1.6:1. Bootstrap values are placed at nodes (mulpars was disabled). 94

24 Meloderma desmazierii L Rhytisma salicinum Lophodermium pinastri Elytroderma deformans Dyblidiopsis pinastri 48 Spathulariaflavida 41I L Cudonia confusa ILL Colpoma quercinum Lirula macrospora 921- Chloroscypha chloromela Chloroscypha seaveri Siphula ceratites Gremmeniella abietina 99r Naemacyclus fimbriatus 37 L Cyclaneusma minus r- Thelebolus crustaceous.

8-61- Ascozonus woolhopensis 100 Rhabdocline parkeri 63 Meria laricis 47 L Fabrella tsugae to Hemiphacidium longisporum Sarcotrochila macrospora Piceomphale hulgarioides 1 Monilinia oxycocci 80 Ciborinia foliicola laxa 76 Sclerotinia scleroliorum Sclerotinia homoeocarpa Rutstroemia firma 58 Claviceps paspali 94 L Hypocrea lutea Nectria cinnabarina 1 )0 965 Microascus trigonosporus 50 L Ceratocystis fimbriata Colletotrichum gloeosporioides 46 100 r Neurospora crassa Chaetomium elatum

100 79 Ophiostoma ulmi Diaporthe phaseolorum 27 91 r Xylaria carpophila L Daldinia concentrica Ncohulgaria premnofila Loramyces juncicola Chalarafusidioides Blumeria graminis 995 Pleospora rudis 83 -L Lophiostoma crenatum 30 Rhytidhysteron rufulum Bottyosphaeria rhodina Phialocephala fortinii Phacidium infestans rMicroglossum viride Leotia lubrica Hymenoscyphus fructigenus Darkera parca Phacidium coniferarum Cvttaria darwinii 95

FIG. 4.2 (continued)

8 Texosporium sancti-jacobi too Thelomma mammosum 20 L Cyphelium inquinans ElSolorina crocea Peltigera neopolydactyla 1 Porpidia crustulata Cladia aggregata 96 L Pilophorus acicularis Stereocaulon ramulosum Lecanora dispersa Sphaerophorus globosus oor Aspergillus fumigatus _ L Eurotium rubrum Elaphomyces maculans Ajellomyces capulatus 92[ Coccidioides iminitis pilosella )0[CaproniaConiosporium perforans Stictis radiata

3 Chaenothecopsis savonica 56 Mycocalicium albonigrum Sphinctrina turbinata

34 CPhaeosclera dematioides Dothidea hippophaeos Morchella esculenta 69 Verpa conica Gyromytra montana Trichophaeopsis bicuspis 80 96 C)tidia leporina Sarcosoma globosum 30 Tuber rufus loot Peziza vesiculosa lodaphanus carneus Ascobolus lineatus 96

16 14 12 g10 8 I 6 4 2 0 0 250 750 1000 Position transitions transversions both

FIG. 4.4. Nucleotide substitutions in the small subunit for 36 inoperculate discomycetes. Distribution of substitutions at 248 variable sites along positions 75 to 1143 species from Leotiales, Rhytismatales, and Thelebolaceae. A 25 by window was used. The minimum number of unambiguous transitions was 112 and the minimum number of unambiguous transversions was 69 (transition/transversion ratio = 1.62). A combination of transitions and transversions were observed at 67 additional sites.

There was no single most parsimonious solution to the branching relationships among the main ascomycete lineages. Bootstrap support at nodes uniting groups higher than class never exceeded 50% in the euascomycetes. The perithecial ascomycete Glade had bootstrap values in excess of 95% in all analyses, but its placement among the ascomycetes was unstable. The apothecial ascomycetes occurred in several different positions. In analysis la, two species of Orbilia (Orbiliaceae, Leotiales) occurred at the base of the euascomycetes, far separated from the main lineage of Leotiales. A monophyletic Glade of Pezizales included members of the families Helvellaceae, Morchellaceae, Otideaceae, Pezizaceae, 97

Sarcosomataceae, and Tuberaceae, but did not include representative genera from Ascobolaceae or Thelebolaceae. Ascobolus lineolatus was placed at the base of the cleistothecial ascomycetes in analysis la, but was at the base of Pezizales in analysis 2. Ascozonus woolhopensis and Thelebolus crustaceous occurred as sister genera in the Leotiales-Rhytismatales Glade, grouping with Cyclaneusma minus and Naemacyclus fimbriatus in all parsimony analyses. A Glade comprised of the discomycete orders Caliciales, Lecanorales, Ostropales, and Peltigerales was in a sister relationship with the cleistothecial orders Elaphomycetales, Eurotiales, and Onygenales (plus Ascobolus lineolatus in analyses 1 and 2). Stictis radiata (Ostropales) was in a derived position in the main Lecanorales Glade, while Porpidia crustulata (Lecanorales) was basal to monophyletic sister clades of Caliciaceae and Peltigerales. Caliciales s.l. was polyphyletic (or paraphyletic if Porpidia crustulata is considered a member of Caliciales). Representatives from Mycocaliciaceae and Sphinctrinaceae formed a monophyletic group separated from Caliciaceae (FIGS. 4.1-4.3). With the exception of Orbiliaceae, genera ordinarily included in Leotiales formed a Glade with Cyttariales, Blumeriales, Thelebolaceae (Pezizales), Rhytismatales, and a monophyletic lineage of perithecial ascomycetes (FIGS. 4.1-4.3). Bootstrap values among the basal nodes of this main inoperculate discomycete Glade did not exceed 50% and several branches collapsed in the consensus trees. The perithecial ascomycete Glade was a paraphyletic grade having Xylariales occupying a basal position, with a branch leading to genera in Diaporthales and Ophiostomatales, and then a second branch leading to two representatives of Sordariales (FIGS. 4.1-4.3). In the most derived position, Hypocreales was in a sister relationship with two taxa from Microascales and Colletotrichum gloeosporioides (Phyllacorales). Neobulgaria bulgarioides (Leotiales) was in a basal position to the perithecial ascomycetes in all most parsimonious trees from analyses 1 and 2.

Familial and generic relationships in Leotiales and RhytismatalesThree families of Leotiales and Rhytismatales could be identified in the strict consensus trees (FIGS. 4.1­ 4.3). These were Hemiphacidiaceae, Rhytismataceae, and Sclerotiniaceae. 98

Hemiphacidiaceae was comprised of Hemiphacidium longisporum, Sarcotrochila macrospora, Fabrella tsugae, Meria laricis, and Rhabdocline parkeri. Sclerotiniaceae was comprised of Ciborinia foliicola, Monilinia oxycocci, M laxa, Sclerotinia homoeocarpa, and S. sclerotiorum. Piceomphale bulgarioides (excluded from Sclerotiniaceae by Holst-Jensen et al., 1997) occupied an intermediate position between Sclerotiniaceae and Hemiphacidiaceae in the majority-rule consensus trees in analyses 1 and 2 (FIGS 4.1 and 4.3). Piceomphale grouped with Hemiphacidium and Sarcotrochila in the neighbor joining analysis (FIG. 4.2). While the Hemiphacidiaceae and Sclerotiniaceae clades were monophyletic, the Rhytismataceae Glade was paraphyletic with two genera provisionally classified in Leotiales and provisionally placed in Geoglossaceae (Cudonia confusa and Spathularia flavida). Loramyces juncicola (Loramycetaceae) was a sister species to the anamorphic fungus, Phialocephala fortinii in the unweighted and neighbor joining trees in analysis 1, but the two species were separated in analysis 2. Darkera parca (Rhytismatales inc. sed.) was also removed from Rhytismataceae in all analyses. Cyclaneusma minus and Naemacyclus fimbriatus (Rhytismataceae) were in a sister relationship to Thelebolus crustaceus and Ascozonus woolhopensis (Thelebolaceae) in the parsimony trees from analyses 1 and 2 (FIGS. 4.1 and 4.3), but the relationship lacked bootstrap support and did not appear in the neighbor joining analysis (FIG. 4.2). Representatives of Leotiaceae grouped with a diversity of inoperculate ascomycete lineages. Leotia lubrica formed a monophyletic Glade with Microglossum viride (Geoglossaceae) and Rutstroemia firma (Leotiales inc. sed) in analysis 1 (FIGS 4.1 and 4.2). In analyses 2 and 3, Rutstroemia occupied a basal position to the Sclerotiniaceae Glade (FIGS 4.3 and 4.4). Gremmeniella abietina (Leotiaceae) grouped with Siphula ceratites (Lecanorales inc. sed). Two species of Chloroscypha (Leotiaceae) grouped with the mitosporic fungus Chalara fiisidioides away from other Leotiaceae genera in the majority-rule consensus tree from the unweighted parsimony analysis (FIG. 4.1). The Chloroscypha species and Siphula ceratites (Lecanorales inc. sed) grouped with a Thelebolus, Ascozonus, and Gremeniella Glade in the weighted parsimony analysis (FIG. 4.3), and with Siphula ceratites in the neighbor joining analysis (FIG. 4.2). Bootstrap support for these relationships was always below 50%. Phacidiaceae, represented by two 99

(putative) species of Phacidium, was polyphyletic in the majority rule consensus tree, and unresolved in the strict consensus tree. Three other species included in the analysis also occupied unique positions in the inoperculate discomycete Glade in all analyses: Hymenoscyphus fructigenus (Hymenoscyphaceae, Leotiales), Blumeria graminis (Erysiphaceae, Erysiphales), and Cyttaria darwinii (Cyttariaceae, Cyttariales).

Structural features and substitution patterns in the small subunit of Leotiales and Rhytismatales The size and position of introns encountered in species sequenced for this study are given in Table 4.2. Intron lengths ranged from 194-386 bp. A 198-264 by intron inserted after position 561 or 564 relative to Saccharomyces cerevisiae, was observed in six Rhytismatales, and a 292-386 by intron after position 563 or 564 was observed in three Leotiales. A 194-217 by intron was also observed after position 366 or 369 in three Rhytismatales. Finally, a 198 by intron was observed after position 101 in Naemacyclus fimbriatus, and a 211 by intron was observed after position 998 in Lirula macrospora. Phylogenetic analyses were not performed on the introns because manual alignment of these regions was difficult to impossible, depending on the phylogenetic distance between the species in question. Introns with similar sizes and insertion sites were slightly easier to align between closely related genera and possible conspecifics (eg. Hemiphacidium and Sarcotrochila, see below). The small subunit data set was observed to have a transitional bias (FIG. 4.4). The ratio of minimum transitions to minimum transversions was 1.6 among the 36 taxa sampled from Leotiales, Rhytismatales, and Thelebolaceae (Thelebolaceae is classified in Pezizales but was included in the analysis because it occurs in the Leotiales-Rhytismatales Glade). The mean and standard deviation pairwise transition:transversion ratio (adjusted for gaps and missing data) among the nine species in the Glade comprised of Rhytismataceae, Cudonia confusa, and Spathularia flavida Glade was 2.01 ± 0.65. Among Hemiphacidiaceae, Fabrella tsugae, Rhabdocline parkeri, and Hemiphacidium longisporum, had an observed pairwise transition:transversion ratio of 1.39 ± 0.45 (Meria laricis was excluded because it differed from R. parkeri only by a single transition, and Sarcotrochila macrospora was excluded because it differed from H. longisporum only by a single transversion). Pairwise transition biases were higher among members of the 100

Sclerotiniaceae Glade. The mean and standard deviation pairwise transition:transversion ratio among Monilinia oxycocci, Ciborinia fohicola, Sckrotinia sclerotiorum, and S. homoeocarpa was 6.53 ± 4.61. (Monilinia laxa was excluded because it differed by 7 transitions and 0 transversions from M oxycocci and by only a single transition from S. sclerotiorumthe transition:transversion ratio is undefined in both cases). Among the Leotiales, Rhytismatales, and Thelebolaceae species sampled, variable sites were not concentrated in either half of the small subunit region sampled (FIG. 4.5). The 5' 525 by had 122 variable sites, 48 of which were parsimony informative, and the 3' 512 by had 126 variable sites, 51 of which were parsimony informative. In analysis 1, the 5' 536 by had 297 variable sites, 193 of which were parsimony informative, and the 3' 536 by had 267 variable sites, 163 of which were parsimony informative. The partial small subunit GC content in the Leotiales, Rhytismatales, and Thelebolaceae species sampled was 45.6%. The partial small subunit sequences were low in cytosine relative to the other three nucleotides, averaging 20.2% C.

4.5 Discussion

The first goal of this study was to construct an nrDNA-based phylogeny to address whether the discomycetes form a monophyletic lineage within the ascomycetes. The high number of equally most parsimonious trees recovered in searches using the small subunit data set prevent a decisive answer to this question. Bootstrap values were lower than 50% at all deep nodes within the euascomycetes. Majority-rule consensus trees were consistent with the interpretation that there are three main lineages of discomycetes: 1) Pezizales and Orbiliaceae, 2) Caliciales s.s., Lecanorales, Peltigerales, Mycocaliciaceae, Sphinctrinaceae, and Ostropales, and 3) Leotiales, Rhytismatales, and Cyttariales (and possibly Erysiphales). In these trees, cleistothecial and perithecial lineages are derived independently from apothecial lineages. The monophyly (or paraphyly) of these main non­ apothecial lineages of ascomycetes can be attributed in part to the sampling method, as 101

there was no conscious attempt to test the monophyly of these groups. Other investigators using the small subunit to study inoperculate discomycetes have observed that increased sampling in the group is attracting the perithecial ascomycetes to the same part of the tree (J. Spatafora, pers. comm.). Bootstrap support for the perithecial ascomycetes is higher than for any supraordinal group, and the internal branch attaching it to other euascomycetes is long, suggesting that its derived position within the inoperculates may be caused by long branch attraction The placement of Blumeria graminis (Erysiphales) as intermediate between two inoperculate discomycetes should not be considered as definitive evidence for its affinity to that group, as only Neobulgaria premnofila separates it from the perithecial ascomycetes in the unweighted parsimony analysis presented here (FIG. 4.1), and only two genera (Neobulgaria and Chalara) separate it from the perithecial ascomycetes in the weighted analysis (FIG. 4.3). Based on these analyses, Blumeria graminis is unlikely to be a member of the cleistothecial ascomycetes. A paraphyletic lineage of discomycetes would support the assertion of Nannfeldt (1932) and more recently Gargas and Taylor (1992) that the ancestral ascomatal form was apothecial. Closure of the apothecium to form cleistothecia, perithecia, or the locules of bitunicate ascomycetes could have occurred independently in several ancestral lineages of apothecial ascomycetes. From a morphological standpoint, it is unlikely that the apothecial ascomycetes have been rendered polyphyletic by a paraphyletic lineage of loculoascomycetes (as in unweighted parsimony analysis 1, FIG. 4.1). Several adaptive advantages to a closed ascocarp can be suggested. Enclosure could have been an adaptation for prevention of desiccation, more sophisticated forms of ascospore discharge, or a defense against predators or parasites. The second goal was to determine the placement of the major discomycete lineages. As in previous studies, the most parsimonious placement of Pezizales was at the base of the euascomycetes (Gargas et al., 1995), although Orbiliaceae may be more basal. Caliciales s. s., Lecanorales, Peltigerales, Mycocaliciaceae, Sphinctrinaceae, and Ostropales formed a Glade with the cleistothecial ascomycete orders Eurotiales, Onygenales and the apothecial order, Elaphomycetales (Wedin and Tibell, 1997). The placement of the 102

Leotiales (excluding Orbiliaceae), Rhytismatales, and Cyttariales Glade among the euascomycetes is unresolved. The placement of Cyttaria darwinii (Cyttariales) at the base of Leotiales had bootstrap support below 50%. This same isolate was included in a small subunit study by

Landvik and Eriksson (1994), where it occurred within a Leotiales Glade represented by

Spathularia flavida, Cudonia confusa, and Leotia lubrica. Korf (1983) summarizes the conflicting morphological and chemical evidence over whether Cyttaria is operculate or inoperculate. Two important issues surround the nature of the ascus tip. Cyttaria possesses an amyloid ring in its ascus tip, a character otherwise unknown in Sarcoscyphineae (Pezizales), a tribe of operculate discomycetes with which it shares similarities in possession of gelatinized hyphal tissues, apothecial size, possession of a "stretching membrane over the developing hymenium", and carotenoid content. Yet the ascus tip of Cyttaria dehisces ascospores through a tear. Korf states that irregularly tearing in the ascus is found in a few other genera of operculate discomycetes, including Ascozonus. Ascozonus has been sampled here, and also occurs in the inoperculate discomycete Glade with another apothecial genus (Thelebolus) whose ascus tip ruptures

irregularly. Kimbrough and Korf (1967) placed Thelebolus in Pezizaceae, but noted that the ascus tip of Thelebolus tears irregularly to liberate ascospores. Later, Kimbrough

(1981) noted that the bitunicate (fissitunicate) nature of the ascus would place the genus among the loculoascomycetes and away from Pezizales. Thelebolaceae is placed in Pezizales in Hawksworth et al. (1995). The strongest contributions that this study makes toward understanding the phylogenetic relationships among the discomycetes is with respect to the familial relationships of Leotiales and Rhytismatales. All new sequences determined from this study were from taxa classified in these two orders. Monophyletic or paraphyletic clades corresponding to Hemiphacidiaceae (Leotiales), Sclerotiniaceae (Leotiales), and Rhytismataceae (Rhytismatales) could be identified in all analyses. Although these three families belonged to a single lineage of inoperculate discomycetes, their interrelationships varied among the different analyses. Sclerotiniaceae was consistently basal relative to the other two families. Hemiphacidiaceae occupied either an intermediate lineage, or was 103

sister to taxa in Leotiaceae (Gremmeniella abietina, Chloroscypha spp.) and Thelebolaceae, all of which in turn were sister to a Rhytismataceae Glade. Hemiphacidiaceae was erected by Korf (1962) to include discomycete fungi with simple apothecial structures possessing a poorly defined excipulum and lacking a covering layer (clypeus) over the hymenium. Members of this family are all foliar parasites of conifers. Ascomota erupt through the host epidermis, throwing back a scale of host tissue. Some members of the family possess an apical pore at the tip of the asci that turns blue in iodine (I+). In this study, the family is monophyletic in all most parsimonious reconstructions, but bootstrap support is low. The family also includes Piceomphale bulgarioides (Leotiales inc. sed.) in the neighbor joining analysis. Hemiphacidiaceae is representated by five of the seven genera generally recognized in the family (Korf, 1962: Reid and Cain, 1963). Didymascella Maire & Sacc. and Korfia Reid & Cain were not sampled. The inclusion of the anamorphic fungus, Mena laricis, in the Hemiphacidiaceae Glade was predicted by morphological similarities among anamorphs and ITS region analysis (Sherwood-Pike et al., 1986; Gernandt et al., 1997). The morphologically similar genera Hennphacidiurn and Sarcotrochila were segregated based on the presence (Sarcotrochila) or absence (Hennphacidium) of an I+ apical pore on the ascus. The small subunit and ITS region sequences of the specimens used here (both isolated by A. Funk from Pinus contorta) are almost identical in sequence. This could be a result of an incorrect determination of an amyloid pore reaction, or could indicate a closer phylogenetic relationship than heretofore recognized. The iodine reaction varies in other genera of inoperculate discomycetes (e.g. Rhabdocline and Chloroscypha (Parker and Reid, 1969; Petrini, 1982). The reliability of the iodine reaction has been explored by Kohn and Korf (1975) and its importance in classification has been disputed (Baral, 1987; Bellemere,1994). The character has not been consistently applied in classification of different groups. The tribe Hemiphacidieae was designated by Korf (1962) for genera of Hemiphacidiaceae with an apical pore that does not turn blue in iodine solution and Sarcotrochileae for genera that possessed the apical pore reaction. Considering the variation in pore reactions within Rhabdocline (designated as Hemiphacidieae), as well as the close morphological similarity between Hemiphacidium (Hemiphacidieae) and 104

Sarcotrochila (the only remaining member of Sarcotrochileae), and molecular similarity between species putatively belonging to either genus reported here, these subtribes should be considered artificial. If further sampling corroborates the high similarity between Hemiphacidium and Sarcotrochila, then the two genera should be merged into one. Rhabdocline has been provisionally classified as Rhytismatales inc. sed. by Hawksworth et al. (1996). This placement may have been based on Rhabdocline including R. pseudotsugae (comprised of two subspecies) lacking an apical pore, as do members of Rhytismatales. However two other Rhabdocline species, R. weirii and R. parkeri, possess I+ ascus pores. The possession of an apical pore seems to be a symplesiomorphic (ancestral) character in the inoperculate discomycetes, one that has been lost in several different lineages. This appears to be the case in Rhabdocline (Gernandt et al., 1997). Symplesiomorphic characters cannot be used to identify monophyletic groups. Furthermore, Rhabdocline occurs in a monophyletic Glade of Hemiphacidiaceae in the parsimony analyses presented here (FIGS. 4.1 and 4.3), is paraphyletic with Hemiphacidium, S'arcotrochila, and Piceomphale bulgarioides (Leotiales inc. sed.) in the distance analysis, and is included in Hemiphacidiaceae in morphological treatments (Korf, 1962). I recommend that Rhabdocline be restored to this family in future classifications of the ascomycetes. Sclerotiniaceae was erected by Whetzel (1945) for inoperculate discomycetes forming apothecia from indeterminate substratal or determinate sclerotial stroma. The group is comprised of a variety of parasites of angiosperms and gymnosperms. Most members have an iodine positive ascal pore. In a recent nrDNA study, Host-Jensen et al. (1997) classified several substratal genera into the family, Rutstroemiaceae. Rutstroemiaceae includes Rutstroemia firma and "Sckrotinia" homoeocarpa, (S. homoeocarpa showed affinities to an exemplar of Poculum henningsianum rather than other species of Sclerotina). The same exemplars of these species were included here. Four species from Sclerotiniaceae (C. foliicola, M laxa, M. oxycocci, and S. sclerotiorum) were consistently monophyletic in the parsimony and distance analyses reported here, but the relationships of three exemplars excluded by Holst-Jensen et al. were uncertain. Sclerotinia homoeocarpa (Rutstroemiaceae) was consistently basal to the 105

Sclerotiniaceae Glade, receiving high bootstrap support for its position there, while the position of Rutstroemia firma varied depending on the analysis. Considering the high bootstrap support for the Rutstroemiaceae Glade that was apparent in the more densely sampled study of Holst-Jensen et al., and because almost 15% of the small subunit sequence was unavailable in this study (an intron prevented sequencing 106 bp), we suggest that the instable placement of Rutstroemia firma in this study is likely to be an artifact. The position of Piceomphale bulgarioides (Leotiales inc. sed.) also varied depending on the analysis. It appeared within Hemiphacidiaceae, basal to Hemiphacidiaceae, and intermediate between Sclerotiniaceae and Hemiphacidiaceae. Hypodermataceae (treated by Darker, 1932; 1967) was placed into Rhytismataceae by Eriksson (1983). Members of this family are common leaf and bark parasites of angiosperms and gymnosperms. Their apothecia are often immersed in plant tissue and typically possess a dark clypeus or covering layer that opens by radial or longitudinal splits to expose the hymenium. In members of the family, the ascus tip lacks an amyloid pore, and often ruptures by an irregular splitting. A Rhytismataceae Glade comprised of Colpoma quercinum, Elytroderma deformans, Lirula macrocarpa, Lophodermium pinastri, Meloderma desmazierii, Rhytisma salicinum, and Tryblidiopsis pinastri consistently appeared in all analyses. The genera sampled include members formerly placed in Hypodermataceae (Colpoma, Elytroderma, Lirula, Lophodermium, Meloderma, and Tryblidiopsis) and Rhytismataceae (Rhytisma). The placement of Rhytisma relative to the former genera of Hypodermataceae was not stable, but did not support the former separation of these families. Livsey and Minter (1993) argued that Ascodichaenaceae and Cryptomycetaceae should be grouped with Rhytismataceae in the order Rhytismatales. These two families have not been sampled here. Their inclusion in subsequent analyses will be important in evaluating the limits of Rhytismatales. More difficult to explain was the paraphyly of Rhytismataceae with two provisional members of Geoglossaceae (Leotiales), Cudonia confusa and Spathularia flavida. Cudonia confusa and Spathularia flavida have I- asci and filamentous ascospores, both characteristics also found in Rhytismataceae. Although the possibility of DNA contamination cannot be ruled out (Cudonia and Spathularia are sometimes 106

parasitized by members of Rhytismataceae), additional species from these two genera have been sequenced, and these agree in their phylogenetic placement with Rhytismataceae (J. L. Platt, unpublished results). If this placement is correct, it must reflect common ancestry with Rhytismataceae, and implies that loss of plant parasitism must have occurred in the Rhytismataceae lineage. Cyclaneusma minus, Naemacyclus fimbriatus (Rhytismataceae), and Darkera parka (Rhytismatales inc. sed.) are removed from the main body of Rhytismataceae within the Leotiales-Rhytismatales Glade. Excluding these taxa from Rhytismataceae will allow for a narrower, more consistent morphological concept for the group. Cyclaneusma and Naemacyclus were well supported sister species. Their placement within the Leotiales- Rhytismatales Glade was unstable, but never monophyletic with the other taxa classified in Rhytismataceae. DiCosmo et al. (1983b) segregated Naemacyclus into Naemacyclus and Cyclaneusma based primarily on differences in ascomatal structure and features of the ascospores. Naemacyclus fimbriatus possesses a distinct, blackened clypeus covering the hymenium and its ascospores are surrounded by a mucilaginous sheath. The possession of a black clypeus over the hymenium, mucilaginous sheaths surrounding the ascospores, and the possession of an anamorph similar to those found in Rhytismataceae (and I- asci) suggests that Rhytismataceae is an appropriate family for Naemacyclus, and it was placed there by DiCosmo et al. (1983a). Cyclaneusma minus has an ill-defined excipulum,lacks a fungal covering layer over the hymenium and its ascospores have gelatinous caps at both ends. However, in Cyclaneusma, the ascocarp is not surrounded by a black clypeus, a character usually encountered in Rhytismataceae (Lophodermella concolor is one exception). Members of the conifer parasitic family, Phacidiaceae, also have a black excipulum, black clypeus, and hyphal bridges similar to those found in Cyclaneusma. Phacidiaceae does not include members with sheathed or septate ascospores, and all members have an iodine positive apical pore, but the overlap in characters pertaining to the excipulum, clypeus, and hyphae points to the difficulties in placing variable but closely related taxa into consistent genera or families (DiCosmo et al., 1983a). This study suggests that these two genera belong to neither Phacidiaceae nor Rhytismataceae, and may be closely enough related for species of Cyclaneusma to be returned to Naemacyclus. 107

Darkera parca is provisionally classified in Rhytismatales inc. sed. by Hawksworth et al., (1995). It was paraphyletic or a sister taxon with Phacidium coniferarum (Phacidiaceae) in most analyses. Darkera was placed in Phacidiaceae by Whitney et al. (1975), and later moved to Hemiphacidiaceae by DiCosmo et al. (1983). The anamorphs of Darkera and Phacidium are similar coelomycetes that produce 1-celled hyalospores with mucilaginous appendages. The conidiomata of Tiarosporella (anamorph of Darkera) are described as pycnidial, and having determinate holoblastic conidiogenous cells, while the conidiomata of Ceuthospora (anamorph of Phacidium) are stromatic, and have conidiogenous cells that exhibit percurrent, enteroblastic proliferation. The two species of Phacidium were polyphyletic in all analyses, with neither belonging to a strongly supported Glade. Further sampling of Phacidiaceae is warranted to determine its familial limits and examine whether Darkera should be included. Phialocephala fortinii, a mitosporic root-endophytic fungus, occurs in the inoperculate discomycete Glade. It is a sister taxon to Loramyces juncicola in the parsimony and distance trees from analysis 1, but the relationship has very weak bootstrap support. Phialocephala fortinii is classified as an anamorphic member of Chaetothyriales (LoBuglio et al., 1996). If P. fortinii is an inoperculate discomycetes, then the ecological range of inoperculate discomycetes might be expanded to include mycorrhizal fungi in the broad sense. The symbiosis between P. fortinii and its hosts involves "dual organs of absorption" (Trappe, 1996) but also behaves pathogenically under some conditions (Currah et al., 1993; Jumpponen et al., 1998). Morphologically, Leotiaceae is a highly heterogeneous group. The five species comprising four genera sampled from Leotiaceae were polyphyletic. Leotia lubrica formed a Glade with Microglossum viride (Geoglossaceae) and sometimes with Rutstroemia firma. Leotia and Microglossum have similar multiguttulate ascospores (Mains, 1956). Chloroscypha and Gremmeniella occurred elsewhere among the inoperculate discomycete Glade, and did not have strongly supported affinities to any other species sampled. Neobulgaria prernnofila was consistently basal to the perithecial ascomycetes, but had low bootstrap support in that position. 108

Furthur research into the phylogenetic relationships of the inoperculate discomycetes is needed to understand the systematics and evolution of the group. The relatively higher bootstrap support at near-terminal nodes compared to those at the paraphyletic backbone of the euascomycetes observed here suggest that a highly conserved molecule will be most useful in discerning higher level relationships among the groups. Phylogenetic reconstructions that sample more genera among the euascomycetes and include the entire small subunit of nrDNA will probably have the greatest short term benefit for understanding the evolution of the group by drawing more precise lines around families and by increasing the accuracy or phylogenetic methods. Phylogenetic reconstructions based on a non-nrDNA based molecule should also be encouraged in order to identify sources of conflict and congruence from independent sources of molecular data, but the payoff from such a project is likely to be distant, as it will take time to sample a comparatively large number of taxa. Future research questions should continue to address whether the inoperculate discomycetes are monophyletic or paraphyletic, and whether the Orbiliaceae should be placed elsewhere, perhaps in a sister order to Pezizales, or Gyalectales. Familial relationships among members Cyttariales, Leotiales, and Rhytismatales Glade need to be addressed further.

4.6. Acknowledgments

I thank Jeff Stone, Jamie Platt, Joseph Spatafora, Arne Holst-Jensen, Linda Kohn, and Richard Hamelin for their assistance with this project. Sequencing was performed by the Central Services Laboratory within the Center for Gene Research and Biotechnology at Oregon State University. Funding was provided in part by a Sigma Xi research grant. 109

4. 7. Literature Cited

Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

Baral, H. 0. 1987. Lugol's solution/IKI versus Melzer's reagent: hemiamyloidity, a universal feature of the ascus wall. Mycotaxon 29: 399-450.

Bellemere, A. 1994. Asci and ascospores in ascomycete systematics. Pp. 111-126. In: Ascomycete systematics, problems and perspectives in the nineties. NATO ASI Series A, Vol 269. Ed., D. L. Hawksworth. Plenum Press, New York, New York.

Berbee, M. L., and J. W. Taylor. 1992a. Dating the evolutionary radiations of the true fungi. Canad. I Bot. 71: 1114-1127.

, and . 1992b. Two Ascomycete classes based on fruiting-body characters and ribosomal DNA sequence. Mol. Biol. Evol. 9: 278-284.

Boudier, J. L. E. 1885. Nouvelle classification naturelle des Discomycetes charnus connus generalement sous le nom de Pezizes. Bull. Soc. Mycol. France 1: 91-121.

. 1907. Histoire et classification des Discomycetes d'Europe. P. Klincksieck, Paris. 222 pp.

Bruns, T., T. White, and J. Taylor. 1991. Fungal molecular systematics. Annual Review of Ecology and Systematics 22: 525-564.

Carbone, I. and L. M. Kohn. 1993. Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae. Mycologia 85: 415-427.

Chase, M. W., Soltis, D. E., Olmstead, R. G. et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastic gene rbcL. Ann. Mo. Bot. Gard. 80: 528-580.

Currah, R. S., A. Tsuneda, and S. Murakami. 1993. Morphology and ecology of Phialocephala fortinii in roots of Rhododendron brachycarpum. Can. I Bot. 71: 1639-1644.

Darker, G. D. 1932. The Hypodermataceae of conifers. Contrib. Arnold Arboretum 1: 1-131. 110

. 1966. A revison of the genera of the Hypodermataceae. Canad. I Bot. 45: 1399-1444.

Dennis, R. W. G. 1978. British Ascomycetes. J. Cramer, Vaduz, Zwitzerland. 585 pp.

DiCosmo, F., T. R. Nag-Raj, and B. Kendrick. 1983a. Prodromus for a revision of the Phacidiaceae and related anamorphs. Canad. J. Bot. 61: 31-44.

H. Peredo, and D. W. Minter 1983b. Cyclaneusma gen. nov., Naemacyclus and Lasiostictis, a nomenclatural problem resolved. Eur. J. For. Path. 13: 206-212.

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

Eriksson, 0. E. 1983. Outline of the ascomycetes 1983. Syst. Ascomycetum 2: 1-37.

Eriksson, 0. E. and D. L. Hawksworth. 1993. Outline of the Ascomycetes 1993. Syst. Ascomycetum 12: 51-257.

Gargas, A., P. T. DePriest, M. Grube, and A. Tehler. 1995. Multiple origin of lichen symbiosis in fungi suggested by SSU rDNA phylogeny. Science (Washington, D.C.) 286: 1492-1495.

and J. W. Taylor. 1995. Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Mot Biol. Eva 12: 208-218.

, and J. W. Taylor. 1995. Phylogeny of discomycetes and early radiations of the apothecial Ascomycotina inferred from SSU rDNA sequence data. Exp. Mycol. 19: 7-15.

Gernandt, D. S., F. J. Camacho, and J. K. Stone. 1997. Meria laricis, an anamorph of Rhabdocline. Mycologia 85: 735-744.

Hamelin, R. C., and J. Rail. 1996. Phylogeny of Gremmeniella spp. based on sequences of the 5.8S rDNA and internal transcribed spacer region. Canad J. Bot. 75: 693­ 698.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp.

Hibbett, D. 1992. Ribosomal RNA and fungal systematics. Trans. Mycol. Soc. Jap. 33: 533-556. 111

Hoist- Jensen, A., L. M. Kohn, K. S. Jakobsen, and T. Schumacher. 1997. Molecular phylogeny and evolution ofMonilinia (Sclerotiniaceae) based on coding and noncoding rDNA sequences. Amer. J. Bot. 84: 686-701.

and T. Schumacher. 1997. Nuclear rDNA phylogeny of the Sclerotiniaceae. Mycologia 89: 885-899.

Jumpponen, A., K. G. Mattson, and J. M. Trappe. 1998. Mycorrhizal functioning of Phialocephala fortinii with Pinus contorta on glacier forefront soil: interactions with soil nitrogen and organic matter. Mycorrhiza 7: 261-265.

Kimbrough, J. W., and R. P. Korf. 1967. A synopsis of the genera and species of the tribe Thelebolaceae (=Pseudoascoboleae). Amer. J. Bot. 54: 9-23.

Kimbrough, J. W. 1981. Cytology, ultrastructure, and taxonomy of Thelebolus (Ascomycetes). Mycologia 73: 1-27.

Kohn L. M. and R. P. Korf. 1975. Variation in ascomycete iodine reactions: KOH pretreatment explored. Mycotaxon 3: 165-172.

Korf, R. P. 1962. A synopsis of the Hemiphacideaceae, a family of Helotiales (discomycetes) causing needle blights of conifers. Mycologia 54: 12-33.

. 1983. Cyttaria (Cyttariales): Coevolution with Nothofagus, and evolutionary relationship to the Boedijnopezizeae. Aust. J Bot. SuppL S'er.10: 77-87.

Landvik, S., 0. E. Eriksson, A. Gargas, and P. Gustafsson. 1993. Relationships of the genus Neolecta (Neolectales ordo nov., Ascomycotina) inferred from 18S rDNA sequences. Sys. Ascomycetum 11: 107-118.

, and . 1994. Relationships of Tuber, Elaphomyces, and Cyttaria (Ascomycotina), inferred from 18s rDNA studies. Pp. 225-231. In: Ascomycete systematics, problems and perspectives in the nineties. NATO ASI Series A, Vol 269. Ed., D. L. Hawksworth. Plenum Press, New York, New York.

. 1996. Neolecta, a fruit-body-producing genus of the basal ascomycetes, as shown by SSU and LSU rDNA sequences. Mycol. Res. 100: 199-202.

, N. Shailer, and 0. E. Eriksson. 1996. SSU rDNA sequence support for a close relationship between the Elaphomycetales and the Eurotiales and Onygenales. Mycoscience 37: 237-241. 112

, K. N. Egger, and T. Schumacher. 1997. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analyses of SSU rDNA sequences. Nord. J. Bot. 17: 403-418.

Livsey, S., and Minter, D.W. 1993. The taxonomy and biology of Tryblidiopsis pinastri. Canad. J. Bot. 72:549-557.

LoBuglio, K. F., M. L. Berbee, and J. W. Taylor. 1996. Phylogenetic origins of the asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes. MoL Phylogenet. Evol. 6: 287-294

Mains, E. B. 1956. North American species of the Geoglossaceae. Tribe Cundonieae. Mycologia 48: 694-710.

Morgan-Jones, G. 1973. Genera coelomycetarum. VII. Cryptocline Petrak. Canad. J. Bot. 51: 309-325.

Nannfeldt, J. A. 1932. Studien Ober die Morphologie and Systematik der nicht­ lichenisierten inoperculaten Discomyceten. Nova Acta Reg. Soc. Sci. Ups. Ser. IV., 8: 1-368.

Nixon, K. C., and J. M. Carpenter. 1993. On outgroups. Cladistics 9: 413-426.

O'Donnell, K., E. Cigelnik, N. S. Weber, and J. M. Trappe. 1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48-65.

Ogawa, H., A. Yoshimura, and J. Sugiyama. 1997. Polyphyletic origins of species of the anamorphic genus Geosmithia and the relationships of the cleistothecial genera: evidence from 18S, 5S and 28S rDNA sequence analysis. Mycologia 89: 756-771.

Parker, A. K., and J. Reid. 1969. The genus Rhabdocline Syd. Canad I Bot. 47: 1533­ 1545.

Petrini, 0. 1982. Notes on some species of Chloroscypha endophytic in Cupressaceae of Europe and North America. Sydowia 35: 206-222.

Reid, J. and R. F. Cain. 1963. New genus of the Hemiphacidiaceae. Mycologia 55: 781­ 785.

Seaver, F. J. 1951 The North American Cuplungi. Vol. 2: Inoperculates. Reprint 1978. Lubrecht & Cramer, Monticello, N.Y.

Sherwood-Pike, M., J. K. Stone, and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canad J. Bot. 64: 1849-1855. 113

Smith, A. B. 1994. Rooting molecular trees: problems and strategies. Biological Journal of the Linnean Society 51: 279-292.

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gillevet. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Spatafora, J. W. and M. Blackwell. 1993. Molecular systematics of unitunicate perithecial ascomycetes: The Clavicipitales-Hypocreales connection. Mycologia 85: 912-922.

Spatafora, J. W. and M. Blackwell. 1994. Polyphyletic origins of Ophiostomatoid fungi. Mycol. Res. 85: 912-922.

. 1995. Ascomal evolution of filamentous ascomycetes: evidence from molecular data. Canad. J. Bot. 73(Suppl. 1): S811-S815

T. G. Mitchell, and R. Vilgalys. 1995. Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of Dematiaceous fungal pathogens. J. Clin. Microbiol. 33: 1322-1326.

Swofford, D. L., G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. Pp. 407-514. In: Molecular systematics. Eds., D. M. Hillis, C. Moritz, and B. K. Mable. Sinauer Associates, Inc., Sunderland, Massachusetts,.

Swofford, D. L. 1998. PAUP: Phylogenetic analysis using parsimony, Version 3.0s +4. Illinois Natural History Survey, Champaign, Illinois.

Sydow, H. and F. Petrak. 1922. Ein Beitrag zur Kenntnis der Pilzflora Nordamericas, insbesondere der nordwestlichen Staaten. Ann. Mycol. 20:178-218.

Trappe, J. M. 1996. What is a mycorrhiza? Pp. 3-6. In: Mycorrhiza in integrated systems from genes to plant development. Proceedings of Fourth European Symposium on Mycorrhiza. Eds., C. Azcon-Aguilar and J. M. Barea. Commission of the European Union, Luxemburg.

Tsumura, Y., Yoshimura, K., Tomaru, N., and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theor. Appl. Genet. 91: 1222-1236.

Wedin, M., and L. Tibell. 1997. Phylogeny and evolution of Caliciaceae, Mycocaliciaceae, and Sphinctrinaceae (Ascomycota), with notes on the evolution of the prototunicate ascus. Canad. I Bot. 75: 1236-1242. 114

Whetzel, H. H. 1945. A synopsis of the genera and species of the Sclerotiniaceae, a family of stromatic inoperculate discomycetes. Mycologia 37: 648-714.

White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols, a guide to methods and applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White. San Diego: Academic Press.

Whitney, H. S., J. Reid, and K. A. Pirozynski. 1975. Some fungi associated with needle blight of conifers. Canad. J. Bot. 53: 3051-3063.

Wulf, Von A., and L. Pehl. 1996. Erster Fund von Fabrella tsugae (Farl.) Kirschst. in der Bundesrepublik Deutschland - eM neuer Nadelpilz an Tsuga canadensis (L.) Carr. Nachrichtenbl. Dent. pflanzenschutzd. 48: 1-4. 115

Chapter 5 Meria laricis, an Anamorph of Rhabdocline

David S. Gernandt Francisco J. Camacho Jeffrey K. Stone Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331-2902

Published in Mycologia 89: 735-744

5.1. Abstract

Meria laricis, a needle cast pathogen of Larix spp. in North America and Europe, has no known ascigerous state, possibly having permanently lost the ability to reproduce sexually and thus representing an obligately asexual mitosporic holomorph. Comparisons of nucleotide sequences from the internal transcribed spacer region of M. laricis, Rhabdocline spp., and several genera of inoperculate discomycetes show that isolates of M laricis form a monophyletic Glade within the ascomycete genus Rhabdocline. Cladistic analysis of infraspecific taxa of Rhabdocline and ordinal placement of the genus are discussed. Results of cladistic analysis suggest that possession of an amyloid ascus pore is an unreliable character for differentiation of species and subspecies. 116

5.2. Introduction

The genus Rhabdocline was erected by Sydow (Sydow and Petrak, 1922) on the basis of specimens from North America collected by J. R. Weir on foliage of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The genus was later emended by Parker and Reid (1969), who divided it into two species groups differentiated by the presence or absence of an iodine-positive ascus pore. The genus is characterized by hypophyllous or epiphyllous, subepidermal hysterothecia that lack both an ectal excipulum and clypeus, and have clavate, 8-spored asci that either possess or lack an I+ apical pore, with hyaline, one- celled ascospores that become two-celled with one brown cell and one hyaline cell before germination (Sydow and Petrak, 1922; Parker and Reid, 1969). Sydow did not designate a type for the genus. Rhabdocline pseudotsugae, the species designated by Parker and Reid (1969) as the genus lectotype, has iodine-negative asci. Parker and Reid (1969) recognized two infraspecific taxa of R. pseudotsugae: ssp. pseudotsugae, which has hypophyllous apothecia and filamentous paraphyses, and ssp. epiphylla, which has mostly epiphyllous apothecia and clavate paraphyses. Parker and Reid (1969) erected a second species, R. weirii, based on the presence of an I+ reaction, and recognized three infraspecific taxa within R. weirii: ssp. weirii, with apothecia occupying half or less than half the width of the needle, ssp. oblonga, with rectangular apothecia occupying the full width of usually 1-yr-old needles, and ssp. obovata, with obovate ascospores and occurring on 2-yr-old and older needles. The species and subspecies described above are only known to occur on Douglas- fir and are all moderate to serious defoliating pathogens of this host. A connection between the Rhabdocline teleomorph and the anamorph genus Meria is provided by Rhabdocline parkeri, an asymptomatic endophyte specific to Douglas-fir that was first described in 1986 by Sherwood-Pike et al. This species has I+ asci and a sporodochial anamorph, Meria parkeri Sherwood-Pike, Stone, & Carroll. The anamorph is morphologically indistinguishable from Meria laricis Vuillemin, a serious defoliating pathogen of Larix species in North America and Europe, although it differs in cultural features noted by Sherwood-Pike et al. (1986). 117

The teleomorph of Rhabdocline parkeri is rare, inconspicuous, and, unlike pathogenic Rhabdocline species, fruits exclusively on abscised needles. Sherwood-Pike et al. (1986) predicted that intensive searching would yield an overlooked teleomorph of M laricis on abscised Larix needles. Sporadic searching has not yielded the sought-after teleomorph. It is hypothesized that M laricis may therefore represent an obligately asexual, "mitosporic holomorph" (Reynolds, 1993). Nucleic acid sequences for the ITS-1, 5.8S, and ITS-2 internal transcribed spacer region of nuclear ribosomal DNA were obtained for Rhabdocline spp., M laricis, and several other inoperculate discomycetes mainly from coniferous hosts. Rhabdocline has been alternately disposed in Hemiphacidiaceae (Korf, 1962), Rhytismataceae (Eriksson and Hawksworth, 1993), and most recently in Rhytismatales incertae sedis (Hawksworth et al., 1995). Because the ordinal and familial placement of Rhabdocline is uncertain, genera within Hemiphacidiaceae, Leotiaceae, Phacidiaceae, and Sclerotiniaceae (Leotiales), and Rhytismataceae (Rhytismatales) were included in the phylogenetic analyses. Maximum parsimony was used to test the hypothesis that M laricis forms a monophyletic Glade with Rhabdocline taxa and to make a preliminary examination of evolutionary relationships within the group.

5.3. Materials and Methods

Fungal isolates -- Collection information for the fungi sampled is given in TABLE 5.1. Rhabdocline parkeri, Meria laricis, and Chloroscypha chloromela were isolated from needles surface-sterilized in 95% ethanol for 60 s and 2% NaOC1 for 5 min and maintained on potato dextrose agar (Difco) supplemented with 0.1% malt extract and 0.1% yeast extract, (PDMYA). Because R. pseudotsugae and R. weirii are difficult to culture, DNA was isolated directly from apothecia dissected from diseased Douglas-fir needles. Needle segments bearing apothecia were ground in liquid nitrogen, DNA was extracted, and the fungal ITS region was selectively amplified. Apothecia of Mitrula elegans and Chloroscypha enterochroma were similarly extracted and amplified. The remaining 118

specimens were provided as cultures from other institutions. Cultures were either grown for 7-21 d in potato dextrose broth (Difco), or were scraped from PDMYA plates prior to DNA extraction. The Fabrella tsugae isolate was isolated from diseased hemlock foliage reported by Wulf and Pehl (1996). The specimens of R. pseudotsugae and R. weirii have been deposited in the OSC Herbarium (accession numbers 55227-55239).

DNA isolation--DNA was extracted from either pure fungal cultures or needles bearing apothecia using the method of Doyle and Doyle (1987). Reagents were provided by Sigma Chemical Company, St. Louis, MO, unless otherwise indicated. Samples were ground in 65 C 2X CTAB (United States Biochemical, Cleveland, OH) buffer supplemented with 2% sodium bisulfite and 2% PVP, extracted twice in chloroform:isoamyl alcohol 24:1, precipitated in isopropanol and 7.5 M ammonium acetate for 60 min at -20 C, spinned, washed in 70% ethanol, and resuspended in Tris- EDTA (10 mM Tris, 1 mM EDTA, pH 8.0).

PCR amplification and DNA sequencing--Primers used for amplification and sequencing of the internal transcribed spacer region of nrDNA were ITS5, ITS4, ITS3, and ITS2 (White et al. 1990). In some strains, presence of an intron in the small ribosomal DNA subunit necessitated the use of an additional forward primer for sequencing. The primer, SSUEND (5' -GAACCTGCGGAAGGATCATTA-3' ), was designed to anneal to a site on the last 21 bases (3' end) of the small subunit. PCR reactions were performed in 100 mt volumes with 2.5 U ReplithermTM DNA polymerase (Epicentre Technologies, Maddison, WI), ReplithermTM buffer, 1.5 mM MgC12, 10 pmol dNTP (10 mM, pH 7.0, Epicentre Technologies, Maddison, WI), 11.7 pmol ITS5, 15 pmol ITS4, 5% DMSO, 1% BSA, and approximately 100 ng sample DNA. PCR amplification reaction conditions were 35 cycles of 94 C denaturing, 55 C annealing, and 72 C extension, each at 60 s. Reactions were terminated following a final extension at 72 °C for ten min. Products were prepared for sequencing by precipitating in 1/2 volume of 5 M ammonium acetate and 1 1/2 volumes of isopropanol. Cycle sequencing with dye-terminator chemistry was TABLE 5.1. Taxa included in the ITS re ion anal ses of Rhabdocline. Isolates are from the United States unless otherwise s ecified.

GenBank Taxon Host Geographic location number Chloroscypha chloromela (W. Phillips & Harkn.) Seaver Sequoia sempervirens (D. Don) Endl. Hwy 101, near Weott, CA U92311 Chloroscypha enterochroma (Peck) Petrini Calocedrus decurrens (Torr.) Florin Peavy Arboretum, near Corvallis, OR U92312 Colpoma quercinum (Pers. ex St.-Am) Wallr. Quercus robur L. Braunschweig, C313 U92306 Cyclaneusma minus Butin Pinus sylvestris L. Kevo, Finland 93197 U92309 Fabrella tsugae (Farl.) Kirsch. Tsuga canadensis (L.) Carr. Herzberg, Germany, F256 U92304 Lophodermium australe Dearn. Pinus palustris Miller Florida U92308 Meria laricis Vuill. 1 Larix occidentalis Nutt. Wenatchee, WA U92298 Meria laricis 2 Larix occidentalis Owlsey Canyon, OR Meria laricis 3 Larix occidentalis Corvallis, OR Meria laricis 4 Larix lyallii Parl. Anthony Lakes, OR Meria laricis 5 Larix decidua Miller Horgenberg Switzerland, CBS 298.52a U92299 Mitrula elegans (Berk.) Fries Florence, OR U92305 Phacidium infestans Karst. Pinus cembra L. Obersulzbachtal, Germany, P431 U92305 Rhabdocline parkeri Sherwood-Pike, Stone, & Carroll 1 Pseudotsuga menziesii (Mirb.) Franco Junction City, OR U92297 Rhabdocline parkeri 2 Pseudotsuga menziesii Jackson Hole, WY U92295 Rhabdocline parkeri 3 Pseudotsuga menziesii Junction City, OR U92296 Rhabdocline parkeri 4 Pseudotsuga menziesii Jackson Hole, WY U92294 Rhabdocline pseudotsugae Syd. 1 Pseudotsuga menziesii MacDonald Forest, Benton Co., OR U92290 Rhabdocline pseudotsugae 2 Pseudotsuga menziesii Corvallis, OR Rhabdocline pseudotsugae 3 Pseudotsuga menziesii Covington, PA U92291 Rhabdocline pseudotsugae ssp. epiphylla Parker & Reid Pseudotsuga menziesii Mt. Hebo, OR U92292 Rhabdocline weirii ssp. obovata Parker & Reid Pseudotsuga menziesii Roseburg, OR U92293 Rhabdocline weirii ssp. weirii Parker & Reid 1 Pseudotsuga menziesii Missoula Co., MT U92300 Rhabdocline weirii ssp. weirii 2 Pseudotsuga menziesii Roseburg, OR U92301 Sclerotinia sclerotiorum (Lib.) de Barvb Brassica sp. Saskatchewan, Canada M96382 Tiarosporella parca (Berk. & Broome) H. S. Whitney, J. Reid & Picea engelmannii Parry ex. Engelm. Mt. Jefferson Wilderness, OR U92302 Pirozynski st. anam. of Darkera parca H. S. Whitney, J. Reid & Pirozynski Tryblidio sis sinastri (Pers.:Fr.) P. Karst. Pinus L. Sweden, CBS 234.83 U92307 a CBS = Centraalbureau voor Schimmelcultures, Baarn, Netherlands. b Sequence obtained from GenBank (Morales et al., 1993) 120

performed using an ABI model 373A fluorescent sequencer. Products were sequenced in both 5' to 3' and 3' to 5' directions.

Sequence analysis -- Sequence contigs were assembled and edited using Genetic Data Environment (GDE) (Smith et al., 1994). Sequence data for the ITS region were aligned using the Pile Up option in Genetics Computer Group (GCG, 1991) (Gap Weight = 1.0, Gap Length Weight = 0.1) and further adjusted by eye in GDE. Sequences were analyzed using PAUP v. 3.0s+4 (Swofford, 1991). Gaps were treated as missing characters and positions where alignment was ambiguous were excluded. Alignments are available from authors on request. An analysis was performed to test the monophyly of Rhabdocline and M laricis (analysis I). The analysis included all taxa in TABLE 5.1 except three isolates of M laricis and one isolate of R. pseudotsugae ssp. pseudotsugae because of their high similarity with other sequences that were retained. A heuristic search that employed mulpars, tree-bisection reconnection and random sequence addition with 100 replicates was performed. Bootstrap values were generated using 500 replicates, with a maximum of 10,000 trees saved for each replicate. Preliminary analyses using different alignment and taxon-sampling schemes had revealed that sequences of M laricis and R. weirii alternately occupied the basal position in the Rhabdocline Glade. For this reason, three rooting techniques were employed to examine more closely the order of evolution among Rhabdocline taxa and M laricis. First, sequences were rooted by including an outgroup (analysis IIA). Fabrella tsugae, Sclerotinia sclerotiorum, and Phacidium infestans were selected as the outgroup based on their proximity to Rhabdocline taxa in the more inclusive analysis and because their ITS regions had the highest nucleotide similarity to Rhabdocline and M laricis. Omission of the more divergent sequences allowed us to compress the alignment and include more positions. Those outgroup characters that were difficult to align were treated as missing data (removed) and all characters of Rhabdocline and Meria were retained (Nixon and Carpenter, 1993). Removing ambiguously aligned outgroup characters and retaining all ingroup characters improved resolution of the Rhabdocline and M laricis Glade while maintaining a more conservative interpretation of positional homology relative to the 121 outgroup. A branch and bound search was performed with PAUP and bootstrap values were generated with 500 replicates. A subsequent analysis was performed on the Rhabdocline and M laricis sequences only, by using either a midpoint root or the most parsimonious connection to a hypothetical ancestor (Lundberg, 1972) to determine polarity (analysis IIB).

5.4. Results

PCR products for the taxa sampled varied in size from 600 to 1050 bp. Because the PCR primers used anneal outside the ITS region, length of the ITS region was shorter, approximately 460-510 by (+/- 5 bp). The ITS-1 region accounted for most of the length variation, ranging from approximately 148-195 by in the 27 taxa sampled. Chloroscypha chloromela and C. enterochroma possessed 35 and 95 by regions respectively at the 5' end of the ITS-1 region. These regions represented one or more indel events relative to all other taxa and were excluded from the analysis. The ITS-2 region was approximately 175 by for all isolates. An insertion homologous to the Group I intron found in several filamentous ascomycetes (Gargas et al. 1995) was observed in M laricis (535 bp), R. parkeri (535 bp), C. chloromela (325 bp), Lophodermium australe (200 bp), M elegans (350 bp), and Dyblidiopsis pinastri (200 bp). The putative intron was in the small subunit approximately 5 by downstream of the 3' end of the ITS5 primer site and accounted for most of the length variation of PCR products used in this study. Two isolates, R. parkeri 3 and the European M laricis isolate, lacked the putative Group I intron. A total of 14 M laricis and ten R. parkeri isolates were PCR-amplified, and all others yielded a 1050 base pair product, indicating presence of the intron. All five R. pseudotsugae and R. weirii PCR products lacked the intron. All introns were checked against DNA databases using Blast (Altschul et al., 1990) and scored high similarities with introns deposited for other taxa, including Hymenoscyphus ericae (XU06868), Dunaliella parva (M62998) and Protomyces inouyei (XD11377). Similarities for the three highest matches ranged from 122

751 R. pseudotsugae ssp. pseudotsugae 3 921 R. pseudotsugae ssp. pseudotsugae 1 Douglas-fir pathogen I- no anamorph 94 R. pseudotsugae ssp. epiphylla R. weirii ssp. obovata RI Douglas-fir pathogen I+ no anamorph 81r M laricis western U.S. larch pathogen M laricis Switzerland Meria holomorph? j R. parkeri 3 coastal Douglas-fir I R. parkeri 1 coastal asymptomatic endophy te 99 R. parkeri 4 interior Meria anamorph R. parkeri 2 interior 100 R. weirii ssp. weirii 1 Douglas-fir pathogen L R. weirii ssp. weirii 2 Rhabdogloeum anamorph 71 Sclerotinia sclerotiorum Fabrella tsugae 86 Colpoma quercinum 62 Thyblidiopsis pinastri Lophodermium australe 100 Chloroscypha chloromela Chloroscypha enterochroma Mitrula elegans 61 Darkera parca Cyclaneusma minus Phacidium infestans 10

FIG. 5.1. ITS region phylogeny of inoperculate discomycetes in analysis I. Single most parsimonious tree found in the heuristic search (533 steps, CI = 0.525, RI = 0.629, and RC = 0.373). The Rhabdocline and Meria taxa formed a monophyletic group. Bootstrap values of 50% or higher are displayed above the branches. The scale bar represents the number of steps along the branches. 123

A. 74 R. pseudotsugae ssp. pseudotsugae eastern U.S. 94 R. pseudotsugae ssp. pseudotsugae western U.S. 96 R. pseudotsugae ssp. epiphylla L R. weirii ssp. obovata 100 R. weirii ssp. weirii 1 I- R. weirii ssp. weirii 2 93 parkeri 4 interior L R. parkeri 2 interior 76 R. parkeri 3 coastal I R. parkeri 1 coastal 82 M laricis western U.S. M laricis Switzerland 80 Sclerotinia sclerotiorum 98 Fabrella tsugae Phacidium infestans

1

B. 74 R. pseudotsugae ssp. pseudotsugae eastern U.S. 94 R. pseudotsugae ssp. pseudotsugae western U.S. 95 R. pseudotsugae ssp. epiphylla 56 R. weirii ssp. obovata 95 M laricis western U.S. M laricis Switzerland 71 r R. parkeri 3 coastal I*R. parkeri 1 coastal parkeri 4 interior 91 LKR. parkeri 2 interior

100 I R. weirii ssp. weirii 1 L R. weirii ssp. weirii 2

FIG. 5.2. Alternative rooting approaches for Rhabdocline. Phylograms generated from analyses HA and IIB of Rhabdocline and Meria laricis. Nodes absent in consensus tree are indicated with an asterisk (*). A. Analysis HA. One of six most parsimonious trees (195 steps, CI = 0.761, RI = 0.813, and RC = 0.659). Outgroup sequences that were difficult to align or uninformative with respect to the ingroup have been excluded. B. Analysis IIB. One of two most parsimonious trees (110 steps, CI= 0.851, RI = 0.900, and RC = 0.785). The node supporting R. parkeri 1 and R. parkeri 3 collapses in the consensus tree. Symplesiomorphic sequence character states identified by comparison with F. tsugae were used to construct a hypothetical ancestor for Lundberg rooting. Subsequent to ingroup analysis, Lundberg and midpoint rooting both placed the root on the branch indicated by the arrow. 124

60-90% and spanned lengths ranging from 30-300 bp. The introns were sequenced in one direction but were not included in the cladistic analyses. Four Meria laricis isolates from the North American hosts, L. occidentalis and L. lyallii were essentially identical; isolate M laricis 1 differed from the other three isolates in only one base substitution. ITS region sequences of the North American isolates and M laricis isolated from European L. decidua differed at 5 positions (all single base substitutions), comparable to the amount of variation between M laricis and Rhabdocline species. Although European and North American M laricis are morphologically indistinguishable, this amount of variation within the ITS is comparable to that found in the ITS-1 region among species of Sclerotiniaceae (Carbone and Kohn, 1993) but much less than the inter- and infraspecific variation observed in ITS region of Fusarium spp. (O'Donnell, 1992). Two ITS region types were recognizable in R. parkeri corresponding to geographical location. The types differed in having two C-T transitions and one A deletion; one isolate, R. parkeri 3, lacked the 535 by insert but was otherwise identical in sequence to isolate R. parkeri 1. After adjustment for gaps, ITS region sequence similarity between the R. parkeri and M laricis isolates examined ranged from 93.7-96.2%. In contrast, the similarity between R. weirii ssp. weirii and R. weirii ssp. obovata was only 88.0-88.3%, the similarity between R. parkeri and R. weirii ssp. obovata was 93.7-95.1%, and the similarity between R. pseudotsugae ssp. pseudotsugae and M laricis was 92.4-92.6%. The M. laricis isolate from Europe had a 96.9% similarity to the North American isolate. After alignment of the 23-taxon dataset, 433 of 623 sequence positions were retained for analysis I. Of those positions retained, 140 were phylogenetically informative, 59 in the ITS-1 region, 7 in the 5.8S region, and 74 in the ITS-2 region. A single most parsimonious tree was found in the heuristic search (FIG. 5.1). The Rhabdocline and M laricis taxa formed a monophyletic group supported by a 99% bootstrap value. Rhabdocline weirii, represented by the two subspecies R. weirii ssp. weirii and R. weirii ssp. obovata, was polyphyletic. Rhabdocline weirii ssp. obovata, was monophyletic with the R. pseudotsugae ssp. pseudotsugae and ssp. epiphylla Glade. Rhabdocline parkeri was paraphyletic. Two branches separated according to host variety 125

(coastal or interior). Several infrageneric relationships had bootstrap values lower than 50%. The most parsimonious tree from analysis I represented F. tsugae, S. sclerotiorum and P. infestans (Leotiales) as sharing a more recent common ancestor with Rhabdocline than C. minus, L. australe, T. pinastri, or C. crispum (Rhytismataceae), but bootstrap support was low. Bootstrap values above 50% supported monophyly of the two species of Chloroscypha, and monophyly of C quercinum, T. pinastri, and L. australe (Rhytismataceae) with respect to the other taxa sampled. Testing the order of evolution within the Rhabdocline and M. laricis Glade by means of outgroup, midpoint, and Lundberg rooting yielded multiple interpretations of their evolutionary relationships (FIG. 5.2). In analysis IIA, outgroup rooting with F. tsugae, S. sclerotiorum, and P. infestans (Leotiales) relied on 88 informative characters and resulted in six equally parsimonious trees (FIG. 5.2a). In four of the six trees, M. laricis occupied the basal position of the Rhabdocline and M laricis Glade. Removing P. infestans from the analysis and retaining either or both F. tsugae and S. sclerotiorum resulted in a single most parsimonious tree agreeing with the topology in FIG. 5.1 (data not shown). The parsimony analysis of Rhabdocline and M. laricis only (analysis IIB) used 66 phylogenetically informative characters. The two subspecies of R. weirii were polyphyletic as they were in analysis I (FIG. 5.2b). Determining polarity using midpoint rooting placed the root on the long internal branch between R. weirii ssp. weirii and the remaining taxa. Fabrella tsugae was selected to construct a hypothetical ancestor because it had the highest range of sequence similarity to the Rhabdocline spp. and M. laricis (84.0-89.5%) in analysis I. Lundberg rooting located the root at the same point as the midpoint root, between the two isolates of R. weirii ssp. weirii and the remaining taxa. Retaining trees that were one step longer than the two most parsimonious trees included a tree where the Lundberg root was placed between M. laricis and the remaining taxa. 126

5.5. Discussion

Recognition of biological relationships between asexual and sexual states of higher fungi and their integration into holomorph taxa has long been a major goal among mycologists. Sexual characters are no longer required to place asexual fungi in the same phylogenetic lineage with their relatives that produce teleomorphs (Taylor, 1993). The results of our analyses support a close relationship between the mitotic holomorph M laricis and species of Rhabdocline. As pointed out by Carbone and Kohn (1993) however, formal taxonomic disposition of a mitotic species in an ascomycete genus, even on the basis of highly supported molecular characters, would require a revision of Article 59 of the International Code of Botanical Nomenclature. Hence, this report presents evidence supporting the connection between M laricis and Rhabdocline predicted by Sherwood-Pike et al. (1986) but does not propose a new nomenclatural combination. The transfer of species parasitic on nematodes formerly classified in Meria to a new genus, Drechmeria (Gams & Jansson, 1987), leaves the genus comprised of the two species considered here: M laricis and M parkeri. As such, Meria represents a homogeneous taxon of anamorphic Rhabdocline. The coelomycete Rhabdogloeum pseudotsugae is the only other mitosporic species having an established anamorph connection to Rhabdocline. Although obviously no less related than the teleomorph taxa, morphological differences, such as conidiomatal type, structure of conidiophores, and presence/absence of conidial appendages, dictate that these anamorph taxa be disposed in separate genera within the current framework for classification of conidial fungi. Although this situation exemplifies difficulties posed by the current artificial system of anamorph classification, Gams (1995) has argued in favor of its continuing utility. We observed variation in branching patterns within the Rhabdocline Glade while refining the sequence alignment and selecting taxa for analysis (FIGS. 5.1 and 5.2). Most importantly, M laricis and R. weirii alternately occupied basal positions depending on the alignment and taxa included in the analysis. This circumstance arose at least in part because of the greater divergence between sequences of R. weirii ssp. weirii and other members of the Rhabdocline and M laricis group. The monophlyetic grouping of M 127

laricis in a single Glade together with Rhabdocline species and subspecies remained consistent, however, regardless of other taxa included in the analysis. To enhance the identification of primitive character states in the Rhabdocline and M laricis group, a single, closely related outgroup was selected for analysis IIA (Smith, 1994). Fabrella tsugae, S. sclerotiorum, and P. infestans (Leotiales) were selected because they were closest to Rhabdocline and M laricis based on sequence similarity and their topology in analysis I. The uncertain ordinal placement of Rhabdocline and limited sampling of genera makes it uncertain whether the sister group to the genus was included in the analysis. The six most parsimonious trees varied in the placement of R. weirii and M laricis relative to the other taxa of Rhabdocline (FIG. 5.2a). Removing one of the three outgroups (P. infestans) resulted in a single most parsimonious tree with R. weirii ssp. weirii basal and R. weirii polyphyletic as in analysis I. These results, together with the observation in analysis IIB that alternate Lundberg rootings were only one step away, suggested that the outgroup, midpoint, and Lundberg rooting did not provide a robust determination of the direction of evolution in the group.

Evolutionary divergence of hosts is one possibility for explaining the speciation and presumed loss of a teleomorph in Meria laricis. Larix and Pseudotsuga are considered sister genera within Pinaceae based on details of fertilization and pollen development (Christiansen, 1972), immunoassays (Prager et al., 1976; Price et al., 1987), nucleotide sequence data from the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (rbcL)(Chase et al., 1993) and chloroplast DNA RFLPs (Tsumura et al., 1995). The date of divergence between Pseudotsuga and Larix is recent relative to other members of the Pinaceae; both occur in North American fossil deposits dated at approximately 45 million years old (Hermann, 1985; Axelrod, 1990).

Rhabdocline parkeri and M laricis may have diverged as a result of congruent host speciation, or alternatively, a more recent host shift may have occurred. The results of this study are equivocal respecting these competing hypotheses. Nucleotide sequence divergence in the ITS region between M laricis and R. parkeri is low, while sequence divergence between Pseudotsuga and Larix ITS regions, particularly the ITS-1 region, is 128

higher (Liston et al., 1996; Gernandt and Liston, unpublished). Generation time, and probably relative evolutionary rates of the ITS region, differ between Rhabdocline taxa and their hosts, but the high degree of sequence similarity between M laricis and R. parkeri favors more recent host-shifting rather than congruent speciation to explain the presence of Rhabdocline and M laricis on closely related hosts. Because development of ascocarps of Rhabdocline species requires at least one year, loss of the ascigerous state in M laricis could have resulted from the deciduous habit of its host. Contrary to this hypothesis, M laricis is the sister group to Rhabdocline in one of the most parsimonious interpretations of analysis IIA. Also, in phylogenetic reconstructions where M. laricis is a derived member of Rhabdocline, the pathogenic species of Rhabdocline were rendered polyphyletic by transposition of R. weirii ssp. weirii from an internal position, where it is paraphyletic with R. weirii ssp. obovata, to the basal position of the Rhabdocline and M laricis Glade. The reliability of ascus characters in ascomycete systematics has been frequently questioned. Tissue layers that give histochemical reactions such as the iodine ("amyloid") reaction may be developmentally non homologous and ascus types within families may be heterogeneous (Bellemere, 1994). Iodine reactions can be variable depending on whether fresh or herbarium specimens are used and on the reagents and pretreatments employed (Baral, 1987). Although instances of variation in iodine reactions within some species have been noted (Kohn and Korf, 1975), bluing of ascal structures in iodine has been considered generally consistent within genera. Rhabdocline is unusual in having species with structural differences that result in variation in iodine reaction; asci of the I" species, R. pseudotsugae, lack a thickened apex and apical pore that is present in the I+ species R. weirii and R. parkeri (Parker and Reid, 1969; Sherwood-Pike et al., 1986). A similar pattern has been observed among species of Chloroscypha. Species having asci with an apical pore also react positively with iodine following KOH pretreatment, but those lacking an apical pore react negatively (Petrini, 1982). The infraspecific taxa recognized by Parker and Reid (1969) grouped as separate, unambiguous clades in our analyses. The phylogenetic grouping of these taxa based on ITS sequences, however, does not agree in all respects with the taxonomic scheme of 129

Parker and Reid (1969), in which the ascus pore/iodine reaction is the primary character used to differentiate species. Rhabdocline weirii ssp. obovata, which has r asci and no anamorph, formed a Glade with the R. pseudotsugae ssp. pseudotsugae, which also lacks an anamorph. Rhabdocline weirii ssp weirii, which has I+ asci and an associated Rhabdogloeum anamorph formed a separate Glade, as did the Meria isolates from Douglas-fir, representing the I+ R. parkeri. Iodine negative asci appear to result from a single loss in speciation of the most derived taxon, R. pseudotsugae. The cladistic patterns in our analyses suggest that the possession of a Rhabdogloeum or Meria anamorph may be a more reliable basis for establishing systematic relationships in Rhabdocline than the possession of an apical pore as identified by iodine reaction. Our analyses suggest that loss of a conidial state occurred prior to speciation of the R. pseudotsugae and R.weirii ssp. obovata group (FIG. 5.1). Disagreement between the phylogenetic hypotheses based on ITS sequences and morphological characters points to a need for further study to resolve this discordance. To date we have been unable to obtain fresh collections of the r R. weirii ssp. oblonga, a taxon recognized by Parker and Reid (1969) with some reservations. This subspecies resembles R. weirii ssp. weirii but is not associated with the epiphyllous Rhabdogloeum pseudotsugae anamorph. It is known primarily from the southwestern U.S., where it may be endemic, and from cultivated Douglas-fir in the northeastern U.S. An unusual I- Rhabdocline associated with a hypophyllous anamorph (Rhabdogloeum hypophyllum Ellis & Gill) also has been collected from the southwestern U. S. (Ellis and Gill, 1945). This species was later transferred to Cryptocline (Morgan-Jones, 1973) but has characteristics of other anamorphs of Rhabdocline, raising the possibility that a third anamorph, Cryptocline hypophyllum (Ellis & Gill) Morgan-Jones, may be associated with the genus Rhabdocline. Both the familial and ordinal placement of Rhabdocline remains uncertain. Korf (1962) included Rhabdocline in the family Hemiphacidiaceae together with the genera Naemacyclus, Didymascella, Sarcotrochila, Fabrella, Lophophacidium, and Hemiphacidium. Korfia was described later (Reid and Cain, 1963). Characteristics shared by these genera are apothecia of simple structure, developing subepidermally on 130

foliage of conifer hosts, and lacking marginal excipular tissue. Late maturation of ascospores characterized by septum formation and cell wall pigmentation accompanied by degeneration of some cells is typical. Genera with ascomata developing beneath an upper covering of fungal tissue, Lophophacidium and Naemacyclus (both noted as questionable inclusions by Korf (1962)), subsequently were transferred to Rhytismataceae (DiCosmo et al., 1983a; Ericksson and Hawksworth, 1993) along with two species originally disposed in Naemacyclus and later segregated as a new genus, Cyclaneusma (DiCosmo et al., 1983b). Although ascocarps of Rhabdocline do not develop beneath a covering layer of fungal tissue, as in Lophophacidium and Cyclaneusma, Eriksson and Hawksworth (1993) transferred Rhabdocline to Rhytismataceae. Considering similarities in ascomatal structure and development and ascospore maturation and germination behavior, Hemiphacidiaceae (Leotiales), as originally proposed by Korf (1962), seems a more suitable taxon to accommodate Rhabdocline than Rhytismataceae. Although our analyses suggest that Rhabdocline may be more closely related to some genera representing Leotiales (Sclerotinia, Fabrella, Phacidium) than those representing Rhytismatales (Colpoma, Tryblidiopsis, Lophodermium, Cyclaneusma, Darkera), they do not strongly support inferences regarding higher order disposition of Rhabdocline and related genera. We should emphasize that the analyses presented here were designed to study relationships among the Rhabdocline taxa and M. laricis and were not designed to address familial and ordinal relationships within Leotiales and Rhytismatales. Therefore the relationships among taxa outside the Rhabdocline and M laricis Glade suggested in FIG. 1, particularly those lacking high bootstrap support, should be considered very tentative. Nucleotide sequence data from the ITS region and small subunit of nrDNA of a broader sampling of Hemiphacidiaceae and Rhytismataceae are being analyzed to address the ordinal rank and other phylogenetic relationships among Rhabdocline and several closely related genera in these two families. Broader sampling of genera of Hemiphacidiaceae and the inclusion of additional characters should result in a more robust phylogeny. 131

5.6. Acknowledgments

We thank Walter Gams, Aaron Liston, Orlando Petrini, Amy Rossman, Barbara Schulz, Joey Spatafora, and an anonymous reviewer for helpful comments on earlier versions of this manuscript. Koren Dennis and Aleta Groenig provided laboratory assistance. All sequencing was performed by the Central Services Laboratory within the Center for Gene Research and Biotechnology at Oregon State University. We are indebted to Ernest Ash III, Brenda Callan, George Carroll, John Dennis, Rolf Kehr, Everett Hansen, Ottmar Holdenrieder, Dan Jones, Alan Kanaskie, Blakey Lockman, Bill Merrill, Catherine Parks, and Craig Schmidt for providing specimens. Funding was provided by a grant from the Oregon State University Research Council.

5.7. Literature Cited

Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.

Axelrod, D. 1. 1990. Environment of the Middle Eocene (45 Ma) Thunder Mountain flora, central Idaho. National Geographic Research 6: 355-361.

Baral, H. 0. 1987. Lugol's solution/IKI versus Melzer's reagent: hemiamyloidity, a universal feature of the ascus wall. Mycotaxon 29: 399-450.

Bellemere, A. 1994. Asci and ascospores in ascomycete systematics. Pp. 111-126. In: Ascomycete systematics, problems and perspectives in the nineties. NATO ASI Series A, Vol 269. Ed., D. L. Hawksworth. Plenum Press, New York, New York.

Carbone, I. and L. M. Kohn. 1993. Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae. Mycologia 85: 415-427.

Chase, M. W., D. E. Soltis, R. G. Olmstead et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Missouri Bot. Gard. 80: 528-580. 132

Christiansen, H. 1972. On the development of pollen and the fertilization mechanisms of Larix and Pseudotsugae menziesii. Silva Genetica 21: 166-174.

DiCosmo, F., T. R. Nag-Raj, and B. Kendrick. 1983a. Prodromus for a revision of the Phacidiaceae and related anamorphs. Canad. J. Bot. 61: 31-44.

., H. Peredo, and D. W. Minter. 1983b. Cyclaneusma gen. nov., Naemacyclus and Lasiostictis, a nomenclatural problem resolved. Eur. J. For. Path. 13: 206­ 212.

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

Ellis, D. E. and L. S. Gill. 1945. A new Rhabdogloeum associated with Rhabdocline pseudotsugae in the Southwest. Mycologia 37: 326-332.

Eriksson, 0. E. and D. L. Hawksworth. 1993. Outline of the Ascomycetes 1993. Syst. Ascom. 12: 51-257.

Gams, W. 1995. How natural should anamorph taxa be? Canad J. Bot. 73: S747-S753.

Gams, W., and H.-B. Jansson. 1987. The nematode parasite Meria coniospora Drechsler in pure culture and its classification. Mycotaxon 22: 33-38.

Gargas, A., P. T. DePriest, and J. W. Taylor. 1995. Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Mol. Biol. Evol. 12: 208-218.

Genetics Computer Group. 1991. Program manual for the GCG package, version 7. 575 Science Drive, Madison, Wisconsin.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp.

Hermann, R. K. 1985. The genus Pseudotsuga: ancestral history and past distribution. Spec. Publ. No. 2b, Forest Research Laboratory, Oregon State University, Corvallis, Oregon.

Kohn L. M. and R. P. Korf. 1975. Variation in ascomycete iodine reactions: KOH pretreatment explored. Mycotaxon 3: 165-172.

Korf, R. P. 1962. A synopsis of the Hemiphacideaceae, a family of Helotiales (discomycetes) causing needle blights of conifers. Mycologia 54: 12-33. 133

Liston, A., W. A. Robinson, J. M. Oliphant, and E. R. Alvarez-Buylla. 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non­ flowering seed plants. Syst. Bot. 21: 109-120.

Lundberg, J. G. 1972. Wagner networks and ancestors. Syst. Zool. 21: 398-413.

Morales, V. M., L. E. Pelcher, and J. L. Taylor. 1993. Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups. Curr. Genet. 23: 490-495.

Morgan-Jones, G. 1973. Genera coelomycetarum. VII. Cryptocline Petrak. Canad. I Bot. 51: 309-325.

Nixon, K. C., and J. M. Carpenter. 1993. On outgroups. Cladistics 9: 413-426.

O'Donnell, K. 1992. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Curr. Genet. 22: 213-220.

Parker, A. K., and J. Reid. 1969. The genus Rhabdocline Syd. Canad. J. Bot. 47: 1533­ 1545.

Petrini, 0. 1982. Notes on some species of Chloroscypha endophytic in Cupressaceae of Europe and North America. Sydowia 35: 206-222.

Prager, E. M., D. P. Fowler, and A. C. Wilson. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

Price, R. A., J. Olsen-Stojkovich, and J. M. Lowenstein. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Syst. Bot. 12: 91-97.

Reid, J. and R. F. Cain. 1963. New genus of the Hemiphacidiaceae. Mycologia 55: 781­ 785.

Reynolds, D. R. 1993. The fungal holomorph: an overview. Pp. 15-25. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

Sherwood-Pike, M., J. K. Stone, and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canad. I Bot. 64: 1849-1855.

Smith, A. B. 1994. Rooting molecular trees: problems and strategies. Biol. J. Linn. Soc. 51: 279-292. 134

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gil levet. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Strauss, S. H., A. H. Doerksen, and J. R. Byrne. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canad. J. Bot. 68: 1502-1510.

Swofford, D. L. 1991. PA UP: Phylogenetic analysis using parsimony, Version 3.0s+ 4 . Illinois Natural History Survey, Champaign, Illinois.

Sydow, H. and F. Petrak. 1922. Ein Beitrag zur Kenntnis der Pilzflora Nordamericas, insbesondere der nordwestlichen Staaten. Ann. Mycol. 20:178-218.

Taylor, J. W. 1993. A contemporary view of the holomorph: nucleic acid sequence and computer databases are changing fungal classification. Pp. 1-13. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

Tsumura, Y., K. Yoshimura, N. Tomaru, and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theor. Appl. Genet. 91: 1222-1236.

White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols, a guide to methods and applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White. San Diego: Academic Press.

Wulf, Von A., and L. Pehl. 1996. Erster Fund von Fabrella tsugae (Farl.) Kirschst. in der Bundesrepublik Deutschland - ein neuer Nadelpilz an Tsuga canadensis (L.) Carr. Nachrichtenbl. Deut. Pflanzenschutzd 48: 1-4. 135

Chapter 6 Analysis of Nuclear Ribosomal DNA Places the Nematode Parasite, Drechmeria coniospora, in Clavicipitaceae (Hypocreales)

David S. Gernandt Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331-2902

6.1. Abstract

Drechmeria coniospora, a nematode endoparasite, was originally described by Drechsler and placed in the hyphomycete form-genus Meria, together with Meria laricis, a foliar parasite of Larix (Pinaceae). Although both species produce 1-celled hyalospores from serial phialides on hyphal conidiomata, the new genus Drechmeria was proposed to accommodate the nematode parasite based on pure culture studies, ecological differences, and having integrated phialides compared to the mainly discrete phialides of M. laricis. Nuclear ribosomal DNA sequences were determined from an accession that continued to produce the diagnostic features of D. coniospora in culture. BLAST comparisons of D. coniospora nrDNA to nucleotide databases revealed high similarities to accessions for species belonging to the perithecial ascomycete order Hypocreales. Parsimony analysis of partial sequences from the small subunit and full ITS region of accessions from the Hypocreales, as well as other species representing the phylogenetic diversity of ascomycetes, placed Drechmeria in the hypocrealean family, Clavicipitaceae. Drechmeria coniospora shares the ecological strategy of animal parasitism also exhibited by some members of Clavicipitaceae. The affinity of Meria laricis to the inoperculate discomycete genus of foliar parasites, Rhabdocline, has already been demonstrated using nrDNA evidence. These findings support the separation of the anamorphic fungi Drechmeria and 136

Meria into distinct genera and indicate that any implied relationship between the two genera is phylogenetically unwarranted.

6.2. Introduction

Drechmeria coniospora (Drechsler) W. Gams & Jansson is an asexual fungus that attacks nematodes (Drechsler, 1941; Barron, 1977). Drechsler (1941) originally placed D. coniospora in the form-genus Meria, along with Meria laricis Vuill., a needle cast pathogen of Larix (Pinaceae). Both species produce 1-celled hyalospores from serial phialides on hyphal conidiomata, but Gams and Jansson (1987) recognized that M coniospora Drechsler produced phialides that were subtended by a conidiophore (integrated) while M laricis produced phialides lacking a subtending conidiophore (discrete). Considering this and the differences in ecological specialization between the two fungi, Gams & Jansson proposed Drechmeria as a separate genus for D. coniospora and for the animal biotrophic fungus, D. harposporioides (Barron and Szijarto) W. Gams & Jansson. The latter species has been reported to attack ciliated protozoans (Barron and Szijarto, 1980) Parasitic and predatory fungi attacking amoebae, nematodes, rotifers, and other small animals have been described from Zoopagales (Zygomycota), Saprolegniales (Oomycota), Agaricales (Basidiomycota) and from several genera of mitosporic Ascomycota. While D. coniospora and M. laricis are both known to be asexual ascomycetes, it remains to be demonstrated whether the historical classification of D. coniospora together with M laricis, based primarily on conidiophore morphology, reflects a close phylogenetic relationship between these fungi. Morphological (Sherwood-Pike et al., 1986) and molecular (Gernandt et al., 1997) information have been used to unite Meria laricis with Rhabdocline, a genus comprised entirely of foliar parasites of Pseudotsuga (Pinaceae). Rhabdocline is an inoperculate discomycete of uncertain familial and ordinal placement, disposed in either Hemiphacidiaceae [Leotiales (Korf, 1962)] or Rhytismatales (Hawksworth et al., 1995). Recent cladistic analysis using nuclear 137

ribosomal DNA (nrDNA) support the former (Gernandt et al., 1997; Gernandt et al., in prep.). Members of these two discomycete orders are comprised predominantly of parasites of plant foliage or bark. With the exception of several genera in Orbiliaceae, nematode predation is not known among the Leotiales (Pfister, 1997). The objective of this study is to use comparative sequence analysis of nuclear ribosomal DNA to determine the phylogenetic position of D. coniospora.

6.3. Materials and Methods

Fungal isolates -The fungi sampled are alphabetized by ordinal classification in Table 6.1 and 6.2 using the system outlined by Hawskworth et al. (1995) except where otherwise specified. All sequences except those for Drechmeria coniospora were obtained from Genbank. A culture of D. coniospora was supplied from Centraalbureau voor Schimmelcultures, Baarn, Netherlands (CBS 596.92). Diagnostic features of the species were determined microscopically prior to DNA isolation.

Sequence determination - -DNA for Drechmeria coniospora was extracted using the method of Doyle and Doyle (1987). Primers used for amplification of the small subunit were NS1 and NS24 (White et al., 1990). NS2 and SR11R (Spatafora et al., 1995) were used as internal sequencing primers. The ITS region was amplified using the primers ITS5 and ITS4 (White et al., 1990). PCR reactions were performed in a 50 p.L volume using 2.5 U TflTM DNA polymerase (Epicentre Technologies, Madison, WI), 1.5 mM MgC12, 7.5 pmol dNTP (10 mM, pH 7.0, Epicentre Technologies, Madison, WI), 10 pmol of each primer, and 5% BSA. PCR amplification included 35 cycles of denaturing at 95 C, annealing at 52 and 55 C for the small subunit and ITS region respectively, and an extension at 72 C, each for 60 s. The amplification was concluded with a final extension step for 7 min at 72 C. PCR products were precipitated in a 0.5 volume of 5 M ammonioum acetate and 1.5 volume of isopropanol. Automated cycle sequencing of 5 138

pmol PCR product was performed using dye-terminator chemistry with an ABI 377 fluourescent sequencer. Sequence reads were determined in both the 5' to 3' and 3' to 5' direction for the small subunit. For the ITS region sequence of D. coniospora, only the ITS5 read was used because the reverse read (3' to 5') was of poor quality.

Sequence analysis -- Sequences were assembled and aligned manually using Genetic Data Environment version 2.2 (Smith et al., 1994). BLAST (Altschul et al., 1990) and gapped- BLAST were used to identify database accessions of high similarity to the SSU and ITS region sequence of D. coniospora. Accessions of high similarity, as well as several representing other orders of ascomycetes, were downloaded from Genbank and used to generate a molecular data set. Sequences were analyzed using PAUP v. 4.d62 (Swofford, 1998). Areas of ambiguous alignment in the ITS region data set were excluded from the analysis and gaps were treated as missing characters. A branch-and-bound search on the original and the bootstrapped ITS region data set were performed. The bootstrap included 500 replicates. For the small subunit data set, a heuristic search was employed that included the following options: mulpars on, tree-bisection reconnection and random sequence addition with 10 replicates. Based on the observed transition:transversion ratio, transversions were weighted 1.2:1 in the small subunit analysis. Bootstrap values were generated using 100 replicates, with a maximum of 500 trees saved for each replicate. Phylogenetic trees were displayed using TREEVIEW (Page, 1996).

6.4. Results

A gapped-BLAST search using the partial SSU sequence of D. coniospora recovered over 110 fungal nrDNA accessions. Seventeen of the 18 highest scoring matches were with genera in Hypocreales. The other match was to an accession designated as Taphrina deformans deposited by Wilmotte et al., 1993). A BLASTn search using the ITS region sequence of D. coniospora retrieved 100 accessions, all members of Hypocreales. Highest scoring matches were with species of Beauveria and 139

Table 6.1. Accessions included in the partial SSU analysis of perithecial ascomycetes.

Order Family Species Genbank no.

Hypocreales Clavicipitaceae sterile epibiont of Baccharis coridifolia D.C. U77179

Hypocreales Clavicipitaceae Atkinsonella hypoxylon (Peck) Diehl U44034

Hypocreales Clavicipitaceae Balansia sclerotica (Pat.) Han. U32399

Hypocreales a Clavicipitaceae Beauveria bassiana (Bals.) Vuill. AF049144

Hypocreales Clavicipitaceae Aciculosporium take Miyake U32404

Hypocreales Clavicipitaceae Cordycepioideus bisporus AF009651

Hypocreales Clavicipitaceae Claviceps paspali Stevens & Hall U32401

Hypocreales Clavicipitaceae Cordyceps capitata (Fr.) Link U44041

Hypocreales Clavicipitaceae Cordyceps militaris (L. ex St. Amans) Link AF049146

Hypocreales Clavicipitaceae Cordyceps ophioglossoides U46881

Hypocrealesa Clavicipitaceae Drechmeria coniospora (Drechsler) Gams &

H.-B Jansson

Hypocreales Clavicipitaceae Echinodothis tuberiformis U44042

Hypocreales Clavicipitaceae Epichloe typhina (Pers.:Fr.) Tul. U32405

Hypocrealesa Clavicipitaceae Neotyphodium coenophialum

(Morgan-Jones & W. Gains) Glenn,

Bacon & Hanlin U45942

Hypocrealesa Clavicipitaceae Tolypocladium inflatum W. Gains AF049154

Hypocrealesa Clavicipitaceae Verticillium lecanii AF049155

Hypocrealesa Clavicipitaceae Verticillium psalliotae Treschow AF049157

Hypocrealesa Hypocreaceae Geosmithia putterillii (Thom) Pitt D88319

Hypocreales a Clavicipitaceae Hirsutella thompsonii F. E. Fisher U32406

Hypocreales Clavicipitaceae Paecilomyces tenuipes D85136

Hypocreales a Hypocreaceae Aphanocladium album (Preuss) W. Gains AF046143 140

Table 6.1. (continued)

Order Family Species Genbank no.

Hypocreales Hypocreaceae Aphysiostroma stercorarium Barassa,

Martinez & Moreno U32398

Hypocreales a Hypocreaceae Cladobotryum obclavatum AF049145

Hypocreales Hypocreaceae Hypocrea lutea (Tode) Petch D14407

Hypocreales Hypocreaceae Hypocrea schweinitzii (Fr.) Sacc. L36986

Hypocreales Hypocreaceae Hypocrella sp. "GJS 89-104" U32409

Hypocreales Hypocreaceae Hypomyces chrysospermus Tulasne M89993

Hypocreales Hypocreaceae Hypomyces polyporinus Peck U32410

Hypocreales Hypocreaceae Nectria cinnabarina (Tode:Fr.) Fr.

"GJS 89-107" U32412

Hypocreales Hypocreaceae Neocosmospora vasinfecta E.F. Sin. U44117

Hypocreales Hypocreaceae Sphaerostilbella (Henn.) Sacc. & D. Sacc.

Hypocreales Hypocreaceae "Taphrina deformans" (Berk.) Tul. X69852

Hypocreales a Hypocreaceae Verticimonosporium diffractum Mats. AF049159

Hypocreales a Phylacoraceae Colletotricum gloeosporioides Penzig M55640

Hypocreales a Stanjemonium spectabile (W. Gams) W. Gains

Hypocreales a Engyodontium album AF049147

Hypocreales a S'tanjemonium fuscescens W. Gains,

Schroers & S. Abdullah AF049148

Hypocreales a Stanjemonium grisellum W. Gains Schroers

& M. Christensen AF049149

Microascales Ceratocystis fimbriata Ellis & Halst. U32418

Microascales Microascaceae Microascus trigonosporus C.W. Emmons &

B.O. Dodge L36987 141

Table 6.1. (continued)

Order Family Species Genbank no.

Microascales a Microascaceae Lomentospora prolificans U43409

Hypocreales Melanosporaceae Melanospora fallax U47842

8 Anamorphic species.

Trichoderma. One isolate was designated Tolypocladium niveum, but this species has been moved to Beauveria (von Arx, 1986). Based on the blast results, an initial parsimony search that included ascomycete taxa outside of Hypocreales, and results from other studies (Spatafora and Blackwell, 1993, 1994; Glenn et al. 1996, Ogawa et al., 1997), three taxa from the closely-related perithecial order, Microascales were chosen as an outgroup in the small subunit analysis. Phylogenetic analysis of the partial small subunit data set placed D. coniospora in a well supported Hypocreales Glade (FIG. 6.1). Colletotrichum gloeosporioides (Phyllacorales) was a sister species to the Hypocreales Glade and a Glade comprised of representatives from three genera of Microascales (Lomentospora prolificans, Microascus trigonosporus, and Ceratocystis fimbriata) formed a monophyletic group with high (99%) bootstrap support. Drechmeria was in a sister relationship with a sterile epibiont from Baccharis coridifolia (Asteraceae)(Bertoni et al., 1997), but bootstrap support was low (42%). The two were included within a larger Clavicipitaceae Glade in all equally most parsimonious trees in preliminary analyses that sampled fewer genera than shown in FIG. 6.1, but bootstrap support was low (results not shown). Clavicipitaceae and Hypocreaceae were not resolved in the strict consensus tree from the weighted parsimony analysis (FIG. 6.1). The patristic distance between D. coniospora and the B. coridifolia epibiont was 97.0%. 142

Table 6.2. Accessions included in the ITS region analysis.

Order Family Species Genbank no.

Hypocreales Clavicipitaceae Atkinsonella hypoxylon (Peck) Diehl U780511U78052

Hypocreales Clavicipitaceae Balansia henningsiana (A. Moller) Diehl U78057/U78058

Hypocreales a Clavicipitaceae Beauveria cylindrospora (W. Gams)

von Arx Z54105

Hypocreales a Clavicipitaceae Beauveria geodes (W. Gains) von Arx U19037

Hypocreales a Clavicipitaceae Beauveria nivea (Rostrup) von Arx Z54114

Hypocreales a Clavicipitaceae Beauveria tundrense Z54108

Hypocreales Clavicipitaceae Cordyceps capitata (Fr.) Link U57668

Hypocreales a Clavicipitaceae Drechmeria coniospora (Drechsler)

Gains & H.-B. _Nilsson

Hypocreales Clavicipitaceae Epichloe typhina (Pers.:Fr.) Tul. L07133

Hypocreales Clavicipitaceae Neotyphodium uncinatum (W. Gains,

Petrini & D. Schmidt) Glenn, Bacon

& Han lin L20305

Hypocreales Hypocreaceae Fusarium sambucinum Fuckel U38279

Hypocreales Hypocreaceae Gliocladium deliquesens AFO20799

Hypocreales Hypocreaceae "Hypocrea rufa" GJS 89-142 X93987

Hypocreales Hypocreaceae Hypocrea schweinitzii (Fr.) Sacc. X93968

Hypocreales Hypocreaceae Nectria albosuccinea

Hypocreales Hypocreaceae Nectria cinnabarina (Tode: Fr.) Fr. L36626

Hypocreales a Hypocreaceae Trichoderma ghanense Doi et al. Z69588

Hypocreales a Hypocreaceae Trichoderma harzianum Rifai U78881

Hypocreales a Hypocreaceae Trichoderma inhamatum Z68187 143

Table 6.2. (continued)

Order Family Species Genbank no.

Hypocreales a Hypocreaceae Trichoderma parceramosum Bissett Z48936

Hypocreales a Hypocreaceae Trichoderma "todica" (=parceramosum) Z48727

Hypocreales a Hypocreaceae Verticillium albo-atrum Reinke &

Berthier L19499

Hypocreales a Hypocreaceae Verticillium tricorpis I. Isaac L28679 a Anamorphic species

The isolate labeled as Taphrina deformans (Genbank # X69852) was a sister taxon to Aphysiostroma stercoreum within the Hypocreaceae Glade, and was dissimilar to other accessions of Taphrina deformans in Genbank (Nos. U00971 and U20376, results not shown). In the ITS region analysis, Drechmeria coniospora was in a moderately-supported Glade (68% boostrap) with the insect-biotrophic genera, Cordyceps capitata, Beauveria spp. and Cordyceps capitata (Clavicipitaceae) (FIG. 6.2). Atkinsonella hypoxylon, Balansia henningsiana, Epichloe typhina and Neotyphodium uncinatum formed a monophyletic group in a sister position to the Glade of D. coniospora, C. capitata, and Beaveria spp. Species of Trichoderma formed a separate Hypocrealean Glade with Gliocladium deliquescens and an isolate tentatively identified as Hypocrea rufa. The relationships among Trichoderma spp. have been treated thoroughly by Kuhls et al., 1997. Two species of Verticillium attached to this Glade on a long branch. The position of Nectria sp. and Fusarium sambucinum was intermediate between the two clades. Drechmeria coniospora had the shortest patristic distance to B. nivea. Inclusion of ambiguously aligned characters and the 5.8S did not change the position of D. coniospora, although the number of equally most parsimonious trees increased (results not shown). In the neighbor joining analysis, D. coniospora was basal to the species of Beauveria, while 144

Verticillium lecanii Verticillium psalliotae Beauveria bassiana 59 Cordyceps militaris Paecilomyces tenuipes

97 Epichloe typhina 65 Neotyphodium coenophialum Atkinsonella hypoxylon r Claviceps paspali L Aciculosporium take Hypocrella sp.

100 Cordycepioideus bisporus Hirsutella thompsonii

6 Aphysiostroma stercorarium 95 Hypocrea schweinitzii 90 non Taphrina Hypocrea lutea Hypomyces polyporinus 71 Sphaerostilbella sp. 97 Verticimonosporium diffractum Cladobotryum obclavatum Aphanocladium album 99 Cordyceps capitata Cordyceps ophioglossoides Drechmeria coniospora EBaccharis epibiont Balansia scierotica Tolypocladium inflatum Engyodontium album

81 Nectria cinnabarina Neocosmospora vasinfecta 100E Stanjemonium grisellum 90 Stanjemonium fuscescens Stanjemonium spectabile Melanospora fallax Geosmithia putterillii Colletotrichum gloeosporioides 74c Lomentospora prolificans 99 Microascus trigonosporus Ceratocystis fimbriata

FIG. 6.1. Phylogenetic tree of perithecial ascomycetes inferred from the small subunit. Majority rule consensus of 500 equally most parsimonious trees using partial SSU sequences (378.0 steps, CI = 0. 473, RI = 0. 687, and RC = 0.425). Transitions were weighted 1.2:1 over transversions. Maxtrees was exceeded. Bootstrap values equal to or greater than 50% are displayed above the branches. 145

97­ Beauveria nivea Beauveria tundrense Beauveria cylindrospora 3 Beauveria geodes 68 Cordyceps capitata Clavicipitaceae Drechmeria coniospora Epichloe typhina Neotyphodium uncinatum 92 L Balansia henningsiana Atkinsonella hypoxylon Nectria cinnabarina Nectria albosuccinea cf. Fusarium sambucinum Gliocladium deliquescens Hypocrea rufa? GJS 89 142 Trichoderma harzianum Trichoderma inhamatum Hypocreaceae Trichoderma ghanense Trichoderma todica Trichoderma parceramosum Trichoderma longibrachiatum Hypocrea schweinitzii

100 Verticillium albo atrum Verticillium tricorpis

FIG. 6.2. Phylogenetic tree of perithecial ascomycetes inferred from the ITS region. One of seven equally most parsimonious trees from a heuristic search of ITS1 and ITS2 sequences. Based on 155 phylogenetically informative characters (480 steps, CIe = 0.616, RI = 0.744, and RC = 0.507). Nodes that collapse in the strict consensus tree are indicated with an asterisk. Bootstrap values equal to or greater than 50% are displayed at the branches. 146

Cordyceps capitata was in a derived sister relationship with B. geodes (results not shown). The sequence similarity between D. coniospora and B. nivea was 88.6 % for ambigous sites plus most of the 5.8S excluded, and 90.0 % with all sites included. The similiarity between D. coniospora and C. capitatum was 87.2 % in the partial alignment and 88.1 % with all sites included.

6.5. Discussion

Analysis of nuclear ribosomal DNA places D. coniospora within Clavicipitaceae. This is consistent with the ecological diversity of the family, which includes parasites of fungi, plants, and animals. Analysis of small subunit sequences identified a sterile Baccharis epibiont as the most closely related sister taxon to Drechmeria. Hypocrealean affinities of this fungus have been previously reported (Bertoni et al., 1997). Resolution of the group was poor using partial small subunit sequences, but indicated that D. coniospora did not have a strong affinity to other genera available in Genbank. More thorough analyses of relationships between Clavicipitaceae and Hypocreaceae are provided by Spatafora and Blackwell (1993), Glenn et al. (1996), Ogawa et al. (1997) and Gams et al. (1998). Other sources of nucleotide sequences in addition to the small subunit of nrDNA are analyzed in most of these studies. In the ITS region tree, Drechmeria was in a basal position of a Glade that included Cordyceps capitata isolated from Elaphomyces and several species of Beauveria. These three genera appeared in the hypocreales Glade in the small subunit analysis, but their relationship with D. coniospora was not strong enough to be resolved in the consensus tree or to receive bootstrap support over 50%. This is probably because more Clavicipitaceae accessions were available for the small subunit analysis. Other genera sampled in the small subunit analysis are parasitic on animals, yet parasitic genera were not confined to a well supported Glade. Cordyceps includes species parasitic on fungi and insects. Beaveria is another anamorphic genus that includes B. 147

bassiana, the causal agent of muscardine disease of silkworm larvae (Beauverie, 1914) and also parasitic on other Lepidoptera and other insects, and the cause of keratitis in humans (MacLeod, 1954). Beauveria and Drechmeria both exhibit holoblastic conidium ontogeny, producing one-celled hyalospores in schizolytic secession from hyphal conidiomata. In Beauveria, proliferation of conidiogenous cells is holoblastic sympodial with maturation by diffuse wall building, while in Drechmeria, proliferation of conidiogenous cells is percurrent enteroblastic followed by replacement apical-wall building (Hawksworth et al., 1995). It is surprising but not unprecedented that such dissimilar modes of conidium ontogeny are represented in two apparently closely related anamorph genera (e.g. Nag Raj and Kendrick, 1993; Wingfield et al., 1991). In general, perithecial ascomycetes include a number of lineages that form associations with animals. Laboulbeniales are arthropod ectoparasites, species of Cordyceps (Clavicipitaceae) parasitize fungi, nematodes, Lepidoptera, and Hymenoptera, and fungi in Microascales and Ophiostomatales have specialized adaptations with insects for dispersal and successful parasitism of plants (eg. Ophiostoma ulmi, the causal agent of Dutch elm disease). Sporothrix schenkii, the causal agent of sporotrichosis, a human mycosis, is also a member of Ophiostomatales. Finally, while the phylogenetic placement of many of the mitosporic genera of predatory fungi has not been demonstrated, at least some members of the form-genus Verticillium have affinities with Hypocreales. Animal symbiosis occurs in all groups of higher fungi, Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. Whether or not it is the ancestral state of the ascomycetes, association with animals appears to occur within a limited number of lineages within the group. The cleistothecial order Onygenales includes most dermatophytes (parasites of skin and keratined tissue) such as Coccidioides iminitis, Blastomyces dermatitidis, and Histoplasma capsulatum. Candida, a form genus that includes the causal agent of candidiasis as well as insect endosymbionts, is an anamorphic member of Saccharomycetales. While Orbiliaceae, currently classified in Leotiales, includes nematode trapping fungi, the other families of inoperculate discomycetes, from which Orbiliaceae seems to be separated (Gernandt et al., in prep.), are comprised of free living fungi (eg. Leotia, Microglossum, and Spathularia), parasites of foliage and bark 148

(eg. Rhytisma, Rhabdocline, and Pezicula), and lichens (eg. Baeomyces and Siphula). Using the ITS region of nrDNA, we previously demonstrated that Meria laricis is an anamorphic member of the inoperculate discomycete genus, Rhabdocline (Gernandt et al., 1997). The phylogenetic analyses of nrDNA presented here demonstrate that the historical placement of Drechmeria coniospora in the asexual genus, Meria, should not be construed to imply a close phylogenetic relationship between Drechmeria and the inoperculate discomycetes to which Meria laricis has been connected.

6.6. Acknowledgments

The author thanks Jeff Stone for encouraging this research and for heroically reviving a contaminated culture of Drechmeria. Jeff Stone and Walter Gams both contributed valuable comments that greatly improved an earlier version of this manuscript. Kerry O'Donnell kindly provided unpublished partial small subunit sequences for many of the species included in this study. Sequencing of Drechmeria coniospora (generously provided by Centraalbureau voor Schimmelcultures) was performed by the Central Services Laboratory within the Center for Gene Research and Biotechnology at Oregon State University. Funding was provided by a Sigma-Xi research grant.

6.7. Literature Cited

Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

Arx, J. A. von. 1986. Tolypocladium, a synonym of Beauveria. Mycotaxon 25: 153-158.

Barron, G. L. The nematode-destroying fungi. Topics in Mycohiology No. 1, 140 pp. Guelph, Ontario. 149

, and E. Szijarto. 1982. A new hyphomycete parasitic on the ciliated protozoans Voticella and Opercularia. Canad. I Bot. 60: 1031-1034.

Beauverie, J. 1914. Les Muscardinesle genre Beauveria Vuillemin. Rev. Gen. Botan. 26: 81-105, 157-173.

Bertoni, M. D., Romero, N., Reddy, P. V., and J. F. White, Jr. 1997. A hypocrealean epibiont on meristems of Baccharis cordifolia. Mycologia 89: 375-382.

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

Drechsler, C. 1941. Some hyphomycetes parasitic on free-living terricolous nematodes. Phytopathology 31: 773-802.

Gams, W., and H.-B. Jansson. 1987. The nematode parasite Meria coniospora Drechsler in pure culture and its classification. Mycotaxon 22: 33-38.

, K. O'Donnell, H.-J. Schroers, and M. Christiansen. 1998. Generic classification of some more hyphomycetes with solitary conidia borne on phialides. Canad. J. Bot. In Press.

Glenn, A. E., C. W. Bacon, R. Price, and R. T. Han lin. 1996. Molecular phylogeny of Acremonium and its taxonomic implications. Mycologia 88: 369-383.

Hawksworth, D. L., Kirk, P. M., Sutton B. C., and D. N. Peg ler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp.

Korf, R. P. 1962. A synopsis of the Hemiphacidiaceae, a family of the Helotiales (Discomycetes) causing needle-blights of conifers. Mycologia 54: 12-33.

Kuhls, K., E. Lieckfeldt, G. J. Samuels, W. Meyer, C. P. Kubicek, and T. Borner. 1997. Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89: 442-460.

MacLeod, D. M. 1954. Investigations on the genera Beauveria Vuill. and Tritiachium Limber. Canad I Bot. 32: 818-890.

Nag Raj, T. R., and W. B. Kendrick. 1993. The anamorph as generic determinant in the holomorph: the Chalara connection in the ascomycetes with special reference to the ophiostomatoid fungi. Pp 61-70 In: M. J. Wingfield, K.A. Seifert, and J. F. Webber (eds.) Ceratocystis and Ophiostoma. Taxonomy, ecology and pathogenicity. APS Press, St. Paul, Minnesota, USA. 150

Ogawa, H., A. Yoshimura, and J. Sugiyama. 1997. Polyphyletic origins of species of the anamorphic genus Geosmithia and the relationships of the cleistothecial genera: evidence from 18S, 5S and 28S rDNA sequence analyses. Mycologia 89: 756­ 771.

Page, R. D. M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358.

Parker, A. K., and J. Reid. 1969. The genus Rhabdocline Syd. Canad J. Bot. 47: 1533­ 1545.

Pfister, D. H. 1997. Castor, Pollux and life histories of fungi. Mycologia 89: 1-23.

Reynolds, D.R. 1993. The fungal holomorph: an overview. Pp. 15-25. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

Sherwood-Pike, M., J. K. Stone, and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canad. J. Bot. 64: 1849-1855.

Smith, A. B. 1994. Rooting molecular trees: problems and strategies. Biological Journal of the Linnean Society 51: 279-292.

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gillevet. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Spatafora, J. W., and M. Blackwell. 1993. Molecular systematics of unitunicate perithecial ascomycetes: the Clavicipitales-Hypocreales connection. Mycologia 85: 912-922

, and . 1994. Polyphyletic origins of Ophiostomatoid fungi. Mycol. Res. 85: 912-922.

, T. G. Mitchell, and R. Vilgalys. 1995. Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of Dematiaceous fungal pathogens. J. Clin. Microbiol. 33: 1322-1326.

Swofford, D. L. 1998. PA UP: Phylogenetic analysis using parsimony, Version 3.0s+ 4 . Illinois Natural History Survey, Champaign, Illinois.

Taylor, J. W. 1993. A contemporary view of the holomorph: nucleic acid sequence and computer databases are changing fungal classification. Pp. 1-13. In: The Fungal 151

Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols, a guide to methods and applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White. San Diego: Academic Press.

Wilmotte, A., Y. Van De Peer, A. Goris, S. Chapelle, R. De Baere, B. Nelissen, J. -M. Neefs, G. L. Hennebert, and R. De Wachter. 1993. Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA analysis. System. Appl. Microbiol. 16: 436-444.

Wingfield, M. J., B. Kendrich, and P. S. van Wyck. 1991. Analysis of conidium ontogeny in anamorphs of Ophiostoma: Pesotum and Phialographium are synonyms of Graphium. Mycol. Res. 95: 1328-1333. 152

Chapter 7

Conclusion

The Relative Roles of Cospeciation, Duplication, Host Switching, and Sorting in the Evolution of Rhabdocline (Hemiphacidiaceae)

7.1. Abstract

Rhabdocline is a member of the ascomycete family, Hemiphacidiaceae, one of several families of inoperculate discomycetes whose members parasitize conifer foliage. Phylogenies generated using sequences from the ITS region of nuclear ribosomal DNA were compared for Rhabdocline and its hosts Pseudotsuga and Larix (Pinaceae). Based on topological and distance-based comparisons and historical biogeography, two, possibly three host switches are evident for Rhabdocline and an asexual member of the genus, Meria laricis. Phylogenetic reconstruction using the ITS region suggests that the most recent common ancestor for Rhabdocline spp. may have been similar to the basal extant genus, R. weirii ssp. weirii. Rhabdocline weirii ssp. weirii exhibits symplesiomorphic characters including an iodine positive apical pore in its ascus tip, both sexual and asexual modes of reproduction, and a pathogenic habit. Species that lack a sexual or asexual state, or that live as asymptomatic endophytes, occupy derived positions in the ITS phylogeny. Two of the hypothesized host switches involve M laricis, which appears to have evolved from an ancestor parasitic on Pseudotsuga menziesii that switched hosts to one or two species of western North American Larix. The second switch may have been a recent host extension to Larix decidua in Europe, but the relatively high degree of ITS region divergence between M laricis from North America compared to Europe weaken this arguement. Topological and distance-based evidence also argues against cospeciation as an explanation of the occurence of Rhabdocline weirii ssp. weirii on the sister species, 153

Pseudotsuga menziesii and Pseudotsuga macrocarpa. A more recent horizontal transfer and subsequent divergence is proposed.

7.2. Introduction

Complex coadaptations among orchids and their pollinators was used by Darwin (1859; 1877) as an argument favoring evolution by natural selection. Since that time, coevolution among phylogenetically distant lineages has been a subject of interest to biologists, who have documented the reciprocal evolution of morphological and genetic (Flor, 1946) adaptations in a diversity of organisms. While one of the mostly widely recognized systems for study has been that between flowering plants and pollinators (Ehrlich and Raven, 1964), there is also a great interest in the evolution of hosts and parasites (Page, 1993). Coevolutionary interactions that are sustained over millions of years result in host- parasite phylogenies that track one another with varying degrees of fidelity. This macroevolutionary facet to coevolution allows us to evaluate the relative importance of codivergence, duplication, horizontal transfer and sorting in the speciation of parasites with their hosts (Page and Charleston, 1998). The relative influence of these factors has been explored for a diversity of symbioses, including pocket gophers with chewing lice (Hafner et al., 1994; Page et al., 1996), trees and shrubs with nitrogen fixing bacteria (Cournoyer et al., 1993) aphids with eubacterial endosymbionts (Munson et al., 1991), and protozoans with eubacterial endosymbionts (Du et al., 1994). Cospeciation involving fungal symbionts has been explored formally between leaf-cutting ants and basidiomycetes (Chapela et al., 1994, Hinkle et al., 1994), rusts and hosts (which often alternate between gymnosperms and angiosperms)(Vogler and Bruns, 1998), and Monilinia with Rosaceae and Ericaceae (Hoist- Jensen et al., 1997.) The goal of this study is to compare phylogenetic reconstructions of foliar parasites in the fungal genus Rhabdocline (Hemiphacidiaceae) with their hosts in the conifer genera Pseudotsuga and Larix (Pinaceae). Phylogenetic reconstructions using the 154 internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) are available for both hosts and parasites. The relative roles of cospeciation of closely related hosts and closely related parasites (codivergence), host switching (horizontal transfer), parasite speciation unaccompanied by complementary host speciation (duplication), and extinction of parasites in some host lineages (sorting) will be evaluated using phylogenies generated using evidence from maximum parsimony, quantification of ITS divergence, morphological characters, biogeography, and known host ranges for Rhabdocline. The group was selected because the hosts are well studied and are comprised of a small number of species, and because the parasites exhibit morphological and ecological diversity together with a high degree of host specificity. Both hosts and parasites are representatives of orders (Coniferales and Leotiales, respectively) that together feature a broader pattern of foliar parasitism. Rhabdocline is a fungal genus that parasitizes foliage of Pseudotsuga and Larix. The genus is concentrated in western North America, but humans have spread it with its host to eastern North America and Europe, where P. menziesii is often grown as a commercial timber species, ornamental, and on Christmas tree farms. Rhabdocline is comprised of seven species and subspecies, including five that cause a needle cast disease of P. menziesii (Douglas-fir) and P. macrocarpa (bigcone Douglas-fir), and one asexual species, Meria laricis that causes a needle cast of three species of (larch) (Parker and Reid, 1969; Gernandt et al., 1997). The seventh species, R. parkeri, is a neutral or mutualistic foliar endophyte of P. menziesii (Sherwood-Pike et al., 1986). The members with a sexual state are distinguished by differences in ascus tip structure, presence of absence of asexual states, ascomata size and position, and ascospore morphology. The genus is a member of Hemiphacidiaceae (Gernandt, in prep.), a family of ascomycetes that includes several genera causing needle blights of conifers (Korf, 1962). In the six species of Rhabdocline with sexual states, ascospores are dispersed by rain in the late spring and early summer. Under favorable conditions, ascospores that land on needles of Pseudotsuga form specialized hyphae that directly penetrate the host cuticle and infect epidermal and subepidermal cells (Brandt, 1960; Stone, 1986). In pathogenic species, the fungi mature for one to two years before producing apothecia that rupture the 155

host-epidermis, throwing back a scale of host tissue. Ascospore release repeats the fungal life cycle, and the mottled needles fall from the tree later in summer. In the endophytic R. parkeri, the intracellular infection remains quiescent for up to five years (Sherwood-Pike et al., 1986). The fungus does not become active until the onset of host senescence, and has only been known to form sexual fructifications from abscised needles on the forest floor. The apothecia of R. parkeri are considerably smaller than those of its pathogenic congeners. In the asexual larch needle cast pathogen, Meria laricis, conidiospores are dispersed by rainfall throughout spring and summer. Needle infections grow and spread rapidly, allowing for multiple infection cycles per season. The fungus reproduces asexually by conidiophores that grow out through the host stomata, releasing conidia (Peace and Holmes, 1933). Needle infection has been observed to start at the lower branches of a tree and spread steadily upward toward the crown through sustained production of conidia. Larix is naturally deciduous, but in individuals infected with M laricis, needles senesce prematurely, dropping from the tree prior to normal abscission. Abscised, infected needles are the source of inoculum the following spring (Sherwood- Pike et al., 1986). The sexual state of Meria laricis may have been lost as an adaptation to the deciduous habit of its host. Sexual reproduction of other Rhabdocline species on P. menziesii requires at least one year to complete the life cycle. The deciduous habit of Larix therefore may not allow enough time for a sexual state to complete a life cycle. Two other members of Rhabdocline possess an asexual state. Rhabdocline parkeri, the endophyte of P. menziesii, has a Meria anamorph that is morphologically similar to M laricis. Rhabdocline weirii ssp. weirii also has an anamorph, Rhabdogloeum pseudotsugae, which has similar conidia to Meria laricis and Meria parkeri, except that the conidia of Rhabdogloeum have a mucilaginous appendage. The conidiomata of Meria and Rhabdogloeum are also quite distinct. Rather than producing phialides that sporulate through host stomata, Rhabdogloeum produces acervular conidiomata that erupt through the host epidermis. 156

The coniferous hosts of Rhabdocline are Pseudotsuga and Larix, two recently derived genera within Pinaceae. The sister relationship between Pseudotsuga and Larix has been demonstrated based on anatomical and developmental features (Christiansen, 1972; Hart, 1987), and molecular phylogenetic analyses using immunological methods (Prager et al., 1976; Price et al., 1987) and DNA sequence variation (Chase et al., 1993, Tsumura et al., 1995; Chaw et al., 1997; Xiao-Quan et al., 1997). Pseudotsuga is comprised of approximately four to five species in western North America and East Asia, and Larix is comprised of ten species, distributed at higher latitudes and altitudes in North America and across Eurasia (Farjon, 1990). Paleontological evidence and current distributions suggest that Larix dispersed widely throughout the Northern Hemisphere while Pseudotsuga remained restricted to East Asia and western North America (LePage and Basinger, 1991; Schorn, 1994). Parsimony analysis of the ITS region suggests that the first major divergence observed among extant species from both genera occurred between the North American and Eurasian species (Gernandt and Liston, in review).

7.3. Materials and Methods

With the exception of two additional representatives from Rhabdocline weirii ssp. weirii, collection information for taxa included in this study have been described elsewhere (Gernandt et al., 1997; Gernandt and Liston, in press). The additional representatives from R. weirii ssp. weirii were both collected in California. One was collected from P. menziesii growing in the Pineridge Ranger District in the Sierra National Forest and the other from P. macrocarpa growing in San Diego County. Methods for DNA isolation, amplification, and sequencing are given elsewhere (Gernandt et al., 1997). Sequences were aligned manually using Genetic Data Environment (Smith et al., 1994). Two other members of Hemiphacidiaceae, Hemiphacidium longisporum (American Type Culture Collection #26761), and Fabrella tsugae, were included as outgroups. Because alignment was straightforward, all sequence positions were retained for the subsequent analysis. Sequences were analyzed using PAUP* 4.0.0d62(Swofford, 1998). Phylogenies for hosts 157

and parasites were based on branch-and-bound searches. Data sets were resampled using the bootstrap method, and additional branch-and-bound searches were performed (500 replicates). Trees were displayed using TREEVIEW version 1.5 (Page, 1996). Phylogenies for parasites and hosts were compared using the experimental program, Tree Map (R. D. M. Page, unpublished).

7.4. Results

In the ITS region analysis of Rhabdocline, 86 of 508 nucleotide positions were parsimony informative. The result of the cladistic analysis was a single most parsimonious tree with moderate-to-high bootstrap support at most nodes (FIG. 7.1). The basal member of the Rhabdocline Glade was R. weirii ssp. weirii, represented by a Glade of three collections from P. menziesii and a lineage leading to a single isolate collected from P. macrocarpa. Rhabdocline parkeri, represented by four isolates, was paraphyletic and separated by geographic origin (whether hosts were from the range of coastal or interior P. menziesii). A Glade comprised of five isolates of M. laricis from three different species of Larix occupied a derived position in a sister relationship to a Glade of P. menziesii pathogens that lack an asexual state. This Glade included the I+ R. weirii ssp. obovata and the I- R. pseudotsugae ssp. pseudotsuga and R. pseudotsugae ssp. epiphylla. Mapping morphological and ecological features onto the tree in FIG. 7.1 reveals that the single internal node lacking bootstrap support over 50% yields R. parkeri paraphyletic rather than monophyletic and requires a loss and regain of pathogenicity in Rhabdocline. A second analysis was performed constraining the four isolates of Rhabdocline parkeri to monophyly. The result was five equally most parsimonious trees two steps longer (190 rather than 188). In all equally most parsimonious trees, the constrained Rhabdocline parkeri isolates were in a sister relationship with Meria laricis. The attachment of this larger Glade to the remaining Rhabdocline taxa varied in the five equally most parsimonious trees. 158

R. weirii obovata Loss of 98 anamorph R. pseudotsugae epiphylla 98 Pseudotsuga Loss of R. pseudotsugae Oregon Loss of ascal pore teleomorph 99 R. pseudotsugae Pennsylvania

M. laricis 5 decidua Pathogenicity gained ??? 85 M. laricis 1

51 M. laricis 4 lyallii 97 Larix M. laricis 3

M. laricis 2 Pathogenicity lost?_ R. parkeri 3 coastal Acervulum lost 79 R. parkeri 1 coastal

R. parkeri 4 interior 95 R. parkeri 2 interior Pseudotsuga R. weirii 4 macrocarpa

100 R. weirii 3 California 80 R. weirii 1 Oregon 89 R. weirii 2 Montana

Fabrella tsugae 100 Hemiphacidium longisporum

FIG. 7.1. Single most parsimonious tree from a branch and bound search of ITS region sequences (188 steps, CIe = 0.793, RI = 0.902, RC = 0.758). Bootstrap values less than 50% not shown. Scale bar represents the number of steps. 159

Loss of M. laricis occidentalis teleomorph Niko M. laricis lyallii Larix Acervulum lost M. lands decidua 1* R. parkeri interior

Pathogenicity lost I R. parkeri coastal

Loss of R. pseudotsugae epiphylla ascal pore

R. pseudotsugae Pseudotsuga R. weirii obovata Loss of anamorph R. weirii coastal

R. weirii interior

R. weirii macrocarpa

Fabrella tsugae

Hemiphacidium longisporum

1

FIG.7.2. One of five most parsimonious trees resulting from an analysis constraining Rhabdochne parkeri to monophyly (184 steps, CIe = 0.780, RI = 0.843, RC = 0.701). For each species and subspecies of Rhabdocline, only one isolates from each host has been included. The trees are two steps longer than the unconstrained tree. Nodes that collapse in the strict consensus tree are denoted with an asterisk. 160

P.macrocarpa R weirii3

P.menziesii coastal .weiriil

P.menziesii interior R.weirii2

P. japonica parkeril P.sinensis

parkeri2 P.wilsoniana

obovata L.laricina

L.occidentalis .pseudotsugae

L.lya1111 R.epiphylla

L.griffithiana M laricisl

L.decidua laricis2 L.kaempferi

laricis3 L.sibirica

Hemiphacidium Pinus

Tsuga Fabrella

FIG. 7.3. A comparison of host and parasite phylogenies. Hosts are shown on the left and fungal parasites on the right. Topologies are adapted from branch and bound analyses using the ITS region for both groups of associates. Lines connect each fungal isolate to its host. Nodes with circles denote host-parasite cospeciation events inferred by TreeMap. 161

For making comparisons between hosts and parasites, a third parsimony analysis was performed that constrained R. parkeri to monophyly and included only one isolate per host for each species or subspecies of Rhabdocline (FIG. 7.2.). The first of five most parsimonious trees was selected for further examination because it depicted R. parkeri and M laricis as a monophyletic Glade, a topology consistent with their mutual possession of a Meria anamorph.

A topological comparison of hosts and parasites is shown in FIG. 7.3. The host tree is based on the phylogenetic analyses reported by Gernandt and Liston (in review) except for the additions of Tsuga and the interior form of Pseudotsuga menziesii. The ingroup topology and outgroup root for the hosts (Pseudotsuga, Larix, Pinus, and Tsuga) match the single most parsimonious tree found in a branch and bound analysis of ITS region data for Pseudotsuga, Larix, and Pinus. Tsuga, also a member of Pinaceae, is included as an outgroup because it is the host of Fabrella tsugae. This placement is consistent with that found in ITS2 phylogenies that sample more widely in Pinaceae (T. Vining, pers. comm.) and from rbcL phylogenies (chapter 2). Divergence of P. menziesii into coastal and interior population has been reported by Li and Adams (1989).

TreeMap was used to enforce cospeciation of Rhabdocline and M. laricis on their respective hosts. Under such conditions, TreeMap interpreted the host-parasite topology as including eight cospeciation events, three duplications, one host switch, and eight sorting events. In the reconstruction, a common ancestor to Fabrella, Hemiphacidium, and Rhabdocline (including Meria) cospeciated on Tsuga, Firms, Pseudotsuga and Larix. The single host-switch involved a shift of the common ancestor of M. laricis onto Pseudotsuga. This switch was followed by a combination of cospeciation and divergence giving rise to R. weirii ssp. obovata, R. pseudotsugae ssp. pseudotsugae, R. pseudotsugae ssp. epiphylla and R. parkeri. When outgroups were excluded, TreeMap interpreted the host-parasite topology as including six cospeciation events, three duplications, one host switch, and seven sorting events. The correlation between host and parasite branch lengths in the outgroup excluded analysis was r = 0.53. A correlation value for the 162 analysis that included the interior form of P. menziesii and Tsuga was not available because ITS region sequences were not available for these two hosts. The low correlation between branch lengths and host-parasite associations indicate weak support for the numbers of cospeciation events, duplications, host switches, and sorting events as reconstructed using topological information in TreeMap. The correlation test allows parasites and hosts to have different rates of evolution, but tests whether the relative rates of substitution among species in the host tree are consistent with the relative rates among species in the parasite tree. For example, when branches between two hosts are short relative to other branches in the host phylogeny, and branches between their corresponding parasites are long relative to other branches in the parasite phylogeny, the correlation value drops. When this occurs, host switches become better explanations for the reconstruction than do cospeciation. When branch lengths between hosts and their corresponding parasites are highly correlated, cospeciation is a more likely explanation than host switching. Patristic distances for several pairs of fungal parasites were much lower than expected if their host associations were the results of cospeciation and sorting events. ITS region sequences for Meria laricis from Larix lyallii and L. occidentalis were identical, and the divergence of these two North American isolates from the European isolate from L. decidua was 3.0%. These levels of divergence are consistent with the level of divergence between congeneric hosts (the patristic distance between L. occidentalis. and L. decidua was 2.0%), but are not consistent when comparisons are made between M laricis and closely related parasites on Pseudotsuga menziesii. The North American and European isolates of M laricis were only 3.7 and 4.8% divergent from the coastal isolates of Rhabdocline parkeri on P. menziesii, but the patristic distance between P. menziesii and L. occidentalis was 20.0%. The patristic distance between the interior and coastal P. menziesii isolates of R. weirii ssp. weirii was 0.2%, and the distances of these two isolates to the R. weirii ssp. weirii from P. macrocarpa were 2.0 and 2.2%, respectively. This is much lower than host patristic distances. Pseudotsuga menziesii and P. macrocarpa ITS sequences were 27.0%. The distances of the three isolates of R. weirii ssp. weirii to the coastal isolate of R. parkeri on P. menziesii ranged from 9.6-9.9%. Therefore, for sorting 163 to be the cause of the presence of R. weirii on two species of Pseudotsuga, substitution rates must have remained very low in all lineages of R. weirii ssp. weirii since the divergence of its two hosts. Substitution rates for other species and subspecies of Rhabdocline must have been very high relative to those in Rhabdocline weirii ssp. weirii.

7.5. Discussion

Mapping morphological and ecological features onto the most parsimonious tree using ITS region data in FIG. 7.1 demonstrates that it is most parsimonious to hypothesize that the common ancestor of extant species of Rhabdocline was a pathogen with both a sexual and asexual state and that possessed an ascus tip with an amyloid pore. Simplifications in reproductive strategies (sexual vs. asexual), and morphological features are apparent from the phylogenetic reconstruction (FIGS 7.1-7.2). These simplifications include the reduction from acervular to hyphomycetous forms, the loss of an amyloid pore in the ascus tip, and the decrease in size of the ascomata (observed in R. parkeri). Endophytism can also be interpreted as a derived ecological strategy in Rhabdocline. Whether asexual reproduction via conidia is a derived feature of the genus Rhabdocline as a whole is open to question. Conidial states are absent in other genera of Hemiphacidiaceae and somewhat rare in the closely related family Sclerotiniaceae. However, genera in other families of inoperculate discomycetes produce acervular conidial states, such as Dermateaceae (eg. the Cryptosporiopsis anamorph of Pezicula and the Cylindrosporium anamorph of Drepanopeziza), Phacidiaceae (eg. the Apostrasseria and Ceuthospora anamorphs of Phacidium), Rhytismataceae (eg. the Leptostroma anamorphs of Lophodermium), and genera whose familial classification is uncertain (eg. the Tiarosporella anamorph of Darkera). It is quite probable that possessing a conidial mode of reproduction, like the presence of an apical pore in the ascus tip, is an ancestral feature that has been repeatedly lost in the inoperculate discomycetes. The single node with bootstrap support below 50% forces a less parsimonious interpretation of the evolution of ecological roles of pathogen and innocuous endophyte. 164

The low bootstrap support suggests that the ITS region data set was not powerful enough to accurately place R. parkeri. The presence of this poorly supported node results in a morphological species (Rhabdocline parkeri) being rendered paraphyletic rather than monophyletic. Constraining R. parkeri to monophyly increases the number of steps in the ITS region tree from 188 to 190. Enforcing the constraint also results in a sister relationship rather than paraphyletic relationship of the two species with similar anamorphs, R. parkeri and Mena laricis. This alternate phylogenetic hypothesis ought to be considered as a more likely alternative to the most parsimonious ITS region tree, as the cost in parsimony on the ITS region data set alone is countered by more parsimonious mapping of both ecological and morphological characters. Before discussing the evolution of Rhabdocline further, two points must be emphasized regarding the taxonomy of genus. Both points stem from the species concept used in the monograph of the genus by Parker and Reid (1969). First, pathogenic species are delimited by the presence or absence of an amyloid ring in the ascus tip. Parker and Reid were aware of the instability of the character in the ascomycetes (see Baral, 1987), but noted that its presence or absence as indicative of two fundamentally different mechanisms of ascospore liberation. In FIGS 7.1 and 7.2, the ITS region phylogeny renders the Rhabdocline taxa with an iodine positive ascus tip paraphyletic. As a result, despite their mutual possession of an I+ pore in the ascus tip, the two subspecies of R. weirii sampled are not closely related (the third subspecies, R. weirii ssp. oblonga was unavailable). From a cladistic perspective, R. weirii ssp. obovata, is more closely allied to I- R. pseudotsugae. While R. weirii ssp. weirii retains the ancestral I+ pore, the data presented here suggest that it belongs to a lineage that diverged after a common ancestor lost the ability to reproduce asexually via conidia. In which case, the species epithet of R. weirii ssp. obovata should be changed. Second, Parker and Reid may have been too conservative in designating only two species of Rhabdocline. The morphological differences between subspecies, as well as the ITS region phylogeny, suggest that subspecies are reproductively isolated and merit species rank. An alternate and better explanation to the reconstruction in TreeMap is that Rhabdocline speciated (duplicated) on Pseudotsuga menziesii and switched hosts from 165

Pseudotsuga to Larix rather than in the opposite direction. This alternate view is based on the derived rather than basal position of M laricis in the Rhabdocline Glade, the high degree of sequence similarity between M laricis and other derived members of Rhabdocline, and that the presumed absence of a sexual state in M laricis could be a consequence of the deciduous nature of its hosts. That ITS regions of M laricis isolates from Larix occidentalis and L. lyallii are identical is not surprising because the two western North American hosts occupy overlapping ranges, with L. lyallii growing at higher elevations than L. occidentalis. Given that M laricis has not been found on other species of Larix, it is also less parsimonious to assume that cospeciation and sorting rather than a host switch is responsible for the association of M. laricis with Larix decidua. Four sorting events are required to explain the absence of M. laricis on L. laricina, L. griffithiana, on the internal node leading to L. sibirica and L. kaempferi, and on the east Asian species of Pseudotsuga. Sorting could be attributable to the presence of heretofore unreported Rhabdocline species on all other species of Larix and Pseudotsuga, but such species have not been found despite some searching and considering that most of their relatives cause noticeable diseases. Host switching rather than cospeciation is also a likely explanation for the occurrence of R. weirii ssp. weirii on hosts P. macrocarpa and P. menziesii. Sequence divergence between the isolates is low relative to that found between other Rhabdocline species, and would have required a substantial decrease in the substitution rate of the parasite as the common ancestor of P. menziesii and P. macrocarpa diverged. The occurrence of R. weirii ssp. weirii on both P. macrocarpa and P. menziesii could be explained by a host switch resulting either from ancient overlap of their natural ranges or from transport of infected material by humans or other animals. Small populations of Pseudotsuga menziesii grow in southern California near the more restricted P. macrocarpa, but the two species are prevented from overlapping by the San Bernadino Mountains. There is fossil evidence of a P. macrocarpa-like form occupying the Willamette Valley of Oregon during the Oligocene (Herman, 1985), suggesting that the range of the big-cone form once extended farther to the north. The occurrence of several species and subspecies of Rhabdocline on Pseudotsuga menziesii is hypothesized to be a result of duplicationspeciation of parasites 166

unaccompanied by speciation of their host. To avert extinction from competition with reproductively isolated but otherwise closely related species, each species must have adapted to a distinct niche. Several different evolutionary trajectories may have been taken to prevent niche overlap, particularly in the three pathogenic Rhabdocline species that have broad distributions. These are R. pseudotsugae ssp. pseudotsugae, R. weirii ssp. weirii, and R. weirii ssp. obovata. Hoff (1988) observed that a Rhabdocline outbreak on seedlings of the interior variety of P. menziesii was least damaging to a low altitude provenance. He suggested that middle and high altitude provenances were resistance because they evolved in an environment where disease pressure was lower. At higher elevations bud flush occurred later in the year when temperatures were higher and rainfall was lower. He also documented that families varied in their resistance to Rhabdocline and that resistance was heritable. These two observations suggest two possible forces driving duplication of Rhabdocline on P. menziesii. First, the evolution of a new resistance gene in the host might provide an opportunity for a pathogen with the fortuitous complementary mutation overcoming the resistance to specialize on that particular host genotype. Second, a pathogen that can adapt to attack hosts that are otherwise escaping disease would also have a new niche to occupy. This could be achieved with an increased tolerance of infective propagules to high temperatures or desiccation, but could also arise by infecting older needles (rather than new growth) earlier in the year when it is wetter and cooler. Ascomatal position may provide niche separation for Rhabdocline pseudotsugae ssp. epiphylla. It is the only pathogenic Rhabdocline with apothecia that develop primarily on the upper surface of needles (the adaxial surface), rather than on the lower surface. This adaptation would presumably expose the pathogen to more sunlight, making it more vulnerable to desiccation. It is noteworthy that R. pseudotsugae ssp. epiphylla is only known from the wettest part of the range of P. menziesii, along the coastal fog belt in Canada, Washington, and Oregon (Parker and Reid, 1969). The innocuous nature of R. parker! can be viewed as a highly successful strategy for avoiding niche overlap. Rhabdocline parker! colonize stands of P. menziesii at very high densities in western Oregon, and has been documented throughout the range of 167

coastal P. menziesii (Carroll and Carroll, 1978, Sherwood-Pike et al., 1986) and from northern populations of interior P. menziesii (Gernandt et al., 1997). This parasite causes no significant loss in fitness to its host, and may even increase host fitness by attacking larvae of the pathogenic gall midge, Contarinia pseudotsugae (Todd, 1987). Therefore, there is no pressure for the host to evolve genetic resistance to infection by this species. At the very least, this would enable R. parkeri to colonize needles of all individuals of P. menziesii that are resistant to infection by pathogenic Rhabdocline species. All the hypothesized host switches from P. menziesii have gone to closely related species or genera, to either P. macrocarpa, or one of three species of the sister genus, Larix. Such switches would require the fewest adaptations for successful infection and reproduction to arise de novo in the parasite. It is logical to predict that host switches will be more likely when the recipient host is 1) closely related to the source host and 2) exposed to a parasite as a result of range overlap or dispersal. With one exception, other genera of Hemiphacidiaceae are also specific in their hosts, especially compared to genera such as Lophodermium in the closely related family, Rhytismataceae. For instance, Fabrella, the closest known genus to Rhabdocline, and Korfia (sequences unavailable) are only known from Tsuga, and Didymascella is known from the Cupressaceae genera Chamaecyparis, Juniperus, and Thuja. Although Herrnphacidium has only been reported from Pinus, the closely related (perhaps congeneric, see chapter 4) Sarcotrochila is parasitic on Abies, Pseudotsuga, Picea, and Pinus. In addition to the macroconidial Meria state of R. parker!, a microconidial, presumably spermatial, state is produced (Sherwood-Pike et al., 1986). Spermatial states are not known from any of the other species of Rhabdocline. The posession of a spermatial anamorph, is probably a more primitive character. Rhabdocline parker!, possesses another primitive character, an apical I+ pore. Spermatial states are also unknown or rare in other Leotialian fungi. Microconidial anamorphs that are presumed to function as spermatia are common in several genera of foliar fungi in Rhytismataceae, such as Lophodermium, Lophodermella, Linda, and Rhytisma. The diversity of anamorph forms in Rhabdocline, together with their presence or absence in connection with 168

particular Rhabdocline species suggests that asexual reproduction has has been an important factor in natural selection and speciation in the genus. Several directions of research still need to be pursued in understanding the systematics and evolution of Rhabdocline. First, R. weirii ssp. oblonga, and other potential anamorphic members of Rhabdocline need to be included in an ITS region phylogeny. Other fungi parasitic on P. menziesii that may belong to this group include the acervular anamorphic fungus Rhabdogloeum hypophyllum, collected from Arizona and New Mexico by Ellis and Gill (1945). Morgan-Jones (1973) transferred R. hypophyllum to Cryptocline. Cryptocline includes several diverse species and is considered to be polyphyletic (Petrini, 1984). Some of these species may be Rhabdocline anamorphs or at the least, derived from the inoperculate discomycetes. Herbarium material from the Ellis and Gill isotype (C. hypophyllum) was examined, but attempts to obtain fresh material from the type locality or to isolate and amplify DNA from the herbarium material were unsuccessful. The putative connection between C. hypophyllum and Rhabdocline thus remains unconfirmed. Although the anamorphs of Rhabdocline are obviously no less related to each other than their congeneric teleomorphs, contemporary concepts in taxonomy of mitosporic fungi dictate that the closely related anamorphs be classified in different form genera. Meria is characterized as a sporodochial hyphomycete with holoblastic conidium formation followed by schizolytic secession and enteroblastic conidiogenous cell proliferation ["phialidic" conidiogenesis Hawksworth et al., (1995)] whereas Rhabdogloeum and Cryptocline are characterized as having acervular conidiomata, holoblastic conidiogenesis, schizolytic secession and enteroblastic-percurrent proliferation ("annelidic" conidogenesis). The absence of appendaged conidia of C. hypophyllum was cited as cause for separation from the distinctly appendaged Rhabdogleoum pseudotsugae by Morgan-Jones (1973). An apparently undescribed, Meria-like fungus has been isolated from asymptomatic needles of Japanese larch (G. C. Carroll pers. com., original cultures lost). If this fungus could be reisolated and is found to be a Rhabdocline, then it could suggest that Meria has cospeciated with Larix rather than undergoing multiple host switches as 169 proposed above. It would also suggest that the genus predates the hypothesized migration of Larix between North America and Asia (Strauss et al., 1990; Gernandt and Liston, 1998). Larix from the same locality should be reexamined in an attempt to reisolate this fungus. The range of the interior form of P. menziesii extends into the southern United States and Mexico, yet not a single Rhabdocline isolate from these regions has been included in the study reported here. Considering the divergence in ITS region sequences found among coastal and interior isolates of R. weirii ssp. weirii and R. parkeri, more isolates from all species sampled in these studies should be sought in the southwest to better document the amount of ITS divergence within morphological species. Rhabdocline and potentially related anamorphs should also be sought from other species of Larix and Pseudotsuga.

The presumed loss of sexual reproduction in Meria laricis can be tested using population level markers such as isozymes, RAPDs, or ISSRs to determine whether segregation of alleles is occurring within natural populations. Such population genetic studies using RAPD markers have documented high genetic diversity among individuals of Rhabdocline parkeri isolated from the same tree (McCutcheon et al., 1993). The discovery of segregation of alleles within populations of M laricis would suggest that the sexual state is present but inconspicuous. Rhabdocline parkeri is likely to be the sister species of M. laricis. If M. laricis is obligately asexual, a study of both these species would provide an excellent opportunity to compare rates and patterns of molecular evolution and population structure in closely related sexual and asexual species.

7.6. Acknowledgements

The author thanks Jeff Stone, Aaron Liston, Joseph Spatafora, Steve Strauss, and Dennis Wilson for stimulating discussions on cospeciation and the morphological and ecological evolution of Rhabdocline. I also thank Greg DeNito for providing specimens 170 of Rhabdocline from California, as well as everyone who provided me with specimens used in this and previous studies of Rhabdocline and its hosts.

7.7. Literature Cited

Baral, H. 0. 1987. Lugol's solution/IKI versus Melzer's reagent: hemiamyloidity, a universal feature of the ascus wall. Mycotaxon 29: 399-450.

Brandt, R. W. 1960. The Rhabdocline needle cast of Douglas-fir. State University College of Forestry, Syracuse University, NY, technical publication no. 84, 66 pp.

Carroll, G. C., and F. E. Carroll. 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56: 3034-3043.

Chapela, I. H., S. A. Rehner, T. R. Schultz, and U. G. Mueller. 1994. Evolutionary history of the symbiosis between fungus-growing ants and their fungi. Science 266: 1691-1694.

Chase, M. W., D. E. Soltis, R. G. Olmstead et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rhcL. Annals of the Missouri Botanical Garden 80: 528-580.

Chaw, S.-M., Zharkikh, A., Sung, H.-M., Lau, T.-C., and W.-H. Li. 1997. Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences. Molecular Biology and Evolution 14: 56-68.

Christiansen, H. 1972. On the development of pollen and the fertilization mechanisms of Larix and Pseudotsuga menziesii. Silva Genetica 21: 166 -174.

Cournoyer, B., M. Gouy, and P. Normand. 1993. Molecular phylogeny of the symbiotic actinomycetes of the genus Frankia matches host-plant infection processes. Molecular Biology and Evolution 10: 1303-1316

Darwin, C. 1859. The origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. Facsimile of the 1st ed. Mentor Books, New York.

. 1877. The various contrivances by which orchids are fertilised by insects. 2d ed. Murray, London. 171

Du, Y., Maslov, D. A., and K.-P. Chang. 1994. Monophyletic origin of (3-division proteobacterial endosymbionts and their coevolution with insect trypanosomatid protozoa Blastocrithidia culicis and Crithidia spp. Proceedings of the National Academy of Science 91: 8437-8441

Ehrlich, P. R., and P. H. Raven. 1964. Butterflies and plants: a study of coevolution. Evolution 18: 586-608.

Ellis, D. E., and L. S. Gill. 1945. A new Rhabdogloeum associated with Rhabdocline pseudotsugae in the southwest. Mycologia 37: 326-332.

Farjon, A. 1990. Pinaceae: drawings and descriptions of the genera Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Koeltz Scientific Books, Konigstein, Federal Republic of Germany.

Flor, H. H. 1946. Genetics of pathogenicity in Melampsora lini . Journal of Agricultural Research 73: 335-357.

Gernandt, D. S., F. J. Camacho, and J. K. Stone. 1997. Meria laricis, an anamorph of Rhabdocline. Mycologia 85: 735-744.

, and A. Liston. 1998. Internal transcribed spacer evolution in Larix and Pseudotsuga (Pinaceae). submitted to American Journal of Botany.

Hafner, M. S., P.D. Sudman, F. X. Villablanca, T. A. Spradling, J. W. Demastes, and S. A. Nadler. 1994. Disparate rates of molecular evolution in cospeciating hosts and parasites. Science 265: 1087-1090.

Hart, J. A. 1987. A cladistic analysis of conifers: preliminary results. Journalof the Arnold Arboretum 68: 269-307.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp.

Hermann, R. K. 1985. The genus Pseudotsuga: ancestral history and past distribution. Forest Research Laboratory, Oregon State University, Corvallis, OR, Special Publication 2b.

Hinkle, G., J. K. Wetterer, T. R. Schultz, and M. L. Sogin. 1994. Phylogeny of the attine ant fungi based on analysis of small subunit ribosomal RNA gene sequences. Science 266: 1695-1697.

Hoff, R.J. 1988. Susceptibility of inland Douglas-fir to Rhabdocline needle cast. USDA Forest Service Research Note, INT-375, pp. 3. 172

Hoist- Jensen, A., L. M. Kohn, K. S. Jakobsen, and T. Schumacher. 1997. Molecular phylogeny and evolution ofMonilinia (Sclerotiniaceae) based on coding and noncoding rDNA sequences. American Journal of Botany 84: 686-701.

Korf, R. P. 1962. A synopsis of the Hemiphacideaceae, a family of Helotiales (discomycetes) causing needle blights of conifers. Mycologia 54: 12-33.

LePage, B. A., and J. F. Basinger. 1991. A new species of Larix (Pinaceae) from the early Tertiary of Axel Heiberg Island, Arctic Canada. Review of Palaeobotany and Palynology 70: 89-111.

Li, P., and W. T. Adams. 1989. Range-wide patterns of allozyme variation in Douglas fir (Pseudotsuga menziesii). Canadian Journal of Forest Research 19: 149-161.

McCutcheon, T. L., G. C. Carroll, and S. Schwab. 1993. Genotype diversity in populations of a fungal endophyte from Douglas-fir. Mycologia 85: 180-186.

Morgan-Jones, G. 1973. Genera coelomycetarum. VII. Cryptocline Petrak. Canadian Journal of Botany 51: 309-325.

Munson, M.A., P. Baumann, M. A. Clark, L. Baumann, N. Moran, D. J. Voegtlin, and B. C. Campbell. 1991. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. Journal of Bacteriology 173: 6321-6324.

Page, R. D. M. 1993. Parasites, phylogeny, and cospeciation. International Journal of Parasitology 23: 499-506.

. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Applic. Biosci. 12: 357--358.

D. H. Clayton, and A. M. Patterson. 1996. Lice and cospeciation: a response to Barker. International Journal of Parasitology 26: 213-218.

, and M. A. Charleston. 1998. Trees within trees: phylogeny and historical associations. Trends in ecology and evolution. In press.

Peace, T. R., and C. H. Holmes. 1933. Meria laricis the leaf cast disease of larch. Oxford Forestry Memoirs 15. 29 pp.

. 1984. Cryptocline arctostaphyli sp. nov., an endophyte of Arcostaphylos uva­ ursi and other Ericaceae. Sydowia Annales Mycologici 37: 238-241. 173

Prager, E. M., D. P. Fowler, and A. C. Wilson. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

Price, R. A., J. Olsen-Stojkovich, and J. M. Lowenstein. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Systematic Botany 12: 91-97.

Qian, T., R. A. Ennos, and T. Helgason. 1995. Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA. Canadian Journal of Forest Research 25: 1197-1202.

Schorn, H. E. 1994. A preliminary discussion of fossil larches (Larix, Pinaceae) from the arctic. Quaternary International 22/23: 173-183.

Sherwood-Pike, M., J. K. Stone, and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canadian Journal Botany 64: 1849­ 1855.

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gillevet, 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Stone, J. K. 1986. Initiation and development of latent infections by Rhabdocline parkeri on Douglas-fir. Canadian Journal of Botany 65: 2614-2621.

Strauss, S. H., A. H. Doerksen, and J. R. Byrne. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canadian Journal of Botany 68: 1502-1510.

Swofford, D. L. 1998. PAUP* version 4.0. Sinauer Assoc., Inc., Sunderland, MA (in press).

Todd, D. 1987. The effects of host genotype, growth rate, and needle age on the distribution of a mutualistic, endophytic fungus in Douglas-fir plantations. Canadian Journal of Forest Research 18: 601-605.

Tsumura, Y., K. Yoshimura, N. Tomaru, and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theoretical and Applied Genetics 91: 1222-1236.

Vogler, D. R., and T. D. Bruns. 1998. Phylogenetic relationships among the pine stem rust fungi (Cronartium and Peridermium spp.). Mycologia 90: 244-257.

Xiao-Quan, W., H. Ying, D. Zheng-Rong, andH. De-Yuan. 1997. Phylogeny of the Pinaceae evidenced by molecular biology. Acta Phytotaxonomica Sinica 35: 97­ 106. 174

Bibliography

Aagaard, J. E., S. S. Vollmer, F. C. Sorensen, and S. H. Strauss. 1995. Mitochondrial DNA products among RAPD profiles are frequent and strongly differentiated between races of Douglas-fir. Molecular Ecology 4: 441-447.

Albert, V. A., Chase, M. W., and B. D. Mishler. 1993. Character-state weighting for cladistic analysis of protein-coding DNA sequences. Annals of the Missouri Botanical Garden 80: 752-766.

Albert, V. A., Backlund, A., Bremer, K., Chase M. W., Manhart, J. R., Mishler, B. D., and K. C. Nixon. 1994. Functional contraints and rbc1., evidence for land plant phylogeny. Annals of the Missouri Botanical Garden 81: 534-567.

Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.

T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.

Alvin, K. L. 1960. Further conifers of the Pinaceae from the Wealden Formation of Belgium. Memoires (Institut Royal des Sciences Naturalles de Belgique) 146: 1­ 39.

Arx, J. A. von. 1986. Tolypocladium, a synonym of Beauveria. Mycotaxon 25: 153-158.

Axelrod, D. I. 1990. Environment of the Middle Eocene (45 Ma) Thunder Mountain flora, central Idaho. National Geographic Research 6: 355-361.

Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campbell, and M. J. Donoghue. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247-277.

Baral, H. 0. 1987. Lugol's solution/IKI versus Melzer's reagent: hemiamyloidity, a universal feature of the ascus wall. Mycotaxon 29: 399-450.

Barron, G. L. The nematode-destroying fungi. Topics in Mycohiology No. 1, 140 pp. Guelph, Ontario. 175

, and E. Szijarto. 1982. A new hyphomycete parasitic on the ciliated protozoans Voticella and Opercularia. Canadian Journal of Botany 60: 1031-1034.

Beauverie, J. 1914. Les Muscardinesle genre Beauveria Vuillemin. Revue Generale de Botanique 26: 81-105, 157-173.

Bellemere, A. 1994. Asci and ascospores in ascomycete systematics. Pp. 111-126. In.. Ascomycete systematics, problems and perspectives in the nineties. NATO ASI Series A, Vol 269. Ed., D. L. Hawksworth. Plenum Press, New York, New , New York.

Berbee, M. L., and J. W. Taylor. 1992a. Dating the evolutionary radiations of the true fungi. Canadian Journal of Botany 71: 1114-1127.

, and . 1992b. Two Ascomycete classes based on fruiting-body characters and ribosomal DNA sequence. Molecular Biology and Evolution 9: 278-284.

Bertoni, M. D., Romero, N., Reddy, P. V., and J. F. White, Jr. 1997. A hypocrealean epibiont on meristems of Baccharis coridifolia. Mycologia 89: 375-382.

Boudier, J. L. E. 1885. Nouvelle classification naturelle des Discomycetes charnus connus generalement sous le nom de Pezizes. Bull. Soc. Mycol. France 1: 91-121.

. 1907. Histoire et classification des Discomycetes d'Europe. P. Klincksieck, Paris. 222 pp.

Bousquet, J., S. H. Strauss, A. H. Doerksen, and R. A. Price. 1992. Extensive variation in evolutionary rate of rbcL gene sequences among seed plants. Proceedings of the National Academy of Science USA 89: 7844-7848.

Brandt, R. W. 1960. The Rhabdocline needle cast of Douglas-fir. State University College of Forestry, Syracuse University, NY, technical publication no. 84, 66 pp.

Brown, G. R., and J. E. Carlson. 1997. Molecular cytogenetics of the genes encoding 18s-5.8s-26s rRNA and 5s rRNA in two species of spruce (Picea). Theoretical and Applied Genetics 95: 1-9.

Bruns, T., T. White, and J. Taylor. 1991. Fungal molecular systematics. Annual Review of Ecology and Systematics 22: 525-564.

Buckler Iv, E. S., and T. P. Holtsford. 1996. Zea Systematics: Ribosomal ITS Evidence. Molecular Biology and Evolution 13: 612-622.

A. Ippolito, and T. P. Holtsford. 1997. The evolution of ribosomal DNA: divergent paralogues and phylogenetic implications. Genetics 145: 821-832. 176

Camacho, F. J., D. S. Gernandt, A. Liston, J. K. Stone, and A. S. Klein. 1997. Endophytic fungal DNA, the source of contamination in spruce needle DNA. Molecular Ecology 6: 983-987.

Campbell, B.C. 1993. Congruent evolution between whiteflies (Homoptera: Aleyrodidae) and their bacterial endosymbionts based on respective 18S and 16S rDNAs. Current Microbiology 26: 129-132.

Campbell, C. S., M. F. Wojciechowski, B. G. Baldwin, L. A. Alice, and M. J. Donoghue. 1997. Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae). Molecular Biology and Evolution 14: 81-90.

Carbone, I. and L. M. Kohn. 1993. Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae. Mycologia 85: 415-427.

Carroll, G. C., and F. E. Carroll. 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56: 3034-3043.

Chapela, I. H., S. A. Rehner, T. R. Schultz, and U. G. Mueller. 1994. Evolutionary history of the symbiosis between fungus-growing ants and their fungi. Science 266: 1691-1694.

Chase, M. W., D. E. Soltis, R. G. Olmstead et al. (42 co-authors). 1993. DNA sequence phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Annals of the Missouri Botanical Garden 80: 528-580.

Chaw, S.-M., H. Long, B. -S. Wang, A. Zharkikh, and W. -H. Li. 1993. The phylogenetic position of Taxaceae based on 18S rRNA sequences. Journal of Molecular Evolution 37: 624-630.

, Zharkikh, A., H.-M.Sung, T.-C. Lau, and W.-H. Li. 1997. Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences. Molecular Biology and Evolution 14: 56-68.

Christiansen, H. 1972. On the development of pollen and the fertilization mechanisms of Larix and Pseudotsuga menziesii. Silva Genetica 21: 166-174.

Cournoyer, B., M. Gouy, and P. Normand. 1993. Molecular phylogeny of the symbiotic actinomycetes of the genus Frankia matches host-plant infection processes. Molecular Biology and Evolution 10: 1303-1316. 177

Critchfield, W. B., and E. L. Little. 1966. Geographic distribution of the pines of the world. U.S. Department of Agriculture, Forest Service. Miscellaneous Publication 991. Washington D.C.

Darker, G. D. 1932. The Hypodermataceae of conifers. Contributions from the Arnold Arboretum 1: 1-131.

. 1966. A revison of the genera of the Hypodermataceae. Canadian Journal of Botany 45: 1399-1444.

Darwin, C. 1859. The origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. Facsimile of the 1st ed. Mentor Books, New York.

. 1877. The various contrivances by which orchids are fertilised by insects. 2d ed. Murray, London.

Dennis, R. W. G. 1978. British Ascomycetes. J. Cramer, Vaduz, Zwitzerland. 585 pp.

DiCosmo, F., T. R. Nag-Raj, and B. Kendrick. 1983a. Prodromus for a revision of the Phacidiaceae and related anamorphs. Canadian Journal of Botany 61: 31-44.

H. Peredo, and D. W. Minter 1983b. Cyclaneusma gen. nov., Naemacyclus and Lasiostictis, a nomenclatural problem resolved. European Journal of Forest Pathology 13: 206-212.

Doerksen, A. H., and K. K. Ching. 1972. Karyotypes in the genus Pseudotsuga. Forest Science 18: 66-69.

Doudrick, R. L., J. S. Heslop-Harrison, C. D. Nelson, T. Schmidt, W. L. Nance, and T. Shwarzacher. 1995. Karyotyping slash pine (Pinus elliottii var elliottii) using patterns of fluorescence in situ hybridization and fluorochrome banding. Journal of Heredity 86: 289-296.

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

Drechsler, C. 1941. Some hyphomycetes parasitic on free-living terricolous nematodes. Phytopathology 31: 773-802.

Du, Y., Maslov, D. A., and K.-P. Chang. 1994. Monophyletic origin of (3- division proteobacterial endosymbionts and their coevolution with insect trypanosomatid protozoa Blastocrithidia culicis and Crithidia spp. Proceedings of the National Academy of Science USA 91: 8437-8441 178

Ehrlich, P. R., and P. H. Raven. 1964. Butterflies and plants: a study of coevolution. Evolution 18: 586-608.

El-Kassaby, Y.A., Colangeli, A.M., and Sziklai, 0. 1983. A numerical analysis of karyotyes in the genus Pseudotsuga. Canadian Journal of Botany 61: 536-544.

Ellis, D. E., and L. S. Gill. 1945. A new Rhabdogloeum associated with Rhabdocline pseudotsugae in the southwest. Mycologia 37: 326-332.

Eriksson, 0. E. 1983. Outline of the ascomycetes 1983. Systema Ascomycetum 2: 1-37.

Eriksson, 0. E. and D. L. Hawksworth. 1993. Outline of the Ascomycetes 1993. Systema Ascomycetum 12: 51-257.

Farjon, A. 1990. Pinaceae: drawings and descriptions of the genera Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Koeltz Scientific Books, KOnigstein, Federal Republic of Germany.

Felsenstein, J. 1993. DNAMLK--DNA maximum likelihood program with molecular clock. http://utmmg.med.uth.tm.../phylip info/dnamlk.doc, version 3.5c.

Flor, H. H. 1946. Genetics of pathogenicity in Melampsora lini . Journal of Agricultural Research 73: 335-357.

Florin, R. 1951. Evolution in cordaites and conifers. Acta Horti Bergiani 15: 285-388.

Gargas, A., P. T. DePriest, M. Grube, and A. Tehler. 1995. Multiple origin of lichen symbiosis in fungi suggested by SSU rDNA phylogeny. Science 286: 1492-1495.

, , and J. W. Taylor. 1995. Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Molecular Biology and Evolution 12: 208-218.

, and J. W. Taylor. 1995. Phylogeny of discomycetes and early radiations of the apothecial Ascomycotina inferred from SSU rDNA sequence data. Experimental Mycology 19: 7-15.

Gams, W. 1995. How natural should anamorph taxa be? Canadian Journal of Botany 73: S747-S753.

W., and H.-B. Jansson. 1987. The nematode parasite Meria coniospora Drechsler in pure culture and its classification. Mycotaxon 22: 33-38.

, K. O'Donnell, H.-J. Schroers, and M. Christiansen. 1998. Generic classification of some more hyphomycetes with solitary conidia borne on phialides. Canadian Journal of Botany. In Press. 179

Gernandt, D. S., F. J. Camacho, and J. K. Stone. 1997. Meria laricis, an anamorph of Rhabdocline. Mycologia 85: 735-744.

, and A. Liston. 1998. Internal transcribed spacer evolution in Larix and Pseudotsuga (Pinaceae). submitted to American Journal of Botany.

Glenn, A. E., C. W. Bacon, R. Price, and R. T. Hanlin. 1996. Molecular phylogeny of Acremonium and its taxonomic implications. Mycologia 88: 369-383.

Goremykin, V., Bobrova, V., Pahnke, J., Troitsky, A., Antonov, A., and W. Martin. Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support the Gnetalean affinities of angiosperms. Molecular Biology and Evolution 13: 383-396.

Hafner, M. S., P.D. Sudman, F. X. Villablanca, T. A. Spradling, J. W. Demastes, and S. A. Nadler. 1994. Disparate rates of molecular evolution in cospeciating hosts and parasites. Science 265: 1087-1090.

Hamelin, R. C., J. Rail. 1996. Phylogeny of Gremmeniella spp. based on sequences of the 5.8S rDNA and internal transcribed spacer region. Canadian Journal of Botany 75: 693-698.

Hart, J. A. 1987. A cladistic analysis of conifers: preliminary results. Journal of the Arnold Arboretum 68: 269-307.

Hawksworth, D. L., P. M. Kirk, B. C. Sutton, and D. N. Pegler. 1995. Ainsworth & Bisby's Dictionary of the Fungi. 8th ed. CAB International, Wallingford, United Kingdom. 616 pp.

Hermann, R. K. 1982. The genus Pseudotsuga: historical records and nomenclature. Forest Research Laboratory, Oregon State University, Corvallis, OR, Special Publication 2a.

Hermann, R. K. 1985. The genus Pseudotsuga: ancestral history and past distribution. Forest Research Laboratory, Oregon State University, Corvallis, OR, Special Publication 2b.

Hershkovitz, M. A., and L. A. Lewis. 1996. Deep-level diagnostic value of the rDNA- ITS region. Molecular Biology and Evolution 13: 1276-1295.

Hibbett, D. 1992. Ribosomal RNA and fungal systematics. Transactions of the Mycological Society of Japan 33: 533-556. 180

Hinkle, G., J. K. Wetterer, T. R. Schultz, and M. L. Sogin. 1994. Phylogeny of the attine ant fungi based on analysis of small subunit ribosomal RNA gene sequences. Science 266: 1695-1697.

Hoist- Jensen, A., L. M. Kohn, K. S. Jakobsen, and T. Schumacher. 1997. Molecular phylogeny and evolution of Monilinia (Sclerotiniaceae) based on coding and noncoding rDNA sequences. American Journal of Botany 84: 686-701.

and T. Schumacher. 1997. Nuclear rDNA phylogeny of the Sclerotiniaceae. Mycologia 89: 885-899.

Hoff, R.J. 1988. Susceptibility of inland Douglas-fir to Rhabdocline needle cast. USDA Forest Service Research Note, INT-375, pp. 3.

Jaeger, J. A., Turner, D. H., and M. Zuker. 1990. Predicting optimal and suboptimal structures for RNA. Methods in Enzymology 183: 281-306.

Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules. pp. 21-123. In: Munro, H. N. (ed.) Mammalian Protein Metabolism. Academic Press, New York.

Kane, R. A., and D. Rollinson. 1994. Repetitive sequences in the ribosomal DNA internal transcribed spacer of Schistosoma haematobiumcchistosoma intercalatum, and Schistosoma mattheei. Molecular Biochemical Parasitology 63: 153-156.

Karvonen, P. and 0. Savolainen. 1993. Variation and inheritance of ribosomal DNA in Pinus sylvestris L. (Scots pine). Heredity 71: 614-622.

A. E. Szmidt, and 0. Savolainen. 1994. Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species. Theoretical and Applied Genetics 89: 969-974.

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120.

Kohn L. M. and R. P. Korf. 1975. Variation in ascomycete iodine reactions: KOH pretreatment explored. Mycotaxon 3: 165-172.

Korf, R. P. 1962. A synopsis of the Hemiphacideaceae, a family of Helotiales (discomycetes) causing needle blights of conifers. Mycologia 54: 12-33.

. 1983. Cyttaria (Cyttariales): Coevolution with Nothofagus, and evolutionary relationship to the Boedijnopezizeae. Australian Journal of Botany Supplement Series 10: 77-87. 181

. 1994. Quoted from Ascomycete systematics: problems and perspectives in the nineties. (David L. Hawksworth, Ed). New York: Plenum Press, pp. 453.

Kuhls, K., E. Lieckfeldt, G. J. Samuels, W. Meyer, C. P. Kubicek, and T. Burner. 1997. Revision of Trichoderma sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA internal transcribed spacer sequences. Mycologia 89: 442-460.

Landvik, S., 0. E. Eriksson, A. Gargas, and P. Gustafsson. 1993. Relationships of the genus Neolecta (Neolectales ordo nov., Ascomycotina) inferred from 18S rDNA sequences. Systema Ascomycetum 11: 107-118.

, and . 1994. Relationships of Tuber, Elaphomyces, and Cyttaria (Ascomycotina), inferred from 18s rDNA studies. Pp. 225-231. In: Ascomycete systematics, problems and perspectives in the nineties. NATO ASI Series A, Vol 269. Ed., D. L. Hawksworth. Plenum Press, New York, New York.

N. Shailer, and 0. E. Eriksson. 1996. SSU rDNA sequence support for a close relationship between the Elaphomycetales and the Eurotiales and Onygenales. Mycoscience 37: 237-241.

K. N. Egger, and T. Schumacher. 1997. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analyses of SSU rDNA sequences. Nordic Journal of Botany 17: 403-418.

LePage, B. A., and J. F. Basinger. 1991. A new species of Larix (Pinaceae) from the early Tertiary of Axel Heiberg Island, Arctic Canada. Review of Palaeobotany and Palynology 70: 89-111.

and . 1995. The evolutionary history of the genus Larix (Pinaceae). In W. C. Schmidt and K. J. McDonald, [compilers], Ecology and management of Larix forests: a look ahead, pp. 19-29. United States Department of Agriculture, Forest Service, Intermountain Research Station, Ogden, UT, General Technical Report GTR-INT-319.

Lewandowski, A. 1997. Genetic relationships between European and Siberian larch, Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species? Plant Systematics and Evolution 204: 65-73.

Li, P., and W. T. Adams. 1989. Range-wide patterns of allozyme variation in Douglas fir (Pseudotsuga menziesii). Canadian Journal of Forest Research 19: 149-161. 182

Liston, A. and J. W. Kadereit. 1995. Chloroplast DNA evidence for introgression and long distance dispersal in the desert annual Senecio flavus (Asteraceae). Plant Systematics and Evolution 197: 33-41.

, W. A. Robinson, J. M. Oliphant, and E. R. Alvarez-Buylla. 1996. Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non­ flowering seed plants. Systematic Botany 21: 109-120.

Liu, J.-S., and C. L. Schardl. 1994. A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Molecular Biology 26: 775-778.

Livsey, S., and Minter, D.W. 1993. The taxonomy and biology of Tiyblidiopsis pinastri. Canadian Journal of Botany 72:549-557.

LoBuglio, K. F., M. L. Berbee, and J. W. Taylor. 1996. Phylogenetic origins of the asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes. Molecular Phylogenetics and Evolution 6: 287­ 294

Lundberg, J. G. 1972. Wagner networks and ancestors. Systematic Zoology 21: 398-413.

Luton, K., D. Walker, and D. Blair. 1992. Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes: Trematoda: Digenea). Molecular Biochemical Parasitology 56: 323-328.

MacLeod, D. M. 1954. Investigations on the genera Beauveria Vuill. and Tritiachium Limber. Canadian Journal of Botany 32: 818-890.

Marrocco, R., M. T. Gelati, and F. Maggini. 1996. Nucleotide sequence of the internal transcribed spacers and 5.8S region of ribosomal DNA in Pinus pinea L. DNA Sequence 6: 175-177.

Martinez, M. 1949. Las Pseudotsugas de Mexico. Anales del Instituto de Biologia 20: 129-184.

McCutcheon, T. L., G. C. Carroll, and S. Schwab. 1993. Genotype diversity in populations of a fungal endophyte from Douglas-fir. Mycologia 85: 180-186.

Millar, C.I. and B.B. Kinloch. 1991. Taxonomy, phylogeny, and coevolution of pines and their stem rusts. In: Rusts of pine. Proceedings of the HIFRO rusts of pine working party conference, September 18-22, 1989, Banff, Alberta, Canada. (Hiratsuka, Y., Samoil, J.K., Blenis, P.V., Crane, P.E., and B.L. Laishley eds.) Information report NOR-X-317, Forestry Canada Northwest Region Northern Forestry Centre, pp. 1-38. 183

Miller Jr., C. N. 1977. Mesozoic conifers. The Botanical Review 43: 217-280.

. 1982. Current status of Paleozoic and Mesozoic conifers. Review of Paleobotany and Palynology 37: 99-114.

. 1988. The origin of modern conifer families. Pp. 448-487 in: Origin and Evolution of Gymnosperms. C. B. Beck, (ed.), Columbia Univ. Press, New York.

Minter, D.W. 1994. Quoted from Ascomycete systematics: problems and perspectives in the nineties. (David L. Hawksworth, Ed). New York: Plenum Press, pp. 453.

Morales, V. M., L. E. Pelcher, and J. L. Taylor. 1993. Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Lepto.sphaeria maculans from different pathogenicity groups. Current Genetics 23: 490-495.

Moran, G. F., and W. T. Adams. 1989. Microgeographical patterns of allozyme differentiation in Douglas-fir from Southwest Oregon. Forest Science 35: 3-15.

Morgan-Jones, G. 1973. Genera coelomycetarum. VII. Cryptocline Petrak. Canadian Journal of Botany 51: 309-325.

Munson, M.A., P. Baumann, M. A. Clark, L. Baumann, N. Moran, D. J. Voegtlin and B. C. Campbell. 1991. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. Journal of Bacteriology 173: 6321-6324.

Nag Raj, T. R., and W. B. Kendrick. 1993. The anamorph as generic determinant in the holomorph: the Chalara connection in the ascomycetes with special reference to the ophiostomatoid fungi. Pp 61-70 In: M. J. Wingfield, K.A. Seifert, and J. F. Webber (eds.) Ceratocystis and Ophiostoma. Taxonomy, ecology and pathogenicity. APS Press, St. Paul, Minnesota, USA.

Nannfeldt, J. A. 1932. Studien fiber die Morphologic and Systematik der nicht­ lichenisierten inoperculaten Discomyceten. Nova Acta Regiae Societatis Scientarum Upsaliensis Ser. IV, 8: 1-368.

Nickrent, D. L., K. P. Schuette, and E. M. Starr. 1994. A molecular phylogeny of Arceuthobium (Viscaceae) based on nuclear ribosomal DNA internal transcribed spacer sequences. American Journal of Botany 81: 1149-1160.

Nixon, K. C., and J. M. Carpenter. 1993. On outgroups. Cladistics 9: 413-426.

, Crepet, W. L., Stevenson, D., and E. M. Friis. 1994. A reevaluation of seed plant phylogeny. Annals of the Missouri Botanical Garden 81: 484-533. 184

O'Donnell, K. 1992. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Current Genetics 22: 213-220.

E. Cigelnik, N. S. Weber, and J. M. Trappe. 1997. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48-65.

Ogawa, H., A. Yoshimura, and J. Sugiyama. 1997. Polyphyletic origins of species of the anamorphic genus Geosmithia and the relationships of the cleistothecial genera: evidence from 18S, 5S and 28S rDNA sequence analysis. Mycologia 89: 756-771.

Page, R. D. M. 1993. Parasites, phylogeny, and cospeciation. International Journal of Parasitology 23: 499-506.

. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Applications in Bioscience 12: 357--358.

, D. H. Clayton, and A. M. Patterson. 1996. Lice and cospeciation: a response to Barker. International Journal of Parasitology 26: 213-218.

, and M. A. Charleston. 1998. Trees within trees: phylogeny and historical associations. Trends in ecology and evolution. In press.

Palmer, J. D. 1992. Mitochondrial DNA in plant systematics: Applications and limitations. In: Molecular Systematics of Plants, P. S. Soltis, D. E. Soltis, and J. J. Doyle, (eds.) Chapman and Hall, New York.

Parker, A. K., and J. Reid. 1969. The genus Rhabdocline Syd. Canadian Journal of Botany 47: 1533-1545.

Patschke, W. 1913. Ober die extra tropischen ostasiatischen Coniferun und ihre Bedeutung fur die pflanzengeographische Gliederung Ostasiens. Botanische Jahrbucher fur Systematik, Pflanzengeschichte und Pflanzengeographie 48: 626­ 776.

Peace, T. R., and C. H. Holmes. 1933. Mena laricis the leaf cast disease of larch. Oxford Forestry Memoirs 15. 29 pp.

Petrini, 0. 1982. Notes on some species of Chloroscypha endophytic in Cupressaceae of Europe and North America. Sydowia 35: 206-222.

. 1984. Cryptocline arctostaphyli sp. nov., an endophyte of Arcostaphylos uva­ ursi and other Ericaceae. Sydowia Annales Mycologici 37: 238-241. 185

Pfister, D. H. 1997. Castor, Pollux and life histories of fungi. Mycologia 89: 1-23.

Pirozynski, K.A, and D. Malloch. 1975. The origin of land plants: a matter of mycotrophism. Biosystems 6: 153-164.

Ponoy, B., Y.-P. Hong, J. Woods, and J. E. Carlson. 1994. Chloroplast DNA diversity of Douglas-fir in British Columbia. Canadian Journal of Forest Research 24: 1824­ 1834.

Prager, E. M., D. P. Fowler, and A. C. Wilson. 1976. Rates of evolution in the Pinaceae. Evolution 30: 637-649.

Price, R. A., J. Olsen-Stojkovich, and J. M. Lowenstein. 1987. Relationships among the genera of Pinaceae: An immunological comparison. Systematic Botany 12: 91-97.

Qian, T., R. A. Ennos, and T. Helgason. 1995. Genetic relationships among larch species based on analysis of restriction fragment variation for chloroplast DNA. Canadian Journal of Forest Research 25: 1197-1202.

Quijada, A., A. Liston, P. Delgado, A. Vazquez-Lobo, and E. R. Alvarez-Buylla. 1998. Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP. Theoretical and Applied Genetics 96: in press.

Reid, J. and R. F. Cain. 1963. New genus of the Hemiphacidiaceae. Mycologia 55: 781­ 785.

Relle, M., Furst, and A.Wild. 1995. Short duplication in a cDNA clone of the rbcL gene from Picea abies. Plant Physiology 107: 1037-1038.

Reynolds, D.R. 1993. The fungal holomorph: an overview. Pp. 15-25. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic Speciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

Rieseberg, L. H., and D. E. Soltis. 1991. Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants 5: 65-84.

Saiki, K.1. 1996. Pinus mutoi (Pinaceae), a new species of permineralized seed cone from the Upper Cretaceous of Hokkaido, Japan. American Journal of Botany 83: 1630-1636.

Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425. 186

Savard, L. P. Li, S. H. Strauss, M. W. Chase, M. Michaud, and J. Bousquet. 1994. Chloroplast and nuclear gene sequences indicate Late Pennsylvanian time for the last common ancestor of extant seed plants. Proceedings of the National Academy of Science USA 91: 5163-5167.

Schnabel, A., J. L. Hamrick, and P. V. Wells. 1993. Influence of Quaternary history on the population genetic structure of Douglas-fir (Pseudotsuga menziesii) in the Great Basin. Canadian Journal of Forest Research 23: 1900-1906.

Schorn, H. E. 1994. A preliminary discussion of fossil larches (Larix, Pinaceae) from the arctic. Quaternary International 22/23: 173-183.

Seaver, F. J. 1951 The North American Cup-fungi. Vol. 2: Inoperculates. Reprint 1978. Lubrecht & Cramer, Monticello, N.Y.

Sherwood-Pike, M., J. K. Stone, and G. C. Carroll. 1986. Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canadian Journal Botany 64: 1849­ 1855.

Smith, A. B. 1994. Rooting molecular trees: problems and strategies. Biological Journal of the Linnean Society 51: 279-292.

Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gillevet. 1994. The genetic data environment, an expandable GUI for multiple sequence analysis. Cabios 10: 671-675.

Spatafora, J. W. and M. Blackwell. 1993. Molecular systematics of unitunicate perithecial ascomycetes: The Clavicipitales-Hypocreales connection. Mycologia 85: 912-922.

, and . 1994. Polyphyletic origins of Ophiostomatoid fungi. Mycological Research 85: 912-922.

. 1995. Ascomal evolution of filamentous ascomycetes: evidence from molecular

data. Canadian Journal of Botany 73 (Suppl . 1): S811-S815

, T. G. Mitchell, and R. Vilgalys. 1995. Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of Dematiaceous fungal pathogens. Journal of Clinical Microbiology 33: 1322-1326.

Springer, M. S. 1995. Molecular clocks and the incompleteness of the fossil record. Journal of Molecular Evolution 41: 531-538.

Stephan, Von B. R. 1980. Testing of Douglas-fir provenances for resistance against 187

Rhabdocline pseudotsugae by infection trials. European Journal of Forest Pathology 10: 152-161.

Stone, J. K. 1986. Initiation and development of latent infections by Rhabdocline parkeri on Douglas-fir. Canadian Journal of Botany 65: 2614-2621.

. 1988. Fine structure of latent infectins by Rhabdocline parkeri on Douglas-fir, with observations on uninfected epidermal cells. Canadian Journal of Botany 66: 45-54.

Strauss, S. H., A. H. Doerksen, and J. R. Byrne. 1990. Evolutionary relationships of Douglas-fir and its relatives (genus Pseudotsuga) from DNA restriction fragment analysis. Canadian Journal of Botany 68: 1502-1510.

Swofford, D. L. 1993. PA UP: Phylogenetic analysis using parsimony, Version 3.0s+ 4 . Illinois Natural History Survey, Champaign, Illinois.

, G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. Pp. 407-514. In: Molecular systematics. Eds., D. M. Hillis, C. Moritz, and B. K. Mable. Sinauer Associates, Inc., Sunderland, Massachusetts.

. 1998. PAUP* version 4.0. Sinauer Assoc., Inc., Sunderland, MA (in press).

Sydow, H. and F. Petrak. 1922. Ein Beitrag zur Kenntnis der Pilzflora Nordamericas, insbesondere der nordwestlichen Staaten. Annales Mycologici 20:178-218.

Takaso, T. 1996. Pollination mechanisms in conifers. Ada Phytotaxonomica et Geobotanica 47: 253-269.

Taylor, J. W. 1993. A contemporary view of the holomorph: nucleic acid sequence and computer databases are changing fungal classification. Pp. 1-13. In: The Fungal Holomorph: Mitotic, Meiotic, and Pleomorphic iSpeciation in Fungal Systematics. Eds., D. R. Reynolds and J. W. Taylor. CAB International, Wallingford, United Kingdom.

Todd, D. 1987. The effects of host genotype, growth rate, and needle age on the distribution of a mutualistic, endophytic fungus in Douglas-fir plantations. Canadian Journal of Forest Research 18: 601-605.

Tsumura, Y., K. Yoshimura, N. Tomaru, and K. Ohba. 1995. Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes. Theoretical and Applied Genetics 91: 1222-1236.

Van Nuess, R. W., J. M. J. Rientjes, C. A. F. M. Van Der Sande, S. F. Zerp, C. Sluiter, J. Venema, R. J. Planta, and H. A. Raue. 1994. Separate structural elements within 188

internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Research 22: 912-919.

Vining, T. F., and C. S. Campbell. 1997. Phylogenetic signal in sequence repeats within nuclear ribosomal DNA internal transcribed spacer 1 in Tsuga. American Journal of Botany 84: 702 (Abstract).

Vogler, D. R., and T. D. Bruns. 1998. Phylogenetic relationships among the pine stem rust fungi (Cronartium and Peridermium spp.). Mycologia 90: 244-257.

Wedin, M., and L. Tibell. 1997. Phylogeny and evolution of Caliciaceae, Mycocaliciaceae, and Sphinctrinaceae (Ascomycota), with notes on the evolution of the prototunicate ascus. Canadian Journal of Botany 75: 1236-1242.

Whetzel, H. H. 1945. A synopsis of the genera and species of the Sclerotiniaceae, a family of stromatic inoperculate discomycetes. Mycologia 37: 648-714.

White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In: PCR Protocols, a guide to methods and applications. Eds., M. A. Innis, D. H. Gelfand, J. J. Snisky, and T. J. White. San Diego: Academic Press.

Whitney, H. S., J. Reid, and K. A. Pirozynski. 1975. Some fungi associated with needle blight of conifers. Canadian Journal of Botany 53 : 3051-3063.

Wilmotte, A., Y. Van De Peer, A. Goris, S. Chapelle, R. De Baere, B. Nelissen, J. -M. Neefs, G. L. Hennebert, and R. De Wachter. 1993. Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA analysis. Systematic and Applied Microbiology 16: 436-444.

Wilson, A. C., Ochman H., and E. M. Prager. 1987. Molecular time scale for evolution. Trends in Genetics 3:241-247.

Wingfield, M. J., B. Kendrich, and P. S. van Wyck. 1991. Analysis of conidium ontogeny in anamorphs of Ophiostoma: Pesotum and Phialographium are synonyms of Graphium. Mycological Research 95: 1328-1333.

Wisconsin Package Version 9.0. 1996. Genetics Computer Group (GCG), Madison, WI.

Wolf, J. A., and H. E. Schorn. 1990. Taxonomic revision of the Spermatopsida of the Oligocene Creede flora, Southern Colorado. US Geological Survey Bulletin 1923. 189

Wulf, Von A., and L. Pehl. 1996. Erster Fund von Fabrella tsugae (Fart.) Kirschst. in der Bundesrepublik Deutschland ein neuer Nadelpilz an Tsuga canadensis (L.) Carr. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 48: 1-4.

Xiao-Quan, W., H. Ying, D. Zheng-Rong, and H. De-Yuan. 1997. Phylogeny of the Pinaceae evidenced by molecular biology. Acta Phytotaxonomica Sinica 35: 97­ 106.

Yeh, L.-C. C., R. Thweatt, and J. C. Lee. 1990. Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common motifs. Biochemistry 29: 5911-5918.

Zuckerkandl, E., and L. Pauling. 1965. Evolutionary divergence and convergence in proteins. Pp. 97-166 in: Evolving Genes and Proteins, V. Brysra and H. J. Vogel (eds.) Academic Press, New York.