A MONOGRAPH OF THE FRESHWATER ASCOMYCETE FAMILY ANNULATASCACEAE: A MORPHOLOGICAL AND MOLECULAR STUDY BY STEVEN EDWARD ZELSKI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology in the Graduate College of the University of Illinois at Urbana-Champaign, 2015 Urbana, Illinois Doctoral Committee: Professor Emerita Carol A. Shearer, Chair, Director of Research Research Professor Andrew N. Miller, Co-Chair, Co-Director of Research Professor Emeritus David S. Seigler Professor Stephen R. Downie ABSTRACT A MONOGRAPH OF THE FRESHWATER ASCOMYCETE FAMILY ANNULATASCACEAE: A MORPHOLOGICAL AND MOLECULAR STUDY Steven Edward Zelski, Ph.D. Department of Plant Biology University of Illinois at Urbana-Champaign, 2015 Carol A. Shearer and Andrew N. Miller, Co-advisors Freshwater fungi are important agents decomposing submerged dead plant material. Roughly ten percent of the known teleomorphic (sexually reproducing) freshwater ascomycetes have been referred to or included in the family Annulatascaceae. Placement in this family is based on characters that include perithecial ascomata, unitunicate cylindrical asci with relatively large J- (Melzer’s reagent negative) apical rings, and the presence of long tapering septate paraphyses. However, the large refractive apical apparati are the distinctive feature of the family. As sparse molecular data were available prior to the beginning of this study, a broad survey of freshwater temperate and tropical areas was conducted to collect these taxa for morphological examination, digital imagery, and extraction of DNA for phylogenetic inference. Thirty-five of roughly 70 described species in 21 genera of Annulatascaceae were assessed molecularly, and forty-five illustrated from holotypes and/or fresh collections. Twelve new taxa with relatively large non-amyloid apical apparati were discovered, only one of which, Longicollum biappendiculatum, belongs in Annulatascaceae. Material examined in this study was preserved as permanent slides, dried specimens, and/or living cultures. Phylogenetic analyses of 4 genes alone and in combination revealed a polyphyletic Annulatascaceae, with a sensu stricto clade consisting of the type, Annulatascus velatisporus, and nine other species. The other twenty-five taxa assessed molecularly were located in clades along the Sordariomycetes tree, basal to the Ophiostomatales, the nearest outgroup to Annulatascaceae in these analyses. Annulatascaceae sensu stricto lacks likelihood support at its root node in single and combined gene analyses, but is strongly supported by Bayesian posterior probability in ii analyses of combined genes. This strongly suggests a familial if not an ordinal relationship of these ten taxa. One limitation of this study was a constraint on the number of genes sequenced. Some taxa only had GenBank sequence data and newly sequenced species did not all provide Mcm7 sequences as the primers are highly degenerate and annealing temperatures were variable. Addition of other genes (e.g. RPB1, TEF1α) to future analyses may increase confidence at both basal and distal nodes for this clade. A second limitation of this study was taxonomic coverage using sequence data. Though great effort was given to collecting, not all Annulatascaceae taxa were encountered. Collecting freshwater ascomycetes entails randomly selecting submerged woody debris and incubating it in moist chambers with the hopes of obtaining these microscopic organism. This is in contrast to collecting macroscopic organisms, which are readily recognizable in the field. Much work remains to be done on taxa thus far known only from the Eastern Hemisphere. Molecular data from these taxa would provide additional resolving power for lineages with large apical rings. iii DEDICATION This is dedicated to my ma n pa, without whose love I might not have been encouraged to explore. iv ACKNOWLEDGEMENTS I would like to start by offering my greatest thanks to my mentor, from my Master’s program to the present, Dr. Carol A. Shearer. Mushrooms intrigued me, fungi fascinated me, and aquatic ascomycetes got me in their thrall. Thank you for opening my eyes to a fascinating field of study. You were strict and kind and that has propelled me to this point. I will be forever in your debt. To my co-advisor Andrew N. Miller, I am in debt to you for allowing me to study under your tutelage. Thank you for knocking off some of the corrosion I had accumulated before joining this program. You demand a steep learning curve (I am glad to no longer hold the doofus award). It has been a remarkable learning experience. To Dr. David Seigler, it has been a pleasure to discuss taxonomy and systematics with you over the last 6 years. Your knowledge and candor were essential to keep me on track. With a kinder committee member and laboratory neighbor, I could not have been blessed. To Dr. Stephen Downie, I must thank you for you advocacy of graduate students and myself especially. I appreciate your systematics knowledge and willingness to serve on my committee. Without Dr. Steve Stephenson I would not have been able to visit Southeast Asia. Molecular data from collections made there have corroborated tropical distributions of many taxa in the course of the work presented here. The invitation to collect at Pea Ridge National Military Park increased the breadth of distribution patterns of some cosmopolitan species of freshwater fungi. What I learned about myxomycete distribution patterns has tempered my ideas about freshwater ascomycete distribution patterns. To my brother Dr. John Janovec, collecting was almost a secondary goal to learning the cultures of your adopted home. We knocked out the collections like true professionals (thankfully the science needed incubation) and you introduced me to the social, economic, and political factors affecting a true v biodiversity hotspot. Thanks to you, I can still feel optimistic about the future of these places we’ve explored. Rock n Roll. To my next door neighbor from 20 years ago, and the first professor I met in graduate school 10 years ago, Dr. Carol Augsperger, I smilingly thank you for being, frankly, awesome. I expect more from my students having taught with you, and am confident that I can get it. My footsteps are lighter, my footprints smaller. Thank you to the Plant Biology Office staff over these years. I would be remiss if I did not mentally hug Carol A. Hall, Martha Plummer, Lisa Boise, Martha J. Forrest, Rayme T. Dorsey, and Laura B. Thurwell. You kept this ship on an even keel. I would also like to thank the undergraduate students who worked in the lab over the years. A lot of the work for this study would have been impossible without their assistance. Zach Weber, Daniel Chavez, Jess Endres and Austin Meyer maintained specimens and cultures for the lab, and in turn for this project. Julia Balto and Christine Do, aside from running the lab, must be acknowledged for co-authoring a published paper. An extra thanks is due Miss Balto, who participated in a month long trip to the Andes and Amazon of Perú. One tough field partner. I would like to thank Vincent P. Hustad for being a consummate travelling companion and collecting partner, a sounding board, and an ally. To the only person with whom I have ever watched a cricket match (or waited for a boat at 4:00 a.m.), Huzefa A. Raja, thanks aren’t enough... vi Finally, I would like to acknowledge the generous funding from the R. Weldon Larimore award, the Herbert Holdsworth Ross award, a month-long INPA fellowship furnished by CNPq, the Lemann grant, and NSF grants DEB 08-44722 and DEB-1214369. vii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ……………………………………………………………………….1 CHAPTER 2: MATERIALS AND METHODS …………………...………………………………….11 Collecting sites ....………………………………...………………………………………………11 Habitat characterization ...…………………………………………..……………………………11 Field Collection and Herbarium Requests ...………………………………………..……………11 Examination of Substrates .............................................................................................................12 Isolation ..........................................................................................................................................13 Imaging ..........................................................................................................................................13 Specimen preservation ...................................................................................................................14 Molecular methods .........................................................................................................................14 DNA extraction .................................................................................................................14 PCR ...................................................................................................................................14 Sequencing ........................................................................................................................15 Taxon sampling .................................................................................................................15 Sequence assembly............................................................................................................15 Phylogenetic analyses .......................................................................................................16 CHAPTER 3: RESULTS ..........................................................................................................................17