ANALYZING ALGAL DIVERSITY IN AQUATIC SYSTEMS USING NEXT GENERATION SEQUENCING __________________________________ A Thesis Presented to The Honors Tutorial College Ohio University __________________________________ In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of Bachelor of Science in Environmental and Plant Biology __________________________________ By Mariah A. Thrush May 2013 This thesis has been approved by The Honors Tutorial College and the Department of Environmental and Plant Biology ______________________________ Dr. Morgan Vis Professor, Environmental and Plant Biology Thesis Advisor ______________________________ Dr. Harvey Ballard Honors Tutorial College, Director of Studies Environmental and Plant Biology ______________________________ Jeremy Webster Dean, Honors Tutorial College 2 ACKNOWLEDGEMENTS I’d like to thank my thesis advisor Dr. Morgan Vis for her time, support and advice throughout my academic career. She helped to keep me on track even as plans changed and changed again. Thanks also go to Dr. Harvey Ballard; he has been my Director of Studies and a source of support and advice since I began at Ohio University. Daryl Lam also spent a great deal of time teaching me genetic techniques and bioinformatics programs, for which I am immensely grateful. I’d also like to thank Vijay Nadella and the staff in the Ohio University Genomics Facility for their work; I wouldn’t have my data without their hard work with this new technology. Thanks to Dr. Kelly Johnson and the Boat of Knowledge program for collecting most of the large river samples for me. I would like to thank my labmates Lauren Fuelling, Emily Johnston, Eric Solamaki, Sam Drerup, and Emily Keil for their help, support, and friendship. A special thanks goes out to Sam Drerup for his help with Hewett Fork field collections, as well as chlorophyll a and phosphorus measurements. The following funding sources made it possible to complete this thesis: Honors Tutorial College Plant Biology Research fund, the Jeanette G. Grasselli Brown Undergraduate Research Award, and a Research and Creative Activity Grant from VP Research Office. I want to thank my family and friends for their continued support through my undergraduate degree, especially my parents, my sister Sam, and my boyfriend Weylin. Your encouragement during the difficult times has meant a great deal to me. 3 TABLE OF CONTENTS Acknowledgements.........................................................................................................3 List of Tables..................................................................................................................5 List of Figures.................................................................................................................6 Introduction.....................................................................................................................8 Chapter One Introduction.......................................................................................................12 Methods.............................................................................................................13 Results...............................................................................................................19 Discussion and Conclusions..............................................................................23 Chapter Two Introduction.......................................................................................................39 Methods.............................................................................................................42 Results...............................................................................................................47 Discussion and Conclusions..............................................................................48 References.....................................................................................................................60 4 LIST OF TABLES Table 1.1: Water chemistry for the Ohio and Muskingum Rivers................................29 Table 1.2: Number of sequences after Prinseq filters were applied..............................30 Table 1.3: Number of sequences for genera included in positive control.....................32 Table 2.1: Primers used for PCR to prepare samples for multiplexing. Shaded bases indicate barcode to separate the different environmental samples during the bioinformatics pipe procedure......................................................................................50 Table 2.2: Water chemistry data for sites along Hewett Fork. Data collected October 2, 2012. Site designations as in Fig. 2.1............................................................................51 Table 2.3: Calculated values for AFDM, Chl. a, and autotrophic index for sites along Hewett Fork. Site designations as in Fig. 2.1................................................................52 5 LIST OF FIGURES Figure 1.1: Positive control consisting of 11 unialgal cultures. The taxonomic groups identified in GenBank represent the unialgal cultures as follows: Cyanobacteria (Oscillatoria, Nostoc, Spirulina), Zygnemophyceae (Spirogyra), Euglenozoa (Euglena, Phacus), and Chlorophyceae (Chlamydomonas, Volvox), Bacillariophyta (Navicula, Synedra) and Synurophyceae (Synura). X, Y, and Z were identified through a GenBank search, but do not represent the unialgal cultures......................................33 Figure 1.2: Ohio River phytoplankton sample composition from single chip data with a total of 19 taxonomic groups identified via a GenBank search.................................34 Figure 1.3: Muskingum River phytoplankton sample composition single chip data with a total of 20 taxonomic groups identified via a GenBank search.........................35 Figure 1.4: Ohio River phytoplankton sample composition from multiplex data with a total of 19 taxonomic groups identified via a GenBank search....................................36 Figure 1.5: Muskingum River phytoplankton sample composition from data with a total of 19 taxonomic groups identified via a GenBank search....................................37 Figure 1.6: Composition of Ohio and Muskingum River phytoplankton single chip samples; sequences identified via a GenBank search in March 2012...........................38 Figure 2.1. Map of Hewett Fork sampling sites. Note that sites HF 137 and HF 129 are upstream of the doser, and all other sites are downstream, and site HF 120 is at the mouth of Carbondale Creek flowing into Hewett Fork............................................................53 6 Figure 2.2: Sites on Hewett Fork. A. HF 095 (not sampled) closer to the doser exhibited an orange deposit covering the stream bottom making sampling impossible. B. HF 010 farther from the doser showed no orange sediments...................................54 Figure 2.3: Filters for AFDM before combustion. Site HF 120 was an orange color due to iron oxide..................................................................................................................55 Figure 2.4: AFDM values for sites along Hewett Fork. Site designations as in Figure 2.1. The linear regression line for the samples is y = -0.9405x + 12.857.....................56 Figure 2.5: Chlorophyll a measurements for sites along Hewett Fork. Site designations as in Figure 2.1. The linear regression for the samples is y = 0.0044x + 0.0515.........57 Figure 2.6: Autotrophic index values for sites along Hewett Fork. Site designations as in Figure 2.1. The linear regression for the samples is y = -18.682x + 222.78............58 Figure 2.7: Agarose gel of PCR products for sites along Hewett Fork. All sites had PCR product, but the bands for HF 129 and HF 120 are not visible on the image. Site designations as in Fig. 2.1.............................................................................................59 7 INTRODUCTION Algae can be broadly defined as photosynthetic, oxygen-producing aquatic bacteria or protists, but there are exceptions to this definition, such as terrestrial algae and some taxa can be heterotrophic (Graham et al. 2009). Algae are a polyphyletic assemblage of organisms; several separate endosymbiotic events – primary, secondary, and tertiary – led to the diversity of algae seen today (Keeling 2010). Depending on where they occur in the water column, algae can be categorized as phytoplankton or periphyton/benthic algae. Phytoplankton is algae that have adapted to live in the open water of any body of water (Graham et al. 2009), while periphyton/benthic algae are unicellular, filamentous, and colonial algae that attach to the stream bottom (benthos) (Graham et al. 2009). Phytoplankton morphology varies in shape and size, ranging from 0.2 µm to 20 mm (Graham et al. 2009). This variation stems from the need to balance buoyancy to stay within the photic zone, surface area for nutrient uptake and deter predation (Reynolds 2006). Periphyton is found attached to the substratum of streams and within the littoral zone of lakes. The size and shape of periphyton are primarily influenced by the physical habitat such as the level of disturbance, current velocity, and substratum, all of which help determine the species present in a given habitat (Graham et al. 2009). Grazing pressures also influences the benthic algal species present, and to what height they will grow (Steinman 1996). Algae (phytoplankton or periphyton) form the base of the food chain