TAXONOMIC STATUS OF GENERA IN THE “NOWAKOWSKIELLA” CLADE (KINGDOM FUNGI, PHYLUM CHYTRIDIOMYCOTA): PHYLOGENETIC ANALYSIS OF MOLECULAR CHARACTERS WITH A REVIEW OF DESCRIBED SPECIES by SHARON ELIZABETH MOZLEY (Under the Direction of David Porter) ABSTRACT Chytrid fungi represent the earliest group of fungi to have emerged within the Kingdom Fungi. Unfortunately despite the importance of chytrids to understanding fungal evolution, the systematics of the group is in disarray and in desperate need of revision. Funding by the NSF PEET program has provided an opportunity to revise the systematics of chytrid fungi with an initial focus on four specific clades in the order Chytridiales. The “Nowakowskiella” clade was chosen as a test group for comparing molecular methods of phylogenetic reconstruction with the more traditional morphological and developmental character system used for classification in determining generic limits for chytrid genera. Portions of the 18S and 28S nrDNA genes were sequenced for isolates identified to genus level based on morphology to seven genera in the “Nowakowskiella” clade: Allochytridium, Catenochytridium, Cladochytrium, Endochytrium, Nephrochytrium, Nowakowskiella, and Septochytrium. Bayesian, parsimony, and maximum likelihood methods of phylogenetic inference were used to produce trees based on one (18S or 28S alone) and two-gene datasets in order to see if there would be a difference depending on which optimality criterion was used and the number of genes included. In addition to the molecular analysis, taxonomic summaries of all seven genera covering all validly published species with a listing of synonyms and questionable species is provided to give a better idea of what has been described and the morphological and developmental characters used to circumscribe each genus. All of the isolates sequenced for the molecular portion of this study have been cryopreserved for future work and a review of cryopreservation of chytrid fungi is included. A modification of a previously published Q-tip method used to cryopreserve fungi for this study is also given. INDEX WORDS: Chytrid, Fungi, Allochytridium, Catenochytridium, Cladochytrium, Endochytrium, Nephrochytrium, Nowakowskiella, Septochytrium, Taxonomic Summary, Morphology, Development, Genera, 18S nrDNA, 28S nrDNA, Bayesian, Parsimony, Maximum Likelihood, Cryopreservation, Q-tip, Glycerol, Liquid Nitrogen TAXONOMIC STATUS OF GENERA IN THE “NOWAKOWSKIELLA” CLADE (KINGDOM FUNGI, PHYLUM CHYTRIDIOMYCOTA): PHYLOGENETIC ANALYSIS OF MOLECULAR CHARACTERS WITH A REVIEW OF DESCRIBED SPECIES by SHARON ELIZABETH MOZLEY B.S., The University of Georgia, 1998 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2005 © 2005 Sharon Elizabeth Mozley All Rights Reserved TAXONOMIC STATUS OF GENERA IN THE “NOWAKOWSKIELLA” CLADE (KINGDOM FUNGI, PHYLUM CHYTRIDIOMYCOTA): PHYLOGENETIC ANALYSIS OF MOLECULAR CHARACTERS WITH A REVIEW OF DESCRIBED SPECIES by SHARON ELIZABETH MOZLEY Major Professor: David Porter Committee: Marshall Darley Robert Kuzoff Charles Mims Zheng-hua Ye Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2005 iv DEDICATION I dedicate my dissertation to my wonderfully supportive husband, Zach Standridge, my family and my extended family that consists of my sister, Laurie Mozley, my mom, Chris Mozley, my dad, Ron Mozley, my grandmothers Sarah I. Mozley and Ethel Baker, my mother- in-law, Patty Standridge, my father-in-law, David Standridge, sister-in-law, brother-in-law, and nephew, Kay, Kirk, and Braxton Standridge and my grandmother-in-law, Betty Corn. v ACKNOWLEDGEMENTS This study was supported by NSF-PEET Grant # DEB-9978094. I would like to thank Dr. Joyce Longcore for providing cultures for this study, for teaching me how to isolate chytrids from the environment and for her advice and expertise. I would also like to express my gratitude to Dr. Jimmy Chambers for allowing me to use several of his chytrid sequences in my phylogenetic analyses and to Dr. Will Blackwell for letting me look at chytrids in his light microscope. I would also like to extend thanks to Dr. Pete Letcher for letting me bug him again and again about zoospore fixation and to Dr. Martha Powell for visiting her lab and coming away with valuable ideas and reprints. Thanks also to Dr. Rytas Vilgalys and Dr. Tim James for allowing me to spend time in the Vilgalys lab asking questions and doing a lot of DNA extraction and sequencing in their wonderful sequencing lab. I would also like to thank the following people for all of their support, advice, encouragement and reminder that life is not all work but can include some play during my time as a graduate student: David and Jean Porter, Celeste, Brian, Avory, and Emerson Leander, Charla, Eric, Griffin and Phoenix Haarbauer, Jason and Monica Watkins, Victoria and Hector Vazquez, Marshall and Priscilla Darley, Rob Specker, Holly Thornton, Jonathan Hulvey and all of the students that have wandered through the Porter lab wanting to know what was growing on the plates on my bench. The following publications have granted permission to use figures pertinent to species of all seven genera in the “Nowakowskiella” clade: Allochytridium, Catenochytridium, Cladochytrium, Endochytrium, Nephrochytrium, Nowakowskiella, and Septochytrium. Chytridiomycetarum Iconographia (Lubrecht and Cramer): Allochytridium expandens Salkin, PLATE 65 figs. 1,6,13,17, vi Catenochytridium carolinianum Berdan, PLATE 72 figs. 1, 2, 11, Catenochytridium laterale Hanson, PLATE 72 figs. 13, 14, 24-28, Catenochytridium marinum (Kobayasi and Ookubo) Karling, PLATE 73 fig. 29, Catenochytridium kevorkiana Sparrow, PLATE 73 figs. 30-31, Catenochytridium oahuensis Sparrow, PLATE 73 figs. 32-35, Cladochytrium tenue Nowakowski, PLATE 101 figs. 1-16, Cladochytrium setigerum Karling, PLATE 101 figs. 22-23, 25-27, Cladochytrium replicatum Karling, PLATE 102 figs. 28, 37, 41-42, 45-47, 51, 53, Cladochytrium aurantiacum Richards, PLATE 102 figs. 48-50, 52, Cladochytrium tainum Shen and Siang, PLATE 102 figs. 54-55, Cladochytrium hyalinum Berdan, PLATE 103 figs. 56, 64- 66, 70, 72, Cladochytrium crassum Hillegas, PLATE 103 figs. 74, 79-80, 82-84, 86-87, Endochytrium operculatum Karling, PLATE 82 figs. 1-3, 12-16, 19-22, Endochytrium ramosum Sparrow, PLATE 82 figs. 23-27, Endochytrium pseudodistomum Schefferl, PLATE 83 figs. 28- 34, Endochytrium digitatum Karling, PLATE 83 figs. 35, 40-45, 48, Endochytrium multiguttulatum Dogma, PLATE 84 figs. 52, 61-64, 65, 67-69, Endochytrium cystarum Dogma, PLATE 84 figs. 70-71, 73-77, 79, 81-83, Nephrochytrium appendiculatum Karling, PLATE 94 figs. 1-5, 7-16, 18, Nephrochytrium stellatum Couch, PLATE 94 figs. 21-26, 28, 31-38, Nephrochytrium aurantium Whiffen, PLATE 95 figs. 40, 43-4547-50, Nephrochytrium amazonenese Karling, PLATE 95 figs. 52, 55-66, Nephrochytrium buttermerense Willoughby, PLATE 95 figs. 68-81, Nowakowskiella elegans Nowakowski, PLATE 105 figs. 1, 2, 7, Nowakowskiella sculptura Karling, PLATE 106 figs. 26, 28-32, 35, 38, Nowakowskiella hemisphaerospora Shanor, PLATE 106 figs. 39-40, 46, 51-52, Nowakowskiella macrospora Karling, PLATE 107 figs. 68, 79, 81, Nowakowskiella multispora Karling, PLATE 108 figs. 101-103, Septochytrium variabile Berdan, PLATE 111 figs. 1, 5, 8, Septochytrium plurilobulum Johanson, PLATE 111 figs. 11, 13, 15, Septochytrium macrosporum Karling, PLATE 112 figs. vii 18-19, 21, 25, Septochytrium marylandicum Karling, PLATE 112 figs. 29,25, Diplophlyctis sexualis Haskins (=Nephrochytrium sexuale (Haskins) Batko), PLATE 92 figs. 22-27, copyright 1977; Torrey Botanical Society (Journal of the Torrey Botanical Society): Nowakowskiella granulata Karling, figs. 1-29, Nowakowskiella elongata Karling, figs. 30-44, Nowakowskiella ramosa Butler, figs. 69A-V, copyright 1944, Septochytrium marilandicum Karling, figs. 9, 16A- B, copyright 1951; Washington Academy of Sciences (Journal of the Washington Academy of Sciences): Nowakowskiella atkinsii Sparrow, figs. 25-26, copyright 1950; E. Schweizerbart ‘ sche Verlagsbuchhandlung (Naegele u. Obermiller) Science Publishers (Nova Hedwigia): Septochytrium willoughbyi Dogma, figs. 5, 8, 10, copyright 1973. The following images were re- drawn for use in this publication: Allochytridium luteum Barr and Désaulniers adapted from Barr and Desáulniers (1987), figs. 3, 5; Catenochytridium hemicysti Knox adapted from Barr, Désaulniers, and Knox (1987), figs. 7, 10; Cladochytrium indicum Singh and Pavgi adapted from Singh and Pavgi (1971), figs. 2, 5, 6, 7; Cladochytrium novoguineense Kobayasi and Konno adapted from Kobayasi and Konno (1971), fig. 3C-I; Cladochytrium salsuginosum Batko and Hassan adapted from Batko and Hassan (1986), figs. 1-5, 8-9; Nephrochytrium bipes Hassan adapted from Hassan (1983), figs. 18, 23, 28; Nowakowskiella keratinophila Hassan and Batko adapted from Hassan and Batko (1986), figs. 1-3, 7; Nowakowskiella methistemichroma Batko and Hassan adapted from Batko and Hassan (1982), figs. 4, 8, 10; Nowakowskiella moubasheriana Hassan adapted from Hassan (1983), figs. 1, 7, 10, 16-17; Nowakowskiella multispora var. longa Kiran adapted from Kiran (1992), figs. 2, 5-6; Septochytrium variabile Berdan adapted from Berdan (1942), fig. 25. viii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES...........................................................................................................................x