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The effect of temperature and pH on the growth and biomechanics of coralline algae Guenther, Rebecca 2016

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  THE EFFECT OF TEMPERATURE AND pH ON THE GROWTH AND BIOMECHANICS OF CORALLINE ALGAE by Rebecca Guenther B.S., University of Wisconsin-Stevens Point 2003 M.Sc., The University of British Columbia 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2016 © Rebecca Guenther 2016 ii  Abstract  Climate change is progressing rapidly and is causing shifts in ecosystem function, species distributions, biodiversity, and abundances worldwide. In this thesis, I explore the physiological and biomechanical responses of red algae in multiple life history stages to climate change.   In Chapter 1, I introduce the looming threat of climate change, and some of the forces driving ocean acidification. I introduce my study system and my study species: rocky intertidal ecosystems and articulated coralline algae. I also describe potential differences in responses to ocean acidification based on life history stage.  Finally, I give an overview of my dissertation and objectives.  In Chapter 2, I investigate the effect that ocean acidification may have on spore stages of red algae. Under reduced pH, I document a reduction in spore settlement of both Pterosiphonia bipinnata and Corallina vancouveriensis, and weakened spore attachment in C. vancouveriensis. Results demonstrate that ocean acidification can negatively impact macroalgal spore adhesion in both calcified and non-calcified algae, but in different phases of their spore adhesion process.  In Chapter 3, I explore the effect of elevated pCO2 and temperature on the growth, calcification, and material properties of two species of articulated coralline algae. I found that increased temperatures and reduced pH were found to negatively affect growth rates of these two species of coralline algae. On the other hand, increased temperature and reduced pH had little influence on the amount of calcium carbonate in the intergenicula, and also had minimal effects on the biomechanical properties.    iii  In Chapter 4, I explore the amount of natural variability of chemistry in tidepools and attempted to relate chemical differences to differences in Corallina vancouveriensis growth, calcification, and biomechanics. In general, I found that organisms within tidepools greatly alter the chemistry of the surrounding water, and these changes are larger in magnitude than what is predicted for global climate change. I also found that, despite extreme changes in chemistry during low tides, C. vancouveriensis was still able to grow all year long.     iv  Preface Chapter 2: Kevin Miklasz designed shear flume and spore settlement apparatus.  I continued work on spore adhesion after Kevin left Friday Harbor Labs.  Emily Carrington assisted with the statistical analysis of detachment data.  I drafted the manuscript, with revisions from Patrick Martone and Emily Carrington.  Chapter 3: Hannah Spohn (Nearshore Ecology FHL 450 student) completed the single-factor temperature experiment.  I helped with data design, experimental monitoring, and data collection.  Chris Harley and Emily Carrington also provided input on experimental design in the multiple factor experiment.  I will draft the manuscript, and will receive feedback from Patrick Martone and Emily Carrington.   Chapter 4: I designed the experiment with input from Patrick Martone, completed field work, and chemical analyses of water samples.  Carolyn Friedman provided use of her total alkalinity titrator.  I will draft the manuscript, and will receive feedback from Patrick Martone and Emily Carrington.            v  Table of Contents Abstract ................................................................................................................................... iiPreface .................................................................................................................................... ivTable of Contents ..................................................................................................................... vList of Tables ......................................................................................................................... viiList of Figures ........................................................................................................................ ixAcknowledgements ................................................................................................................ xi1 Introduction ..................................................................................................................... 11.1 Background ........................................................................................................... 11.2 Study system: rocky intertidal ecosystem ............................................................ 31.3 Sensitive life stages of red algae ........................................................................... 81.4 Dissertation overview and objectives ................................................................... 92 Ocean acidification delays and weakens adhesion of red algal spores ..................... 142.1 Synopsis .............................................................................................................. 142.2 Introduction......................................................................................................... 152.3 Methods .............................................................................................................. 192.4 Results ................................................................................................................ 262.5 Discussion ........................................................................................................... 313 The effects of temperature and pH on the growth, calcification, and biomechanical properties of two species of articulated coralline algae ...................................................... 363.1 Synopsis .............................................................................................................. 363.2 Introduction......................................................................................................... 363.3 Methods .............................................................................................................. 45 vi  3.4 Results ................................................................................................................ 613.5 Discussion ........................................................................................................... 764 Seasonal analysis of tidepool chemistry and putative effects on Corallina physiology ............................................................................................................................... 844.1 Synopsis .............................................................................................................. 844.2 Introduction......................................................................................................... 844.3 Methods .............................................................................................................. 894.4 Results ................................................................................................................ 974.5 Discussion ......................................................................................................... 1145 Conclusion .................................................................................................................... 1215.1 Synopsis ............................................................................................................ 1215.2 Effects of ocean acidification on spores of red algae ....................................... 1225.3 Effects of multiple climate variables on adult stages of coralline algae ........... 1255.4 Variability in carbonate chemistry in situ ......................................................... 130References ............................................................................................................................. 133         vii  List of Tables Table 2.1: Table summarizing water chemistry for spore adhesion experiments. ................ 23Table 2.2: Measured Total Alkalinity (mol/kg) in experimental treatments ....................... 24Table 2.3: Generalized linear model results for settlement time of P. bipinnata and C. vancouveriensis....................................................................................................................... 28Table 2.4: Results of exponential rise to maximum non-linear regression on final detachment of P. bipinnata and C. vancouveriensis spores. . .................................................................... 30Table 3.1: Experimental carbonate chemistry conditions in specimen chambers. . .............. 60Table 3.2: Results of linear mixed effect model on single-factor (temperature) experiment on fronds of C. vancouveriensis and C. tuberculosum. . ............................................................. 73Table 3.3: Results of linear mixed effect model on single-factor (pH) experiment on fronds of C. vancouveriensis and C. tuberculosum. . ........................................................................ 74Table 3.4: Results of linear mixed effect model on multi-factor (temperature x pH) experiment on fronds of C. vancouveriensis and C. tuberculosum. . ..................................... 75Table 4.1: Two-way ANOVA results of chemistry analyses between small, medium, and large tidepool. . ..................................................................................................................... 106Table 4.2: Two-way ANOVA results of chemistry analyses between sound water and tidepool water (pooled tidepools). ........................................................................................ 107Table 4.3: One-way ANOVA results between sound water and tidepool water at end of tide (pooled tidepools) ................................................................................................................. 107Table 4.4: Two-way ANOVA results on apical linear growth of C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools. ......................... 109 viii  Table 4.5: Two-way ANOVA results of percent calcium carbonate of  C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools. .............. 111Table 4.6: Two-way ANOVA results on measurements of biomechanical properties of C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools. ............................................................................................................................... 113  ix  List of Figures Figure 1.1: Articulated coralline algae morphology.. ............................................................ 12Figure 1.2: Line drawings of Corallina and Calliarthron showing morphological differences between the two genera. ......................................................................................................... 13Figure 1.3: Map of San Juan Islands, red star indicates collection and field study site (Dead Man Bay) ................................................................................................................................ 13Figure 2.1: Spore settlement apparatus and shear flume. ...................................................... 25Figure 2.2: The effect of pH on algal spore settlement time and detachment. ...................... 29Figure 3.1:  Single-factor temperature experiment set-up in seawater table. ........................ 55Figure 3.2: OAEL culture system set-up and carbonate chemistry manipulation. ................ 56Figure 3.3: Specimen preparation and grip attachment of C. vancouveriensis and C. tuberculosum in Instron tensometer........................................................................................ 57Figure 3.4: Measurements of temperature and pH in specimen chambers. ........................... 58Figure 3.5: Visualization of Calcofluor stain under black light. ........................................... 59Figure 3.6: Growth, percent calcium carbonate and biomechanical properties of C. vancouveriensis and C. tuberculosum under control conditions for six weeks ...................... 66Figure 3.7: Apical linear growth and percent calcification of C. vancouveriensis and C. tuberculosum in single-factor temperature experiment. ......................................................... 67Figure 3.8: Biomechanical properties of C. vancouveriensis genicula in single factor temperature experiment.  ........................................................................................................ 68Figure 3.9: Apical linear growth and percent calcification of C. vancouveriensis and C. tuberculosum in single-factor pH experiment. ....................................................................... 69 x  Figure 3.10: Biomechanical properties of C. vancouveriensis and C. tuberculosum genicula in the single-factor pH experiment ......................................................................................... 70Figure 3.11: Apical linear growth and percent calcification of C. vancouveriensis and C. tuberculosum in multi-factor temperature by pH experiment.. .............................................. 71Figure 3.12: Biomechanical properties of C. vancouveriensis and C. tuberculosum genicula in multi-factor temperature by pH experiment. ...................................................................... 72Figure 4.1: Photographs of studied tidepools.. ...................................................................... 96Figure 4.2: Tidepool temperatures in small, medium, and large tidepools in the summer and in the winter. ......................................................................................................................... 102Figure 4.3: Tidepool chemistry.. ......................................................................................... 103Figure 4.4: Chemical difference between tidepool and sound water. ................................. 104Figure 4.5: Comparison of tidepool chemistry at the end of isolation time in the summer and winter to predicted values for 2100 and pre-industrial values. ............................................. 105Figure 4.6: Apical linear growth of C. vancouveriensis by tidepool size ........................... 109Figure 4.7: Percent calcium carbonate of apical intergenicula of field collected C. vancouveriensis by tidepool size. ......................................................................................... 110Figure 4.8: Biomechanical properties of genicula of C. vancouveriensis. .......................... 112        xi  Acknowledgements  Completing this dissertation was by no means a solo endeavor and I have many people to thank for their help and support.    First and foremost, I would like to thank both my advisor, Dr. Patrick Martone and also Dr. Emily Carrington.  As the majority of my PhD research was completed at Friday Harbor Labs, I want to thank Patrick for being willing to advise me from afar and making the time to skype with me on a regular basis about my scientific progress.  I would also like to thank Dr. Emily Carrington, for “adopting” me into your lab while I was at Friday Harbor.  The support of these two mentors was pivotal in me completing this research, and they not only provided me with scientific and funding support, but also support on a personal level.  I would also like to thank my committee members: Dr. Christopher Harley and Dr. Mary O’Connor, for providing feedback and advise throughout my PhD. I would also like to thank Dr. Robert DeWreede for helping extensively with the revisions to this dissertation, and also Dr. Paul Harrison and Dr. Catriona Hurd for their feedback on this dissertation.  Many thanks also to all the members of both the Martone and Carrington labs, both past and present, for providing feedback and fruitful discussions over the years.    The work included in this dissertation was funded through many sources, including the National Science Foundation, The University of British Columbia, and several graduate student fellowships provided by the University of Washington-Friday Harbor Laboratories. I would also like to thank the San Juan County Land Bank for allowing me to collect and complete my research at Dead Man Bay Preserve, on San Juan Island.    I would like to give a big thank you to everyone at Friday Harbor Laboratories: all of the resident and visiting scientists, the office staff, maintenance staff, housekeeping staff, and  xii  the dining hall staff. I feel lucky to have been able to do my research at such a great marine lab, with the help and support of so many people.   Finally, I would like to thank my family and my friends for your constant support and encouragement.  I couldn’t have done this without you. I want to especially thank Dr. Kathy Ann Miller, as she was a driving force in my interest in phycology and excited my passion for algae.    1   1 Introduction 1.1 Background Since the industrial revolution, over 500 billion tons of atmospheric CO2 has been absorbed by the world’s oceans (Sabine et al. 2004, Bala 2013). Once absorbed into the ocean, dissolved CO2 dissociated into carbonic acid, shifting the carbon equilibrium and resulting in a decrease in seawater pH and lower saturation states for carbonate minerals (Feely et al. 2004). This process is termed ocean acidification. Currently, the oceans absorb approximately 2 of the 6 Gt C (gigaton carbon) per year of anthropogenically produced CO2 (Prtner 2008). In the 400,000 years before the industrial period, atmospheric CO2 fluctuated between 200 and 280 ppm (Feely et al. 2004). In 2016, the average monthly atmospheric CO2 concentration reached 400 ppm, which hasn’t occurred in at least 800,000 years (Jackson et al. 2016). Between 2004 and 2013, global CO2 emissions have continued to grow at a rate of 2.5% per year (Friedlingstein et al. 2014). Since pre-industrial times, the pH of ocean surface waters has decreased by approximately 0.1 units (Caldeira & Wickett 2003, Orr et al. 2005) and is predicted to decrease a further 0.3-0.4 units by 2100 (Feely et al. 2009, International Panel on Climate Change 2013). As the oceans absorb more and more CO2, their buffering capacity is reduced, which results in less CO2 absorbed into the ocean and more remaining in the atmosphere. Unfortunately, CO2 inputs are not halting; the present increase in CO2 is approximately 100-fold faster than at the end of the last ice age (Parry et al. 2007).   The changes in carbonate chemistry occurring in the world’s oceans are not constant across habitats and regions and variable responses due to ocean acidification are anticipated  2  (Hofmann et al. 2010, Hofmann et al. 2011). Organismal responses may be influenced by the natural variability in carbonate chemistry that they experience in situ (Hofmann et al. 2010, Hofmann et al. 2011). For example, the pH in intertidal tidepools can vary by up to 3 pH units over the course of one tidal cycle (Raven 2011) and organisms living in upwelling zones experience wide fluctuations in carbonate chemistry, but on a seasonal basis rather than a daily basis (Feely et al. 2008).  Because species living in these habitats have evolved with naturally variable levels of pH, they are likely to be more resistant to changes in ocean pH due to ocean acidification than their open ocean and subtidal counterparts (Hofmann et al. 2011).  Ocean acidification is only one component of climate change that is expected to influence organisms living in the ocean. Several other abiotic factors are predicted to change in concert with ocean acidification, such as increases in upwelling, storminess, nutrient loads, UV exposure, and temperature (Harley et al. 2006). Many researchers have documented effects of ocean acidification alone on marine organisms, and many are starting to incorporate the response of multiple stressors, such as temperature, nutrient availability and UV exposure. For example, when exposed to both ocean acidification and increased temperature, there may be a larger impact on marine organisms than either of these factors individually (Anthony et al. 2008, Martin & Gattuso 2009). Additive, synergistic or antagonistic biological responses are all possible. In additive cases, organismal responses to multiple factors are fairly predictable from the effects of each factor in isolation (Darling & Côtè 2008). Synergistic and antagonistic responses are more complicated, however, where biological responses may be greater or less than the sum of effects of each factor in isolation (Crain et al. 2008, Darling & Côtè 2008). The testing of multiple climate variables  3  concurrently is important in order to make accurate predictions about climate change, since combined effects can be unpredictable based on single-factor experiments.  Understanding the complicated effects of ocean acidification and climate change on marine organisms is critical, if we intend to mitigate climate impacts. All organisms are expected to be impacted by changes in ocean water chemistry, and predicting the effects of future climate change will require consideration of interactions between multiple climate variables, environmental heterogeneity, species-specific effects, competitive interactions, and community and population dynamics.     1.2 Study system: rocky intertidal ecosystem The intertidal zone of temperate, rocky shores is one of the most stressful habitats on Earth (Denny 2006). As the tide rises and falls, organisms must contend with both marine and terrestrial conditions on a daily basis. Such fluctuations of submergence and emergence bring extreme swings in physiological challenges for many intertidal organisms (Davison & Pearson 1996, Helmuth & Hofmann 2001). Intertidal organisms living in tidepools may be able to avoid stresses experienced when the tide recedes. In tidepools, organisms avoid desiccation (Dethier 1980) and experience less variable temperature and light regimes (Metaxas & Scheibling 1993). Some intertidal organisms have been shown to be more tolerant of increasing temperatures than their subtidal counterparts (Tomanek & Somero 1999, Harley et al. 2006); however, intertidal organisms may be living at their physiological limits and may not be able to physiologically adjust to changing conditions expected with climate change (Tomanek & Somero 1999, Harley et al. 2006).   4  Ocean acidification and climate change will have far reaching impacts on many marine organisms, and algae are no exception. Algae are major constituents of marine intertidal communities; they are major primary producers and form important habitat structures. Unlike most other marine organisms, macroalgae are unique in that they may benefit from extra CO2 in the seawater, and in some cases this may ameliorate the negative effects due to ocean acidification. Algae can use either bicarbonate or CO2 as a substrate for photosynthesis, and concentrations of both of these will increase under ocean acidification.  For the most part, ocean acidification has been found to have a widely variable effect on the physiology of macroalgae (Gao & Zheng 2010, Israel & Hophy 2002, Hofmann et al. 2012, Harley et al. 2012) but has led to faster growth rates in some species (Gao et al. 1991, 1993, Kübler et al. 1999, Iglesias-Rodriguez et al. 2008). In the intertidal zone, algae must contend with large hydrodynamic forces with every crashing wave, and so the strength of their materials is important in determining whether such hydrodynamic forces will dislodge and/or fragment algae. The changing pH and/or temperature of seawater has been shown to affect material properties of several marine organisms (Madin et al. 2008, Gaylord et al. 2011, Newcomb et al. 2015, Collard et al. 2015) and may lead to differences in material properties of coralline algae. Ocean acidification could result in weaker tissues being produced in future climate conditions, leading to increased dislodgement and breakage. Unlike animals and invertebrates, algae are non-mobile and cannot re-locate to mitigate these forces. Therefore, they must develop tissues that are strong enough and flexible enough to endure these hydrodynamic stresses.  5  1.3 Study species: Articulated coralline algae Rising acidity and elevated seawater CO2 levels are changing the environment for marine life worldwide, particularly for organisms that rely on calcification. Higher levels of CO2 may reduce the ability of calcifiers to maintain and build new carbonate skeletons (Hurd et al. 2009). Within the calcifiers, the specific type of calcium carbonate polymorph (aragonite, calcite or high-magnesium calcite) deposited may impact the response to ocean acidification (Kroeker et al. 2010). The cell walls of coralline algae are composed of high-magnesium calcite, which is the most soluble form of CaCO3 (Martin & Gattuso 2009, Kroeker et al. 2010), and species using this form of calcium carbonate are predicted to be most impacted by ocean acidification. Changes to coralline algae’s calcareous composition would not only impact the algae, but would indirectly affect the organisms that rely on the ecosystems that coralline algae help create and maintain. In the corallines, both biological and chemical processes mediate calcification (Borowitzka & Larkum 1987). Calcification and photosynthesis are linked, such that photosynthesis stimulates calcification (Pearse 1972, Pentecost 1978, Smith & Roth 1979, Borowitzka 1981). Calcification in coralline algae has been suggested to occur by the ‘trans calcification enzymatic mechanism (McConnaughey & Whelan 1997). In this mechanism of calcification, bicarbonate ions from the seawater are taken up and subsequently converted into CO2 for photosynthesis, and this then produces the carbonate used in calcification (McConnaughey & Whelan 1997, Roleda et al. 2012b, McCoy et al. 2016). Due to varying mechanisms of calcification, it is somewhat unknown how coralline algae calcification will respond to changes in pCO2. Egilsdottir et al. (2013) found that pCO2 did not affect respiration, gross primary production, and calcification rates of Corallina elongata from  6  tidepools in either light or dark.  But, this differs from Corallina officinalis in which calcification rates showed a parabolic response of skeletal accretion to pCO2 (Smith & Roth 1979, Ries et al. 2009). Interestingly, a comparison of three different morphotypes of coralline algae from different habitats demonstrated that intertidal species that regularly experience large fluctuations in pCO2 and pH are not necessarily more resistant to elevated pCO2 than their subtidal counterparts (Noisette et al. 2013). There is great variation in algal response to the abiotic factors driving calcification in coralline algae, even among closely related species and growth forms. This variability in responses by coralline algae may stem from differences between gross and net calcification rates under different levels of pCO2.  Net calcification is determined by two factors; the amount of gross calcification and the amount of dissolution (Andersson & Gledhill 2013). While gross calcification may increase under ocean acidification due to greater availability of bicarbonate and CO2, increased dissolution might also occur as the saturation state for calcite in the seawater declines (Roleda et al. 2012b).  Coralline algae play a critical role in establishing and modifying habitats and ecosystems. Not only do they induce the settlement and recruitment of many species in shallow waters, but they also provide habitats for a large number of organisms (Martin & Gattuso 2009). Many invertebrate larvae deliberately settle on coralline algae, including echinoderms, mollusks, annelid worms, and soft corals (Johnson et al. 1991, Gee 1965, Morse & Morse 1984, Sebens 1983, Lasker & Kim 1996).  Coralline algae occur worldwide (Johansen 1981) and can inhabit a wide range of habitats from the high intertidal zone (Spongites tumidum) to more than 300 meters deep, representing the deepest recorded living macroalgae (Littler 1972).  In temperate and subtropical regions, coralline algae act as  7  ecosystem engineers that promote marine biodiversity (Foster 2001). For example, they play a large part in cementing coral reefs and in colonizing bare rocks, building an environment for animals and other algae to live (Steneck & Adey 1976, Kelaher et al. 2002).  The fronds of articulated corallines are composed of alternating calcified intergenicula and uncalcified genicula fronds arising from a crustose base (See Figure 1.1 & 1.2). The main growth that occurs in articulated coralline algae is linear branch elongation, which occurs when apical cells synchronously elongate and divide (Adey 1970). Cell walls of coralline algae are impregnated with calcium carbonate, except the cell walls of genicula which decalcify during development. The genicula are evenly distributed tiers of un-calcified cells (Adey 1970) that give the plant a jointed structure and provide flexibility in crashing waves (Martone & Denny 2008, See Figure 1.1).  Since corallines deposit high-magnesium calcite, they are predicted to be negatively impacted by ocean acidification (Martin & Gattuso 2009, Kroeker et al. 2010). Yet, some corallines have been shown to increase calcification rates under low pH (Kroeker et al. 2010, Basso 2012) and in the intertidal coralline, Corallina elongata, increased pCO2 did not affect respiration, gross primary production, or calcification (Egilsdottir et al. 2013).   This resistance of some species to ocean acidification may be due to their ability to control pH at the site of calcification (Hurd et al. 2011). In the seawater immediately surrounding the organism, the pH is often different than that of the mainstream water (Vogel 2003). In this diffusive boundary layer, photosynthesis, respiration, and calcification alter the water chemistry: photosynthesis increases pH, while calcification and respiration reduce pH (Hurd et al. 2011).  Hurd et al. (2011) found that, for turf-forming coralline algae, pH in the light was approximately 0.5 units higher and approximately 0.35 units lower in the dark  8  within the diffusive boundary layer. The variation in pH within the diffusive boundary layer along with the lower ambient pH will likely affect organismal performance (Hurd et al. 2011). These fluctuations in pH within the boundary layer will likely be regulated by the interaction between water movement, organism morphology, and physiological processes of photosynthesis, respiration, and calcification (Hurd et al. 2011).    1.4 Sensitive life stages of red algae Many studies have focused on the effects of ocean acidification on adult stages of organisms, but there has been increased interest in the effects on early life stages (Hofmann et al. 2010), which are thought to be the most vulnerable to environmental perturbation and the most susceptible to ocean acidification (Kurihara 2008, Hofmann et al. 2010, Timmins-Schiffman et al. 2013). Negative impacts of ocean acidification have already been demonstrated for early life stages of many invertebrate taxa, including echinoderms, bivalves, corals, and crustaceans (Kurihara 2008, Gibson et al. 2011, Chan et al. 2015), but the effects of ocean acidification on early life stages of macroalgae is relatively unexplored.   Roleda et al. (2012a) found that increased CO2 ameliorates the negative effect of lowered pH on germination of kelp spores and Jorve-Hoos (2015) documented damage to kelp zoospores and overall reductions in microscopic life history stages due to temperature and pH. Within the corallines, a decrease in coralline algal recruits under reduced pH was also found (Roleda et al. 2015). Macroalgal spores represent sensitive life stages that could be greatly impacted by climate change. Once mature, spores are released, encased within a mucilage layer (Johansen 1981, Boney 1975).  Mucilages are particularly important in the attachment of red algal  9  spores compared to spores of brown or green algae since red algal spores do not have flagella and are for the most part non-motile (Fletcher & Callow 1992, Maggs & Callow 2001) with the exception of some slight amoeboid movement (Pickett-Heaps et al. 2001). Flagellar movement helps spores from other groups locate the substrate once in the boundary layer (Fletcher & Callow 1992), but red algal spores must rely on mucilage alone. Flagella also help with the initial adhesion of green and brown algal spores (Henry & Cole 1982, Fletcher & Peters 1981), whereas, in the red algae the mucilage that the spore is released with aids in spore attachment (Fletcher & Callow 1992).  The life cycles of red algae often alternate between free-living gametophyte and sporophyte stages, and the relative proportion of these life stages in the environment may reflect functional differences among microscopic stages. For example, some populations of coralline algae have been found to be dominated by sporophytes (Kain 1982, De Wreede & Klinger 1988, Fierst et al. 2005). While this may result from differential mortality of one free-living phase over the other, it may also reflect differential survival of one spore type over another, or differences in fertilization success (Santelices 1990, Santelices 2002, Fierst et al. 2005). That is, if diploid spores (i.e., carpospores) are more resistant to ocean acidification than haploid spores (i.e., tetraspores), this could lead to a dominance in sporophytes.    1.5 Dissertation overview and objectives In this dissertation, I investigate the potential impacts of climate stressors on several life stages of articulated coralline algae will be. This dissertation is broken down into three chapters addressing the following questions: (1) What are the effects of ocean acidification  10  on spore attachment in calcified and un-calcified algal species?, (2) What are the effects of ocean acidification and seawater warming on the growth, calcification, and material properties of two species of articulated coralline algae (C. vancouveriensis and C. tuberculosum)? and (3) What is the variability in carbonate chemistry and temperature experienced by intertidal coralline algae growing in tidepools across the seasons in temperate marine waters, and do these differences in chemistry and physical factors affect growth, calcification and material properties of C. vancouveriensis? In Chapter 2, I explore the effects of reduced pH on  spore adhesion in two species of algae, one calcified (Corallina vancouveriensis) and one un-calcified (Pterosiphonia bipinnata) to determine if effects of ocean acidification on spore stages are limited to calcified species. I found that the two species were both affected in their spore adhesion process. Reduced pH delayed spore settlement of both P. bipinnata and C. vancouveriensis, but the effect was stronger in P. bipinnata. Reduced pH also weakened spore attachment in C. vancouveriensis. In Chapter 3, I explore the effects of reduced pH and increased temperature on the growth, calcification, and material properties of two species of coralline algae, Corallina vancouveriensis and Calliarthron tuberculosum.  I performed single factor temperature and pH experiments, and then combined these factors in one experiment to determine if effects of one stressor added in predictable ways. I found that in general, both of these species are quite resistant to ocean acidification and temperature increases but that C. tuberculosum was more resistant than C. vancouveriensis.    In Chapter 4, I document the variability in carbonate chemistry and temperatures in tidepools dominated by C. vancouveriensis and relate these differences to growth,  11  calcification and material properties between the seasons. I found that the chemistry within tidepools fluctuates widely over the course of the low tide, but that overall, C. vancouveriensis was unaffected by these changes.  In Chapter 5, I discuss the implications of my work, summarize main conclusions from my research chapters, and highlight areas requiring further study.           12    Figure 1.1: Articulated coralline algae morphology. Fronds arise from a crustose base.  Genicula are uncalcified tiers of cells and intergenicula are calcified coralline tissue.      13   Figure 1.2: Line drawings of Corallina and Calliarthron showing morphological differences between the two genera.   Figure 1.3: Map of San Juan Islands, red star indicates collection and field study site (Dead Man Bay)     14  2 Ocean acidification delays and weakens adhesion of red algal spores 2.1 Synopsis The reduction in ocean pH due to ocean acidification has been shown to have wide and variable effects on many marine organisms (Hendriks et al. 2010, Kroeker et al. 2010) and it is generally thought that early life stages are more vulnerable to ocean acidification than adult phases (Hofmann et al. 2010). This is likely true for macroalgae, where reproductive spores are an important and sensitive stage, affecting survival to adulthood. For example, previous work has demonstrated that early algal life stages are highly susceptible to climate stresses, primarily increased UV, because of their small size and simple physiological capabilities (Henry & Cole 1982, Dring et al. 1996, Coelho et al. 2000). Unfortunately, the specific effect of ocean acidification on macroalgal spores is largely unknown. Few studies have assessed the impact of climate change on early life stages of macroalgae, although recent studies have started to reveal negative impacts of pCO2 and temperature on kelp zoospore germination and survivorship (Roleda et al. 2012a, Gaitán-Espitia et al. 2014, Jorve-Hoos 2015).  Within the corallines, reduced pH negatively impacted the recruitment of the coralline alga, Arthrocardia corymbosa (Roleda et al. 2015), and I hypothesize that this climate stress may disrupt the spore adhesion process in red macroalgae.  A novel shear flume was developed to examine the effect of elevated pCO2 on two aspects of spore adhesion, settlement time and attachment strength, in two intertidal  15  rhodophyte macroalgae, one calcified (Corallina vancouveriensis) and one non-calcified (Pterosiphonia bipinnata).  Reduced pH delayed spore settlement of both P. bipinnata and C. vancouveriensis, but the effect was stronger in P. bipinnata.  In contrast, reduced pH weakened spore attachment in C. vancouveriensis, but had no effect on the attachment strength of P. bipinnata.  Results demonstrate that ocean acidification can negatively impact macroalgal spore adhesion in two distinct ways, and that these effects differ among species.  Slower, weaker spore adhesion could negatively impact macroalgal populations, independent of the effects of ocean acidification on mature thalli.    2.2 Introduction As the world’s oceans absorb increasing levels of atmospheric carbon dioxide, causing a reduction in surface ocean pH, ocean acidification is expected to affect many marine organisms and have far-reaching ecological impacts (Hendriks et al. 2010, Kroeker et al. 2010, Porzio et al.  2013). The effects of ocean acidification are highly variable and difficult to anticipate, as both positive and negative responses have been documented (Ries et al. 2009, Hendriks et al. 2010, Kroeker et al. 2010, Porzio et al. 2011, Pfister et al. 2014).  It is predicted that calcified macroalgal species will fare worse under ocean acidification than uncalcified macroalgal species, at least during adult life history phases (Kroeker et al. 2010, James et al. 2014, Roleda et al. 2015). Although many studies have focused on the effects of ocean acidification on adult populations, there has been increased interest in the effects on early life stages of marine organisms (Hofmann et al. 2010), which are thought to be the most vulnerable to environmental perturbation and the most susceptible to ocean acidification (Kurihara 2008, Hofmann et al. 2010, Timmins-Schiffman et al.  16  2013). However, there are few studies comparing the effects of ocean acidification on reproductive spores of calcified and uncalcified species. Like spores of other red algae, spores of coralline algae are uncalcified but begin calcification when cells begin dividing (Vesk & Borowitzka 1984, Pentecost 1991). For the first time, this study compares the effects of ocean acidification on calcified and uncalcified macroalgal species in a life history stage that exists prior to calcification. A few studies have assessed the effects of ocean acidification on kelp zoospores (Roleda et al. 2012a, Jorve-Hoos 2015) and Roleda et al. (2015) also document reduced growth in the recruits of the coralline alga, Arthrocardia corymbosa, due to ocean acidification.  However, many questions remain to be answered about the effects of climate change on algal spores, including differential effects on calcifying and non-calcifying species.  There are few studies comparing the vulnerability of young and adult life history stages of algae to ocean acidification (But see Jorve-Hoos 2015, Nielsen et al. 2014, Roleda et al. 2015).  However, Nielsen et al. (2014) document an increased sensitivity of juveniles of Fucus serratus to the combined stress of increased temperature and high copper concentration than adult life-history stages. Negative impacts of ocean acidification have already been demonstrated for early life stages of many invertebrate taxa, including echinoderms, bivalves, corals, and crustaceans (Kurihara 2008, Gibson et al. 2011, Chan et al. 2015), but the effects of ocean acidification on early life stages of macroalgae are understudied.   The establishment and persistence of benthic macroalgal communities is contingent upon the successful settlement and attachment of algal spores (Santelices 1990, Fletcher & Callow 1992, Taylor et al. 2010). Spores in the intertidal zone of rocky shores must contend  17  not only with hydrodynamic forces imposed by breaking waves but also with environmental stresses such as increased temperature and desiccation. The settlement and attachment of algal spores has been shown to respond to changes in several abiotic factors (Chamberlain & Evans 1973, Steinhoff et al. 2011), but the effect of reduced seawater pH on these processes is unknown.  The speed and strength with which spores attach is important for their survival, ultimately determining whether or not seaweeds can settle, grow, and reproduce again (Gaylord et al. 2006, Taylor et al. 2010). When algal spores are released, they are dispersed by ocean currents until they sink and come into contact with a surface within the boundary layer (Maggs & Callow 2001, Taylor et al. 2010, Fletcher & Callow 1992). Although flow conditions are much reduced within boundary layers, shear force is typically the greatest force acting on small objects located near surfaces (Vogel 1994). Shear force is generated on a surface as a result of the viscosity of a moving fluid and its interaction with the no-slip boundary condition (Vogel 1994, Miklasz 2012). Attached to a surface, algal spores have to resist shear stresses imposed by a gradient in water velocity to avoid being dislodged (Vogel 2003).   Red algae produce two different types of mucilaginous secretions to assist in spore settlement and attachment (Chamberlain & Evans 1973, Boney 1975, Boney 1981, Fletcher & Callow 1992). Spores are initially released from a sporangium enveloped by mucilage composed of acidic sulphated polysaccharides (Boney 1981). This mucilage initially adheres red algal spores to the substrate, and the curing rate of this mucilage determines settlement time (Boney 1975, Fletcher & Callow 1992). Once settled, spores discharge a second type of adhesive mucilage, composed mainly of glycoproteins, through the plasma membrane of the  18  spore (Fletcher & Callow 1992). This mucilaginous adhesive spreads out from the spore bodies to form a pad, permanently attaching the spores to the substrate (Chamberlain & Evans 1973, Chamberlain 1976), and the quantity and quality of this secondary mucilage influences the strength with which spores attach (Fletcher & Callow 1992). Mucilage function depends upon gel-formation, which likely varies with seawater pH (Reynolds 2007), yet, little is known about the impact of ocean acidification on mucilage and spore adhesion. I used a novel spore settlement apparatus and shear flume that allowed fine-scale differences in time on spore adhesion to be evaluated.  I used this spore settlement apparatus and shear flume to assess the impact of reduced pH on settlement time and adhesive strength of two co-occurring rhodophyte species: one calcified, Corallina vancouveriensis and one non-calcified, Polysiphonia bipinnata. I aimed to determine whether ocean acidification affected both species similarly. C. vancouveriensis grows in mid-intertidal tidepools, whereas P. bipinnata grows on mid-intertidal rocks, where it is exposed to air at low tide in our study region (the Salish Sea region of Washington). Although calcified macroalgae are thought to be more vulnerable to ocean acidification (Kroeker et al. 2010), coralline spores have not been studied with regard to climate stress. 2.2.1 Hypotheses 1. A reduction in pH will delay settlement time and final adhesion strength in C. vancouveriensis and P. bipinnata. 2. Since spores are not calcified, it is expected that spores produced by the fleshy alga P. bipinnata and the calcified alga C. vancouveriensis will be similarly susceptible to reduced pH  19  3. A reduction in pH will delay settlement time and final adhesion strength in C. vancouveriensis and P. bipinnata 4. Since spores are not calcified, it is expected that spores produced by the fleshy alga P. bipinnata and the calcified alga C. vancouveriensis will be similarly susceptible to reduced pH  2.3  Methods Specimens of reproductive Pterosiphonia bipinnata and Corallina vancouveriensis were collected from the mid to low intertidal zone from San Juan Island, Washington (Deadman Bay: 480'48.93"N, 1238'56.14"W) and immediately transported to Friday Harbor Laboratories (FHL) and maintained in an outdoor seawater table for no more than one week before testing. P. bipinnata assays were performed in August and September of 2012 and 2013.  C. vancouveriensis assays were carried out in January through February and May through July of 2013 and also in June of 2014 and 2015 for settlement assay and in March 2014 for final adhesion assay. Reproductive parent fronds were held at ambient levels of seawater pH/pCO2 and were only exposed to experimental conditions while releasing spores. Spore release and settlement was performed in the Ocean Acidification Environmental Laboratory (OAEL) at FHL, allowing precise control of pH and temperature in a flow-through system (O’Donnell et al. 2013, see Methods in Chapter 3). Approximately 50L of seawater was adjusted to the target pH level. Two pH treatments were established by bubbling CO2 (7.75, 7.30 total scale) at 11C and confirmed with carbonate water chemistry analyses (Table 2.1 & 2.2). Specifically, spectrophotometric pH was determined as per standard operating procedures (SOP6) outlined in Dickson et al. (2007) and total alkalinity  20  was measured using an open cell titrator as outlined in SOP 3b (Dickson et al. 2007). Ambient surface seawater pH values for the Salish Sea region are approximately 7.8 (Murray et al. 2015), and pH 7.3 has been documented in tidepools at the collection site (Guenther, unpubl. data), and in near-shore environments in Washington state (Wootton & Pfister 2012).   A novel spore settlement apparatus and shear flume was developed to examine algal spore adhesion over small time scales. The spore settlement apparatus comprised a carriage system that slowly (1-2.5 cm/hr.) drove the reproductive parent algal frond across a glass settlement plate (Figure 2.1 A). Spores were steadily released and fell onto a large glass plate (1/4” deep, 3” wide, 24” long).  It is possible that settlement times and adhesion strengths in situ may be different than what was documented here due to biofilms and/or rugosity of the natural substrate on which spores settle. The working section was at one end of the plate, defined by two 14 cm long parallel lines drawn down the center, and spaced 1.5 cm apart. Spores landing on the working section of the plate have a decreasing gradient of settlement time, determined by the rate the parent was driven across the plate and the time the plate was allowed to set.  The shear flume was designed to release a tall column of water that flushed quickly across a channel covering the working section of the spore settlement plate. The height of this water column was varied to create a range of shear stresses (Figure 2.1 B), calculated for each column type according to (Schultz et al. 2000), using the height of the channel, the pressure gradient across the working section (measured with a manometer), and the length of the working section.  Previous studies demonstrated that most spores are removed in the first 10 seconds of exposure to shear regardless of the shear force, and longer exposure times  21  result in little additional detachment of spores (Christie et al. 1970, Miklasz 2012) and so trials were limited to 15 seconds.  Prior to each test, the shear flume was fitted on the settlement plate with clamps and the released spores were photographed using a microscope connected to a camera, using the lines drawn on the settlement plate for reference.   A low shear stress (1 Pa) was then applied and the remaining spores were photographed again, supplying data for the settlement time assay. This process of photographing the remaining spores on the plate after the application of shear was repeated for increasing shear stresses (4, 7, 17, 20 Pa), supplying the data for the adhesive strength assay.  At each position and at each shear stress applied, percent attachment (settlement time) and detachment (adhesive strength) was quantified through photo-analysis in ImageJ. For settlement time data, spore presence or absence was recorded after a low (1 Pa) shear stress was applied. A generalized linear model logistic regression was used to determine the effect of pH and time on spore settlement time (R version 3.1.2, Table 2.3, Figure 2.2 A & B). Attachment strength was then estimated as the proportion of spores that were detached by a given shear stress applied in increasing increments. To control for differences in settlement time between the two species, we allowed P. bipinnata spores to settle for 35-48 hours and C. vancouveriensis spores to set for 5-10 hours, since maximum settlement was found in these time frames. These time frames were chosen based on the logistic regression analysis of settlement time. For P. bipinnata, replication was achieved by repeating the experiment in time since one frond moved across entire settlement plate in each run. Due to limitations with C. vancouveriensis spore release, several fronds were used to provide sufficient spore set for this assay. In this case, each frond that released spores was considered a replicate, and this was repeated across several days. Any attempts to induce spore release  22  (osmotic, temperature, or light) proved to be unsuccessful. This study examined only the effect of reduced pH on haploid spores (tetraspores) produced by sporophytes, and did not investigate impacts on gametes or diploid spores (carpospores). An exponential rise to maximum non-linear regression was used to estimate detachment as a function of shear stress for each species (SigmaPlot 11.0, Table 2.4, Figure 2.2 C & D).      23   Table 2.1: Table summarizing water chemistry for spore adhesion experiments. Spore assay is either initial (settlement time assay) or final (adhesive strength assay), pH treatment is control (7.8) or low (7.3). pH(before) and temperature(before) are Durafet pH and temperature readings before releasing spores onto settlement plate. pH(after) and temperature(after) are Durafet pH and temperature readings after assay was completed.  Values are means ± S.E.M.          24  Table 2.2: Measured Total Alkalinity (mol/kg) in experimental treatments     25    Figure 2.1: (A) Spore settlement apparatus used to settle algal spores for variable amounts of time; showing continual release of spores as a mature plant is moved just above the settlement plate and (B) the shear flume and the replaceable columns used to generate different shear stresses. The flume was attached on top of the settlement plate where spores were released creating a channel over the working section through which the outflow of water passed.  26  2.4  Results In general, C. vancouveriensis spores settled much faster than P. bipinnata spores.  For example, C. vancouveriensis reached 70% settlement in only 2.5-8 hours while P. bipinnata needed 35-43 hours to reach 70% settlement (Figure 2.2A & B).  At this first stage of spore adhesion, there was a significant interaction between time and reduced pH, such that in P. bipinnata, it took approximately 21 hrs. for spores to reach 50% settlement under control pH (7.8) and approximately 32 hrs. to reach 50% settlement under low pH (7.3) (Figure 2.2A, Table 2.3).  In C. vancouveriensis, both time and reduced pH also caused a significant delay in initial settlement, but the two factors did not interact (Figure 2.2 B, Table 2.3). Spores of C. vancouveriensis took approximately 10 hrs. to reach 80% settlement under control pH (7.8) and approximately 14 hrs. under low pH (7.3).  Interestingly, the magnitude of this difference was similar between the two species; it took spores of both species approximately one-third longer to settle under low pH as compared to control pH.  A decline in pH also resulted in weaker attachment of C. vancouveriensis spores, while P. bipinnata spores were unaffected.  Since final spore attachment is a rapid process, it was expected that spore detachment would level out at higher shear stresses, as has been shown in previous work on red algal spore adhesion (Miklasz 2012).  Exponential rise-to-maximum, non-linear regressions fit the data well (Table 2.4, R2 = 0.84-0.99, Table 2.4) and provided estimates of two parameters, the maximum amount of detachment and the initial dependence of force on detachment.  Approximately 50% of spores of both species detached in this assay; and while P. bipinnata spores were unaffected by pH (Figure 2.2 C), low pH increased detachment of C. vancouveriensis spores to 70% between 1 and 5 Pa, and higher shear forces did not result in increased dislodgement (Figure 2.2 D).  Although maximum  27  detachment was similar in the two species, the dependence of detachment on low shear was different.   Under control pH conditions, P. bipinnata spores resisted up to10 Pa before 40% detachment, whereas 40% of C. vancouveriensis spores detached at only 5 Pa (Figure 2.2 C & D).       28  Table 2.3: Generalized linear model results for settlement time of P. bipinnata and C. vancouveriensis     29   Figure 2.2: The effect of pH on algal spore settlement time (A-B) and detachment (C-D) for 7.30 pH (red) and 7.75 pH (black). (A) P. bipinnata (n = 614-663); (B) C. vancouveriensis (n = 335-477) settlement time.  Each point is one spore scored as a 0 (not attached) or 1 (attached) after one low shear stress (1 Pa).  Lines are logistic regressions (ln[Y/(1-Y)] = a +bX), where a is the intercept, b is the slope, and X is time. Dashed lines are 95% confidence intervals; (C) P. bipinnata detachment after 35-48 hours of settlement time (symbols are mean of n = 5-9, bars are S.E.M.); (D) C. vancouveriensis detachment after 5-10 hours of settlement time (symbols are mean of n = 9-11, bars are S.E.M).  Lines are an exponential rise to maximum non-linear regression of percent spore detachment as a function of shear stress (y = a * (1-exp(b * x), where a is the maximum detachment and b is the initial rate of increase of detachment with shear stress.      30  Table 2.4: Results of exponential rise to maximum non-linear regression (R2 = 0.84-0.99) on final detachment of P. bipinnata and C. vancouveriensis spores. p-value (parameter) represents the significance of that parameter in the regression.  p-value (treatment) represents a t-test comparing parameter estimates between the two treatments (7.75 and 7.30 pH).     31  2.5  Discussion It is predicted that calcified macroalgal species will fare worse under ocean acidification than uncalcified macroalgal species, at least in adult phases (Kroeker et al. 2010, James et al. 2014, Roleda et al. 2015). To the author’s knowledge, there are no studies comparing the effects of ocean acidification on the spores of calcified and uncalcified species, which are universally uncalcified and perhaps equally vulnerable. The first hypothesis, that a reduction in pH will delay settlement and final adhesion strength in C. vancouveriensis and P. bipinnata, was partially supported.  Reduced pH negatively affected spore adhesion in these two species of red algae, but in different phases of the adhesion process. Reduced pH delays the settlement of both P. bipinnata and C. vancouveriensis spores, and weakens the final attachment of C. vancouveriensis spores. The second hypothesis, that spores produced by C. vancouveriensis and spores produced by P. bipinnata, would be equally susceptible to reduced pH was also partly supported.  As stated previously, the two species responded to reduced pH in different phases of the spore attachment process. In P. bipinnata, only settlement time was affected by reduced pH. On the other hand, C. vancouveriensis spores were affected by reduced pH in both settlement time and attachment strength.  Additionally, the two species had very different settlement times; however, the magnitude of the delay in settlement time due to reduced pH was similar for both species.  In the wave-swept intertidal zone, spore settlement is inherently uncertain. If spores take too long to settle, there is an increased likelihood that they will be swept out to sea or cast ashore before settling on suitable substrata; if spores attach too weakly, they are more likely to be dislodged by waves. In general, algal spores are viable in the water column for  32  only a few days before senescing (Santelices 1990). Even though spores of C. vancouveriensis settle much more rapidly (5-10 hours) than P. bipinnata spores (35-43 hours), fewer of these spores may be able to survive high shear stresses in reduced pH conditions. In contrast, any P. bipinnata spores that are able to settle, despite being delayed, are likely to have an attachment that is insensitive to reduced pH. In this manner, differing effects of ocean acidification on the settlement times and attachment strengths of spores have the potential to impact the future composition, diversity and abundance of seaweed populations along the shore.   The life cycle of red algae generally involves an alternation of free-living gametophyte and sporophyte stages. Many seaweed populations are dominated by sporophytes (Kain 1982, De Wreede & Klinger 1988, Fierst et al. 2005), which may result from differential mortality of one free-living phase over the other, differential survival of one spore type over another, or differences in fertilization success (Santelices 1990, Santelices 2002, Fierst et al. 2005). Reduced spore success has the potential to impact both gametophyte and ultimately sporophytes of seaweed populations. For example, reduced spore success may affect gametophyte density in the environment, potentially limiting subsequent fertilization (Santelices 1990). This study examined only the effect of reduced pH on haploid spores (tetraspores) produced by sporophytes, and did not investigate impacts on gametes or diploid spores (carpospores). Diploid spores may be equally susceptible, and therefore results demonstrated here may represent an underestimate of the impact of ocean acidification on overall spore success. Several biological processes have been shown to be impacted by ocean acidification, but calcification and therefore calcified species, are often predicted to be among the most  33  sensitive (Kroeker et al. 2010).  Growth and calcification of coralline algae have been shown to decrease in reduced pH seawater (James et al. 2014, Roleda et al. 2015).  However, spores of coralline algae are uncalcified, and only begin calcification when cells begin dividing (Vesk & Borowitzka 1984, Pentecost 1991). It is unknown when cell division begins in C. vancouveriensis, and it could possibly happen within the time frame that assays were performed in this study. Jones and Moorjani (1973) found differences in the onset of calcification between two calcified species; Corallina officinalis spores calcified much sooner than Jania rubens spores. However, J. rubens is an epiphytic species while C. officinalis attaches to rocky substrates, and Jones and Moorjani (1973) hypothesize that the difference in settlement times could be due to the ecology of the species.  Since J. rubens is an epiphytic species and has a restricted range of hosts, spores need to attach faster than C. officinalis spores, which can settle on rocky substrate (Jones and Moorjani, 1973). In addition, although mucilage is important for the initial adherence of Corallina spores to a substrate, calcification may play a role in final attachment (Jones and Moorjani, 1973). If this is the case, it may explain why reduced pH resulted in higher spore dislodgement but had a lesser effect on settlement time of C. vancouveriensis spores.  Although the investigation on effects of ocean acidification on spores of non-calcifying groups is somewhat lacking in the literature, there has been a fair amount of research done on kelp zoospores. For example, high pCO2 negatively affected the development of germination tubes in kelp zoospores (Gaitán-Espitia et al. 2014) and caused a decrease in the germination of kelp zoospores (Roleda et al. 2012a). Negative effects on spores of calcifying algae have also been documented. High pCO2 inhibited both spore production and growth of early life history stages in the calcifying red alga, Lithophyllum  34  incrustans (Cumani et al. 2010). Similarly, Bradassi et al. (2013) found that small changes in pH resulted in increased abnormalities of early life stages of Phymatolithon leormandii and also documented reductions in growth rate in abnormal thalli as compared to normal thalli. The effects of ocean acidification on early life stages have begun to be studied, but many questions remain, and further investigation into the effects on both calcifying and non-calcifying algae is warranted.  The delay and weakening of spore adhesion in reduced pH documented in this study is likely due to the effect of seawater chemistry on spore mucilage. Mucilage is not only central to adhesion, but it also aids in the dispersal and buoyancy of spores (Boney 1981, Fletcher & Callow 1992, Maggs & Callow 2001). Boney (1981) suggests that the mucilage surrounding spores of intertidal algae also acts as a physical buffer, protecting spores from desiccation and maintaining ionic equilibrium. Little is known about the specifics of how ocean acidification will interact with mucilages, however, it is likely that changes in seawater pH will affect the ionic environment surrounding the spore and mucilage body. In order for spores to stick to the substrate, extracellular mucilages must thicken into a gel by cross-linking polymer chains by divalent cations, such as calcium or magnesium (Campbell1982). Because pH determines the degree of protonation associated with the extracellular mucilage, reduced pH seawater may reduce the amount and extent of cross-linking (Campbell 1982).  In conclusion, ocean acidification negatively affected spore adhesion in two species of red algae, suggesting that reduced pH may affect the life cycles of both calcified and fleshy red seaweeds. These impacts may negatively impact seaweed populations via spore 'malfunction', despite the viability of adult algal thalli. Since a loss at any stage of spore attachment translates into a reduction of fitness, variable impacts on spore adhesion in  35  different algal species may result in far-reaching changes in seaweed diversity and abundance along our shores. However, given the shear numbers of spores released by one individual, the negative effects on spore adhesion documented in this study may or may not have significant effects on the population.  Future studies should address whether increased spore mortality results in changes in population dynamics.       36  3 The effects of temperature and pH on the growth, calcification, and biomechanical properties of two species of articulated coralline algae 3.1 Synopsis In this chapter, I examined the effect of pH and temperature, both individually and in combination, on the growth, calcification and biomechanical properties of two species of articulated coralline algae (Corallina vancouveriensis and Calliarthron tuberculosum).  Increased temperatures and reduced pH were found to negatively affect growth rates in both species (30-89% reduction), but had little influence on the amount of intergenicular calcium carbonate or on the biomechanical properties of genicula of these species.  Results suggest that although these corallines exhibit reduced growth under climatic stressors, they will maintain the integrity of their tissues.   3.2 Introduction 3.2.1 Climatic stressors Since the industrial revolution, approximately half of the anthropogenically produced CO2 has been absorbed into the world’s oceans (Sabine & Feely 2007), which equates to over 500 billion tons of CO2 (Sabine et al. 2004). Once absorbed into the ocean, dissolved CO2 shifts the carbon equilibrium and results in a decrease in seawater pH, an increase in pCO2, and lower saturation states for carbonate minerals (Feely et al. 2004). Ocean acidification is only one component of climate change. Several other abiotic factors are  37  predicted to increase in concert with ocean acidification, such as upwelling, storminess, nutrient loads, UV exposure, and temperature (Harley et al. 2006, Feely et al. 2008). Responses due to ocean acidification alone may not be significant. In situ, organisms are exposed to multiple stressors simultaneously due to climate change and, the responses of marine organisms to multiple stressors may be more severe than responses to any single factor alone (Anthony et al. 2008, Martin & Gattuso 2009, Harley et al. 2012, Gunderson et al. 2016).  The changes in carbonate chemistry occurring in the world’s oceans are not constant across habitats and regions (McNeil & Matear 2008, Hofmann et al. 2011). This variability can result from both abiotic and biotic factors not specifically related to anthropogenic inputs of CO2.  For example, temperature influences the carbonate chemistry of the water. Carbon dioxide is more soluble in colder waters, and so more CO2 is absorbed in polar regions compared to tropical regions (McNeil & Matear 2008). Biological processes, such as photosynthesis and respiration, can also affect the chemistry of seawater (Hurd et al. 2009).  Photosynthesis involves CO2 removal from the surrounding seawater and if this process is sufficiently high, this can increase seawater pH. The process of respiration, on the other hand, releases CO2 and results in more acidic waters. Thus, variability in carbonate chemistry conditions can be caused by both biotic and abiotic factors and these factors can interact to produce unpredictable changes in carbonate chemistry (Murray et al. 2015).  Seawater temperature has been shown to affect a multitude of metabolic processes in algae.  Photosynthesis (Davison 1991, Guenther & Martone 2014), growth (Fortes & Lüning 1980), and calcification (Jokiel & Coles 1990) have all been shown to depend on temperature. In general, as temperature increases, organisms exhibit a positive response until  38  they reach some thermal maximum, and this is termed a thermal response curve.  At this point, increased temperature becomes detrimental and starts damaging metabolic processes (Davison 1991, Kordas et al. 2011). When this occurs, seaweeds may reallocate resources to protection and repair, but this could potentially slow growth, delay development, and lead to mortality (Davison & Pearson 1996, Harley et al. 2012). For example, algae can synthesize heat shock proteins for protection against increasing temperature (Li & Brawley 2004, Henkel & Hofmann 2008), which may compete with other vital physiological processes. Within the marine calcifiers, the specific type of calcium carbonate polymorph (aragonite, calcite or high-magnesium calcite) deposited may impact the response to ocean acidification. Organisms using aragonite or high-magnesium calcite (the more soluble forms of CaCO3) are thought to be more susceptible to ocean acidification in regards to calcification than organisms depositing the less soluble form, calcite. Many studies have documented reduced abundance (Kuffner et al. 2008), growth (Kuffner et al. 2008, Kroeker et al. 2010, Cornwall et al. 2013), and calcification (Anthony et al. 2008, Büdenbender et al. 2011) in corallines under future ocean acidification scenarios. Surprisingly, however, in a meta-analysis by Kroeker et al. (2010), calcification in organisms with high-magnesium calcite was not always negatively affected by ocean acidification. This may be owing to the fact that corallines are able to use the bicarbonate ion and/or respired CO2 to calcify rather than utilizing carbonate ions directly from the seawater (Roleda et al. 2012b, Koch et al. 2013). Variability in responses to ocean acidification may be owing to different mechanisms of calcification (Price et al. 2011). Recently, the origin of the carbonate ion that is used to form calcareous structures has been re-examined (Roleda et al. 2012b). Previously, it was assumed that the carbonate ion deposited in calcium carbonate structures originated directly  39  from the seawater. In this scenario, calcification rates would be directly linked to calcium carbonate saturation states.  However, recent work suggests that bicarbonate is the substrate for calcification in many groups, including coralline algae (Jokiel 2011, Roleda et al. 2012b).  In this scenario, the bicarbonate ion is either acquired directly from seawater or derived from respired CO2, which is subsequently converted to bicarbonate by carbonic anhydrase (Jokiel 2011, Roleda et al. 2012b). In the corallines, both biological and chemical processes mediate net calcification (Borowitzka & Larkum 1987). Calcification and photosynthesis are linked, such that photosynthesis stimulates calcification (Pearse 1972, Pentecost 1978, Smith & Roth 1979, Borowitzka 1981). Net calcification is therefore determined by two factors; the amount of gross calcification and the amount of dissolution. The amount of gross calcification is biologically mediated (and partly chemically mediated), but the rate of dissolution is strictly chemically mediated by the saturation state of the seawater with respect to the calcium carbonate polymorph.  The seawater saturation state for calcium carbonate is calculated by:   = [Ca2+][CO32-]/Ksp This equation relates the calcium and carbonate ion products to the solubility product expected when seawater is in equilibrium with the carbonate mineral.  When  = 1, seawater is in equilibrium with the carbonate mineral. When  > 1 the seawater is supersaturated with respect to the carbonate mineral, and when  < 1, seawater is undersaturated with respect to the carbonate mineral. If saturation states for the specific CaCO3 are above 1, calcification is thermodynamically favored. If the saturation state is below 1, dissolution of the CaCO3 skeleton is expected (Feely et al. 2009).      40  Many calcifying organisms possess mechanisms by which they can counteract ocean acidification effects. For example, some macroalgae can regulate the pH of the diffusive boundary layer (Hurd et al. 2011, Roleda et al. 2012b). Photosynthesis results in a higher pH and respiration results in a lower pH in the diffusive boundary layer compared to the surrounding seawater.  Organisms that are able to regulate pH at the site of calcification are less likely to be impacted by ocean acidification. Hurd et al. (2011) found that for Sporolithon durum, a rhodolith forming coralline, the pH within the diffusive boundary layer was different from that of the surrounding seawater, and was regulated by photosynthesis and respiration. During photosynthesis, the pH in the diffusive boundary layer increases, depending on photosynthetic rate and light availability enabling the alga to regulate pH at the site of calcification and mitigate impacts of ocean acidification. The opposite occurs at night, when the pH of the diffusive boundary layer decreases with increasing respiration, and  dissolution may be enhanced depending on organismal metabolism. The extent of the protection provided by the diffusive boundary layer also relates to water motion (Hurd 2015), as the diffusive boundary layer breaks down at relatively low water velocities, so this process may only counteract ocean acidification effects in slow moving water (Cornwall et al. 2014, Hurd 2015). Additionally, if  there is an increasing metabolic demand to maintain the pH at the site of calcification, less energy may be allocated to other processes such as growth, development, and reproduction (Beardall & Giodano 2002).   It is difficult to draw broad conclusions about responses to ocean acidification for species that differ in physiology, collection location, time of year, taxonomic group, or life history stage (Hofmann et al. 2011, Harley et al. 2012). Environmental changes that are occurring due to climate change are not a continual increase or decrease, but are variable and  41  complex (Helmuth et al. 2006, Wootton et al. 2008).  Extremes, ranges, and patterns of variability are predicted to have different biological impacts (Harley et al. 2012).  The impact of ocean acidification and rising seawater temperatures may be related to the variability in pH and temperature in their natural environment (Hofmann et al. 2011, Harley et al. 2012).  For example, organisms living in highly variable habitats, such as tidepools and regions of upwelling may be adapted to abrupt changes in carbonate chemistry and temperature of the seawater (Gooding et al. 2009, Yu et al. 2011), such that an individual or population that has been exposed to stressful conditions in the past may be better able to survive them in the future (Padilla-Gamino & Carpenter 2007).  Comeau et al. (2014) found that physiological responses of the nongeniculate coralline, Porolithon onkodes, to elevated pCO2 were location specific, varying with carbonate chemistry conditions at the collection site.  On the other hand, Noisette et al. (2013) compared the responses of three different forms (i.e. morphotypes) of coralline algae to ocean acidification and found that species from habitats with naturally strong pH fluctuations were not necessarily more resistant to elevated pCO2 than species from stable habitats.  Yet, only one species per morphotype was tested, making it difficult to determine whether observed effects were due to species, morphotype or habitat (McCoy & Kamenos 2015).  The uncertain effects of interactions between different climatic stressors, the wide variation in species responses, and environmental conditions make predicting responses to climate change challenging.    3.2.2 Biomechanical Properties Algae living on wave-swept shores experience high hydrodynamic forces with every crashing wave (Denny et al. 2003, O’Donnell 2005, Martone 2006). Hydrodynamic forces  42  can dislodge and/or fragment algae, impacting community structure in the intertidal zone (Denny 1994, Denny et al. 2003). Unlike most animals and invertebrates, algae are non-mobile and cannot re-locate to mitigate these forces. Therefore, they must develop tissues that are strong enough and flexible enough to endure these hydrodynamic stresses. There are several ways that seaweeds can resist hydrodynamic forces; they can (1) make stronger tissues (Martone 2007, Speck & Burgert 2011), or (2) increase cross-sectional area of load-bearing tissues (Martone 2007, Starko & Martone 2016), or (3) reconfigure in flow (Boller & Carrington 2007, Martone et al. 2012).  Articulated coralline algae produce segmented thalli that are calcified, resulting in a relatively rigid thallus that is also flexible. Calcified regions (intergenicula) are separated by flexible uncalcified tissues (genicula). The genicula act as joints and provide flexibility, allowing these algae to resist hydrodynamic forces and grow upright.  However, genicula are the most likely part of the alga to fail when stretched in tension (Martone 2006).  When a wave imposes a force on coralline thalli, the genicula stretch in tension or in bending (Martone & Denny 2008). Previous work has shown that genicula are exceptionally strong to support the calcified tissue and not break (Martone 2006, Martone 2007, Martone & Denny 2008, Janot & Martone 2016).  Specifically, Martone (2006) found that, in tension, the tissue genicular tissue in Calliarthron is approximately 10% stronger than kelp tissues, and approximately 35% stronger than Mastocarpus stellatus, the next strongest tissue found in red algae to date (Martone 2006, Martone 2007).  How the mechanical properties of algal tissues are affected by ocean acidification has yet to be thoroughly investigated, however, changing the pH of the seawater may affect the synthesis of cell wall components and/or matrix polysaccharides in genicula.  This may result in production of weaker tissues during future climate conditions, leading to increased  43  dislodgement and breakage.  In a crustose coralline, Lithothamnion glaciale, increasing CO2 resulted in cell wall thinning and increased cell size (Ragazzola et al. 2012). Such changes in cellular properties could lead to a change in material properties, making tissues weaker, and possibly easier to break.  In this chapter, I examine the effects of ocean acidification and increased temperature on two species of coralline algae: Corallina vancouveriensis and Calliarthron tuberculosum.  Both species are commonly found in the intertidal zone throughout the Northeast Pacific (Foster 1975, Padilla 1984).  Corallina vancouveriensis has a wide distribution, from the Aleutian Islands to the Galapagos Islands (Abbott & Hollenberg 1976).  The environmental tolerance of Corallina vancouveriensis is reflected in its habitat; it is strictly found in the intertidal zone where it frequently grows out of tidepools or near the surface of tidepools (Padilla 1984). Furthermore, it is the only articulated coralline species that can survive emergence for an extended period of time (Abbott & Hollenberg 1976), likely due to its ability to retain water within its finely branched thalli during low tide (Guenther & Martone 2014). Calliarthron tuberculosum, on the other hand, occurs commonly from British Columbia, Canada to Los Angeles County, California (Gabrielson et al. 2011) and is most abundant subtidally (Konar & Foster 1992), suggesting that it is well adapted to cooler temperatures and generally is exposed to less variable environmental conditions than C. vancouveriensis.  C. tuberculosum does, however, extend up into the low intertidal zone if immersed in large tidepools where extreme temperatures and pH fluctuations are somewhat muted.  C. vancouveriensis was found to be more resistant to increasing temperature and desiccation (Guenther & Martone 2014), and so, C. vancouveriensis was predicted to be more resistant to adverse environmental conditions than Calliarthron tuberculosum.  44  3.2.3 Hypotheses In this chapter, I explore the effect of increased temperature and reduced pH, both singly and in combination, on the growth, calcification, and biomechanics of two species of articulated coralline algae. Specifically, I test the following hypotheses:  1. Single factor Temperature Experiment: A typical dose response curve is expected for rates of growth and calcification and also for material properties, such that increasing temperature will result in positive changes in growth, calcification, and biomechanical properties up to a point, after which reductions are expected. The negative effects of high temperatures on mid-intertidal Corallina are expected to be less than those on low intertidal Calliarthron.  Mid-intertidal Corallina is expected to be more resistant to high temperature than low-intertidal Calliarthron.  2. Single factor pH Experiment: Growth, calcification and material properties will be negatively affected by decreasing pH for both species, but mid-intertidal Corallina is predicted to be more resistant to reduced pH than low intertidal Calliarthron.   3. Multi-factor Temperature by pH Experiment: Corallina and Calliarthron growth rates are predicted to be positively affected by increasing temperature regardless of pH.  Increased temperature is not hypothesized to have an effect on calcification, while reduced pH is predicted to have a strong effect on calcification. Increased temperature is not hypothesized to have an effect on biomechanical properties, while reduced pH is expected to produce weaker, more extensible, and less stiff fronds.        45  3.3 Methods 3.3.1 Specimen Collection and Preparation For all experiments, Corallina vancouveriensis and Calliarthron tuberculosum were collected from Deadman Bay (4830’48.09”N, 1238’49.38”W), San Juan Island, Washington and immediately transported back to Friday Harbor Laboratories (FHL). I tested my hypothesis by exposing mid-intertidal samples of Corallina vancouveriensis and low intertidal Calliarthron tuberculosum to replicated treatments of increased pH and temperature. After collection, specimens were held in flow-through seawater tanks for less than one week before preparing specimens for experimental treatments. The number of plants collected was dictated by the number of experimental replicates. Plants were divided so that three fronds from each plant went into each experimental treatment (Figure 3.1A). Fronds were stained with Calcofluor White prior to putting specimens into experimental chambers in order to record growth (See Section 3.3.6 and Martone 2010 for methods).  Coralline fronds were grown under defined pH and temperature conditions in FHL’s Ocean Acidification Environmental Laboratory (OAEL) for the single-factor pH experiment and for the multiple factor temperature-by-pH experiment (Figure 3.1 B). The single factor temperature experiment was performed in a seawater table at FHL (Figure 3.1A).  Light was supplied by Chroma 50 full spectrum fluorescent lights (2 fixtures of 2 lights per culture system, 52.8 ± 0.8 mol photons m-2s-1) during all experiments. Fronds periodically became fouled with diatom growth, and so, were removed from experimental treatments and scrubbed with a soft brush approximately once per week for the entire experimental period.  46  Following culture under controlled laboratory treatments, rates of growth, calcification, and biomechanical assays were completed (See sections 3.3.6, 3.3.7, 3.3.8, respectively).  3.3.2 Species Differences C. vancouveriensis and C. tuberculosum fronds were compared for baseline differences in growth, calcification, and biomechanics between the two species. Fronds from the multi-factor temperature by pH experiment (Section 3.3.5) were used for this assay. Only fronds from the control pH (~7.8) and medium temperature (13C) were used for this assay.   3.3.3 Single-factor Temperature Experiment Calliarthron tuberculosum and Corallina vancouveriensis samples were grown for four weeks (April 18, 2015 – May 15, 2015) under controlled temperature conditions in a seawater table at FHL (Figure 3.1 A). Samples (~10-20 fronds per plant) were collected from three different plants of each species. They were then divided up into four treatment groups: 12ºC, 15ºC, 18ºC, and 22ºC. Each temperature treatment had three interdependent replicate specimen chambers, A, B and C, where A held three C. vancouveriensis fronds and three C. tuberculosum fronds each from one plant, B held fronds from a second pair of plants, and held fronds from a third pair of plants (Figure 3.1A). Fronds from both species were grown together, but any potential species interactions were assumed to be negligible. Specimen chambers held approximately 4L of seawater, and seawater within the chamber was replenished at a rate of 2.5L/hour. A small aquarium pump was placed within the specimen chambers to ensure adequate water mixing and the chambers were held in an empty seawater table at FHL. Chambers were rotated every week to randomize for any location effects  47  within the seawater table. Temperature in specimen chambers was monitored continuously with iButtons (Maxim Integrated Products) and was also manually measured daily.  Temperature was controlled by heat exchangers contained within three 5 gallon header tanks each manipulated to the target temperature (15C, 18ºC and 22ºC), and the fourth temperature treatment was ambient seawater (~12C).  The two highest temperature treatments (18ºC and 22ºC) were maintained with an aquarium heater and a temperature controller. A third bucket was maintained at room temperature (15ºC).  Each bucket was filled with freshwater and seawater was pumped through coiled tubing that was submersed in the bucket to ensure that specimen chambers received stable temperature seawater.  Seawater passed through the coils, was heated to the target temperature, and was then pumped into the specimen chambers that held the algal fronds (Figure 3.1 A).  Flow into specimen chambers was controlled by sprinkler heads, and each specimen chamber also had an aquarium pump to ensure adequate water movement.  Temperature loggers (ibuttons) were placed into each specimen chamber to monitor long term temperature levels. Specimen chambers were also rotated every week, scrubbed for diatoms, and put into clean chambers as needed.   At the end of the 4 weeks, linear growth and calcification was measured for fronds of both species in all temperature treatments (see section 3.3.6 & 3.3.7 for methods).  Biomechanical properties were measured only for C. vancouveriensis (See section 3.3.8 for methods) due to equipment malfunction.   3.3.4 Single-factor pH experiment Fronds of C. vancouveriensis and C. tuberculosum were maintained for six weeks (November 6, 2013 – December 18, 2013) under a range of 6 pH levels (8.0, 7.8, 7.6, 7.4,  48  7.2, 7.0, n = 6) following the methods of O’Donnell et al. (2013). Briefly, seawater entering the OAEL was filtered through a series of filters, the smallest of which was 0.2 m, scrubbed of CO2 and sterilized with UV. Filtered seawater was then pumped into the lab and into each culture system (cooler). A float valve in the culture system stopped the flow of water when a specified volume of water was reached. Culture systems in the OAEL are coupled into pairs on each table and each table has one temperature and pH analyzer that controls both culture systems on that table. This analyzer monitored pH and temperature of each culture system using a separate Durafet III pH electrode. The pH and temperature of each culture system was displayed in real time on the analyzer’s home screen, and all analyzers are networked so that pH and temperature in all culture systems are monitored continuously.  Carbonate chemistry in the culture system was manipulated by bubbling a CO2: air gas mixture into the culture system through a Venturi injector. Water within the culture system was pumped through a heat exchanger and chiller system to maintain water temperature. The temperature was then fine-tuned with an aquarium heater within the culture system using temperature controllers within the pH/temperature analyzer (Figure 3.1 B). Each analyzer used a feed-back loop between the analyzer and the Durafet pH electrode to control the amount of CO2: air gas mixture pumped into the culture system, to achieve precise control of pH.  Culture systems were designed as flow-through systems in which treatment water is modified in a reservoir, essentially each treatment had one mixing tank. The water from this reservoir was then pumped into eight individual specimen chambers where experimental organisms are held (Figure 3.1 B). Manipulated seawater from the culture system was pumped into the specimen chambers through a water distribution system. This system was composed of a network of sprinklers that dripped manipulated seawater to each specimen  49  chamber. Specimen chambers held approximately 4L of seawater, and seawater within the chamber was replenished at a rate of 2.5L/hour. A small aquarium pump was placed within the specimen chambers to ensure adequate water mixing. Fronds from both species were grown together, and so, any potential species interactions were assumed to be negligible.  Outflow from the chambers was connected through a rack and tubing, and this water drained directly out of the culture system. In this way, water between the specimen chambers did not mix and outflow drained directly out of the culture system and not back into the manipulated culture system water. pH and temperature was continuously measured in the culture system water (mixing reservoir), and was measured daily in specimen chambers.  Weekly water samples from culture system seawater were collected for carbonate chemistry analysis (spectrophotometric pH, Total alkalinity, Table 3.1) and measured within 24 hours. Using the program CO2calc (version 1.3.0, Hansen et al. 2010), pCO2 and carbonate saturation states were calculated from measured temperature, salinity, pH and total alkalinity values. The same CO2calc constants were used as outlined above (Section 3.3.4). Additionally, pH (Durafet electrode) and temperature were monitored in specimen chambers throughout the experiment (Figure 3.3 B).  After five weeks, linear growth, calcification, and biomechanical assays were conducted on fronds from each treatment (See sections 3.3.6, 3.3.7, 3.3.8).  3.3.5 Multi-factor temperature x pH experiment Fronds of C. vancouveriensis and C. tuberculosum were maintained for six weeks (June 9, 2014 – July 21, 2014) under three temperatures (10°C, 13°C, 16°C) and two pH levels (7.4, 7.8) in the OAEL (See Section 3.3.4 for methods). For each treatment of  50  temperature and pH, specimens were held in eight interdependent replicate specimen chambers. Each specimen chamber held approximately 4L of seawater, and seawater within the chamber was replenished at a rate of 2.5L/hour. A small aquarium pump was placed within the specimen chambers to ensure adequate water mixing. Fronds from both species were grown together, but any potential species interactions were assumed to be negligible. Treatments (n = 6) were rotated among experimental culture systems once per week, to randomize for any culture system effects, such that each culture system held each treatment for one week. pH and temperature was continuously measured in the culture system water (mixing reservoir) and was measured daily in specimen chambers.    Water samples were collected from culture system seawater once per week for carbonate chemistry analysis (spectrophotometric pH, Total alkalinity, Table 3.1) and measured within 24 hours.  Using the program CO2calc (version 1.3.0, Hansen et al. 2010), pCO2 and carbonate saturation states from measured values of temperature, salinity, pH and total alkalinity were calculated. The following CO2calc constants were used: CO2 constants (K1, K2 from Mehrbach et al. 1973), KHSO4 (Dickson 1990), pH scale (total scale (mol/kg-SW), Total Boron (Lee et al. 2010), and Air-sea flux (Wanninkhof 1992). Additionally, pH (Durafet electrode), temperature, and salinity were monitored in specimen chambers throughout the experiment (Figure 3.3 C).  At the end of the six week experimental period, linear growth, calcification, and biomechanical assays were completed on fronds from each treatment (See sections 3.3.6, 3.3.7, 3.3.8)     51  3.3.6 Linear Growth Assay  Linear growth was assessed using the Calcofluor White method described in Martone (2010).  Briefly, before each experiment, fronds were stained with a 0.05% solution of Calcofluor white (Sigma-Aldrich, Fluorescent Brightener 28) for approximately 1 hour.  Adequate light was supplied during the staining process to ensure that the staining was successful, since fronds stained more readily while photosynthesizing (Martone, pers. comm).   At the end of the experiment, the stained tissue (i.e. the previous position of the apical meristem) was visualized by exposing fronds to black light and taking long exposure photographs (~10-13 sec.). Photographs were then analyzed for linear growth of newly deposited coralline tissue in ImageJ by measuring the length from the Calcofluor stain mark to the tip of the coralline (Figure 3.4). Approximately 5-10 tips from each species in each specimen chamber were measured. Measured lengths were then divided by the number of days since the stain was applied, and averaged to yield an average growth rate for each plant. These values were then averaged across replicates to calculate the average linear growth for each experimental treatment (See Martone 2010).  3.3.7 Calcium Carbonate Assays  The amount of calcium carbonate in calcified tissues (intergenicula) was determined by decalcifying segments in HCl. Intergenicula that had formed during the experiment (new growth) and also intergenicula that were formed before the experiment (old growth) were each harvested for this assay. Approximately 10-30 intergenicula per plant were harvested for each measurement. Intergenicular segments were first dried (68°C overnight) and  52  weighed (g), then de-calcified in 1N HCl for at least 12 hours, and then rinsed with distilled water, dried, and re-weighed.  Percent calcium carbonate was calculated as follows:    		  						    3.3.8 Biomechanical Assays Biomechanical properties (breaking stress, breaking strain, and modulus) of uncalcified tissues (genicula) were measured in tension using an Instron 5565 tensometer fitted with a temperature controlled water bath (12C) and submersible pneumatic grips (40 psi). Measurements of genicular cross-sectional area and genicular length were used to standardize force and length measurements from the tensometer. For C. vancouveriensis, apical frond sections with approximately 10-20 genicula were mounted into the grips with thin foam (2-3mm) and fine sandpaper to reduce slippage (Figure 3.2). The sample was then extended at 0.2 mm/sec until the frond failed at one geniculum. The cross-sectional area of the geniculum that broke was measured under a dissecting microscope (40X), and a neighboring genicula was measured for length in the same manner. Initial length of C. vancouveriensis was determined by multiplying by the number of genicula between the tensometer grips by the length of the measured geniculum to yield the total genicular length tested.  Since C. tuberculosum has larger genicula, it was feasible to test only the first apical geniculum. Each calcified intergeniculum connected to the apical geniculum was glued into  53  small divots that were drill into aluminum T-bars using Zap-a-Gap glue and kicker. The T-bars were then mounted into the pneumatic grips for tensile testing (Figure 3.2 B).   The extension and load values provided by the tensometer were used to construct stress versus strain curves from which breaking stress (MPa), breaking strain (mm/mm), and modulus (MPa) were measured for each plant from each treatment. The strength, or breaking stress, of the material was defined as the amount of force applied at breakage, divided by the cross sectional area of the geniculum. Extensibility, or breaking strain, was the maximum extension of the genicula at breakage, divided by the initial length of the genicula. The stiffness, or tensile modulus, of the genicula was quantified as the initial slope of the stress-strain curve. Since all biomechanical parameters were normalized to the size and shape of the sample, these tests evaluated the properties of the genicular material regardless of thallus morphology.   3.3.9 Statistical analyses Differences between baseline parameters (growth, calcification, and biomechanics) between C. vancouveriensis and C. tuberculosum were analyzed with two-tailed t-tests (Excel 2010) on data from 13C/7.8 pH treatment of Temperature x pH experiment. A repeated measures subjects-by-treatment ANOVA design was used to analyze data from all experiments, using linear mixed effects models (R v.3.1.2). The model included the response variable (growth, calcification, or biomechanical parameter), the fixed categorical variables representing treatment (temperature, pH, or temperature x pH), and a random categorical variable representing block (plant #). Marginal fitting of terms was employed to obtain Type III Sum of Squares. Tukey HSD tests were used (R version 3.1.2) to separate out  54  treatments for effects that were found to be significant at a significance level of alpha < 0.05).       55   Figure 3.1:  Single-factor temperature experiment set up in seawater table.      56   Figure 3.2: OAEL culture system set-up and carbonate chemistry manipulation.      57    Figure 3.3: Specimen preparation and grip attachment of (A) C. vancouveriensis and (B) C. tuberculosum in Instron tensometer.    58   Figure 3.4: Measurements of temperature and pH in specimen chambers. (A) Single-factor temperature experiment. Daily average of temperature, averaged across the three chambers (ibutton data); (B) Single-factor pH experiment. Weekly average of pH, measured by Durafet electrode. Lines are each chamber value; (C-E) Multi-factor (pH x temperature) experiment (Also see Table 3.1). Weekly measurements of pH, measured by Durafet electrode.  Lines are values from each chamber.      59   Figure 3.5: Visualization of Calcofluor stain under black light. White arrows show location of calcofluor white stain established at beginning of experiment and distal tissue represents growth that occurred thereafter.  60  Table 3.1: Experimental carbonate chemistry conditions in specimen chambers. pH (total scale) is mean (n = 5 -6) ± S.E.M across all chambers (measured by Durafet probe, calibrated with spectrophotometric pH every week).  pCO2 and carbonate saturation state (calcite) were calculated in CO2calc using measured temperature, salinity, pH, and total alkalinity values.     61  3.4 Results 3.4.1 Species differences C. vancouveriensis and C. tuberculosum differed in their growth and biomechanical properties but not percent calcium carbonate after growth in control conditions in the lab (fronds from the temperature x pH experiment, 7.8 pH/13C treatment). C. tuberculosum grew slightly faster (0.097 ± 0.002 mm/day, Mean ± S.E.M throughout) than C. vancouveriensis (0.087 ± 0.003 mm/day, Figure 3.6 A, p = 0.02). There was no significant difference in the percentage of calcium carbonate in the two species (Figure 3.6 B, p = 0.08), as C. tuberculosum intergenicula were composed of 89.2 ± 0.2% calcium carbonate while C. vancouveriensis intergenicula were composed of 88.1 ± 0.5% calcium carbonate (Figure 3.6 B). Genicula of C. vancouveriensis were three times stronger and four times stiffer, but 25% less extensible than genicula of C. tuberculosum (Figure 3.6 C-E, p < 0.01 for breaking stress and breaking strain, p < 0.001 for modulus). The breaking stress of C. vancouveriensis genicula was 40.4 ± 5.8 MPa while the breaking stress of C. tuberculosum genicula was 13.3 ± 1.1 MPa. C. tuberculosum genicula extended 0.85 ± 0.07 mm/mm before breaking while C. vancouveriensis only extended 0.57 ± 0.03 mm/mm before breaking. C. vancouveriensis and C. tuberculosum had modulus values of 113.3 ± 15.5 MPa and 25.7 ± 3.3 MPa, respectively.   3.4.2 Single Factor: Temperature  For C. vancouveriensis, the growth rate in the 22C treatment was 4-5 times lower than for all other temperature treatments (Figure 3.7 A, Table 3.2, p < 0.0001). For C. tuberculosum, the growth rate in the 18C treatments was reduced by half as compared to the 12C and 15C treatments and no growth was found in the highest temperature of 22C  62  (Figure 3.7 B, Table 3.2, p < 0.0001). C. vancouveriensis fronds grew 0.081 ± 0.004 mm/day in the 12C, 15C, and 18C temperature treatments, but growth was greatly reduced (~89% reduction) in the highest temperature treatment (22C), down to 0.01 ± 0.0002 mm/day.  C. tuberculosum fronds grew 0.111 ± 0.003 mm/day in the 12C and 15C treatments, but grew 50% less in the two higher temperature treatments (18C and 22C): C. tuberculosum fronds grew 0.05 ± 0.01 mm/day in the 18C treatment and there was no evidence of growth in the 22C treatment. Additionally, many C. tuberculosum fronds in the 22C treatment bleached and lost pigment while this was not observed in fronds of C. vancouveriensis.   Temperature treatments did not affect the accretion of calcium carbonate of apical intergenicula of C. vancouveriensis (~87% CaCO3, Figure 3.7C, Table 3.2) or the dissolution of calcium carbonate (~90%, Figure 3.7 E, Table 3.2). Increased temperature also had no effect on the accretion of calcium carbonate in apical intergenicula of C. tuberculosum (~90%, Figure 3.7 D, Table 3.2) nor did it result in dissolution of tissue that was laid down before the experiment (~90%, Figure 3.7 F, Table 3.2).  Temperature treatments significantly affected the strength of C. vancouveriensis genicula (Figure 3.8 A, Table 3.2), such that the strongest genicula (35.6 ± 3.1 MPa) were recorded in fronds from the 15C and 18C temperature treatments. At 10C and 16C, strength values were 16.3 ± 3.1 MPa, approximately half as strong as genicula in the 15C and 18C temperature treatments.   There was no significant effect of temperature on the extensibility and modulus of C. vancouveriensis (Figure 3.8 B & C, Table 3.2). Breaking strains of C. vancouveriensis were 0.37 ± 0.05 mm/mm and modulus values were 110.2 ± 8.9 MPa.   63  3.4.3 Single Factor: pH  Reduced pH significantly slowed the growth of C. vancouveriensis (Figure 3.9 A, Table 3.3). For C. tuberculosum the only effect of pH was enhanced growth at 8.0 compared to all other pH treatments (Figure 3.9 B, Table 3.3). C. vancouveriensis fronds grew similarly (0.073 ± 0.003 mm/day) from pH 8 to pH 7.4 treatments, but had reduced growth in pH 7.2 (0.05 ± 0.003 mm/day) and pH 7.0 (0.03 ± 0.002 mm/day) treatments. Comparison of pH 8.0 and pH 7.0 showed a 63% reduction in growth. C. tuberculosum fronds grew significantly faster in pH 8 than all other pH treatments (Figure 3.9 B), which did not differ significantly from each other. Growth of C. tuberculosum fronds were 0.10 ± 0.003 mm/day in pH 8, and 0.074 mm/day ± 0.002 in pH 7.8-7.0. C. tuberculosum growth was reduced ~30% from 8.0 pH to 7.0 pH.  Decreased pH caused a reduction in percent calcium carbonate in C. vancouveriensis (Figure 3.9 C & E, Table 3.3), but not in C. tuberculosum (Figure 3.9 D & F, Table 3.3).  Additionally, there was no significant difference between growth that occurred prior to the experiment (old growth) and growth that occurred during the experiment (new growth) (Table 3.3). C. vancouveriensis fronds had a 0.12% reduction in calcium carbonate from pH 8 to pH 7. C. tuberculosum, on the other hand, only had a 0.04% reduction in calcium carbonate from pH 8 to pH 7. Reduced pH had no significant effects on the biomechanical properties of C. vancouveriensis (Figure 3.10 A, C & E, Table 3.3) or on C. tuberculosum (Figure 3.10 B, D, & F, Table 3.3).      64  3.4.4 Multi-factor: pH x temperature  Reduced pH and increased temperature affected the growth of both C. vancouveriensis (Figure 3.11 A, Table 3.4) and C. tuberculosum fronds (Figure 3.11 B, Table 3.4). There was an interactive effect of temperature and pH (Table 3.4) on growth of C. vancouveriensis fronds but only temperature affected the growth rate of C. tuberculosum fronds. In fronds of C. vancouveriensis, growth was increased under 16C as compared to 10C and 13C treatments (from 0.09 ± 0.003 to 0.10 ± 0.003 mm/day) under control pH conditions. Under reduced pH, C. vancouveriensis fronds grew 0.068 ± 0.002 mm/day, and temperature treatments had no effect on growth (Figure 3.11 A, Table 3.4). In fronds of C. tuberculosum, rising temperature increased growth rates (from 0.08 ± 0.002 mm/day in the 10C treatment to 0.12 ± 0.004 mm/day in the 16C treatment). The only effect of pH was documented in the 13C treatment, where growth was reduced to 0.08 ± 0.005 mm/day in low pH compared to 0.10 ± 0.002 mm/day in the control pH treatment (Figure 3.11 B). Reduced pH decreased the percentage of calcium carbonate in apical intergenicula of C. vancouveriensis at the highest temperature of 16C (Figure 3.11 C, Table 3.4). At this temperature, calcium carbonate was reduced by ~5% in the low pH as compared to control pH. At the control pH and 16C, calcification was slightly reduced as compared to the lower temperatures (but not significantly).  However, when pH was reduced to 7.3, there was a significant reduction in calcification at this low pH (Figure 3.11 C, Table 3.4). In fronds of C. tuberculosum, percent calcium carbonate was reduced slightly at 16C compared to 10C and 13C but there was no effect of pH for any of the three temperatures tested (Figure 3.11 D, Table 3.4).  65  Percent calcium carbonate of C. vancouveriensis intergenicula that occurred prior to the experiment (old growth) was affected by both increased temperature and reduced pH (Figure 3.11 E, Table 3.4). The effect of pH was strongest at 10C, at this temperature there was a slight but significant reduction in percent calcium carbonate due to pH. There was no reduction in calcification at the other two temperatures due to pH. C. tuberculosum calcification that occurred prior to the experiment was unaffected by temperature, pH, or the interaction between the two (Figure 3.11 F, Table 3.4).  There were also no significant effects of temperature or pH on the strength of genicula of C. vancouveriensis (Figure 3.12 A, Table 3.4) or C. tuberculosum (Figure 3.12 B, Table 3.4).   There was an interactive effect on the extensibility of genicula of C. vancouveriensis (Table 3.4, Figure 3.12 C). Extensibility of C. vancouveriensis genicula was unaffected by temperature but at 16C it increased at low pH (0.68 ± 0.06 mm/mm) compared to control pH (0.51 ± 0.04 mm/mm) (Figure 3.12 C, Table 3.4). There were no significant interactive effects of increased temperature and reduced pH on the extensibility of C. tuberculosum fronds (Figure 3.12 D, Table 3.4).   The trends seen in the modulus measurements of C. vancouveriensis genicula were similar, but opposite, to the trends in extensibility. There was a significant interaction between pH and temperature (Table 3.4, Figure 3.12 E). However, this effect was most pronounced at 16C, where reduced pH affected genicular stiffness of C. vancouveriensis (Figure 3.12 E), and resulted in a reduction in stiffness from 148 ± 19MPa (control pH) to 86 ± 12 (reduced pH). The combination of increased temperature and reduced pH did not affect the modulus of C. tuberculosum genicula (Figure 3.12 F, Table 3.4).  66    Figure 3.6: Growth, percent calcium carbonate and biomechanical properties of C. vancouveriensis and C. tuberculosum under control conditions for six weeks (Data from temperature x pH experiment; fronds grown at 7.8 pH and 13C).  (A) Growth (mm/day); (B) Percent calcium carbonate; (C) Strength (MPa); (D) Breaking strain (mm/mm);  (E) Modulus (MPa).  Bars are means ± S.E.M, n = 7-8, p-value on each panel indicates results of t-test comparing the two species.    67   Figure 3.7: Apical linear growth in mm/day and percent calcification of C. vancouveriensis and C. tuberculosum in single-factor temperature experiment (12C, 15C, 18C, 22C). (A) C. vancouveriensis growth under four temperature treatments (12C, 15C, 18C, 22C), n = 3; (B)  C. tuberculosum growth under four temperature treatments (12C, 15C, 18C, 22C),  n = 3;  (C) Percent calcium carbonate of C. vancouveriensis fronds grown during the experiment (new growth), n = 3; (D) Percent calcium carbonate of C. tuberculosum intergenicula grown during the experiment (new growth), n = 3;  (E) Percent calcium carbonate of C. vancouveriensis fronds grown prior to the experiment (old growth), n = 3; (F) Percent calcium carbonate of C. tuberculosum fronds grown prior to the experiment (old growth), n = 3. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).   68   Figure 3.8: Biomechanical properties of C. vancouveriensis genicula in single factor temperature experiment (12C, 15C, 18C, 22C). (A) Strength (MPa) of C. vancouveriensis fronds in 12C, 15C, 18C and 22C treatments, n = 3; (B) Extensibility (mm/mm) of C. vancouveriensis genicula in 12C, 15C, 18C and 22C treatments, n = 3;  (C) Modulus (MPa) of C. vancouveriensis genicula 12C, 15C, 18C and 22C treatments, n = 3. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).  69   Figure 3.9: Apical linear growth in mm/day and percent calcification of C. vancouveriensis and C. tuberculosum in single-factor pH experiment. (A) Apical linear growth (mm/day) of C. vancouveriensis fronds under pH treatments of 8.0, 7.8, 7.6, 7.4, 7.2, 7.0, n = 6; (B) Apical linear growth (mm/day) of C. tuberculosum fronds under pH treatments of 8.0, 7.8, 7.6, 7.4, 7.2, 7.0, n = 6; (C) Percent calcium carbonate of C. vancouveriensis intergenicula grown during the experiment (new growth), n = 6; (D) Percent calcium carbonate of C. tuberculosum intergenicula grown during the experiment (new growth), n = 6; (E) Percent calcium carbonate of C. vancouveriensis intergenicula grown prior to the experiment (old growth), n = 6; (F) Percent calcium carbonate of C. tuberculosum intergenicula prior to the experiment (old growth, n = 6. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).   70   Figure 3.10: Biomechanical properties of C. vancouveriensis and C. tuberculosum genicula in the single-factor pH experiment (8.0, 7.8, 7.6, 7.4, 7.2, 7.0). (A) Strength (MPa) of genicula of C. vancouveriensis under pH treatments, n = 3 – 6; (B) Strength of genicula of C. tuberculosum under pH treatments, n =5 – 6; (C) Extensibility (mm/mm) of C. vancouveriensis genicula under pH treatments, n = 3 – 6; (D) Extensibility (mm/mm) of genicula of C. tuberculosum under pH treatments, n = 5 – 6;  (E) Modulus (MPa) of genicula of C. vancouveriensis under pH treatments, n = 3 – 6; (F) Modulus (MPa) of genicula of C. tuberculosum under pH treatments, n = 5 – 6. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).   71   Figure 3.11: Apical linear growth (mm/day) and percent calcification of C. vancouveriensis and C. tuberculosum in multi-factor temperature (10C, 13C, 16C) by pH (7.8, 7.3) experiment. Dark bars are 7.8 pH treatment and white bars are 7.3 pH treatment. (A) Apical linear growth in mm/day of C. vancouveriensis fronds in multi-factor temperature by pH experiment, n = 8; (B) Apical linear growth in mm/day of C. tuberculosum fronds in multi-factor pH experiment, n = 8; (C) Percent calcium carbonate of C. vancouveriensis intergenicula grown during the experiment (new growth), n = 8; (D) Percent calcium carbonate of C. tuberculosum intergenicula grown during the experiment (new growth), n = 8; (E) Percent calcium carbonate of C. vancouveriensis intergenicula grown prior to the experiment (old growth), n = 8; (F) Percent calcium carbonate of C. tuberculosum intergenicula grown prior to the experiment (old growth), n = 8. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).    72    Figure 3.12: Biomechanical properties of C. vancouveriensis and C. tuberculosum genicula in multi-factor temperature (10C, 13C, 16C) by pH (7.8, 7.3) experiment. Dark bars are 7.8 pH treatment and white bars are 7.3 pH treatment. (A) Strength (MPa) of C. vancouveriensis genicula in the multi-factor temperature by pH experiment, n = 8; (B) Strength (MPa) of C. tuberculosum genicula in multi-factor temperature by pH experiment, n = 7 – 8; (C) Extensibility (mm/mm) of C. vancouveriensis genicula in multi-factor temperature by pH experiment, n = 8; (D) Extensibility (mm/mm) of C. tuberculosum genicula in multi-factor temperature by pH experiment, n = 7 – 8; (E) Modulus of C. vancouveriensis genicula in multi-factor temperature by pH experiment, n = 8; (F) Modulus of C. tuberculosum genicula in multi-factor temperature by pH experiment, n = 7 – 8. In all panels, bars are ± S.E.M and letters indicate significant differences between experimental treatments (Tukey’s HSD posthoc tests).    73   Table 3.2: Results of linear mixed effect model on single-factor (temperature) experiment on fronds of C. vancouveriensis and C. tuberculosum. Asterisk represents significant effect at alpha < 0.05 level.    74    Table 3.3: Results of linear mixed effect model on single-factor (pH) experiment on fronds of C. vancouveriensis and C. tuberculosum. Asterisk represents significant effect at alpha < 0.05 level.   75  Table 3.4: Results of linear mixed effect model on multi-factor (temperature x pH) experiment on fronds of C. vancouveriensis and C. tuberculosum. Asterisk represents significant effect at alpha < 0.05 level.     76  3.5 Discussion  Results of this study demonstrate that responses of organisms to climatic stressors are species-specific. Both species of coralline algae tested were collected from similar habitats, are closely related taxonomically, and are the same morphotype (articulated coralline algae), yet, these algae had very different responses to future climate conditions and also demonstrated different biomechanical properties under control conditions. Several other studies have demonstrated that effects of warming and acidification can vary between closely related species (Noisette et al. 2013), between strains of the same species (Langer et al. 2009), and among different habitats of the same species (Comeau et al. 2014). Therefore, responses to climate change are hard to predict, and caution should be exercised when making broad predictions about the effect of climate change on macroalgae based on taxonomic group or habitat.  It was hypothesized that mid-intertidal Corallina would be more resistant to high temperature and low pH than low-intertidal Calliarthron. However, the responses of the two species were contrary to predictions. C. vancouveriensis was found to be more sensitive to these stressors than C. tuberculosum, perhaps highlighting previous conclusions that intertidal organisms often live dangerously close to their physiological limits (Stillman & Somero 2000). However, specimens of C. tuberculosum in this study were collected from an intertidal tidepool, where conditions are highly variable (See Chapter 4).  Therefore, it is possible that since this population is exposed to widely fluctuating conditions in tidepools, and that it was better adapted to increases in temperature and decreases in pH than its subtidal counterparts would be.   77  Previous studies have shown a negative effect of global warming and ocean acidification on recruitment (Kuffner et al. 2008, Roleda et al. 2015), growth (Jokiel et al. 2008, Kram et al. 2016), and calcification (Gao et al. 1993, Semesi et al. 2009, Martin et al. 2013) of coralline algae. It was hypothesized that a typical dose response curve would result for rates of growth, calcification and material properties, such that increasing temperature would result in positive changes in growth, calcification, and biomechanical properties up to a point, after which reductions were expected. However, the strongest negative effect of increased temperature was documented in the growth assay only, with calcification and material properties being relatively unaffected. Growth rates were similar under lower temperatures, but sharply decreased when a thermal maximum was reached. This thermal maximum was different between the two study species. In C. vancouveriensis, the growth rate in the highest temperature treatment (22C) was 4-5 times lower than for all other temperature treatments, and in C. tuberculosum, the growth rate in the 18C treatments was reduced by half as compared to the 12C and 15C treatments and no growth was found in the highest temperature of 22C. The results for C. vancouveriensis in this study are similar to results found for Corallina officinalis (Colthart & Johansen 1973), where C. officinalis was found to have a thermal optimum between 12C and 18C.  Temperature had no significant effect on the percent calcium carbonate of C. vancouveriensis and C. tuberculosum fronds in either accretion or dissolution. This study, however, documented the final amount of calcium carbonate, and not the rate of calcification as is measured in many studies (Gao et al. 1993, Semesi et al. 2009, Martin et al. 2013).  Martin et al. (2013) showed that maximum calcification rates were affected by temperature  78  in the coralline L. cabiochae, but this effect depended on pCO2 level and season. Since corallines deposit high magnesium calcite, their calcification is hypothesized to be susceptible to temperature (Chave 1954). The substitution of magnesium into the calcite skeleton is an endothermic process, and therefore, more magnesium is incorporated into the calcium carbonate skeleton at higher temperatures (Nannini et al. 2015). This higher amount of magnesium in the calcite skeleton would make these corallines more susceptible to dissolution. Although this study documented no differences in the final percentage of calcium carbonate, it is possible that they had reduced calcification rates but also decreased dissolution, maintaining the same amount of calcium carbonate. Support for this hypothesis is provided by the slower growth rates at higher temperatures. It is also possible that the calcium carbonate had a higher percentage of magnesium incorporated into the calcium carbonate skeleton, but this was not explicitly tested.  It was hypothesized that growth, calcification, and material properties of both species would be negatively affected by reduced pH. This hypothesis was supported in that reduced pH significantly slowed the growth and reduced the amount of calcium carbonate of C. vancouveriensis. However, the hypothesis that Corallina would be more resistant than Calliarthron to reduced pH was not supported. Instead, C. tuberculosum was more resistant to reduced pH than C. vancouveriensis. Reduced pH significantly slowed the growth and reduced the amount of calcium carbonate of C. vancouveriensis, and this effect was only documented at very low pH levels of 7.2 and 7.0 (for growth) and 7.0 (for percent calcium carbonate). The only effect on C. tuberculosum was enhanced growth at 8.0 as compared to all other pH treatments and there was no pH effect on percent calcium carbonate. In a recent study by Kram et al. (2016), C. vancouveriensis growth rates were significantly negatively  79  affected by elevated pCO2, while other species of coralline algae were unaffected. Kram et al. (2016) document this decrease in growth at an elevated pCO2 level of approximately 900 µatm, but in my study, reductions in growth were not documented until the pCO2 level was above 1700 µatm (7.2 pH). Although the two studies used the same species, the level at which a response was documented was very different. One likely reason for this difference between our studies could be the habitat in which the corallines were collected and previously adapted. In the study by Kram et al. (2016), specimens were collected either subtidally or from the low intertidal zone while in the current study, specimens were collected from the low to mid-intertidal zone.  It has been shown that organisms from highly fluctuating environments are better able to adapt to climatic changes (Schaum & Collins 2014), therefore species living in such environments are more likely to adapt better to climatic changes.  The specimens from this study were all collected from tidepool environments, which are highly variable in temperature and carbonate chemistry (See Chapter 4). If these corallines were already adapted to extreme variability in pH and other carbonate chemistry conditions, it is possible that strong effects due to ocean acidification were not documented due to their previous adaptation to highly variable conditions.  It’s possible, however, that if the same species of corallines were collected from stable habitats, such as low intertidal or subtidal areas, a stronger effect due to reduced pH and increased temperature may have been documented. Future research could address differences in species responses due to environmental variability. The hypothesis that C. vancouveriensis growth rates will be positively affected by increasing temperatures regardless of pH was not supported. When temperature and pH were combined, growth rates of C. vancouveriensis fronds increased with temperature under  80  control pH, but growth was reduced at all temperatures under low pH. The hypothesis that C. tuberculosum growth rates will be positively affected by increasing temperatures regardless of pH also was only partially supported. In fronds of C. tuberculosum, rising temperature increased growth rates, but the only effect of pH was documented in the 13C treatment. The hypothesis that increased temperature will not have an effect on calcification, and that reduced pH will have a strong effect on calcification was not supported. In regards to calcification, pH slightly decreased the percentage of calcium carbonate of C. vancouveriensis, but only at the highest temperature treatment.  In fronds of C. tuberculosum, percent calcium carbonate was reduced slightly at 16C compared to the 10C and 13C temperature treatments but there was no effect of pH for any of the three temperatures tested. Previous studies have documented an increased negative effect on coralline algae with warming and elevated pCO2 (Martin & Gattuso 2009, Diaz-Pulido et al. 2012). This trend was not found in this study, and it is hypothesized that the increase in temperature ameliorated the negative effect due to reduced pH.   The hypothesis that increased temperature will not affect biomechanical properties, while reduced pH was expected to produce weaker, more extensible, and less stiff fronds was not supported. Overall, the effects of temperature on biomechanical properties of C. vancouveriensis were slight. Temperature did affect the strength of C. vancouveriensis fronds, such that the strongest fronds were recorded in the 15C and 18C treatments, as compared to the 12C and 22C treatments. However, temperature did not significantly affect any of the other biomechanical properties (extensibility or modulus) of C. vancouveriensis. Reduced pH had no significant effects on any of the biomechanical properties of C.  81  vancouveriensis or on C. tuberculosum. And when temperature and pH were combined, there were no effects on the strength of either C. vancouveriensis or C. tuberculosum. There was an interactive effect on the extensibility of genicula of C. vancouveriensis, such that extensibility increased under low pH at 16C. The trends seen in the modulus measurements of C. vancouveriensis, were similar, but opposite, to the trends in extensibility, such that genicular stiffness of C. vancouveriensis was reduced under low pH and 16C. No effects due to either temperature or pH were found in fronds of C. tuberculosum. To the author’s knowledge, this is the first study to assess whether algal biomechanical properties change with abiotic factors, such as temperature and pH. Future studies should start to incorporate this assay into the suite of responses measured in climate change studies.   It is well documented that physiological trade-offs exist (Carrington 2002, Wood et al. 2008, Ragazzola et al. 2013, McCoy & Ragazzola 2014) whereby species are able to maintain certain biological processes (here, calcification and biomechanical properties) while other processes are affected (growth). Organisms living in stressful conditions, such as high temperature and low pH, may devote more energy to maintain materials, leaving less energy for growth and reproduction. Results shown here suggest that although these two coralline algae maintain the quality of the material that they produce, they grow at a slower rate. Similarly, Cornwall et al. (2013) found that the coralline, Arthrocardia corymbosa, had reductions in growth with reduced pH, but that other metabolic processes such as photosynthesis, recruitment and elemental composition were unaffected. The conservation of material quality in corallines is contrary to what was seen in the mussel Mytilus trossulus (O’Donnell et al. 2013, Newcomb 2015), perhaps because the cells in corallines are regulating cell wall composition, whereas byssal threads are deposited externally and then  82  not further regulated by the organism. This study is one of the first to look at the effects of environmental factors on biomechanical properties of seaweed; more studies are needed to determine if corallines, or algae, in general will be resistant to climate change by maintaining the quality of their tissues.   In conclusion, these two species of coralline algae were found to respond differently to climatic stressors of increasing temperature and decreasing pH; C. tuberculosum was found to be less sensitive to these stressors than C. vancouveriensis. Overall, the main effects were seen in the reduction in growth. Other parameters tested were only slightly affected, if at all. C. vancouveriensis had an 89% reduction in growth over the temperature range studied, and a 63% decrease over the pH range studied. C. tuberculosum had a 50% reduction in growth from 12-18C, no growth at 22C, and a 30% reduction over the pH range studied. These reductions in growth could have several ecological implications. If corallines start to grow slower, this may have implications for competitive ability (McCoy & Pfister 2014), such that they may not be able to compete for space as well under climatic stressors as compared to ambient conditions. Additionally, if these species are re-allocating energy to maintain material properties and amount of calcium carbonate, it is likely that other processes not tested here will also be affected. One likely scenario is a reduction in the amount of energy allocated towards reproduction. If these algae reduce reproductive output to maintain tissue integrity, this could have far reaching effects on species abundance and composition along our shores.  Global warming and ocean acidification are expected to have an effect on species distributions (Harley et al. 2012, Bijma et al. 2013) and may result in a shift in abundances and geographical distributions (Dez et al. 2012). For example, Brodie et al. (2014)  83  documented different thermal optimums for different species of calcifying algae, and so, their abundances and distributions may already be changing due to warming and acidification. Future studies should assess the combined effects of warming and ocean acidification on additional species of coralline algae from variable habitats to tease apart where the strongest effects due to climate change will be. Additionally, long term data sets on the abundance and distribution of corallines are needed to determine if climatic factors are affecting species abundances and distributions.     84  4 Seasonal analysis of tidepool chemistry and putative effects on Corallina physiology 4.1 Synopsis Fluctuations in water chemistry that occur in tidepools are extreme. For example, fluctuations in pH and pCO2 that occur over one low tide cycle can be as great as the total documented changes in these parameters from pre-industrial times to those predicted for the year 2100. Yet coralline algae, which are thought to be highly susceptible to ocean acidification, often thrive in these habitats. I investigated the effects of carbonate chemistry and temperature in tidepools on C. vancouveriensis growth, calcification, and biomechanical properties. I found that, despite extreme changes in chemistry during low tides, this species of coralline algae was still able to grow all year long. Fronds of C. vancouveriensis also maintained calcium carbonate deposition and had no change in material properties due to differences in chemistry between the seasons. Results suggest that this species of coralline algae is able to maintain tissue quality and integrity despite the extreme chemical conditions in tidepools.  4.2 Introduction Organisms living in the intertidal zone must contend with both marine and terrestrial conditions as the tide rises and falls, making the intertidal zone of temperate rocky shores one of the most stressful habitats on Earth (Denny 2006). Intertidal organisms that live in tidepools, however, may be able to avoid certain low tide stresses. For example, in tidepools,  85  organisms avoid desiccation (Dethier 1980) and experience more muted variation in temperature and light regimes (Metaxas & Scheibling 1993) than intertidal organisms that occur out of tidepools. Although tidepool organisms are somewhat buffered by low tide stresses by being immersed, they still experience generally large fluctuations in water chemistry such as pH, temperature, and dissolved oxygen, pCO2 that may be stressful (Daniel & Boyden 1975, Truchot & Duhamel-Jouve 1980, Bjork et al. 2004). For example, the changes that occur over the course of one low tide can exceed changes in carbonate chemistry predicted from pre-industrial times to predicted values for the year 2100. As we begin to explore how marine organisms may fare under ocean acidification conditions in the future, tidepools may provide a tractable model system in which to study the responses of seaweeds to ocean acidification.  Coralline algae are found throughout intertidal habitats in the NE Pacific (Abbott & Hollenberg 1976) where they are an especially important component of the intertidal flora; they provide key ecological functions by cementing carbonate fragments into reef structures (Adey 1998, Bjork et al. 1995), and provide refugia for invertebrates (Coull & Wells 1983, Dommasnes 1969, Gibbons 1988). Corallines are predicted to be especially susceptible to low pH (Kuffner et al. 2008, Büdenbender et al. 2011, Kroeker et al. 2013) due to the fact that they calcify and possess the most soluble form of calcium carbonate (high-magnesium calcite). However, Guenther & Martone (2014) found that C. vancouveriensis was able to resist several low tide stressors and was able to quickly recover from desiccation and increased temperature once the tide returned. Although C. vancouveriensis is commonly found growing in the intertidal zone in tidepools, it is one of the only corallines that can survive emersion during low tide (Abbott & Hollenberg 1976).   86  Conditions in tidepools fluctuate widely, reflecting differences that occur during alternations of flooding and isolation. While isolated at low tide, biological processes of photosynthesis, respiration, and calcification all affect water chemistry within the tidepool (Daniel & Boyden 1975, Morris & Taylor 1983, Bjork et al. 2004). Additionally, tidepools that are isolated at night experience different chemical changes from tidepools that are isolated during the day. For example, when tidepools are isolated at night, respiration primarily drives chemical changes: organisms release CO2 and use oxygen, which raises the level of pCO2, while decreasing pH and dissolved oxygen within the tidepool. When tidepools are isolated during the day, photosynthesis overrides respiration: algae in the tidepool use CO2 and bicarbonate for photosynthesis and release oxygen, thereby raising the pH and dissolved oxygen levels while reducing the pCO2 within the tidepool. If oxygen levels in tidepools get sufficiently high, photorespiration processes may override photosynthetic processes due to the enzyme RuBisCO’s affinity for oxygen over carbon dioxide. If oxygen levels get sufficiently high, RuBisCO will use oxygen rather than carbon dioxide and this leads to photorespiration and an inhibition of photosynthesis (Burris 1977, Lloyd et al. 1977).   Although the changes in tidepool total alkalinity are somewhat less studied, several authors document changes in total alkalinity as tidepools are isolated at low tide (Truchot & Duhamel-Jouve 1980, Morris & Taylor 1983, Moulin et al. 2011). It is generally accepted that changes in total alkalinity are driven by calcification and dissolution of calcium carbonate (Smith & Key 1975, Chisholm & Gattuso 1991), and not by photosynthesis and respiration (Sournia 1982). An increase in total alkalinity indicates dissolution and a decrease in total alkalinity indicates calcification (Chisholm & Gattuso 1991). However, since  87  photosynthesis and respiration are two processes that control pH, rates of calcification are tightly coupled to rates of photosynthesis and respiration (Smith & Roth 1979, Borowitzka 1981, Gao et al. 1993, De Beer & Larkum 2001, Martin et al. 2013). Photosynthetic processes use CO2, thereby raising the pH, and this increases the CaCO3 saturation state, favoring calcification (Gao et al. 1993) while respiration releases CO2 and lowers the pH, and favors dissolution (De Beer & Larkum 2001). Physical factors in tidepools have been shown to vary widely over the course of low tides due to biological processes, with daily fluctuations in oxygen saturation, alkalinity, and pH (Pyefinch 1943, McGregor 1965, Daniel & Boyden 1975, Morris & Taylor 1983, Moulin et al. 2011). For example, higher oxygen values have been recorded in tidepools isolated during the day-time due to photosynthesis and lower values during night-time (Huggett & Griffiths 1986). Daily fluctuations in pO2 and pCO2 have been documented in tidepools, and these fluctuations depended on season (Morris & Taylor 1983), height of the tidepool along the intertidal gradient (Daniel & Boyden 1975), and pool depth (Goss-Custard et al. 1979).   The composition and abundance of algae growing in tidepools correlates with pool height, topography, and shading by surrounding rock outcrops (Johnson & Skutch 1928). Additionally, the number of species present in tidepools relates to tidepool depth and volume, such that deeper tidepools have more plant, algal and invertebrate species (Droop 1953, Pajunen 1977, Ranta 1982, Fairweather & Underwood 1991). Other studies have shown that pool area, depth or volume determine the biomass, species number and abundance of species in tidepools (Marsh et al. 1978, Bennett & Griffiths 1984, Mgaya 1992).   88  In this study, I aim to document whether growth rates, percent calcification and biomechanics of an abundant intertidal calcifying alga, C. vancouveriensis, correlated with tidepool size, as variation in pH was expected to depend upon tidepool volume. In tidepools, the change in pH over the course of the tide is primarily controlled by biological processes of photosynthesis and respiration, and so, it was expected that larger tidepools would have a smaller change in pH over the course of the low tide. Another objective of this research is to document the chemical variation (temperature, pH, pCO2, total alkalinity) that occurs in these pools during low-tide isolations during day-time (summer) and night-time emersions (winter) and whether these chemical changes could be correlated to growth, biomechanics, and calcification.      4.2.1 Hypotheses 1. Growth rates, percent calcification, and material properties of C. vancouveriensis will vary with season such that faster growth rates, greater calcification and stronger and more extensible materials will occur in the summer.   2. There will be differences in growth, calcification, and material properties of C.vancouveriensis across tidepool size within each season. The fastest growth rates, most calcification, and strongest materials are expected to occur in the largest tidepool, due to muted shifts in pH at low tide. 3. In the summer, pH and temperature will increase, pCO2 will decrease, and total alkalinity will remain the same during low tide.  The opposite trends are expected in the winter.    89  4.3 Methods 4.3.1 Field Site and Specimen Preparation Research was conducted at Dead Man Bay (4830’48.09”N, 1238’49.38”W), on San Juan Island, using the Ocean Acidification Environmental Laboratory at Friday Harbor Laboratories. I measured water chemistry (temperature, salinity, dissolved oxygen, pH, and total alkalinity) in three differently sized tidepools dominated by Corallina vancouveriensis. These tidepools were at the same approximate tide height (+0.3-0.6 m above MLLW), and were located within 50 m of each other. However, the medium sized-tidepool had some shading effects due to near-by rock out-croppings.  In each tidepool, C. vancouveriensis fronds were stained with Calcofluor White so that growth could be measured at the end of the month (See Methods in Section 4.3.3 and Martone 2010). Stained C. vancouveriensis fronds were collected at the end of the month to measure growth (See Section 4.3.3), percent calcium carbonate (See Section 4.3.4) and material properties (See section 4.3.5).    In each tidepool, two ibuttons (Maxim Integrated products, San Jose, CA) were secured to the tidepool amongst the coralline algae. The ibuttons were waterproofed with parafilm, and two fingers of rubber gloves.  First, the ibutton was wrapped in parafilm, then the ibutton was placed within the finger of a rubber glove and tied, and this was then placed within another finger of a rubber glove and tied again.  This waterproofed ibutton was then placed in a falcon tube which had holes drilled in it (to allow water to flow through). The falcon tube was then secured to rock in the tidepool using Z-spar marine adhesive.  In this manner, waterproofed ibuttons could be collected and replaced throughout the experiment.   90  Tidepool volume and biomass of corallines was estimated for each tidepool. Water was pumped out of tidepools and measured in 5 gallon buckets. Density of coralline cover was calculated with a 25 cm by 25 cm quadrat with 5 cm by 5 cm subdivided squares. The number of squares filled with coralline algae was estimated. To estimate biomass of corallines in each pool, corallines within one small square were harvested, and dried in oven (68C).  From this, the density of corallines (grams dry weight/L seawater) in each tidepool was calculated.    4.3.2 Water sample collection and analyses Water samples were collected throughout the low tide (1 hr. isolation, 3 hr. isolation, and end of tide) from each tidepool and from the ambient Puget Sound water (Haro Strait, WA), hereafter referred to as sound water (Figure 4.1). Water samples were analyzed for carbonate chemistry and oxygen parameters four to five times per month in both the winter and in the summer (January and August). Seawater samples for pH and total alkalinity were collected in 12 oz. glass bottles, and poisoned with 200 L HgCl2. Samples were analyzed within one week of collection. Carbonate chemistry analyses of seawater samples were completed in the analytical chemistry lab in the Ocean Acidification Environmental Laboratory at Friday Harbor Labs. Spectrophotometric pH was measured using m-cresol dye with an Ocean Optics USB 2000 spectrophotometer, according to the methods in Dickson et al. (2007). Certified reference materials (CRM, Dickson Laboratory) were used to verify the accuracy of spectrophotometric measurements. Total alkalinity was determined using an endpoint titration method with a Mettler-Toledo DL115 titrator and measurements were calibrated with certified reference materials (CRM, Dickson Laboratory). pCO2 was  91  calculated using the program, CO2calc (version 1.3.0, Hansen et al. 2010), using the measured values of pH, total alkalinity, temperature and salinity of each sample. The following CO2calc constants were used: CO2 constants (K1, K2 from Mehrbach et al.1973), KHSO4 (Dickson 1990), pH scale (total scale (mol/kg-SW), Total Boron (Lee et al. 2010), and Air-sea flux (Wanninkhof 1992). Dissolved oxygen samples were collected in volume-calibrated 130 mL DO flasks, and immediately fixed with 1 mL manganous chloride solution followed by 1 mL of alkaline sodium hydroxide-sodium iodide reagent, as per the Carpenter method (See Codispoti 1988 for detailed methods).  Dissolved oxygen was determined by endpoint titration with a Metrohm 665 dosimat with a 5 mL burette.  pH and pCO2 data from all tidepools during the summer and winter were compared to predicted values for 2100 and pre-industrial times. The average pH of coastal seawater during sample months was 7.80 ± 0.03, and pH is predicted to decrease by 0.2-0.3 (Feely et al. 2004, Orr et al. 2005, International Panel on Climate Change 2013), so I predicted an approximate value of 7.5 pH for San Juan waters in 2100 (Figure 4.4). For pre-industrial values, I used a value of 8.0 pH, since pH is predicted to have declined 0.1 units since pre-industrial times (Feely et al. 2004, Orr et al. 2005, International Panel on Climate Change 2013).   pCO2 is predicted to increase by 600 units (IPCC predictions from 400-1000 for 2000-2100). The calculated average sound pCO2 measured in this study was 721 ± 42 atm, so I predicted a value of ~1300 atm for San Juan waters in 2100. I used a pre-industrial pCO2 value of 280 atm (Feely et al. 2009).      92  4.3.3 Linear Growth Assay Linear growth was assessed using the Calcofluor White method described in Martone (2010). Briefly, at the beginning of each field month (January or August), approximately 10 plants of C. vancouveriensis were stained with a 0.05% solution of Calcofluor white (Sigma-Aldrich, Fluorescent Brightener 28) for approximately 1 hour. Small ziploc bags were used (bottoms off cut off) and the bag was secured around the base of the coralline clump using zip-ties. The stain was then poured into the ziploc bag and allowed to set for approximately 30 minutes. The location of stained plants in the tidepool was marked with Z-spar marine epoxy. At the end of the month, the stained fronds of C. vancouveriensis were collected, and the stained tissue (i.e. the previous position of the apical meristem) was visualized by exposing fronds to black light (Sky City International, 20W) and taking long exposure photographs (~10-13 sec.). Photographs were then analyzed for linear growth of newly deposited coralline tissue in ImageJ by measuring the length from the Calcofluor stain mark to the tip of the coralline. Approximately 5-10 tips from each specimen of C. vancouveriensis was measured, divided by the number of days since the stain was applied, and averaged to yield an average daily growth rate over the course of the month for each plant. These values were then averaged across plants to calculate the average growth (mm/day) of C. vancouveriensis fronds in each tidepool.  4.3.4 Calcium Carbonate Assay  The amount of calcium carbonate in calcified tissues (intergenicula) was determined by decalcifying segments in 1N hydrochloric acid (HCl). Apical intergenicula that had  93  formed during the experimental month were collected, and approximately 10-30 apical intergenicula per plant were harvested for each measurement. Intergenicular segments were first dried (68°C overnight) and weighed (g), then de-calcified in 1N HCl for at least 12 hours, and then rinsed with distilled water, dried, and re-weighed. Percent calcium carbonate was calculated as follows:    		  						    4.3.5 Biomechanical Assays  Biomechanical properties (breaking stress, breaking strain, and modulus) of uncalcified tissues (genicula) were measured in tension using an Instron 5565 tensometer fitted with a temperature controlled water bath (12C). Measurements of genicular cross-sectional area and genicular length were used to standardize force and length measurements from the Instron. Approximately 10-20 genicula of C. vancouveriensis were tested at the same time (Frond: 10-5 cm; genicular material: 2.5-7 mm), apical sections of fronds were mounted into the grips with thin foam (2-3mm) and fine sandpaper to reduce slippage (See Chapter 3, Figure 3.2 A). Tensile tests then stretched fronds until one geniculum failed. The cross-sectional area of the genicula that broke was measured under a dissecting microscope (40X), and a neighboring genicula was measured for length in the same manner. This number was multiplied by the number of genicula between the Instron grips to yield the total starting length of genicula pulled. The temperature controlled water bath was then raised and the test started.    94   The pressure to the grips was ~40 psi and the tensometer pulled the specimen at 0.2 mm/sec until failure. The Instron provided extension and load values, and stress-strain curves were constructed from these values. Strength, or breaking stress, of the material was defined as the amount of force applied at breakage, divided by cross sectional area of the geniculum. Extensibility, or breaking strain, is the maximum extensibility of the genicula at breakage, this change in length is divided by the initial length of the genicula. Stiffness, or tensile modulus, of the genicula was quantified using the initial slope of the stress-strain curve. Breaking stress, breaking strain, and modulus were measured for each plant from each treatment.   4.3.6 Statistical Analyses  Chemistry data was analyzed with two-way ANOVAs in R (The R Foundation for Statistical Computing, version 3.1.2).  First, a two-way ANOVA was performed between the small, medium, and large tidepool to find any differences between the tidepools in pH, total alkalinity, dissolved oxygen and pCO2.  Isolation time and tidepool size were fixed factors, and two separate ANOVAs were run, one for each season.  Since no effect of tidepool size was found in either ANOVA, data for the tidepools was pooled. A second two-way ANOVA was then run between tidepool data and sound water data, with Isolation time (1 hr., 3 hr., or end of tide) and Type (Tidepool or Sound water chemistry) for each season individually to determine differences in pH, total alkalinity, dissolved oxygen and pCO2.  Additionally, a one-way ANOVA was performed between sound water and tidepool water at the end of tide, for each season individually.   95  To analyze growth data, percent calcium carbonate of apical intergenicula and biomechanical properties, a two way ANOVA on growth data was performed, with season and tidepool size as fixed factors. For all statistical tests, marginal fitting of terms was used.     96    Figure 4.1: Photographs of studied tidepools.  Small tidepool was 0.75 meters long x 0.45 meters wide x 0.15 meters deep.  Medium tidepool was 1.5 meters long x 0.80 meters wide x 0.15 meters deep. Large tidepool was 3.7 meters long x 1.10 meters wide x 0.30 meters deep. Red line indicates approximately 0.5 meters in length. Red star indicates position of water sampling for sound water samples (Haro Strait, WA).    97  4.4  Results 4.4.1 Tidepool size and density of coralline algae The small tidepool was 0.75 meters long x 0.45 meters wide x 0.15 meters deep and had approximately 19 L of seawater and an approximate density of 4.6 g/L of coralline algae.  The medium tidepool was 1.5 meters long x 0.80 meters wide x 0.15 meters deep and had approximately 66 L of seawater, and an approximate density of 1.7 g/L of coralline algae. The large tidepool was 3.7 meters long x 1.10 meters wide x 0.30 meters deep and had approximately 78 L of seawater, and an approximate density of 1.7 g/L of coralline algae.   4.4.2 Temperature Tidepool temperatures were highly variable during periods of low tide. During the winter, when low tides occur at night, tidepools either got cooler or warmer than sound seawater. Temperatures got approximately 4C colder than ambient and 3C warmer than ambient and ranged from ~4C to ~14C (Figure 4.2 A-C). Winter tidepool temperatures were less variable than summer tidepool temperatures (Figure 4.2 A-C). In the summer, tidepools were always warmer than ambient seawater (Figure 4.2 D-F). In the summer months, low tide occurs during the day, and so, tidepool temperatures always increased relative to sound water. At times, tidepools heated to approximately 30C, nearly 20C warmer than ambient temperatures of sound water.  4.4.3 pH  On average, pH decreased in tidepools during night-time low tide from 7.9 to 7.5 (Figure 4.3 A) in the winter, and increased in the summer from 7.6 to 8.4 (Figure 4.3 B). The  98  average pH of sound water was 7.9 in the winter and 7.7 in the summer. The magnitude of these changes over the course of a low tide is similar to the changes in pH predicted to occur from now to the year 2100 (see Figure 4.5). There was no significant effect of isolation time on pH during winter low tides (Table 4.1, p = 0.06), but there was a significant effect of isolation time on pH during summer low tides (Table 4.1, p = 0.03). In both seasons, there was no significant effect of tidepool size on pH (Table 4.1, Winter: p = 0.56, Summer: p = 0.95). When tidepool data were pooled, no significant difference was found between sound seawater pH and tidepool seawater pH (Table 4.2, Winter: p = 0.30; Summer: p = 0.39).  When looking only at the end of the tide, there were significant differences in pH between tidepools and sound (Table 4.3, Winter: p = 0.03; Summer: p = 0.01). At the end of the tide, pH was ~0.1 units below that of the sound water in the winter. In the summer, the pH of the tidepools at the end of the tide was approximately 0.5 above that of the sound water (Figure 4.4).  4.4.4 Total Alkalinity  There was no significant effect of tidepool size on the range change in total alkalinity in either season (Figure 4.3 C & D, Table 4.1). When total alkalinity data for the three tidepools was pooled, there was a significant interaction between isolation time and type (Table 4.2, p = 0.009) in the winter, when the total alkalinity of the sound was relatively constant over time but there was an effect of isolation time on tidepool total alkalinity. However, there were no significant effects of isolation time or type (sound water v. tidepool water) found in the summer (Table 4.2, p = 0.09). At the end of the tide, there was a significant difference between the tidepools and the sound water (Figure 4.4, Table 4.3,  99  Winter: p = 0.03; Summer: p = 0.01). In the winter, total alkalinity in the pools increased approximately 150 units over the course of the tide, as compared to sound water (Figure 4.4 C). In the summer, total alkalinity decreased approximately 350 units over the course of the tide as compared to sound water (Figure 4.4 D).    4.4.5 Dissolved Oxygen During winter night-time low tides, dissolved oxygen decreased over the course of the tide (Figure 4.3 E, Table 4.1, p = 0.007), but there was no effect of tidepool size (Table 4.1, p = 0.60). There was a significant difference between the tidepools (pooled data) and the sound at the end of the low tide, tidepools had approximately 4 mg/L less oxygen than sound water (Figure 4.4 E, Table 4.3, p < 0.0001).   During summer day-time low tides, there was a significant effect of isolation time on dissolved oxygen levels (Table 4.1, p = 0.01) and dissolved oxygen levels increased over the course of the tide (Figure 4.3 F). Tidepools had approximately 13 mg/L more oxygen than sound water (Figure 4.4 F) by the end of the low tide. When tidepool data was pooled, there was a significant interaction between isolation time and type (Table 4.2, p = 0.01), due to sound water being fairly constant in oxygen levels over the course of sampling. At the end of the tide, there were highly significant differences in dissolved oxygen between the tidepools and the sound (Table 4.3, p < 0.0001).   4.4.6 pCO2 During winter night-time low tides, pCO2 values increased over the course of the tide in tidepools while sound water remained relatively constant (Figure 4.3 G), and on average  100  these values were approximately 600 units above that of sound water (Figure 4.4 G). There was a significant effect of isolation time (Table 4.1, p = 0.02) but not tidepool size (Table 4.1, p = 0.57) on levels of pCO2. When data from the tidepools were combined, there was a significant interaction between isolation time and type (Table 4.2, p = 0.04), indicating that tidepools had significantly more pCO2 than tidepools. At the end of the low tide, tidepools had significantly more pCO2 than sound water (Table 4.3, p = 0.01) During summer day-time low tides, pCO2 levels in tidepools were much higher than sound water at the beginning of the low tide (Figure 4.3 H). However, as isolation time increased, pCO2 values in the tidepools decreased (Figure 4.3 H). By the end of the low tide, tidepools had approximately 300 atm less pCO2 than of sound water (Figure 4.4 H). There was no significant effect of isolation time, tidepool size or the interaction between the two between the three differently sized tidepools (Table 4.1). Likewise, there was no significant effect of isolation time or type (sound v. tidepools) on pCO2 when tidepool data were pooled (Table 4.2). No significant differences in pCO2 were found between tidepools and the sound at the end of the tide (Table 4.3).   4.4.7 Growth Rates Growth rates of C. vancouveriensis were slower in the winter than in the summer (Figure 4.6). In the winter, growth rates were not different across tidepool size (Figure 4.6, Table 4.4). In the summer, however, there was a difference between the smallest tidepool and the two larger tidepools (Table 4.4, Tidepool x Season: p = 0.001). In the winter, growth rates were approximately 0.04 ± 0.002 mm/day (mean ± S.E.M). In the summer, C.  101  vancouveriensis in the smallest tidepool had a growth rate of 0.06 ± 0.007 mm/day while in the medium and large tidepool, growth rates were 0.08 ± 0.002 mm/day.    4.4.8 Percent Calcium Carbonate  There were no changes in percent calcium carbonate of apical intergenicula of C. vancouveriensis between the summer and winter, nor between the differently sized tidepools (Figure 4.7, Table 4.5). On average, new growth intergenicula of C. vancouveriensis were approximately 86.2 ± 0.4% calcium carbonate.   4.4.9 Biomechanical Properties  There were no significant differences between the seasons or the tidepools in any of the biomechanical properties (breaking stress, breaking strain, modulus) of fronds of C. vancouveriensis (Figure 4.8, Table 4.6). On average, the strength of C. vancouveriensis genicula was 35 ± 2 MPa; the extensibility was 0.44 ± 0.03 mm/mm, and the modulus was 129 ± 10 MPa.     102    Figure 4.2: Tidepool temperatures in small, medium, and large tidepools in the summer and in the winter.  (A) Winter, small tidepool; (B) Winter, medium tidepool; (C) Winter, large tidepool; (D) Summer, small tidepool; (E) Summer, medium tidepool; (F) Summer, large tidepool.  Solid line represents ocean seawater temperature (measured at FHL Weather Station) and red symbols are tidepool temperatures. Apparent vertical lines of red symbols indicate increasing or decreasing temperatures during one low tide.    103   Figure 4.3: Tidepool chemistry. Dashed line is sound water, black line is small tidepool, dark grey is medium tidepool, and light grey line is small tidepool. n = 4 – 5, error bars are ± S.E.M. (A) pH (total scale) in winter; (B) pH (total scale) in summer; (C) Total alkalinity in winter; (D) Total alkalinity in summer; (E) Dissolved oxygen in winter; (F) Dissolved oxygen in summer; (G) pCO2 (calculated) in winter; (H) pCO2 (calculated) in summer.   104   Figure 4.4: Chemical difference between tidepool and sound water at 1 hr. isolation, 3 hr. isolation, and at the end of the low tide. Negative value indicates that the tidepool is less than sound water for that parameter, and positive value indicates that the tidepool has a greater value than sound water, n = 4 – 5, average difference ± S.E.M. (A) Difference in pH (total scale) in winter; (B) Difference in pH (total scale) in summer; (C) Difference in total alkalinity in winter; (D) Difference in total alkalinity in summer; (E) Difference in dissolved oxygen in winter; (F) Difference in dissolved oxygen in summer; (G)Difference in pCO2 (calculated) in winter; (H) Difference in pCO2 (calculated) in summer.   105     Figure 4.5: Comparison of tidepool chemistry at the end of isolation time in the summer and winter to predicted values for 2100 and pre-industrial values. (A) Average end of tide summer and winter pH ± S.E.M., from tidepool measurements, and comparison to predicted pre-industrial and predicted for the year 2100 pH levels; (B) Average calculated end of the tide pCO2 ± S.E.M, from tidepool measurements and comparison to predicted pre-industrial and predicted for the year 2100 pCO2 values.   106  Table 4.1: Two-way ANOVA results of chemistry analyses (pH, Total Alkalinity, Dissolved oxygen, and pCO2) between small, medium, and large tidepool. Asterisk represents significant effect at alpha < 0.05 level.    107  Table 4.2: Two-way ANOVA results of chemistry analyses (pH, Total Alkalinity, Dissolved oxygen, and pCO2) between sound water and tidepool water (pooled tidepools).   108  Table 4.3: One-way ANOVA results between sound water and tidepool water at end of tide (pooled tidepools)   109   Figure 4.6: Apical linear growth of C. vancouveriensis in mm/day by tidepool size.  Dark bars are growth during the winter, n =6-8. White bars are growth during the summer, n = 5-6. Bars are S.E.M and letters indicate results of Tukey’s posthoc test and different letters represent significant differences.   Table 4.4: Two-way ANOVA results on apical linear growth of C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools.   110   Figure 4.7: Percent calcium carbonate of apical intergenicula of field collected C. vancouveriensis by tidepool size.  Dark bars are percent calcium carbonate of intergenicula that grew during the winter, n = 7. White bars are percent calcium carbonate of intergenicula that grew during the summer, n = 5-7. Bars are S.E.M.     111  Table 4.5: Two-way ANOVA results of percent calcium carbonate of C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools.   112   Figure 4.8: Biomechanical properties of genicula of C. vancouveriensis. Dark bars are measurements taken during the winter and white bars are measurements taken during the summer. (A) Breaking stress (MPa), n = 5 - 8; (B) Breaking strain (mm/mm), n = 5 – 8; (C) Modulus (MPa), n = 5 - 8.  Bars are ± S.E.M.    113  Table 4.6: Two-way ANOVA results on measurements of biomechanical properties of C. vancouveriensis fronds collected during the winter and the summer in differently sized tidepools.      114  4.5  Discussion 4.5.1 Tidepool Chemistry As expected, tidepool chemistry was highly variable, and differed in the summer and the winter. Unexpectedly, tidepool size had no effect on tidepool chemistry, and so, the hypothesis that variations in growth, calcification, and biomechanics within one season due to fine-scale differences in the chemistry between the tidepools could not be tested. However, this study did illustrate the extreme nature of chemistry conditions in tidepools. The changes in carbonate chemistry that occur over one tide cycle parallel changes predicted due to ocean acidification over the next 100 years. The tidepool coralline, C. vancouveriensis, was found to be resistant to the fluctuating low tide stressors of increased temperature and changed carbonate chemistry, but did exhibit reduced growth rates both in the winter and in the smallest tidepool in the summer. Yet, C. vancouveriensis fronds preserved the quality of their tissues, and had no changes in calcification or material properties between the seasons.  For the most part, tidepool chemistry followed my predictions. In the summer, tidepool temperatures were much warmer, pH and dissolved oxygen increased and pCO2 decreased. In the summer, temperatures were likely warm enough to inhibit the growth of the corallines in the tidepool based on results of Chapter 3. Temperatures in the studied tidepools in the summer exceeded 25C, and growth rates were much reduced in this species when grown at a constant temperature of 22C (See Chapter 3). In the winter, tidepool temperatures varied, but were more muted compared to the summer and oscillated only a few degrees above and below sound seawater. With increased isolation time in the winter, pH and dissolved oxygen decreased and pCO2 increased.   115  The changes found in tidepool total alkalinity was not predicted, but consistently increased during nocturnal emersions (when respiration is dominating) and decreased during day-time emersions (when photosynthesis is dominating), and similar results have been found in other studies (Truchot & Duhamel-Jouve 1980, Morris & Taylor 1983, Moulin et al. 2011). Since changes in alkalinity often indicate dissolution and calcification (Chisholm & Gattuso 1991), but no difference in calcium carbonate between the seasons in the differently sized tidepools was detected, this difference in total alkalinity was unexpected. If during low tide, dissolution was occurring during nocturnal emersions and calcification was occurring day-time emersions, it would be expected that this would result in differences in percent calcium carbonate in fronds of C. vancouveriensis between the summer and winter months, regardless of calcification that may be occurring at high tide. However, it is possible that C. vancouveriensis in this study had a greatly reduced calcification rate and/or dissolution during night time emersions and a higher calcification rate during day time emersions, while maintaining the same amount of calcium carbonate in the algal material. One possibility is that C. vancouveriensis conserves the percentage of calcium carbonate, but grows at a slower rate when conditions for calcification are not ideal. Other studies have found seasonal differences in calcification rate; McCoy et al. (2016) found that Corallina frondescens and Corallina vancouveriensis deposited more calcium carbonate during the day than at night, and also when submerged at high tide compared to when emerged at low tide. Cornwall et al. (2013) also found that the coralline alga, Arthrocardia corymbosa, had reduced net calcification in diurnally fluctuating pH conditions, and calcification was generally lower at night than during the day, but additionally, reduced pH in darkness further reduced net calcification rates. My study did not specifically assess calcification rates, and only  116  documented the final percentage of calcium carbonate. It is possible that the changes in total alkalinity in the tidepools was reflective of calcification and dissolution processes occurring during low tide, but more studies are needed to specifically assess this question.   4.5.2 Growth In the summer, C. vancouveriensis fronds grew slower in the smallest tidepool relative to the larger two tidepools. It was also found that the smallest tidepool had a denser population of corallines (~4 g/L) than the other two tidepools (~2 g/L). The density of the corallines in this tidepool may have contributed to the slower growth rates. One possibility is that self-shading effects in the corallines in this tidepool contributed to the slower growth rates. Another possibility is that with this higher density of corallines and therefore more oxygen via photosynthesis released into boundary layer, that excess oxygen could have resulted in photoinhibitory conditions (Mass et al. 2010). Yet, no difference in dissolved oxygen in the small pool relative to larger pools was detected, so if this were the case, it would be confined to the boundary layer. Another possibility is that the corallines in the pool had depleted resources such as nutrients, carbon species, or calcium ions, which translated into slower growth rates, but this was not quantified.    Between the seasons, growth rates were slower in the winter than in the summer. This is most likely due to the longer photoperiod, higher light levels in the summer and higher temperatures, and not chemistry factors. However, I was not able to explicitly test this hypothesis. The slower growth in the winter relative to the summer and the similarity in winter growth rates between the three pools are likely due to light factors (photoperiod and light quantity/quality) and/or colder temperatures. During the day-time in the winter,  117  tidepools were submerged and therefore receiving the same amount of light. During the day-time in the summer, however, the tidepools are isolated and receiving a greater amount of sunlight and are also exposed to warmer temperatures at low tide relative to high tide.    4.5.3  Calcification Despite the wide variation in tidepool chemistry between the summer and the winter, apical intergenicula of C. vancouveriensis fronds had the same percentage of calcium carbonate. This result suggests that either C. vancouveriensis is able to maintain pH at site of calcification (in the region of the cell wall) as has been found for other calcified macroalgae (Hurd et al. 2011), or that calcification is proceeding by another mechanism than simply harvesting carbonate ions from the seawater. Roleda et al. (2012b) suggested that coralline algae utilize bicarbonate to calcify, and not carbonate ions. If this is the case, it is not surprising that no differences in percent calcium carbonate were detected. In fact, C. vancouveriensis was able to maintain percent calcium carbonate of hard structures despite fluctuating pH, pCO2, and total alkalinity. This is similar to results reported in Chapter 3 where C. vancouveriensis maintained percent calcium carbonate even under adverse conditions. Yet, unlike the stable lab conditions presented in Chapter 3, field conditions here were fluctuating. Cornwall et al. (2013) found that for the coralline alga Arthrocardia corymbosa, fluctuating conditions were more stressful than stable conditions and resulted in growth reductions. However, in this study, the fluctuating environment of tidepools wasn’t found to be more stressful than stable conditions (Chapter 3).  Similar values for growth rates, percent calcium carbonate, and biomechanical properties were found in this chapter (fluctuating conditions) as in Chapter 3 (stable conditions). This may be due to the return of  118  the high tide after a few hours of adverse conditions offering some respite and time to recover as has been shown for photosynthesis in this species (Guenther & Martone 2014).   4.5.4 Biomechanical properties Biomechanical properties of genicula were unaffected by the wide fluctuations in tidepool chemistry. Although, biomechanical testing was not performed on newly formed genicula, other results (Chapter 3) show that mechanical changes in older genicula are still possible (see Figure 3.9 A). Thus, the data suggest that C. vancouveriensis is able to maintain tissue integrity, despite the extreme conditions in tidepools. Biomechanical results for C. vancouveriensis from this study are similar to those presented by Janot & Martone (2016) and similar values for strength, strain, and extensibility were found despite the corallines being collected from different locations (British Columbia v. Washington). However, this study represents one of the first studies to demonstrate the resilience of coralline material properties to abiotic factors.  In conclusion, tidepools were found to be highly variable in their chemistry while isolated during low tide, and these changes were influenced by the timing of the tides and likely influenced by the biological activity within the tidepools. The changes that occur in tidepools are as great (or greater) than differences in open water chemistry from pre-industrial times to predicted levels for 2100. Organisms that live in tidepools are exposed to rapidly changing chemistry that occurs over just a few hours that is equal in magnitude for changes predicted over the next 100 years. Tidepool organisms must endure these rapid and extreme changes every day, but are only exposed to them for a few hours at a time.   The tidepool coralline, C. vancouveriensis, was found to be tolerant to the chemical  119  changes that occur during low tide. Despite the adverse conditions associated with low tide, C. vancouveriensis exhibited positive growth rates and maintained calcified structures and material properties in both seasons. Chemistry in tidepools fluctuates with the tide, and organisms are returned to ambient conditions when the tide returns. Therefore, it is also possible that C. vancouveriensis was only growing and calcifying during high tide, and was shutting down these processes while isolated at low tide for the short period of time when conditions were too extreme. However, results in Chapter 3 suggest that C. vancouveriensis maintains positive growth; with minimal changes in calcification and material properties under long-term (~2 months) low pH conditions.   In the future, the pH of ambient water is projected to decrease 0.2-0.3 units (Feely et al. 2004, Orr et al. 2005, International Panel on Climate Change 2013).  This may have implications for marine organisms and, in particular, tidepool organisms that already experience wide fluctuations in chemistry during isolation at low tide. Intertidal organisms may be living at their physiological limits and may not be able to physiologically adjust to changing conditions expected with climate change (Tomanek & Somero 1999, Harley et al. 2006). For example, in the future, when tidepools are isolated at low tide at night, the starting pH of the water will be lower and the respiration of plants and animals will drive the pH down even farther.  More research is needed to determine if results presented here are generalizable across all corallines, or if they are specific to the species or the habitat where C. vancouveriensis is found. Previous literature has documented a strong negative response of coralline algae to acidification and warming (Kuffner et al. 2008, Anthony et al. 2008, Jokiel et al. 2008, Ragazzola et al. 2012), but many of these were corallines from relatively stable  120  habitats. The lack of response of C. vancouveriensis in this study to warming and acidification may relate to its previous habitat and acclimation. It has been suggested that organisms from habitats with variable pH may be more resistant to changes in pH in the future (Hofmann et al. 2011). However, Cornwall et al. (2013) found that for the calcifying alga, Arthrocardia corymbosa, fluctuating pH was more detrimental to relative growth rates than stable pH.  Nevertheless, C. vancouveriensis apparently survives wide fluctuations in chemistry every day, suggesting that at least one intertidal coralline species is well-suited to resisting daily changes in carbonate chemistry. Whether C. vancouveriensis’ demonstrated ability is common or unique among the corallines remains an open question.     121  5 Conclusion 1.1 Synopsis Climate change is progressing rapidly and is causing shifts in ecosystem function, species distributions, biodiversity, and abundances worldwide.  The field of climate change ecology has made great strides toward understanding how organisms will be affected by climate change and how different climate variables will interact, but many challenges still lie ahead.  Climate change has impacted organisms from the tropics to the polar regions, in both terrestrial and aquatic habitats. It is challenging to predict how species and communities will respond to climate change due to a multitude of factors, species-specific effects, interactive effects of multiple stressors, and the uncertain ability of organisms to acclimate to rapidly changing climate variables. It is also critically important that we begin to elucidate the effects of climate change on sensitive life stages, such as macroalgal spores, and how these effects may scale up to later life history stages.  The environment is complex, and correspondingly, so are the biological responses to changing climate variables.  Therefore, there is a need for studies that not only assess the effects of individual and combined stressors on adult organisms, but also examine the effects on different stages of an organism’s life cycle.  Additionally, there is a need for studies that consider the natural variability in climate factors, and how this may relate to biological responses. In this thesis, I addressed these issues in the rocky intertidal ecosystem with a group of calcifying algae that are ecologically important. I broadly asked: What are the effects of ocean acidification on coralline algae across different life history stages, and what are the conditions that they experience in situ? I divided this thesis into three separate  122  components: (1) What are the effects of ocean acidification on potentially sensitive life stages of red algae? (2) What are the effects of ocean acidification and increased temperature, both singly and in combination, on mature specimens of coralline algae? and (3) What is the range of variability and extremes across the seasons experienced by coralline algae growing in tidepools at my study site?   In this chapter, I will summarize major conclusions from my three research chapters and discuss potential limitations of my studies and how this relates to the interpretation of my results and conclusions.  I will recommend areas of needed future work and possible improvements for future studies. Furthermore, I will consider my results in the broader context of ecological responses to climate change.   5.2 Effects of ocean acidification on spores of red algae Results from Chapter 2 show that ocean acidification is detrimental to reproductive spores of both calcified and non-calcified algae.  This result is significant because studies on adult life stages of non-calcified macroalgae have shown either a positive or no response to elevated CO2 (Israel & Hophy 2002, Swanson & Fox 2007, Connell & Russell 2010). My study highlights that non-calcareous algae can be negatively affected by ocean acidification and shows that spores of algae represent a sensitive life stage in both calcifying and non-calcifying groups. Due to the smaller size and still-developing physiological capability of algal spores, it has been suggested that microscopic early life stages may be more sensitive to environmental stressors than mature macroscopic stages (Henry & Cole 1982, Dring et al. 1996, Coelho et al. 2000).  123  Although the investigation on effects of ocean acidification on spores of non-calcifying groups is somewhat lacking in the literature, there has been a fair amount of research done on kelp zoospores. For example, high pCO2 negatively affected the development of germination tubes in kelp zoospores (Gaitán-Espitia et al. 2014) and caused a decrease in the germination of kelp zoospores (Roleda et al. 2012a). Negative effects on spores of calcifying algae have also been documented. High pCO2 inhibited both spore production and growth of early life history stages in the calcifying red alga, Lithophyllum incrustans (Cumani et al. 2010). Similarly, Bradassi et al. (2013) found that small changes in pH resulted in increased abnormalities of early life stages of Phymatolithon leormandii and also documented reductions in growth rate in abnormal thalli as compared to normal thalli. The effects of ocean acidification on early life stages have begun to be studied, but many questions remain, and further investigation into the effects on both calcifying and non-calcifying algae is warranted.  In this study, reduced pH negatively affected spore adhesion in the two species of red algae studied, but in different phases of the adhesion process. Reduced pH delayed the settlement of P. bipinnata spores, and weakened the attachment of C. vancouveriensis spores. These results suggest that although C. vancouveriensis will be likely able to settle in low pH conditions, fewer C. vancouveriensis spores may be able to survive hydrodynamic forces in reduced pH conditions. In contrast, P. bipinnata spores that are able to settle, despite being delayed, are likely to have an attachment that is insensitive to reduced pH.  The spore adhesion process is complex, and ocean acidification was shown here to affect different phases of the spore adhesion process, such that not all species will be affected by ocean acidification in the same way. However, given the shear numbers of spores released by  124  one individual, the negative effects on spore adhesion documented in this study may or may not have significant effects on the population.  Future studies should address whether increased spore mortality results in changes in population dynamics.  This study examined only the effect of reduced pH on haploid spores (tetraspores) produced by sporophytes, and did not investigate impacts on gametes or diploid spores (carpospores). It is possible that ocean acidification may affect diploid and haploid spores differently due to differences in spore size and/or differential susceptibility of gametophytes compared to sporophytes. If diploid spores are equally susceptible, results demonstrated here may represent an underestimate of the impact of ocean acidification on overall spore success. Future studies should assess whether impacts on carpospores success are comparable to effects on tetraspores presented in this thesis. Differential success between the two types of spores could lead to differential mortality of one-living phase over the other, differential survival of one spore type over another, or differences in fertilization success (Santelices 1990, Santelices 2002, Fierst et al. 2005). Reduced spore success has the potential to impact both gametophyte and ultimately sporophytes of seaweed populations. Thus, ocean acidification may impact the future composition, diversity, and abundance of seaweed populations on the shore not by affecting adult stages, but by affecting the different stages of the spore adhesion process and by possibly differentially affecting one type of spore over the other (tetraspores v. carpospores). Furthermore, coralline algae may allocate less energy to reproduction under future climate scenarios to maintain the quality of their tissues and this may result in fewer spores being produced or a lower quality of spore being produced, which could further affect the composition and abundance of seaweed communities.   125  One limitation of the research conducted in Chapter 2 was that the parent algae used were not grown under conditions of ocean acidification.  The parent frond, and therefore the spores that that frond produced, were grown under ambient conditions in the field.  In considering this, two possibilities arise: either the spores used in this study were more fit because they were formed under ambient levels of pH/pCO2 so results presented here may represent an underestimate of the impact, or else spores grown under low pH would be somehow better adapted and more resistant to low pH conditions, and so results here may represent an overestimate of the impact.  Future research should concentrate on whether results here are species-specific, or if they may be generalizable across certain algal groups or phases of the spore adhesion process.  This information would allow more accurate predictions of the effects of climate change on algal reproduction and the ultimate effects on seaweed population abundance and demographics.   5.3 Effects of multiple climate variables on adult stages of coralline algae  In Chapter 3, I explored the effects of multiple abiotic variables on adult stages of two species of articulated coralline algae. I used a factorial design, and manipulated pH and temperature both singly and in combination. I found that these two species of coralline algae are resistant to changes in pH and temperature, and only exhibited strong negative responses when the pH was greatly reduced, or the temperature was significantly raised. I also found that within the range of temperatures and pH levels used in these experiments, effects were not additive and were mostly driven by temperature, and not pH. Surprisingly, the species that was originally predicted to be more resistant to reduced pH (C. vancouveriensis) was  126  actually more sensitive. Results from Chapter 3 suggest that species responses are not necessarily predictable, and that responses of closely related species to climate variables can differ greatly in magnitude.  Experiments from Chapter 3 show that these two species of coralline algae maintain the quality of the material that they produce under low pH, but in the case of C. vancouveriensis, grow at a slower rate. This reduction in growth could have several ecological implications. If corallines grow more slowly, they may have reduced competitive ability for space (McCoy & Pfister 2014). Under future climatic conditions, it has been proposed that corallines could either become more abundant as other species are lost due to ocean acidification (Wootton 2001), or become less abundant due to negative impacts of climate change (Nelson 2009, Ragazzola et al. 2012, McCoy 2013, McCoy & Pfister 2014).  Another consideration is that if these species are re-allocating energy to maintain material properties and amount of calcium carbonate, then other processes not tested here may be affected.  For example, if corallines devote more resources to maintaining growth and tissue quality, they may experience a reduction in reproductive allocation which could have far reaching effects on species abundance and composition along our shores.   The effects of fluctuating versus stable pH (as occurs in many environments) in different life stages is one avenue for future research. Roleda et al. (2015) followed the growth and development of the juvenile life history stage of the coralline alga, Arthrocardia corymbosa, which had recruited into experimental conditions during the prior experiment of Cornwall et al. (2013). Adults of A. corymbosa were cultured under static and fluctuating pH conditions, to simulate pH fluctuations observed within a kelp forest (Cornwall et al. 2013). Interestingly, Roleda et al. (2015) found that juveniles of A. corymbosa were less affected by  127  the fluctuating/low pH treatment as compared to adults from Cornwall et al. (2013). In the Cornwall et al. (2013) experiment, the growth rate of adults in the fluctuating/low pH treatment was close to zero. However, in the subsequent experiment by Roleda et al. (2015) on juveniles, a positive growth rate was recorded. This result suggests that juveniles may be less susceptible to fluctuating, low pH than adults. Yet, this may be due to a positive carry-over effect on the next generation after exposing reproductive adults to fluctuating and low pH. It is possible that the exposure of fertile A. corymbosa sporophytes fluctuating and low pH induced a preconditioning response to their spores to tolerate lower pH. Carry-over effects of pH conditions from adult to progeny are a topic that should be further investigated in future studies.   Coralline algae have been shown to have both positive and negative effects to ocean acidification and rising temperature.  For the most part, negative effects are reported (Kuffner et al. 2008, Anthony et al. 2008, Jokiel et al. 2008, Ragazzola et al. 2012) but has led to faster growth rates and increased calcification in some species (Gao et al. 1991, 1993, Kübler et al. 1999, Iglesias-Rodriguez et al. 2008) and no response in others (Egilsdottir et al. 2013).  However, many of these studies were performed on crustose forms, and so the contrasting results could be due to taxonomic differences.  Additionally, many of these studies were performed on tropical species (Kuffner et al. 2008, Anthony et al. 2008, Martin & Gattuso 2009). Since tropical waters are fairly stable in their pH as compared to temperate kelp beds (Hofmann et al. 2011) and much more stable than tidepool habitats (Chapter 4), these species are not exposed to highly fluctuating pH, as are the species studied in this thesis. It is possible that the lack of negative results on adult thalli shown in this thesis is due their previous acclimation to tidepool habitats. The species studied in this thesis were  128  collected from tidepool environments that are highly variable (Chapter 4), and it is possible that lab culture was a much less stressful environment. Experiments that mimic pH fluctuations predicted for rock pools in a future ocean would certainly be justified.  A major contribution of this chapter is that responses to future climate conditions may not be generalizable across taxonomic groups.  Even closely related corallines from similar habitats were shown to have a different magnitude of response to ocean acidification (Noisette et al. 2013). Additionally, responses were counter to predictions (i.e. C. tuberculosum was predicted to be more sensitive, but it was more resistant). In many meta-analyses, species are grouped in broad taxonomic groups, such as ‘calcifying algae’. However, this grouping encompasses members in various lineages of algae that use different mechanisms to calcify and deposit different polymorphs of calcium carbonate, and so, predictions based on this wide taxonomic grouping may not be informative. I recommend that meta-analyses use more specific groupings, such as ‘crustose coralline algae’, ‘articulated coralline algae’, ‘green calcifying algae’. Additionally, it may be useful to start separating groups out based upon the habitat, and the variability within that habitat that organisms live in.     One aspect that should be considered when interpreting my results from Chapter 3 is the magnitude of change in experimental manipulations, and the previous history of the specimens that were used in the experiments. First of all, the pH levels used in my experiments were rather extreme. Although I did measure tidepool pH’s of ~7.3 on more than one occasion, the organisms are only exposed to that low pH for a short period of time, never continuously over 1-2 months. I used very low pH values in my experiment in order to document if there was any effect of pH at all (i.e. if I only used pH values of 8-7.6 and found  129  no effect, is it because there is no effect of pH or that I didn’t test a wide enough range of pH values to detect the effect). The lowest pH value of 7.0 used in my experimental manipulations is not likely to occur in the near future due to anthropogenic climate change, but I wanted to detect if there would be any effect due to pH at an extreme. Although I documented reduced growth and calcification at this extremely low pH, specimens of both species were able to survive this treatment and exhibited positive growth and calcification. This implies that even under the most drastic predicted future scenarios of climate change, these species are likely to persist.  An additional consideration is that I collected both of these species from intertidal tidepools. C. vancouveriensis only grows in the intertidal zone and does not occur subtidally.  C. tuberculosum, on the other hand, is more commonly found subtidally and less commonly in the intertidal zone. In my studies, I collected C. tuberculosum from tidepools for feasibility of collection; however, it is possible that I was testing a very resilient intertidal population. Since this population was able to persist in the intertidal zone, specimens may have been more acclimated to changing abiotic conditions than their subtidal counter parts.    Another detail to consider when interpreting my results is that the experimental manipulations were unnaturally stable, and did not fluctuate as occurs naturally in the field. Cornwall et al. (2013) found that for another species of coralline algae, Arthrocardia corymbosa, fluctuating conditions were more stressful than stable conditions. Given this, two possibilities arise; that fluctuating conditions would have been more stressful and my results represent an underestimate of the impact or that these species of coralline algae are resistant to both fluctuating and stable conditions. One avenue for future research is to assess the effects of fluctuating versus stable conditions on these species. I hypothesize that since these  130  species of coralline algae are exposed to wide fluctuations in nature that they might be resistant to fluctuating conditions and that they would respond similarly under fluctuating conditions as under stable conditions.   5.4 Variability in carbonate chemistry in situ Results of Chapter 4 highlight the extreme changes in chemistry that occur in tidepools during low tide isolation. Organisms in tidepools are exposed to changing chemistry conditions on a daily basis that can be greater in magnitude than those predicted for climate change. However, the changing chemistry that occurs in tidepools is a biological signal, driven by processes of photosynthesis and respiration, and not by anthropogenic climate change. We often consider the effects of chemistry on organisms; however, this study highlights how organisms can greatly alter the chemistry of the water around them.  C. vancouveriensis was shown to be very tolerant of these low tide stresses, and continued to grow. For the most part, C. vancouveriensis maintained positive growth year round and percent calcium carbonate and material properties were unaffected. This suggests, similar to results in Chapter 3, that although C. vancouveriensis grows slower in the winter, it maintains the quality of the tissue it produces.  One major limitation of this study was that I was not able to separate out responses during high tide versus responses at low tide. One possibility is that C. vancouveriensis was stressed from changing chemistry at low tide, experienced reduced physiological activity during low tide isolation, but then recovered during high tide. Another possibility is that C. vancouveriensis was resistant to changes in chemistry and was able to maintain physiological processes at both low tide and high tide. I was not able to capture the physiological activity  131  of C. vancouveriensis at high tide, and it’s possible that C. vancouveriensis is only physiologically active when the tide is in, and essentially shuts down at low tide.  If possible, future research should explore how physiology differs at low tide compared to high tide.   I was also not able to tease apart the effects of tidepool chemistry in the same season, based on differences in tidepool size. I expected differently sized tidepools to result in a gradation in carbonate chemistry but this was not found to be the case. The three tidepools studied did not differ significantly in any of the chemical parameters studied. However, differences in tidepool chemistry probably relate more to the amount and kind of biomass in the tidepool than the actual size of the tidepool. During day-time isolations, the density of photosynthesizing organisms in the tidepool dictates the amount of chemical change within the tidepool. During nocturnal isolations, the amount of respiring organisms in the tidepool dictates the amount of chemical change within the tidepool. Moreover, this study was unavoidably pseudo-replicated, as the three tidepools occurred on the same shore within 50 meters of each other. Ideally, future studies should evaluate several tidepools of each size along a few distinct coastlines. Additionally, future studies could experimentally manipulate the amount of biomass within tidepools to control the rate of chemical change in tidepools during isolation.  In summary, global climate change has the potential to affect seaweeds in multiple stages of their life history. While adult stages of articulated corallines were sometimes tolerant of changing climate variables and perhaps not as sensitive as previously thought, reproductive spores were found to be sensitive to elevated pCO2. On a broader scale, the consequences climate change on the abundance and persistence of these species will likely depend on how the effects on the spore stages of these algae scale up; and many questions  132  remain to be answered. Will spores developed under future climate scenarios be of lower quality or will they be pre-conditioned for improved survival in a changed ocean? Will spore performance translate into altered fitness of adults? Are haploid and diploid spores equally vulnerable? 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