Nutritional stoichiometry and growth of filamentous green algae (Family Zygnemataceae) in response to variable nutrient supply A Thesis Submitted to the Committee on Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Faculty of Arts and Science TRENT UNIVERSITY Peterborough, Ontario, Canada © Copyright by Colleen Middleton 2014 Environmental and Life Sciences M.Sc. Program September 2014 ABSTRACT Nutritional stoichiometry and growth of filamentous green algae (Family Zygnemataceae) in response to variable nutrient supply. Colleen Middleton In this study, I investigate the effects of nitrogen (N) and phosphorus (P) on the nutritional stoichiometry and growth of filamentous green algae of the family Zygnemataceae in situ and ex situ . I found a mean of Carbon (C):N:P ratio of 1308:66:1 for populations growing in the Kawartha Lakes of southern Ontario during the summer of 2012. FGA stoichiometry was variable, with much of the variation in algal P related to sediment P ( p < 0.005, R 2 = 0.58). Despite large variability in their cellular nutrient stoichiometry, laboratory analysis revealed that Mougeotia growth rates remained relatively consistent around 0.28 day -1. In addition, Mougeotia was found to be weakly homeostatic with respect to TDN:TDP supply (1/H NP = 0.32). These results suggest that FGA stoichiometry and growth rates are affected by sediment and water N and P. However, they will likely continue to grow slowly throughout the summer despite variable nutrient supply. Keywords: Filamentous green algae, Mougeotia , Spirogyra , Zygnemataceae, nutrient supply, sediment nutrients, dissolved nutrients, nutritional stoichiometry, growth rates, chlorophyll concentration, homeostatic regulation. ii ACKNOWLDEGEMENTS My Masters has been so much more about the journey than the final product, and I need to thank all the people who have supported me in various aspects along the way. Starting from the beginning, I would first have to thank my grandparents and the rest of the Middleton clan for raising me to appreciate the beauty and balance of nature on the Kawartha Lakes. Because of adventures at the cottage, I knew that I wanted to preserve these lakes for future generations to enjoy as I have. Fundamental in my pursuit of environmental stewardship was the acquisition of knowledge, which led me to begin my Masters at Trent University. I thank all of my teachers for challenging me and sharing their wisdom. I especially thank my supervisor Dr. Paul Frost, for guiding me throughout my Masters. Paul is able to break down complex ecosystem processes into their basic elements (literally!), and brought me back to the “K.I.S.S.” principle. Qualified scientific advice was also provided from start to finish by my supervisory committee: Dr. Eric Sager and Dr. Jennifer Winter. My regular field assistants were Emily Porter-Geoff, Catharine Monaghan, Clay Prater, and Shayla Larson. Rain or shine, they joined me in my search for “elephant snot”. A number of family members and friends also joined me on the boat: Tim Racette, Trina Kaus, Andres Raun, Sean Middleton, Marianne Meyer, and Erwin Meyer. I owe my most sincere appreciation to my long-term lab members and friends: Dr. Emily Porter-Geoff, Nicole Wagner, Clay Prater, Charlotte Narr, Dr. Keunyea Song, and Andrew Scott for helping me with project design, laboratory analysis, and editing. Thank you also Peter Lin and Ryan Franckowiak for help with statistics, Kyle Borrowman for helping me set up a laboratory experiment in the wee hours, and Andrea Hicks for her mentorship in the early stages of my Masters. Just as important is the emotional support I received throughout the course of my thesis, especially towards the end. I am eternally grateful to my mom (Marianne Meyer), Leah Ensing, Jessica Middleton, Sean Middleton, Robert Sainsbury, Heather Leech, Trina Kaus, Eva Dwyer, Heather Hodgson, and Linda Cardwell. Finally, thank you to members of the Kawartha Lake Stewards Association and other concerned lake-users for their questions, interest, and support. It is because of people like you that important environmental issues get recognized and addressed. This work was supported by funds provided by the Natural Sciences and Engineering Research Council of Canada, the Kawartha Lake Stewards Association, and the David Schindler Professorship at Trent University. iii I dedicate my thesis to my father, Dr. Bill Middleton, who recently passed away. While his life’s work involved human health, I have no doubt that his critical thinking, strong work ethic, high expectations of me, and love of the cottage have transferred to my success in completing my M.Sc. in Environmental and Life Sciences. May his carbon, nitrogen, and phosphorus continue to travel the globe forever more. iv TABLE OF CONTENTS Pg Abstract ii Acknowledgements iii Table of Contents v List of Tables vii List of Figures viii General Introduction 1 Chapter 1: Patterns and drivers of filamentous green algae (Zygnemataceae) stoichiometry in the Kawartha Lakes of Southern Ontario 5 Abstract 6 1. Introduction 7 2. Methods 9 2.1 Study area 9 2.2 Study design 9 2.3 Sampling methods 10 2.4 Chemical analysis 11 2.5 Statistical analysis 12 3. Results 12 3.1 FGA stoichiometry 12 3.2 Algal stoichiometric variability over space and time 13 3.3 Water and sediment as sources of nutrients 14 4. Discussion 14 Chapter 2: Stoichiometric and growth responses of a freshwater filamentous green alga ( Mougeotia spp. ) to varying nutrient supplies 25 Abstract 26 1. Introduction 28 2. Methods 30 2.1 Algal collection and purification 30 2.2 Experimental procedure 31 2.3 Chemical anlysis 33 2.4 Homeostasis calculation 33 2.5 Field study 33 2.6 Statistical analysis 35 v 3. Results 35 3.1 Elemental composition of Mougeotia 35 3.2 Responses of Mougeotia to increasing media N concentrations 36 3.3 Responses of Mougeotia to increasing media P concentrations 36 3.4 Homeostatic regulation of Mougeotia elemental composition 37 3.5 Elemental composition of lake-derived Mougeotia 38 4. Discussion 38 General Conclusion 52 Literature Cited 56 vi LIST OF TABLES Pg Table 2.1. Summary of Mougeotia’s stoichiometric and physiological traits created by varying media nutrient concentrations in the lab. Elemental ratios are molar ratios. 45 Table 2.2. Standard major axis regression statistics for the N and P experiments on Mougeotia stoichiometry 46 Table 2.3. Summary of Mougeotia’s stoichiometry and water chemistry from the Kawartha Lakes. 47 vii LIST OF FIGURES Pg Figure 1.1. Map of Kawartha Lakes sampling sites with location map overlay. Spatial sites (n = 6 per lake) are shown with black squares and temporal sites (n = 3 per lake) are shown with white squares. Gray squares show sites used in both studies. 19 Figure 1.2. Boxplot showing alga stoichiometry for two different genera of filamentous green algae: Mougeotia (MG, n = 71) and Spirogyra (SP, n = 22) for spatial and temporal study data combined. Boxplots show the median (horizontal line), 25 th and 75 th percentiles (box limits), range (whiskers), and outliers (points). 20 Figure 1.3. Boxplot showing algal stoichiometry for six different lakes (n = 6 sites per lake, up to 3 samples per site). Boxplots show the median (horizontal line), 25 th and 75 th percentiles (box limits), range (whiskers), and outliers (points). 21 Figure 1.4. Scatter plot showing mean algal stoichiometry for Pigeon Lake (gray squares, n = 3 sites, up to 3 samples per site) and Balsam Lake (open circles, n = 2 sites, up to 3 samples per site) over time. Error bars are standard deviations. 22 Figure 1.5. Linear regression showing the effect of water stoichiometry on algal stoichiometry for the spatial study. Data is fitted with the line of best fit. 23 Figure 1.6. Linear regression showing the effect of sediment stoichiometry on algal stoichiometry for the spatial study. Data is fitted with the line of best fit. 24 Figure 2.1. Nitrogen (N) and phosphorus (P) concentrations in the growth media at the start of each experiment. Also provided are the molar N:P ratios bracketing the range of values present in each experiment. 48 Figure 2.2. The effect of media N concentration on Mougeotia ’s stoichiometric (C:N, C:P, and N:P ratios, by mol) and physiological (MSGR (day -1), Chl ( µg·L -1) and C:Chl ratio) responses at high and low P levels. Low P is shown with open circles and a dashed trend line and high P is shown with open triangles and a solid trend line. It is also noted where slopes between high and low P concentrations were common ( p > 0.05) or significantly different ( p < 0.05). 49 Figure 2.3. The effect of media P concentration on Mougeotia ’s stoichiometric (C:N, C:P, and N:P ratios, by mol) and physiological (MSGR (day -1), Chl ( µg·L -1) and C:Chl ratio) responses at high and low N levels. Low N is shown with open circles and a solid trend line and high N is shown with open triangles and a dashed trend line. It was noted where slopes between high and low N concentrations were common ( p > 0.05) or significantly different ( p < 0.05). 50 viii Figure 2.4. The effect of media N:P, N concentration, and P concentration on Mougeotia N:P ratio, Mougeotia %N, and Mougeotia %P of cultured Mougeotia . Shown also for each is the line of best fit and regression statistics. Note that the slope of the media N:P to Mougeotia N:P relationship is equal to 1/H. 51 ix GENERAL INTRODUCTION Excessive algal growth is associated with a suite of environmental, social, and economic problems.