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Molecular Identity and Nutrient Limitation of Lyngbya Wollei Mat Communities in North Florida Freshwater Systems

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Molecular Identity and Nutrient Limitation of Lyngbya Wollei Mat Communities in North Florida Freshwater Systems MOLECULAR IDENTITY AND NUTRIENT LIMITATION OF LYNGBYA WOLLEI MAT COMMUNITIES IN NORTH FLORIDA FRESHWATER SYSTEMS Jennifer Jendro Joyner A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Sciences Chapel Hill 2007 Approved by: Marc J. Alperin R. Wayne Litaker Rachel T. Noble Hans W. Paerl Stephen C. Whalen © 2007 Jennifer Jendro Joyner ALL RIGHTS RESERVED ii ABSTRACT JENNIFER JENDRO JOYNER Molecular Identity and Nutrient Limitation of Lyngbya wollei Mat Communities in North Florida Freshwater Systems (Under the direction of Hans W. Paerl) Lyngbya wollei (Speziale ex Gomont) is a filamentous cyanobacterium that is capable of nitrogen fixation. Within Florida springs, L. wollei is an opportunistic invader that can smother both native and invasive submerged aquatic vegetation with its thick benthic mats (up to 1-1.5 kg dry weight m-2), and it has been shown to produce toxins (neurotoxins and hepatotoxins). Blooms of L. wollei persist in many recreational and drinking water supplies within the southeastern United States. The inability to find a statistically significant relationship between L. wollei biomass in Florida springs and nutrient concentrations (nitrogen, phosphorus) has led to the hypothesis that the present standard for identification of freshwater L. wollei may in fact encompass multiple species. There is a need to accurately identify this cyanobacterium and identify limiting nutrients, the input of which may be controlled by water managers in an effort to reduce L. wollei biomass. We sequenced partial 16S rDNA and nifH genes from L. wollei mats that were identified using the current taxonomic description for L. wollei. The 16S and nifH datasets contained diverse sequences. The majority of sequences were found to be from L. wollei filaments and unidentified cyanobacteria. Of the unidentified cyanobacteria, six 16S sequences corresponded (>98% similarity) to a known toxin-producing Phormidium sp. The combined phylogenetic analyses of the 16S rDNA and nifH genes, in conjunction with iii morphological analysis (cell width and length), indicates that the current L. wollei description in the literature represents two or possibly three species. No correlation was found between the species divisions and dissolved inorganic nitrogen concentration and N:P for the collection sites. To determine limiting nutrients for effective mitigation of L. wollei biomass in Florida springs, four dissolved inorganic nutrient addition bioassays were employed over the course of one year. Primary productivity results showed that L. wollei primary productivity was limited by both nitrogen and phosphorus. Therefore, L. wollei biomass will most likely be reduced by lowering nitrogen and phosphorus loading in affected springs. iv To my husband, Alan v ACKNOWLEDGEMENTS I would like to begin by thanking my advisor, Dr. Hans Paerl, for this opportunity. This dissertation would not have been possible without his guidance and the guidance of my committee: Marc Alperin, Wayne Litaker, Rachel Noble, and Steve Whalen. Mike Piehler, Tim Steppe, and Juli Dyble also had many conversations with me about the design and methods of this project. Much of this project was conducted on site in Florida. I would like to thank the Ocala National Forest Seminole Ranger District for repeatedly allowing me to enter the Ocala National Forest to conduct my week-long and overnight experiments and leave with cyanobacteria samples and spring water. The Park Rangers at Silver Glen Springs and Alexander Springs were especially helpful and cheerful. This work was sponsored by the St. Johns River Water Management District and the Florida Department of Health. John Burns (CyanoLab) is the one who first introduced me to the nuisance Lyngbya spp. in Florida springs. We went on two Lyngbya collecting trips across Florida, and John also conducted a solo Lyngbya hunt. All of these samples were used in the analyses in Chapter II and III. Aga Pinowska (Jan Stevenson lab) donned scuba gear with an accompanying “alligator lookout” and gathered Lyngbya samples that were used in Chapter II and III as well. Bill Frazier also informed me of Lyngbya’s presence in City Lake, High Point, North Carolina. Bill allowed Juli, Bonnie, and I to use his labs at the High Point Water Treatment Facility to conduct research. Thank you to Bill, John, and Aga for your contributions to this project. So many people in the Paerl lab offered technical support and comradery. Lou Anne Cheshire, Karen Rossignol, Juli Dyble, Tim Steppe, Amy Waggener, Ben Peierls, Jeremy Braddy, Pam Wyrick, Patrick Sanderson, John Fear, Lexia and Rich Weaver, and Bonnie Chang, thank you for everything! In addition to the Paerl lab, several other staff members at IMS went out of their way to make many of my tasks as a graduate student and research assistant so much easier. These people include Kar Howe, Jean Stack, Laura White, Kellie Albus, and Claude Lewis. I have also been fortunate to have wonderful neighbors that always kept my spirits high: Kristen, Val, and Ben. Thank you for our walks, talks, and shared TV addictions! The people who never grew tired of hearing about my highs and lows throughout this time were my family: Mom, Dad, Kristen, Daniel, Grandma, Debbie, Doug, Lucas, Amelia, Brenda, Billy, Karen, Atari, and Rodriguez. Thank you, Mom and Dad, for always loving me and wanting the best for me. Finally, thank you, Alan, for your endless support and love. You and I have learned a lot about life here in Morehead City, and I look forward to many more adventures together. vi TABLE OF CONTENTS LIST OF TABLES............................................................................................................ xi LIST OF FIGURES .......................................................................................................... xii CHAPTER I. INTRODUCTION .......................................................................................1 Cyanobacteria ..............................................................................................1 Nitrogen fixation..........................................................................................3 Molecular tools ............................................................................................4 Lyngbya wollei.............................................................................................5 Florida springs .............................................................................................7 Dissertation goals.........................................................................................9 References..................................................................................................13 II. CYANOBACTERIAL AND EUBACTERIAL SPECIES ASSOCIATED WITH LYNGBYA WOLLEI MATS IN FLORIDA AND NORTH CAROLINA (US) FRESHWATER SYSTEMS...............16 Introduction................................................................................................16 Materials and Methods...............................................................................17 Sampling locations.........................................................................17 Sample collection...........................................................................18 DNA extraction..............................................................................18 PCR amplification..........................................................................18 vii Phylogenetic analysis.....................................................................19 Sequence identification..................................................................20 Cell measurements.........................................................................21 Results........................................................................................................21 16S rRNA sequences .....................................................................21 nifH sequences ...............................................................................23 Discussion..................................................................................................24 16S rDNA ......................................................................................25 nifH ................................................................................................27 General conclusions.......................................................................28 References..................................................................................................42 III. MORPHOLOGICAL AND GENETIC EVIDENCE THAT LYNGBYA WOLLEI (SPEZIALE EX GOMONT) FOUND IN FLORIDA SPRINGS ENCOMPASSES AT LEAST TWO SPECIES..........................................................................................45 Introduction................................................................................................45 Materials and Methods...............................................................................47 Sample collection...........................................................................47 DNA extraction..............................................................................47 PCR amplification..........................................................................47 Phylogenetic analysis.....................................................................48 Morphometric analysis...................................................................48
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