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Evans, Joshua 7-19-17 Transcriptional Regulation of Select Light-Harvesting Genes during Photoacclimation in Lympha mucosa gen. et sp. prov. (Batrachospermales, Rhodophyta) A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Joshua R. Evans August 2017 © 2017 Joshua R. Evans. All Rights Reserved. 2 This thesis titled Transcriptional Regulation of Select Light-Harvesting Genes during Photoacclimation in Lympha mucosa gen. et sp. prov. (Batrachospermales, Rhodophyta) by JOSHUA R. EVANS has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by Morgan L. Vis Professor of Environmental and Plant Biology Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT EVANS, JOSHUA R., M.S., August 2017, Environmental and Plant Biology Transcriptional Regulation of Select Light-Harvesting Genes during Photoacclimation in Lympha mucosa gen. et sp. prov. (Batrachospermales, Rhodophyta) Director of Thesis: Morgan L. Vis The strictly freshwater red algal order Batrachospermales has undergone numerous taxonomic rearrangements in the recent past to rectify the paraphyly of its largest genus Batrachospermum. These systematic investigations have led to the description of new genera and species as well as re-circumscription of some taxa. Specimens collected from two locations in southeastern USA were initially identified as being allied to Batrachospermum sensu lato, but could not be assigned to any previously described species. Comparison of DNA sequence data for two gene regions and morphology with other batrachospermalean taxa resulted in the proposal of a new monospecific genus Lympha mucosa gen. et sp. prov. to accommodate these specimens. A phylogeny of L. mucosa showed it is sister to the genus Volatus, but has morphological similarities with Batrachospermum sections Turfosa and Virescentia. This new taxon adds to the freshwater red algal diversity of southeastern USA, a region already known for biodiversity and high endemism of the aquatic flora and fauna. Lympha mucosa occurs in open and shaded sites of temperate streams and is abundant during summer months. Although most freshwater red algal taxa are considered shade-adapted, many species exhibit differences in photosynthetic rates and characteristics that indicate they have a much greater ability to acclimate to higher irradiances. Specimens of L. mucosa were collected from open (sun-acclimated) or 4 shaded (shade-acclimated) sites and were exposed to low (<20 µmol photon m-2 s-1) or high (220 µmol photon m-2 s-1) for 72h in controlled conditions to examine photoacclimation. To observe regulation for this process at the transcriptional level, the L. mucosa plastid genome was assembled to provide sequence data for photosynthetic genes involved with light harvesting machinery. Of the six light-harvesting genes selected, two involved with photosystem I and one involved with phycoerythrin synthesis were downregulated at high light. This is the first evidence of transcriptional regulation as a potential mechanism for acclimation to varying irradiances in a freshwater red alga. 5 DEDICATION This work is dedicated to my parents, Karen and Harry, without whom I would not be on this journey, and to my nephews. “No man ever steps in the same river twice, for it’s not the same river and he’s not the same man” - Heraclitus 6 ACKNOWLEDGMENTS A number of people have helped me shape and conduct this project and I would still be at the starting line without them. I would first like to acknowledge my advisor, Dr. Morgan Vis. During my short time in her lab, my ability to conduct critical research and be independent has become stronger because of her knowledge and support, and I have gained many new skills that I will take with me on the next step of my career. I am honored to have been her student, and in the future, I hope to be even half the mentor she was for me. Many thanks go to members of my thesis committee, Drs. Sarah Wyatt and Harvey Ballard, who have taken the time to critically evaluate my work and provide constructive feedback. I am indebted to Dr. Wyatt, who generously provided me with space and encouragement to learn something new and expand my skills base, and for shaping some of my thoughts on education. I thank Dr. Ballard for helpful comments, specifically with the second chapter of this thesis, and for graciously inviting us to his field accommodations. Colin Kruse is thanked for taking the time to troubleshoot RNA extractions with me, even if it meant not working with Arabidopsis. I appreciate his collaborative spirit and desire to help others. Dr. Erin Murphy is thanked for generously allowing me to use her qPCR machine. Additional acknowledgements go to past and present members of the Vis lab (Emily Keil-Loudner, Danny Wolf, Nathan St. Amour, Lexie Redmond) for their assistance and support. Dr. Bill Broach, Al Meyers, and Anne Sternberger are thanked for helpful discussions. I would like to acknowledge the support and expertise provided by the Ohio University Genomics Facility in completion of NGS sequencing. This research was partially funded through Original Works Grants provided by the Ohio University Graduate Student Senate. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments ............................................................................................................... 6 List of Tables ...................................................................................................................... 9 List of Figures ................................................................................................................... 10 Chapter 1: Introduction ..................................................................................................... 11 References ................................................................................................................... 13 Chapter 2: Adding to the Freshwater Red Algal Diversity in North America: Lympha mucosa gen. et sp. prov. (Batrachospermales, Rhodophyta). ........................................... 17 Introduction ................................................................................................................. 17 Methods....................................................................................................................... 19 Sample Collection ................................................................................................. 19 DNA Extraction and Amplification ...................................................................... 19 Phylogenetic Analyses .......................................................................................... 20 Morphological Measurements .............................................................................. 21 Results ......................................................................................................................... 22 Molecular Results ................................................................................................. 22 Morphological Results .......................................................................................... 23 Taxonomy ............................................................................................................. 23 Discussion ................................................................................................................... 26 References ................................................................................................................... 28 Chapter 3: Plastid Genomics and Relative Expression Analysis of Light-Harvesting Genes in Lympha mucosa Exposed to Low and High Irradiances .................................... 37 Introduction ................................................................................................................. 37 Methods....................................................................................................................... 42 Sample Collection and Experimental Design ....................................................... 42 DNA Extraction and Sequencing .......................................................................... 44 Plastid Assembly and Annotation ......................................................................... 45 RNA Extraction .................................................................................................... 46 Gene Selection and RT-qPCR Primer Design ...................................................... 47 cDNA Synthesis and RT-qPCR Conditions ......................................................... 49 8 Gene Expression Analysis .................................................................................... 50 Results ......................................................................................................................... 51 Plastid Genome ..................................................................................................... 51 Gene Expression Analyses .................................................................................... 53 Discussion ..................................................................................................................
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