A REVIEW OF THE PRYMNESIOPHYTA, EMPHASIZING THE MORPHOLOGY AND SYSTEMATICS OF HYMENOMONAS STEIN (1878) AND PLEUROCHRYSIS PRINGSHEIM (1955) Cory Dashiell A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Biology and Marine Biology University of North Carolina Wilmington 2010 Approved by Advisory Committee Dr. Wilson Freshwater Dr. Richard Dillaman Dr. Alison Taylor Dr. J. Craig Bailey Chair Accepted by Dr. Roer Dean, Graduate School TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii ACKNOWLEDGMENTS ................................................................................................ iv DEDICATION.....................................................................................................................v LIST OF TABLES............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER 1 ........................................................................................................................1 Background Information .........................................................................................1 CHAPTER 2 - PHYLOGENY...........................................................................................11 Introduction............................................................................................................11 Methods .................................................................................................................14 Results ...................................................................................................................18 Discussion..............................................................................................................23 CHAPTER 3 – CONFOCAL MICROSCOPY..................................................................32 Introduction............................................................................................................32 Methods .................................................................................................................33 Results ...................................................................................................................35 Discussion..............................................................................................................37 LITERATURE CITED ......................................................................................................39 FIGURE LEGENDS .........................................................................................................75 ii ABSTRACT The systematics of several isolates belonging to the Hymenomonadaceae and Pleurochrysidaceae (Prymnesiophyceae) were newly examined or reexamined. A new species, Pleurochrysis dimidius, is described on the basis of light- and electron microscopic observations and comparative analyses of nuclear 18S rRNA and plastid rbcL gene sequences. Results confirm that the Hymenomonadaceae and Pleurochrysidaceae are sister taxa within the Coccolithales. Two isolates identified as Hymenomonas spp are re-classified as Pleurochrysis elongata and P. pringsheimii comb. nov. An updated classification for these two families of coastal coccolithophorids is presented. iii ACKNOWLEDGMENTS I would like to acknowledge the hard work of my advisor, Dr. J. Craig Bailey. Without him this thesis would not have been possible. I would like to thank my committee members, Dr. Dillaman, Dr. Freshwater, and Dr. Taylor, for their time, advice, and valuable suggestions, Mark Gay for all of his help with my microscopy, and my friends and family for their support throughout my time at UNC Wilmington. This work was supported by NSF grant 0328316 awarded to JCB. The culture of Pleurochrysis dimidius was a gift from the late Dr. Paul Krugrens. iv DEDICATION I would like to dedicate this thesis to my parents. They have always been there for me and encouraged me to do my best. Without their love and encouragement I wouldn’t have been able to become the person that I am today. v LIST OF TABLES Table Page 1. GenBank accession numbers for the 18S RNA species of haptophytes included in the study.......................................................................................................................56 2. Revised classification for the Hymenomonadaceae and Pleurochrysidaceae based on information obtained in this study .........................................................................62 3. Staining specificity of the lectins used in this study for the three Pleurochrysis spp. ................................................................................................................................63 vi LIST OF FIGURES Figure Page 1. Diagrammatic representation of cell structures of coccolithophores.....................66 2. Line drawings of coccoliths in selected Pleurochrysidaceae and Hymenomonadaceae..............................................................................................67 3. Light micrographs of isolates examined in this study ...........................................68 4. TEM micrographs of Pleurochrysis dimidius sp. nov ...........................................69 5. Strict consensus tree depicting relationships among haptophyte species based upon 18S rRNA gene sequences............................................................................70 6. Maximum likelihood tree depicting relationships among haptophyte species based upon rbcL gene sequences.....................................................................................71 7. Maximum likelihood tree depicting relationships among species belonging to the Hymenomonadaceae and Pleurochrysidaceae based upon 18S rRNA gene sequences ...............................................................................................................72 8. DIC images of nonmotile diploid and haploid life history phases of Pleurochrysis species....................................................................................................................73 9. Four patterns of binding specificity seen in the species of Pleurochrysis.............74 vii CHAPTER 1 - BACKGROUND INFORMATION ON THE PRYMNESIOPHYTA Prymnesiophyceae The phylum Prymnesiophyta is divided into two classes, the Pavlovophyceae and the Prymnesiophyceae (which includes the coccolithophorids) and encompasses organisms colloquially known as haptophytes (Hibberd 1972, Hibberd 1976, Nicholls 2002). There are over 200 marine species of haptophytes but very few freshwater representatives (Jordan and Green 1994, Jordan and Chaimberland 1997). Less than 12 freshwater species are known and are placed in seven genera (Hymenomonas, Chrysochromulina, Acanthoica, Anacanthoica, Pavlova, Diacronema, and Exanthemachrysis) (Nicholls 2002). Hymenomonas roseola is the only freshwater coccolithophorid reported from North America (Lackey 1939, Meyer and Brook 1968, Smith 1950, Stoermer and Sicko-Goad 1977). Haptophytes possess chloroplasts that lack a girdle lamellum and most contain chlorophylls a and c1/c2, β-carotene, diadinoxanthin, and diatoxanthin. Important fucoxanthin derivatives (19’-hexanoxyfucoxanthin and 19’-butanoyloxyfucoxanthin) and chlorophyll c3 divide the phylum into four main subgroups (Jeffrey and Wright 1994, Jordan et al. 1995). Haptophytes possess flagellae that may be equal or unequal in length and lack mastigonemes, except in the Pavlovophycidae where fibrous hairs and knobscales are present on the longer flagellum (Jordan and Chamberlain 1997). Eyespots are lacking in most haptophytes but are present in the Pavlovophycidae. Most species have fibrillar, unmineralized body scales, and many species also possess calcified scales known as coccoliths (Jordan and Chamberlain 1997). 1 As the name of the group implies, many haptophytes posses a haptonema that is emergent or nonemergent (i.e., reduced). The haptonema is a structure unique to haptophytes and Christensen (1962) used the haptonema and features of the cell covering to segregate the haptophytes from the Chrysophyceae. The haptonema comprises a protrusion supported by microtubules covered by plasmallema scales and is inserted at the anterior end of the cell between the flagella. The number of microtubules making up the haptonema varies from three to eight among species (Billard and Inouye 2004). The presence of a haptonema is plesiomorphic for haptophytes and has been adapted for several uses. Haptonema are known to be used to adhere to substrata and in some species of Chrysochromulina the haptonema has been implicated in prey capture. The haptonema is also sometimes used in collision avoidance responses and may be chemotactic (Graham and Wilcox 2000, Kawachi and Inouye 1995, Kawachi et al. 1991). Haptonema vary in length, with some species possessing a long haptonema capable of coiling or uncoiling, whereas others possess a reduced (short) bulbous haptonema (Billard and Inouye 2004). Although some haptonema are capable of coiling and uncoiling, the structure does not beat like a flagellum (Graham and Wilcox 2000). Species that lack haptonema and have lost them secondarily typically possess intracellular microtubules indicating its former presence (Jordan and Chamberlain 1997). The haptonema