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Photosynthetic Picoplankton and Bacterioplankton in the Central Basin of Lake Erie During Seasonal Hypoxia

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Photosynthetic Picoplankton and Bacterioplankton in the Central Basin of Lake Erie During Seasonal Hypoxia PHOTOSYNTHETIC PICOPLANKTON AND BACTERIOPLANKTON IN THE CENTRAL BASIN OF LAKE ERIE DURING SEASONAL HYPOXIA Audrey R. Cupp A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2006 Committee: George S. Bullerjahn, Advisor R. Michael L. McKay Scott O. Rogers ii ABSTRACT George S. Bullerjahn, Advisor In Lake Erie’s central basin, a hypoxic region commonly termed the dead zone forms during late summer. Previous work has demonstrated an abundance of photosynthetic picocyanobacteria despite this lack of oxygen. High-throughput sequencing of over 400 cyanobacterial and eubacterial 16S rDNA amplicons has characterized some of the major members of the microbial community both during and prior to the dead zone formation. In July, the bacterial communities mainly consisted of two unique clusters of Gram-positive Actinobacteria, with a smaller percentage of Flexibacter-Cytophaga-Bacteroides , α, β, and γ Proteobacteria . Cyanobacteria in the form of photosynthetic picoplankton was found at 16.5 m in July, but was absent from the 20 m library. During hypoxia in August, a community shift was demonstrated with a decrease in the Flexibacter-Cytophaga-Bacteroides , an increase in number and diversity of cyanobacteria, and an increase in an α-Proteobacterial cluster. Diurnal oxygen production in the hypolimnion of Lake Erie was exhibited by in situ probes and showed actively photosynthetic picoplankton producing oxygen. Cyanobacterial 16S libraries showed an increase in diversity of photosynthetic picoplankton in August compared to July. The vast majority of clones similar to Synechococcus sp. MH301 were found in July with only a small percentage of clones from other groups. Conversely, during hypoxia, an increase of diversity was shown to exist. These differences in bacterial community members indicate the cycling of oxygen may influence the community structure in Lake iii Erie. Novel primers specific for the cpeB gene in phyoerythrin-rich (PE-rich) cyanobacteria were used to study diversity of potential marine-like forms, indicating a new way to phylogenetically study PE-rich cyanobacteria. iv ACKNOWLEDGEMENTS I would first like to acknowledge George Bullerjahn, my advisor and mentor for the past two years. I have learned so much, and I hope to be able to apply what I have learned to my future endeavors. I would also like to thank Mike McKay and Scott Rogers for serving on my committee, and for helping me become a better scientist, and in the process, a better writer. Without the help of my other lab members (Natasha, Masha, Linda, and Irina) I would have never finished my projects. I will never forget their knowledge and willingness to help. I would also like to thank some members from the University of Tennessee, especially Steven W. Wilhelm and Johanna Rinta-Kanto for their help with the chlorophyll and nutrient data. Last but not least, I would like to thank my parents, Beryl and Deborah Cupp, and my friends for their support and love throughout my college career. There is no way I could have endured this without you all. v TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION........................................................................................... 1 Rise and Evolution..................................................................................................... 1 Marine Cyanobacterial Diversity............................................................................... 2 Freshwater Photosynthetic Picoplankton (Ppicos)..................................................... 3 Lake Erie as a Model Ecosystem............................................................................... 5 Invasive Species Issues in Lake Erie ......................................................................... 6 Lake Erie Anoxia ....................................................................................................... 6 Lake Erie Microbial Community............................................................................... 7 CHAPTER II. METHODS ................................................................................................... 10 Sampling ............................................................................................................ 10 DNA Extraction and PCR Amplification .................................................................. 10 Cloning and Sequencing of Fragments...................................................................... 11 Sequence Alignment and Phylogenetic Analysis ...................................................... 12 Culturing of Picocyanobacteria.................................................................................. 12 CHAPTER III. RESULTS.................................................................................................... 14 Oxygen and Lake Conditions..................................................................................... 14 Eubacterial Libraries.................................................................................................. 14 Uncultured Actinobacteria and α-proteobacteria Clusters........................................ 18 Ppico Libraries........................................................................................................... 18 Cultured Ppico Sequences.......................................................................................... 21 cpeB Library ............................................................................................................ 22 vi CHAPTER IV. DISCUSSION.............................................................................................. 24 Eubacterial Library Diversity .................................................................................... 24 Ppico Libraries........................................................................................................... 30 cpeB Library ............................................................................................................ 32 REFERENCES ...................................................................................................................... 64 vii LIST OF TABLES/FIGURES Table Page 1 Primers with corresponding sequences and amplicon lengths used in the study....... 34 2 Characterization of clones using the Ribosomal Database Project (RDP) using the Project Classifier........................................................................................................ 35 3 16S rDNA sequences identified in Lake Erie prior to and during seasonal hypoxia as taken from the eubacterial libraries. ...................................................................... 36 4 Reference sequences used in phylogenetic trees as taken from the GenBank database ............................................................................................................ 42 5 Table describing major species found in universal 16S libraries using BLAST search from samples collected in Lake Erie during 2005.......................................... 45 viii Figure Page 1 Estimated yearly hypoxia in Lake Erie Central Basin............................................... 46 2 Map of select Lake Erie master stations .................................................................... 47 3 1.5% agarose gel visualizing all PCR products from August samples...................... 48 4 Graphs depicting dissolved oxygen and temperature in Lake Erie............................ 49 5 Comparison of chlorophyll a concentrations between July and August using varying filter sizes...................................................................................................... 50 6 Bacterial assignment of clones from Station 84 July................................................. 51 7 Bacterial assignment of clones from Station 84 August............................................ 52 8 Neighbor-joining phylogenetic tree using universal 16S rRNA sequences obtained from Lake Erie in July at 16.5 m depth ..................................................................... 53 9 Neighbor-joining phylogenetic tree from 16S rRNA sequences obtained from Lake Erie in July at 20 m depth ................................................................................. 54 10 16S rRNA neighbor-joining phylogenetic tree from samples taken from August at 16.3 m ............................................................................................................ 55 11 16S rRNA neighbor-joining phylogenetic tree with major groups of bacteria from August at 22 m depth ................................................................................................. 56 12 16S rRNA neighbor-joining phylogenetic tree of the major clusters of Ppicos from 16.5 m in July.................................................................................................... 57 13 16S rRNA neighbor-joining phylogenetic tree of the major clusters of Ppicos from 20 m in July....................................................................................................... 58 14 16S rRNA neighbor-joining phylogenetic tree of the major clusters of Ppicos from 16.3 m in August............................................................................................... 59 ix 15 16S rRNA neighbor-joining phylogenetic tree of the major clusters of Ppicos from 22 m in August.................................................................................................. 60 16 Percentage of
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