Physiology and Biogeochemistry of Corals Subjected to Repeat Bleaching and Combined Ocean Acidification and Warming

PHYSIOLOGY AND BIOGEOCHEMISTRY OF CORALS SUBJECTED TO REPEAT BLEACHING AND COMBINED OCEAN ACIDIFICATION AND WARMING

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Verena Schoepf, M.S. Graduate Program in Geological Sciences

The Ohio State University 2013

Dissertation Committee: Professor Andréa G. Grottoli, Advisor Professor Lawrence A. Krissek Professor John W. Olesik Professor Ozeas S. Costa

Abstract

Rising atmospheric CO2 concentrations threaten coral reefs worldwide by causing ocean warming and acidification. When seawater temperatures are unusually high, corals lose a significant portion of their vital algal endosymbionts and/or photosynthetic pigments making them appear pale or white – a process referred to as coral bleaching. As corals get most of their carbon from the algal endosymbionts, the breakdown of this symbiosis significantly weakens coral and can lead to widespread mortality if bleaching is severe. Bleaching events have been predicted to become annual events sometime later this century. Despite this knowledge, the impacts of annual bleaching on coral physiology, biogeochemistry, and overall resilience remain largely unknown. For the first time, annually recurring bleaching was simulated on ecologically relevant timescales by subjecting three Caribbean coral species (Orbicella faveolata, Porites astreoides, and

Porites divaricata) to experimental coral bleaching (+1°C for 2.5 weeks) in two consecutive years. Impacts on their physiology and biogeochemistry were assessed in great detail immediately after repeat bleaching as well as after short and long term recovery. We show that repeat bleaching can dramatically alter thermal tolerance of the coral holobiont (i.e., animal host and endosymbiont). Species such as P. divaricata will be able to rapidly acclimate to frequent temperature stress and persist on future coral

ii reefs, whereas others such as P. astreoides will show increasing bleaching susceptibility and may thus face significant decline.

Coral skeletal carbon isotopes are important paleo-climate proxies and have the potential to record past bleaching events. However, they are often confounded by strong kinetic isotope effects that can mask the bleaching signal in the skeleton and compromise overall accuracy of the proxy. A proposed data correction to remove kinetic isotope effects was tested for the first time using bleached corals. In addition, it was tested if photosynthesis to respiration (P/R) ratios can be reliably calculated from coral isotopes.

We found that the data correction did not effectively remove kinetic isotope effects, and that isotope-based P/R ratios are in poor agreement with P/R ratios measured by respirometry. Therefore, the data correction should not be routinely applied to paleo- climate reconstruction, and P/R ratios should be obtained by respirometry only.

While the potential of some coral species to acclimate to frequent bleaching stress is encouraging, corals will also have to cope with increasingly more acidic seawater, which compromises calcification rates. Yet, the combined effects of ocean acidification (OA) and warming on coral physiology remain poorly understood. We therefore conducted a controlled tank experiment where four Pacific coral species

(Acropora millepora, Pocillopora damicornis, Montipora monasteriata, and Turbinaria reniformis) were maintained at seawater pCO2 levels and temperatures expected by the end of this century (741 μatm, 29.0°C). Surprisingly, three of the four species studied were able to maintain calcification rates under these conditions. Further, we show for the first time that coral energy reserves were not catabolized to sustain calcification rates

iii under OA conditions, which has important implications for coral health and bleaching resistance.

Overall, the findings from this dissertation research highlight that some coral species will be able to survive predicted near-future ocean acidification and warming.

However, as seawater pCO2 increases beyond 700 μatm and coral bleaching becomes more frequent and intense, temperature- and CO2-sensitive species will likely face significant demise. Since this is likely to have dramatic consequences for coral reef diversity and overall reef functioning, the future of coral reefs beyond the 21st century may be uncertain.

Dedication

This dissertation is dedicated to Michael and Inge.

Acknowledgements

I would like to thank my advisor, Andréa Grottoli, for her support, guidance, and patience throughout my PhD studies. She was a great mentor and always there to support me. I would also like to thank the members of my dissertation committee –Larry Krissek, John Olesik, and Ozeas Costa – for their support and guidance during the preparation of this dissertation. Special thanks go to everybody at the stable isotope biogeochemistry laboratory, and Yohei Matsui in particular, for their support, advice, and guidance throughout my time at OSU. I am grateful for funding from the Ohio State University Presidential Fellowship and Helen M. and Milton O. Lee Fellowship, the Papousek donation from the Rotary Club Innsbruck-Austria, the PADI Foundation, Geological Society of America, and American Association of Petroleum Geologists, as well as OSU School of Earth Science graduate teaching assistantships. The research presented here was funded by the National Science Foundation. Laboratory, field, and writing assistance were provided by: S. Levas, J. Baumann, T. Huey, D. Borg, E. Zebrowski, J. Scheuermann, M. McBride, M. Berzins, M. Munster, M. Aschaffenburg, and M. McGinley. Logistical support in Mexico was provided by R. Iglesias- Prieto, A. Banaszak, S. Enriquez, R. Smith, and the staff of the Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico. Special thanks go to T. Melman and the staff at Reef Systems Coral Farm for their continued support and patience.

Vita

2005 ...... B.S. Biology, University of Innsbruck, Austria 2008 ...... M.S. Zoology, University of Innsbruck, Austria 2009 - 2012 ...... Graduate Teaching Associate, School of Earth Sciences, The Ohio State University 2012 to present ...... Presidential Fellow, The Ohio State University

Publications

Schoepf V, Herler J, Zuschin M (2010) Microhabitat use and prey selection of the coral- feeding snail Drupella cornus in the northern Red Sea. Hydrobiologia, 641:45-57.

Fields of Study

Major Field: Geological Sciences Area of Emphasis: Marine Biogeochemistry

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Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgements ...... vi Vita ...... vii List of Tables ...... xii List of Figures ...... xix

1. Introduction...... 1 1.1 Coral Biology ...... 1 1.2 Coral Reefs and Climate Change ...... 1 1.2.1 Coral Bleaching ...... 3 1.2.1.1 Physiology of Bleached Corals ...... 4 1.2.1.2 Recovery From Bleaching ...... 6 1.2.1.3 Repeat Bleaching ...... 7 1.2.2 Stable Carbon, Nitrogen, and Oxygen Isotopes in Bleached and Recovering Corals ...... 9 1.2.2.1 Stable Carbon Isotopes (δ13C) ...... 9 1.2.2.2 Stable Nitrogen Isotopes (δ15N) ...... 11 1.2.2.3 Stable Oxygen Isotopes (δ18O) ...... 13 1.2.3 Coral Reefs and Ocean Acidification ...... 13 1.2.3.1 The Effects of Ocean Acidification on Coral Calcification ...... 15 1.2.3.2 The Effects of Ocean Acidification on Coral Energy Reserves ...... 15 1.3 Dissertation Outline ...... 16 1.4 References ...... 20

2. The Effect of Repeat Bleaching on the Physiology and Biogeochemistry of Three Caribbean Coral Species ...... 33

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2.1 Abstract ...... 34 2.2 Introduction ...... 35 2.3 Material and Methods ...... 39 2.3.1 Coral Collection ...... 39 2.3.2 Repeat Bleaching Experiment ...... 40 2.3.3 Physiological Analyses ...... 42 2.3.4 Isotopic Analyses ...... 46 2.3.5 Statistical Analyses ...... 47 2.4 Results...... 49 2.4.1 Bleaching and Mortality Status ...... 49 2.4.2 Physiology ...... 49 2.4.3 Tissue and Skeletal Isotopes ...... 52 2.5 Discussion ...... 54 2.5.1 Orbicella faveolata ...... 55 2.5.2 Porites astreoides ...... 58 2.5.3 Porites divaricata ...... 61 2.5.4 Implications for the Future of Coral Reefs ...... 64 2.6 References ...... 66 2.7 Tables ...... 77 2.8 Figures ...... 90

3. Kinetic and Metabolic Isotope Effects in Coral Skeletons: A Re-Evaluation using Experimental Coral Bleaching as a Case Study ...... 99 3.1 Abstract ...... 100 3.2 Introduction ...... 101 3.3 Material and Methods ...... 106 3.3.1 Hawaii Bleaching Experiments ...... 106 3.3.2 Mexico Bleaching Experiment ...... 107 3.3.3 Physiological Analyses ...... 108 3.3.4 Isotopic Analyses ...... 109 3.3.5 Statistical Analyses ...... 113 3.4 Results...... 114 3.4.1 Isotope correlations ...... 115 ix

3.4.2 Measured and isotope-based P/R ratios ...... 116 3.5 Discussion ...... 118 3.5.1 Presence of kinetic isotope effects ...... 118 13 3.5.2 Evaluation of the δ Cs data correction ...... 119 3.5.3 Comparison of measured and isotope-based P/R ratios ...... 121 3.5.4 Implications for paleo-climate reconstruction ...... 123 3.6 References ...... 125 3.7 Tables ...... 131 3.8 Figures ...... 143

4. Coral Energy Reserves and Calcification in a High-CO2 World at Two Temperatures ...... 148 4.1 Abstract ...... 149 4.2 Introduction ...... 150 4.3 Material and Methods ...... 153 4.3.1 Experiment ...... 153 4.3.2 Monitoring of Seawater Chemistry ...... 156 4.3.3 Laboratory analyses ...... 156 4.3.4 Statistical analyses ...... 159 4.4 Results...... 159 4.4.1 Calcification ...... 160 4.4.2 Chlorophyll a and endosymbiont density ...... 161 4.4.3 Energy reserves and tissue biomass ...... 162 4.4.4 Effects of parent colony ...... 163 4.5 Discussion ...... 163 4.6 References ...... 171 4.7 Tables ...... 181 4.8 Figures ...... 193

5. Summary and Future Research ...... 197 5.1 Summary ...... 197 5.2 Future Research ...... 200

List of References ...... 203 x

Appendix A: Chapter 2 Raw Data ...... 228

Appendix B: Chapter 3 Raw Data ...... 246

Appendix C: Chapter 4 Raw Data ...... 263

List of Tables

Table 2.1. Parent colony (=genotype) collection dates, depths, and locations...... 77

Table 2.2. Results of two-way ANOVAs for gross photosynthesis (P), day respiration (R), and night respiration of O. faveolata, P. astreoides, and P. divaricata at 0 month recovery. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Genotype (G) was a random factor with 9 levels (1-9). Significant p-values (p≤0.05) are highlighted in bold...... 78

Table 2.3. Results of two factorial MANOVAs assessing the effects of coral species (S) and bleaching status (T) on the composition of captured zooplankton by size and taxa. The effect of species (S) was fixed and fully crossed with three levels (O. faveolata, P. astreoides, P. divaricata). Bleaching status was fixed and fully crossed with two levels (non-bleached, repeat bleached). Significant p-values (p≤0.05) are highlighted in bold...... 79

Table 2.4. Results of two-way ANOVAs for feeding rate of O. faveolata, P. astreoides, and P. divaricata at 0 month recovery. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Genotype (G) was a random factor with 9 levels (1-9). Significant p-values (p≤0.05) are highlighted in bold...... 80

repeat bleached 31.6°C). Genotype (G) was a random factor with 9 levels (1-9). Significant p-values (p≤0.05) are highlighted in bold...... 81

Table 2.6. Results of three-way ANOVAs for chlorophyll a, lipid, protein, carbohydrate content, tissue biomass, and calcification rates of O. faveolata, P. astreoides, and P. divaricata. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Recovery (R) was fixed and fully crossed with 3 levels (0, 1.5, and 11 months). Genotype (G) was a random factor with 9 levels (1-9). Post hoc Tukey tests were used when main terms – but no interaction terms - were significant. Significant p-values (p≤0.05) are highlighted in bold...... 82

13 13 13 13 15 15 Table 2.7. Results of three-way ANOVAs for δ Ch, δ Ce, δ Ch-δ Ce, δ Nh, and δ Ne of O. faveolata, P. astreoides, and P. divaricata. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Recovery (R) was fixed and fully crossed with 3 levels (0, 1.5, and 11 months). Genotype (G) was a random factor with 9 levels (1-9). Post hoc Tukey tests were used when main terms – but no interaction terms - were significant. Significant p-values (p≤0.05) are highlighted in bold...... 85

13 18 Table 2.8. Results of three-way ANOVAs for δ Cs and δ Os of O. faveolata, P. astreoides, and P. divaricata. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Recovery (R) was fixed and fully crossed with 3 levels (0, 1.5, and 11 months). Genotype (G) was a random factor with 9 levels (1-9). Post hoc Tukey tests were used when main terms – but no interaction terms - were significant. Significant p-values (p≤0.05) are highlighted in bold...... 88

Table 3.1. Seawater dissolved inorganic carbon (DIC) concentration and isotopic 13 composition (δ CDIC) from Kaneohe Bay, Hawaii, from September 2006 to August 2007. Stdev = standard deviation...... 131

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13 Table 3.2. Correlation analyses of original skeletal carbon isotope (δ Csorig) versus 18 13 13 13 skeletal oxygen isotopes (δ Os), δ Csorig versus δ C of the animal host (δ Ch), 13 13 13 and corrected δ Cs (δ Cscorr) versus δ Ch for the Hawaiian coral species Porites compressa, Montipora capitata, and Porites lobata. Analyses were computed (A) pooled for species and treatments, and (B) individually for each treatment and recovery interval. Significant p-values (p≤0.05) are highlighted in bold. Treat. = treatment, NB = non-bleached, BL = singly bleached, 0, 1, 1.5, 4, 5, 8, 11 = months of recovery, N = sample size, r = Spearman’s correlation coefficient. .. 132

13 Table 3.3. Correlation analyses of original skeletal carbon isotope (δ Csorig) versus 18 13 13 13 skeletal oxygen isotopes (δ Os), δ Csorig versus δ C of the animal host (δ Ch), 13 13 13 and corrected δ Cs (δ Cscorr) versus δ Ch for the Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata. Analyses were computed (A) pooled for species and treatments, and (B) individually for each treatment and recovery interval. Significant p-values (p≤0.05) are highlighted in bold. Treat. = treatment, NB = non-bleached, BL = repeat bleached, 0, 1.5, 11 = months of recovery, N = sample size, r = Spearman’s correlation coefficient. .. 134

Table 3.4. Correlation analyses of measured (meas.) and isotope-based P/R ratios for the Hawaiian coral species Porites compressa, Montipora capitata, and Porites 13 lobata. Isotope-based P/R ratios were computed with both δ Csorig (orig. P/R) and 13 18 δ Cscorr (corr. P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. N = sample size, r = Spearman’s correlation coefficient...... 136

Table 3.5. Results from paired t-tests comparing measured (meas.) and isotope-based P/R ratios from Hawaiian coral species Porites compressa, Montipora capitata, and 13 Porites lobata. Isotope-based P/R ratios were computed with both δ Csorig (orig. 13 18 P/R) and δ Cscorr (corr. P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery xiv

intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. df = degrees of freedom...... 137

Table 3.6. Correlation analyses of measured (meas.) and isotope-based P/R ratios for the Mexican coral species Orbicella faveolata, Porites astreoides, and Porites 13 divaricata. Isotope-based P/R ratios were computed with both δ Csorig (orig. P/R) 13 18 and δ Cscorr (corr. P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. N = sample size, r = Spearman’s correlation coefficient. .. 138

Table 3.7. Results from paired t-tests comparing measured (meas.) and isotope-based P/R ratios from Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata after 0 month of recovery from repeat bleaching. Isotope-based 13 13 P/R ratios were computed with both δ Csorig (orig. P/R) and δ Cscorr (corr. P/R). 18 Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Significant p-values (p≤0.05) are highlighted in bold. df = degrees of freedom...... 139

13 13 Table 3.8. Correlation analyses of original skeletal carbon isotope (δ Csorig) versus δ C 13 13 13 13 of the algal endosymbiont (δ Ce), and corrected δ Cs (δ Cscorr) versus δ Ce for (A, B) the Hawaiian coral species Porites compressa, Montipora capitata, and Porites lobata, and (C, D) the Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata. Analyses were computed (A, C) pooled for species and treatments, and (B, D) individually for each treatment and recovery interval. Significant p-values (p≤0.05) are highlighted in bold. Treat. = treatment, NB = non-bleached, BL = singly or repeat bleached, 0, 1, 1.5, 4, 5, 8, 11 = months of recovery, N = sample size, r = Spearman’s correlation coefficient...... 140

Table 4.1. Average conditions for each of the 6 treatments representing three pCO2 levels at two temperature regimes (ambient, elevated = ambient + 2.5°C). Mean ± 1 SE are shown. Sample size was 25 for all measurements. Temp. = Temperature. .. 181

three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold)...... 182

Table 4.3. Results of 8 two-way ANOVAs for average chlorophyll a concentrations and symbiont density. Four species (Acropora millepora, Pocillopora damicornis,

Montipora monasteriata, Turbinaria reniformis) were compared at three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold)...... 185

Turbinaria reniformis) were compared at three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold)...... 188

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repeat bleaching. Coral ID consists of species (OF, PA, or PD), year (2= repeat bleaching), treatment (NB=control, BL=repeat bleached), recovery (00, 02, 11 months), and genotype (1-9). Treatment had two levels (1=control, 2=repeat bleached), whereas genotype had nine levels (1-9). Outliers that were excluded from the data set during statistical analyses are indicated by an asterisk. Dots indicate missing measurements due to missing or small fragments……………232

13 Table A.3. Chapter 2 raw data for stable carbon isotopes of the animal host (δ Ch) 13 13 endosymbiont (δ Ce), their difference (δ Ch-e), and the stable nitrogen isotopes 15 15 of the animal host (δ Nh) and endosymbiont (δ Ne) for Orbicella faveolata, Porites astreoides, and Porites divaricata after 0, 1.5, and 11 months of recovery after repeat bleaching. Coral ID consists of species (OF, PA, or PD), year (2= repeat bleaching), treatment (NB=control, BL=repeat bleached), recovery (00, 02, and 11 months), and genotype (1-9). Treatment had two levels (1=control, 2=repeat bleached), whereas genotype had nine levels (1-9). Outliers that were excluded from the data set during statistical analyses are indicated by an asterisk. Dots indicate missing measurements due to missing or small fragments. ……..239

13 Table B.1. Chapter 3 raw data for skeletal carbon isotopes (δ Cs), oxygen isotopes 18 13 (δ Os), transformed (=transf.) δ Cs, measured (=meas.) P/R ratios, and isotope- based P/R ratios calculated with δ18Oeq after Grossman and Ku 1986 and Maier 2004 for Orbicella faveolata, Porites astreoides, and Porites divaricata at 0, 1.5, and 11 months of recovery repeat bleaching. Coral ID consists of species (OF, PA, or PD), year (2= repeat bleaching), treatment (NB=control, BL=repeat bleached), recovery (00, 02, and 11 months), and genotype (1-9). Year had one level (2=repeat bleaching), treatment had two levels (1=control, 2=repeat bleached), and genotype had nine levels (1-9). No outliers were excluded from the data set during statistical analyses. Dots indicate missing measurements due to missing or small fragments. ……………………………………………………247

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13 Table B.2. Chapter 3 raw data for skeletal carbon isotopes (δ Cs), oxygen isotopes 18 13 (δ Os), transformed (=transf.) δ Cs, measured (=meas.) P/R ratios, and isotope- based P/R ratios calculated with δ18Oeq after Grossman and Ku 1986 and Maier 2004 for Porites compressa, Montipora capitata, and Porites lobata after single bleaching. Coral ID consists of species (PC, MC, PL), treatment (NB=control, BL=bleached), recovery (0, 1 or 2, 4 or 5, and 8 or 11 months), and genotype (01- 12). Treatment had two levels (1=control, 2=bleached), and genotype had twelve levels (1-12). Outliers that were excluded from the data set during statistical analyses are indicated by an asterisk. Dots indicate missing measurements due to missing or small fragments. ……………………………………………………255

BL=29.0°C), CO2 level (LC=382 μatm, MC=607 μatm, and HC=741 μatm), and genotype (1-6). Temperature (=Temp.) had two levels (1=26.5°C, 2=29.0°C),

CO2 had three levels (1=382 μatm, 2=607 μatm, and 3=741 μatm), and genotype had six levels (1-6). Outliers that were excluded from the data set during statistical analyses are indicated by an asterisk. Dots indicate missing measurements due to missing or small fragments. …………………………….264

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List of Figures

Figure 2.1. Scheme of the experimental design of the repeat bleaching experiment including timeline. Numbers in brackets indicate sample size. Blue color represents ambient seawater temperature, whereas red color represents elevated temperature during the bleaching treatments. The three species used in this experiment were Orbicella faveolata, Porites astreoides, and Porites divaricata. Gray fields indicate coral samples collected during the repeat bleaching experiment. White fields indicate coral samples collected to assess their response to single bleaching, which was published in Levas (2012)...... 90

Figure 2.2. Photographs of representative coral fragments of (A-C) Orbicella faveolata, (D-F) Porites astreoides, and (G-I) Porites divaricata following 0, 1.5, and 11 months of recovery after repeat bleaching stress. Repeat bleached corals are shown on the left, whereas non-bleached control corals are shown on the right.. 91

Figure 2.3. Bleaching and mortality status for repeat bleached (A) Orbicella faveolata, (B) Porites astreoides, and (C) Porites divaricata at 0, 1.5, and 11 months of recovery...... 92

Figure 2.4. Average (A) gross photosynthesis, (B) day respiration, and (C) night respiration for Orbicella faveolata, Porites astreoides, and Porites divaricata immediately after the repeat bleaching treatment (=0 month recovery). Averages are shown ± 1 SE. Sample size ranges from 7-9...... 93

Figure 2.5. Proportion of zooplankton capture assemblage by (A) size class and (B) zooplankton group for all species combined immediately after the repeat xix

bleaching treatment (=0 month recovery). Proportions were calculated from a total of 84 zooplankton captures. (C) Average feeding rate of Orbicella faveolata (OF), Porites astreoides (PA), and Porites divaricata (PD) at 0 month recovery. Averages are shown ± 1 SE. Sample size ranges from 8-9...... 94

Figure 2.6. Average contribution of (A) photophotophotoautotrophically-derived carbon to animal respiration (CZAR), (B) heterotrophically-derived carbon to animal respiration (CHAR), and (C) total carbon to animal respiration (CTAR) for Orbicella faveolata (OF), Porites astreoides (PA), and Porites divaricata (PD) immediately after the repeat bleaching treatment (=0 month recovery). Averages are shown ± 1 SE. Dashed lines indicate where 100% of the daily metabolic energy demand is met. Sample size ranges from 8-9...... 95

Figure 2.7. Average chlorophyll a, lipid, protein, carbohydrate, tissue biomass, and calcification rates of (A-F) Orbicella faveolata, (G-L) Porites astreoides, and (M- R) Porites divaricata at 0, 1.5, and 11 months of recovery. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a recovery interval. Sample size ranges from 5-9...... 96

13 Figure 2.8. Average stable carbon isotopes of the animal host (δ Ch) and endosymbiont 13 (δ Ce), the difference between the stable carbon isotopes of animal host and 13 endosymbiont (δ Ch-e) , and the stable nitrogen isotopes of the animal host 15 15 (δ Nh) and endosymbiont (δ Ne) of (A-E) Orbicella faveolata, (F-J) Porites astreoides, and (K-O) Porites divaricata at 0, 1.5, and 11 months of recovery. For 13 δ Ch-e, heterotrophy contributes more to the fixed carbon pool when the difference is <0, whereas photosynthesis contributes more when the difference is ≥0. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a specific recovery interval. Sample size ranges from 3-9...... 97

13 Figure 2.9. Average stable carbon isotopes of the skeleton (δ Cs) and stable oxygen 18 isotopes of the skeleton (δ Os) of (A-B) Orbicella faveolata, (C-D) Porites astreoides, and (E-F) Porites divaricata at 0, 1.5, and 11 months of recovery. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a specific recovery interval. 18 Sample size ranges from 3-9. Note that the axis for δ Os is reversed...... 98

18 13 Figure 3.1. Plot of skeletal δ Os vs. δ Csorig for (A) non-bleached and singly bleached Hawaiian corals, and (B) non-bleached and repeat bleached Mexican corals. KIE marks the trend along which kinetic isotope effects occur. Eq represents isotopic 18 equilibrium composition based on two different methods to calculate δ Oeq (Grossman and Ku 1986, Maier 2004). Wi and Su represent winter and summer isotopic equilibrium composition, respectively. Resp and P indicate the carbon isotopic offset from the KIE line due to respiration and photosynthesis, respectively. Slow and Fast refer to calcification rates. NB = non-bleached control, BL = bleached, PC = Porites compressa, MC = Montipora capitata, PL = Porites lobata OF = Orbicella faveolata, PA = Porites astreoides, PD = Porites divaricata. Please see text for further explanation...... 143

18 18 13 13 Figure 3.2. Plots of skeletal δ O (δ Os) vs. (A-F) original skeletal δ C (δ Csorig) and 13 13 (G-L) corrected skeletal δ C (δ Cscorr) for non-bleached and singly bleached Porites compressa, Montipora capitata, and Porites lobata from Hawaii throughout 8-11 months of recovery. ● = 0 month of recovery, ● = 1 or 1.5 months of recovery, ● = 4 or 5 months of recovery, and ● = 8 or 11 months of recovery. NB = non-bleached control, BL = singly bleached. – = KIE line leading 18 18 to δ Oeq after Grossman and Ku (1986), --- = KIE line leading to δ Oeq after Maier (2004). + = outliers excluded from statistical analyses...... 144

throughout 11 months of recovery. ● = 0 month of recovery, ● = 1.5 months of recovery, and ● = 11 months of recovery. NB = non-bleached control, BL = 18 repeat bleached. – = KIE line leading to δ Oeq after Grossman and Ku (1986), --- 18 = KIE line leading to δ Oeq after Maier (2004)...... 145

Figure 3.4. Correlations of measured and isotope-based P/R ratios for non-bleached and bleached Porites compressa, Montipora capitata, and Porites lobata from Hawaii. 13 Isotope-based P/R ratios were computed with both (A-C) δ Csorig (original P/R) 13 18 and (D-F) δ Cscorr (corrected P/R). Further, they were computed using δ Oeq values either after Grossman and Ku (1986) (filled symbols) or after Maier (2004) (open symbols). Dotted line indicates perfect agreement of isotope-based and measured P/R ratios. Treatments and recovery intervals were pooled for each species. r = Spearman’s correlation coefficient when correlation was statistically significant. + = outliers excluded from statistical analyses...... 146

Figure 3.5 Correlations of measured and isotope-based P/R ratios for non-bleached and repeat bleached Orbicella faveolata, Porites astreoides, and Porites divaricata from Mexico at 0 month of recovery. Isotope-based P/R ratios were computed 13 13 with both (A-C) δ Csorig (original P/R) and (D-F) δ Cscorr (corrected P/R). 18 Further, they were computed using δ Oeq values either after Grossman and Ku (1986) (filled symbols) or after Maier (2004) (open symbols). Dotted line indicates perfect agreement of isotope-based and measured P/R ratios. Treatments were pooled for each species. r = Spearman’s correlation coefficient when correlation was statistically significant...... 147

Figure 4.1. Photos of representative coral fragments from (A) Acropora millepora, (B) Pocillopora damicornis, (C) Montipora monasteriata, and (D) Turbinaria reniformis. Rectangles indicate subsamples taken from each fragment for lipid, protein/carbohydrate, and tissue biomass analyses. The remaining tissue was airbrushed for chlorophyll a and endosymbiont density measurements...... 193

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shown for three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate significant differences between 26.5 and 29.0°C within a given

pCO2 level (determined by a posteriori slice tests). The letters a and b indicate

results of the post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 5 and 6. Statistical details can be found in Table 4.2...... 194

three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate

significant differences between 26.5 and 29.0°C within a specific pCO2 level (determined by a posteriori slice tests). The letters a and b indicate results of the

post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 5 and 6. Statistical details can be found in Table 4.3...... 195

three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate

significant differences between 26.5 and 29.0°C within a specific pCO2 level (determined by a posteriori slice tests). The letters a and b indicate results of the

post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 4 and 6. Statistical details can be found in Table 4.4...... 196

xxiii

1. INTRODUCTION

1.1 CORAL BIOLOGY

Reef-building corals belong to the phylum Cnidaria, order Scleractinia (“stony corals”). Most scleractinians are colonial and their polyps are interconnected by living tissue. Each polyp has tentacles for prey capture, an oral opening, and a gastrovascular cavity. The basal/aboral epidermis consists mostly of calicoblastic cells that deposit calcium carbonate as an aragonite skeleton. Most corals live in endosymbiosis with unicellular, photosynthetic dinoflagellates of the genus Symbiodinium (formerly referred to as zooxanthellae). The algal endosymbionts transfer photosynthetically fixed carbon to the coral host, providing up to 100% of its daily energy requirements (e.g. Muscatine et al. 1981; Edmunds, Spencer Davies 1986; Brown et al. 1999). The other major food sources to corals are heterotrophic from feeding on dissolved and particulate organic matter, bacteria, and zooplankton, which provides up to 46% of their daily energy requirements (e.g. Anthony 2000; Palardy et al. 2008; Houlbreque, Ferrier-Pages 2009; Levas 2012).

1.2 CORAL REEFS AND CLIMATE CHANGE

In the present world of global climate change, the future of coral reefs is at risk (e.g. Pandolfi et al. 2003; Sheppard 2003; Hoegh-Guldberg et al. 2007; Pandolfi et al. 2011; Wild et al. 2011; Frieler et al. 2012). A combination of both anthropogenic and natural threats has caused an effective loss of 19% of the world’s reefs, threatens another

35% over the coming decades (Wilkinson 2008), and has put one third of reef corals at elevated risk of extinction (Carpenter et al. 2008). Among these threats, global climate change is the most powerful, least predictable, and least understood (Wilkinson 2008).

Rising levels of atmospheric CO2 cause increases in sea surface temperatures, which in turn are responsible for mass bleaching events where coral communities region- or worldwide lose a significant portion of their vital algal endosymbionts and/or photosynthetic pigments (Hoegh-Guldberg, Smith 1989; Jokiel, Coles 1990; Glynn 1993, 1996; Brown 1997b; Hoegh-Guldberg 1999; Fitt et al. 2001; Baker et al. 2008). As healthy corals often meet most of their daily energy requirements via photosynthesis by their endosymbionts, bleaching threatens their survival and significantly contributes to their worldwide decline. Mass bleaching events have increased in frequency over the past decades (Hoegh-Guldberg 1999; Eakin et al. 2009) and, at the current rate of global warming (~0.2°C/decade), are predicted to occur every 5 years for the majority of reefs worldwide by 2030 (Donner 2009) or when global mean temperatures increase 1.5°C relative to pre-industrial times (Frieler et al. 2012). Yet, the effects of repeat bleaching on coral physiology, recovery capacity, and resilience remain largely unknown.

In addition, increases in atmospheric CO2 concentrations pose another threat to coral reefs by causing acidification of the ocean’s surface waters. About one third of anthropogenic CO2 released into the atmosphere over the past 200 years has been absorbed by the ocean (Sabine et al. 2004a), where it changes the carbonate chemistry of the seawater and ultimately lowers the pH (Caldeira, Wickett 2003). This is of particular concern for calcifying marine organisms because more acidic seawater compromises calcification rates and ultimately enhances dissolution of calcium carbonate (Kleypas et al. 1999; Guinotte, Fabry 2008; Hofmann et al. 2010; Kroeker et al. 2010; Chan, Connolly 2012; Andersson, Gledhill 2013). Over the coming decades, coral reefs will have to cope with both ocean acidification (OA) and warming simultaneously. Recent studies suggest that elevated pCO2 may lower bleaching thresholds (Anthony et al. 2008) and that OA and warming can have complex interactive effects on coral physiology (Reynaud et al. 2003; Anthony et al. 2008; Muehllehner, Edmunds 2008; Harvey et al.

2013), but their combined effects on overall coral health and resilience remain largely unknown.

1.2.1 Coral Bleaching

Coral bleaching is defined as the loss of a significant portion of a coral’s algal endosymbionts and/or photosynthetic pigments (Hoegh-Guldberg, Smith 1989; Jokiel, Coles 1990; Glynn 1993, 1996; Brown 1997b; Hoegh-Guldberg 1999; Fitt et al. 2001; Baker et al. 2008). As corals obtain most of their color from their endosymbionts, this process lets the white skeleton show through the coral tissue, thus making the coral appear pale to white or “bleached”. Bleaching events can be caused by a variety of factors including higher than normal sea surface temperatures (e.g., >1°C maximum summer temperatures) (Glynn 1993; Brown 1997b, 1997a; Lesser 2004), high irradiance (Brown, Dunne 2008), cold-water episodes (Gates et al. 1992), salinity changes (Goreau 1964; Kerswell, Jones 2003), sedimentation stress (Bak 1978; Dollar, Grigg 1981; Rogers 1990), exposure to UV radiation (Gleason, Wellington 1993; Banaszak, Lesser 2009) and air (Brown et al. 1994), bacterial and other infections (Kushmaro et al. 1996), and elevated seawater pCO2 (Pecheux 2002; Anthony et al. 2008). Thermal stress in combination with high irradiance levels is viewed as the principal cause of large-scale mass bleaching events (Hoegh-Guldberg et al. 2007; Baker et al. 2008; Lesser 2011). While all these factors can cause bleaching independently, they can also interact by lowering the threshold temperature at which bleaching occurs (Lesser 2004, 2006). Anthony et al. (2008) reported a synergistic interaction between high temperature and high pCO2 that lowers thermal bleaching thresholds – but this was questioned by Dunne (2010). Consequences of coral bleaching are numerous and range from immediate to more prolonged effects capable of affecting entire coral reef communities as well as ecosystem structure and functioning. Bleaching results in reduced photosynthesis rates (Iglesias-Prieto et al. 1992 ; Lesser 1996; Rodrigues, Grottoli 2007; Levas 2012),

3 calcification (Leder et al. 1991; Mendes, Woodley 2002; Rodrigues, Grottoli 2006; Levas 2012), tissue biomass (Porter et al. 1989; Fitt et al. 1993; Rodrigues, Grottoli 2007; Levas 2012), energy reserves (Porter et al. 1989; Fitt et al. 1993; Rodrigues, Grottoli 2007; Levas 2012), and increased susceptibility to diseases (Bruno et al. 2007; Lesser 2007). It can lead to partial or full mortality depending on the severity of the bleaching event. Longer-term effects include interrupted or compromised reproduction (Szmant, Gassman 1990; Ward et al. 2000), changes in the abundance of other reef organisms such as corallivores (Glynn 1985; Baird 1999) and obligate coral associates (Glynn et al. 1985; Iglesias-Prieto et al. 2003), and enhanced reef erosion (Glynn 1988, 1994).

1.2.1.1 Physiology of Bleached Corals

Metabolism. Due to decreases in endosymbiont density and/or photosynthetic pigments, photosynthesis (P) is significantly reduced in bleached corals (Porter et al. 1989; Fitt, Warner 1995; Jones 1997; Lesser 1997; Grottoli et al. 2006; Rodrigues, Grottoli 2007; Rodrigues et al. 2008; Levas 2012). Less is known about changes in respiration (R) in bleached corals. P:R ratios of bleached corals often decrease (Jokiel, Coles 1977), which can be due to decreases in P (Porter et al. 1989; Fitt, Warner 1995; Rodrigues, Grottoli 2007) or decreases in both P and R (Porter et al. 1989; Rodrigues, Grottoli 2007). Reduced P results in less photosynthetic carbon being translocated from the algal endosymbiont (Symbiodinium sp.; formerly referred to as zooxanthellae) to the coral host (Hughes et al. 2010), which can be quantified as decreases in the per cent Contribution of Zooxanthellae acquired carbon to daily Animal Respiration (CZAR) (Muscatine et al. 1981; Grottoli et al. 2006; Levas 2012). Consequently, bleached corals have to compensate for the loss of autotrophic carbon by employing strategies to conserve energy and/or utilize alternative carbon sources in order to survive and recover from bleaching. Decreasing respiration (i.e., metabolic rate) has been interpreted as one possible strategy to conserve energy in bleached corals (Rodrigues, Grottoli 2007). Other

4 possible strategies include decreases in calcification rates, catabolization of energy reserves, and increases in heterotrophy (see below).

Calcification. Calcification increases with increasing temperatures up to an optimum (e.g. Jokiel, Coles 1977; Reynaud et al. 2003), then decreases or stops when temperatures rise above the maximum summer temperatures causing bleaching stress (Jokiel, Coles 1977; Leder et al. 1991; Mendes, Woodley 2002; Al-Horani et al. 2005; Rodrigues, Grottoli 2006; Levas 2012). As calcification is an energetically costly process that may take up to 30% of a coral’s energy budget (Allemand et al. 2011), decreasing calcification during times of stress such as coral bleaching is generally considered a necessary energetic adjustment to maximize chances of survival and recovery. However, some studies conclude that calcification is energetically cheap and thus responds to environmental stress less than tissue biomass (Anthony, Fabricius 2000; Anthony et al. 2002).

Energy Reserves, Tissue Biomass, and Heterotrophy. Healthy corals acquire up to 100% of their daily energy needs from their endosymbiotic algae as photosynthetically fixed carbon (Muscatine et al. 1981; Edmunds, Spencer Davies 1986) and up to 46% from feeding on zooplankton (Grottoli et al. 2006; Palardy et al. 2008; Levas 2012). Any excess is stored in the host tissue as lipids, which represent the dominant long term energy reserve in corals (Edmunds, Spencer Davies 1986). The two other major energy reserve pools are carbohydrates and protein. Carbohydrates turn over rapidly in corals while structural protein is catabolized only under extreme conditions (Fitt et al. 1993; Rodrigues, Grottoli 2007). Energy reserves and tissue biomass typically decrease in bleached corals (Porter et al. 1989; Fitt et al. 1993; Grottoli et al. 2004; Grottoli et al. 2006; Rodrigues, Grottoli 2007; Thornhill et al. 2011; Levas 2012). However, the Hawaiian coral species Montipora capitata was able to maintain lipid content when bleached (Grottoli et al. 2004), and in a different experiment recovered depleted energy reserves and tissue biomass within 6 weeks after bleaching due to its capacity to

5 dramatically increase feeding rates under these conditions (Grottoli et al. 2006; Rodrigues, Grottoli 2007; Palardy et al. 2008). Further, fed corals were less susceptible to bleaching compared to starved corals (Hoogenboom et al. 2012), and more likely to maintain lipid and protein concentration when subjected to temperature stress (Tolosa et al. 2011). This shows that heterotrophic plasticity and access to food play a critical role in promoting resilience to and recovery from bleaching (Grottoli et al. 2006; Palardy et al. 2008; Tolosa et al. 2011; Hoogenboom et al. 2012). The amount of heterotrophic carbon from zooplankton feeding contributing to a coral’s energy budget can be quantified by calculating the per cent Contribution of Heterotrophically acquired carbon to daily

Animal Respiration (CHARzoo) (Grottoli et al. 2006; Palardy et al. 2008; Levas 2012; Levas et al. 2013). In addition to feeding on zooplankton, corals acquire heterotrophic carbon also from other sources such as bacteria and dissolved and particulate organic matter in the water column (Anthony 2000; Houlbreque, Ferrier-Pages 2009). Levas (2012) showed that healthy corals tend to release dissolved organic carbon (DOC), whereas bleached corals uptake DOC. By calculating the per cent contribution of DOC to a coral’s energy budget (CHARdoc), he showed that DOC is a significant source of heterotrophic carbon in bleached corals, contributing up to 16% of a coral’s energy budget. Thus, zooplankton, DOC and picoplankton combined with CZAR, can provide a detailed estimate of the relative importance of both auto- and heterotrophy in bleached and healthy corals.

1.2.1.2 Recovery From Bleaching

Depending on the severity of the bleaching event, corals can recover from bleaching, and both endosymbiont and host physiology play an essential role in this process. From the endosymbiont perspective, recovery occurs when (1) the remaining endosymbionts reproduce asexually and colonize the bleached tissue (Hayes, Bush 1990; Fitt et al. 1993; Jones et al. 2000), (2) chlorophyll a concentrations recover (Rodrigues, Grottoli 2007), (3) new endosymbionts are recruited from the water column (Baker

2001), or (4) a combination of all three, resulting in the recovery of photosynthesis rates. From the perspective of the coral host, recovery also depends on replenishing energy reserves, restoring tissue biomass, and calcification rates, which can take significantly longer than recovering photosynthesis rates (Rodrigues, Grottoli 2007; Levas et al. 2013). Availability of a healthy zooplankton community is crucial because access to food plays a critical role in promoting resilience to and recovery from bleaching (Grottoli et al. 2006; Rodrigues, Grottoli 2007; Palardy et al. 2008; Tolosa et al. 2011; Hoogenboom et al. 2012). Both photosynthetically and heterotrophically derived carbon are essential for recovery from bleaching events because recent work indicates that corals depend on photosynthesis in endosymbionts to meet daily metabolic needs and to provide energy for calcification, and depend on heterotrophy for tissue growth (Hughes et al. 2010). However, these results are from research on a limited number of species only, and from corals that have been only bleached once (not repeat bleached). Since recovery from bleaching can take more than 8 months (e.g. Fitt et al 1993, Rodrigues and Grottoli 2007), annually repeated bleaching may overwhelm the capacity of corals to recover before the next bleaching event, which would have severe consequences for coral reef resilience. It therefore needs to be determined if other species in other regions, such as the Caribbean where coral reefs are in serious decline (Gardner et al. 2003), will display similar response mechanisms and if repeat bleaching will have different effects on coral physiology and recovery than single bleaching does.

1.2.1.3 Repeat Bleaching

Despite the widespread knowledge that mass bleaching events will occur every 5 years for most coral reefs worldwide and are expected to occur bi-annually in the Caribbean by 2030 based on model predictions (Sheppard 2003; Donner et al. 2007; Donner 2009), almost no studies exist to date examining the effects of repeat bleaching on coral physiology and resilience. There is, however, mounting evidence that prior

7 exposure to thermal stress can result in acclimation and/or potentially adaption of the coral holobiont. For example, when bleaching response was compared across natural bleaching events (4-5 years apart), corals were found to be more resistant in later bleaching events (Guest et al. 2012; Penin et al. 2012), even when temperature stress was more severe (Maynard et al. 2008). Further, corals that were pre-exposed to thermal stress 1-2 weeks (Middlebrook et al. 2008) or 1-3 years (Haslun et al. 2011) prior to a simulated bleaching event, were found to be more tolerant to bleaching stress and to have more effective photoprotective mechanisms (Middlebrook et al. 2008). Similarly, Goniastrea aspera corals seemed to have acquired cross-protection against thermal bleaching after being exposed to solar radiation stress several months earlier (Brown et al. 2002). Reproduction of the temperate coral Oculina patagonica experiencing annual bleaching was not compromised, whereas it was significantly reduced in colonies of the same species experiencing bleaching for the first time (Armoza-Zvuloni et al. 2011). Forereef Siderastrea siderea corals were found to be more vulnerable to ocean warming than backreef or nearshore corals (inferred from lower skeletal extension rates), because they lack the high thermal variability experienced by their backreef and nearshore conspecifics (Castillo et al. 2012). The importance of thermal history in determining bleaching susceptibility was also confirmed by a study that showed that estimating bleaching thresholds from local historical sea surface temperature variability delivered the highest predictive power (Donner 2011). Nevertheless, experiments simulating annually repeated bleaching on ecologically relevant timescales are still lacking, and are desperately needed to determine (1) how frequent thermal stress will impact the physiology and resilience of both endosymbiont and animal host, (2) if their response and recovery differs from that to single bleaching, and (3) which underlying mechanisms promote acclimation and increased temperature tolerance or conversely are associated with reduced tolerance to elevated temperature.

1.2.2 Stable Carbon, Nitrogen, and Oxygen Isotopes in Bleached and Recovering Corals

1.2.2.1 Stable Carbon Isotopes (δ13C)

13 Skeletal carbon isotopes. Stable carbon isotopes of the coral skeleton (δ Cs = per mil deviation of 13C: 12C of the sample relative to 13C: 12C of Vienna Peedee Belemnite limestone) are important paleoclimate proxies for cloud cover (total amount of light), seasonality (temperature and amount of light), and nutrient/zooplankton levels (e.g. Fairbanks, Dodge 1979; Gagan et al. 1994; Swart et al. 1996b; Grottoli 1999; Grottoli, Wellington 1999; Grottoli 2002). They are primarily influenced by metabolic isotope effects, which are driven by light-related photosynthetic and respiratory modulation of the coral internal dissolved inorganic carbon (DIC) pool (Fairbanks, Dodge 1979; Swart 1983a; McConnaughey 1989; McConnaughey et al. 1997; Grottoli 1999; Grottoli, 13 13 Wellington 1999; Grottoli 2002). Skeletal δ C (δ Cs) decreases in healthy corals when light/photosynthesis decreases (e.g. Grottoli, Wellington 1999; Grottoli 2002) and thus also in bleached corals, where photosynthesis is significantly compromised (Porter et al. 1989; Grottoli et al. 2004; Rodrigues, Grottoli 2006; Levas et al. 2013). However, 13 bleached corals do not always show this expected decrease in δ Cs (Leder et al. 1991; Rodrigues, Grottoli 2006; Hartmann et al. 2010; Levas 2012). Feeding on isotopically depleted zooplankton also decreases skeletal δ13C (Grottoli, Wellington 1999; Grottoli 2002; Rodrigues, Grottoli 2006), but usually influences skeletal δ13C less than changes in light or photosynthesis (Grottoli, Wellington 1999; Grottoli 2002). Skeletal δ13C may also vary due to changes in isotopic composition of seawater DIC (Swart et al. 1996b), dissolved organic matter and bacteria (Sorokin 1973; Muscatine, Porter 1977), spawning (Gagan et al. 1994, 1996), and kinetic isotope effects associated with CO2 hydration and hydroxylation during calcification (McConnaughey 1989a, 1989b; Omata et al. 2005).

13 During recovery from bleaching, δ Cs is expected to gradually become more enriched as photosynthesis rates recover, and to approach the values of non-bleached 13 corals. However, δ Cs of recovering corals can surpass that of non-bleached controls (Rodrigues, Grottoli 2006), because it starts approaching isotopic equilibration with seawater under significantly compromised calcification rates (McConnaughey 1989; 13 Rodrigues, Grottoli 2006). This is consistent with the finding that δ Cs of faster growing 13 corals is dominated by metabolic isotope effects, whereas δ Cs of slower growing corals is dominated by kinetic isotope effects (McConnaughey 1989; Omata et al. 2005). In 13 cases of severe bleaching, δ Cs may therefore not record decreases in photosynthesis due to bleaching (Leder et al. 1991; Rodrigues, Grottoli 2006; Levas 2012). Heikoop et al. 18 (2000a) proposed a simple data transformation using skeletal oxygen isotopes (δ Os) to 13 18 separate kinetic and metabolic isotope effects in δ Cs because δ Os is influenced by 13 kinetic but not metabolic isotope effects. Kinetic isotope effects affect δ Cs more than 18 δ Os, typically resulting in carbon being approximately three times more depleted in its heavy isotope than oxygen (McConnaughey 1989a, 1989b). However, the data correction as well as the assumptions of the underlying model (McConnaughey 1989a, 1989b) have never been tested using controlled, replicated experiments where both calcification and 13 photosynthesis were measured directly. Further studies are needed to assess δ Cs responses of corals during temperature stress and recovery from bleaching to better understand how metabolic and kinetic isotope effects affect the bleaching response in the skeleton, and if they influence skeletal carbon isotopes in a predictable manner that allows for a simple data correction as proposed by Heikoop et al. (2000a).

13 13 Tissue carbon isotopes. The δ C of the algal endosymbiont (δ Ce) tracks changes in light/photosynthesis because the endosymbiont incorporates relatively more 12C compared to 13C when photosynthesis rates are low (Muscatine et al. 1989a; Rodrigues, Grottoli 2006). As a consequence, corals living at greater depth as well as bleached and 13 recovering corals tend to have lower δ Ce values than healthy, shallow-water corals

(Muscatine et al. 1989a; Rodrigues, Grottoli 2006). However, this is not always the case (e.g. Levas 2012; Levas et al. 2013). 13 13 The δ C of the host tissue (δ Ch) varies with changes in both photoautotrophy and heterotrophy (e.g. Muscatine et al. 1989a; Rodrigues, Grottoli 2006). As the majority of a healthy coral’s metabolic needs are met by photoautotrophic carbon (e.g. Muscatine et al. 13 1981), δ Ch typically decreases as light and/or photosynthetic rate decreases, but not always (Rodrigues, Grottoli 2006; Levas 2012; Levas et al. 2013). Further, both zooplankton and oceanic particulate and dissolved organic carbon (POC, DOC) have strongly depleted δ13C values ranging from -14 to -25‰ (Rau et al. 1989; Rau et al. 1990; Guo et al. 2003). Therefore, as heterotrophic carbon input becomes increasingly more important in corals living at greater depth (though not always: Grottoli 1999) as well as in 13 bleached and recovering corals, δ Ch can become even more depleted (Muscatine et al. 1989a; Grottoli et al. 2006; Rodrigues, Grottoli 2006). Host tissue and endosymbiont δ13C are typically within 2‰ of each other (Muscatine et al. 1989a; Grottoli et al. 2004; Rodrigues, Grottoli 2006; Levas 2012) due to translocation of photoautotrophic C to the animal host, and depleted by as much as 13‰ relative to the skeleton (McConnaughey et al. 1997; Grottoli et al. 2004; Rodrigues, Grottoli 2006). The difference in δ13C of the host tissue and the algal endosymbiont is considered diagnostic of the relative contribution of photosynthesis and feeding to the fixed carbon pool (Muscatine et al. 1989a; Grottoli et al. 2004; Rodrigues, Grottoli 2006; Levas 2012). Similarly, skeletal δ13C reflects the net integrated changes in proportion of photosynthesis and feeding (Grottoli, Wellington 1999; Grottoli 2000). Therefore, coral stable carbon isotopes can be useful indicators of the physiological changes occurring during bleaching and recovery (Rodrigues, Grottoli 2006), and can be used to study the impacts of repeat bleaching in corals.

1.2.2.2 Stable Nitrogen Isotopes (δ15N)

The stable nitrogen isotopes of coral tissue (δ15N = per mil devation of 15N: 14N of the sample relative to 15N: 14N of air) are useful tracers of nutrient history and pollutant 11 sources in coral reef ecosystems (e.g. Heikoop et al. 2000b; Hoegh-Guldberg et al. 2004). However, relatively little is known about how bleaching and recovery from bleaching affects coral δ15N. Generally, asymbiotic corals have higher δ15N values than symbiotic corals because they mainly feed on particulate organic nitrogen sources already enriched in 15N (Yamamuro et al. 1995; Muscatine et al. 2005). In contrast, symbiotic corals also acquire dissolved inorganic nitrogen (DIN) from seawater via their endosymbionts, which is depleted in 15N (Muscatine et al. 2005). Due to tight recycling of nutrients within the coral holobiont (Muscatine, Porter 1977) and the large uptake of isotopically 15 15 depleted DIN, both the host tissue (δ Nh) and endosymbiont (δ Ne) nitrogen isotopes are depleted compared to asymbiotic corals (Muscatine et al. 2005), and within 1-2‰ of each other (Hoegh-Guldberg et al. 2004; Rodrigues, Grottoli 2006; Levas 2012). Further, the presence of symbiotic cyanobacteria in some corals (Lesser et al. 2004), which fix atmospheric nitrogen depleted in 15N, may provide an alternative – but not mutually exclusive – explanation of why symbiotic coral δ15N is more depleted than asymbiotic δ15N. 15 In the context of coral bleaching, δ Nh values hypothetically should be similar to those of asymbiotic corals, and that they gradually approach δ15N values of symbiotic corals during recovery. This is in fact observed for some (Rodrigues, Grottoli 2006), but not all coral species (Rodrigues, Grottoli 2006; Levas 2012) and may depend on species- specific differences in bleaching mechanisms (Rodrigues, Grottoli 2006). The nitrogen 15 isotopic composition of the endosymbiont (δ Ne) is thought to reflect changes in the uptake of dissolved inorganic nitrogen (DIN) from seawater (Hoegh-Guldberg et al. 2004). Since corals recovering from bleaching require large amounts of nitrogen to rebuild chlorophyll a and endosymbiont densities, their uptake rate of DIN may increase, 15 15 resulting in more enriched δ Ne values. Indeed, δ Ne of bleached corals was found to be significantly enriched compared to non-bleached controls during the early phase of recovery in both Hawaiian and Caribbean coral species (Rodrigues, Grottoli 2006; Levas 2012). Since coral δ15N appears to be a useful proxy of increased DIN uptake during

12 recovery from bleaching, it can be used to determine if similar processes are involved in the recovery of repeat bleached corals, which has not been studied to date.

1.2.2.3 Stable Oxygen Isotopes (δ18O)

18 18 16 Stable oxygen isotopes of the coral skeleton (δ Os = per mil deviation of O: O of the sample relative to 18O: 16O of Vienna Peedee Belemnite limestone) are reliable recorders of sea surface temperature (SST) and salinity (SSS) (e.g. Fairbanks, Dodge 1979; Gagan 18 et al. 1994; Swart et al. 1996a; Druffel 1997; Grottoli, Eakin 2007). Generally, δ Os increases when as salinity increases, and decreases as temperature increases. As coral 18 13 bleaching is caused by periods of elevated SST, δ Os in combination with δ Cs has been suggested to be a multi-proxy signal for past bleaching events (e.g. Porter et al. 1989). 18 Although δ Os has the potential to record elevated SST during bleaching episodes (Porter et al. 1989), this is often not the case due to compromised skeletal growth (Leder et al. 1991; Rodrigues, Grottoli 2006; Levas 2012), concurrent changes in seawater δ18O due to changes in the ratio of evaporation to precipitation, and/or the relatively short duration of the bleaching event (Levas 2012). Overall, the stable C, N, and O isotopes of coral tissue and skeleton are useful proxies of the environmental and physiological changes occurring during coral bleaching and recovery from bleaching, and can therefore be useful tools to study the physiological response of corals to repeat bleaching, and how it differs from their response to single bleaching.

1.2.3 Coral Reefs and Ocean Acidification

Today’s atmospheric CO2 concentrations are about 30% higher than their natural maxima over the past 800,000 years (Siegenthaler et al. 2005; Lüthi et al. 2008) and are increasing at an annual rate of 0.5% (IPCC 2007), which is 200 times faster than any changes that occurred during the last glacial cycles (Siegenthaler et al. 2005; Lüthi et al.

2008). About one third of anthropogenic CO2 released into the atmosphere over the past

200 years has been absorbed by the ocean (Sabine et al. 2004a), where it reacts with seawater and forms weak carbonic acid (H2CO3). Most of the carbonic acid quickly + - dissociates into protons (H ) and bicarbonate (HCO3 ), thereby lowering the pH and altering the carbonate chemistry of seawater (Gattuso et al. 1999; Kleypas et al. 1999; Caldeira, Wickett 2003; Feely et al. 2004). As pH decreases, some of the carbonate ions 2- + (CO3 ) combine with H released from the dissociation of dissolved CO2 to form 2- - bicarbonate, thus decreasing [CO3 ] and increasing [HCO3 ]:

As a consequence, the calcium carbonate saturation state of seawater (ΩCaCO3) decreases, which means that less carbonate ions are available for calcifying marine organisms to form calcium carbonate (CaCO3):

[ ][ ]

2- 2+ Ω is largely determined by [CO3 ] because [Ca ] is nearly conservative in seawater (Kleypas et al. 1999). Present-day tropical surface waters are supersaturated (Ω > 1) with respect to all mineral phases of CaCO3, but more so for calcite (Ωcalc = 5 to 6) than for aragonite (Ωarag = 3 to 4) (Kleypas et al. 1999). Aragonite is the metastable form of

CaCO3 that is precipitated by modern scleractinian corals.

As atmospheric CO2 continues to be absorbed by the ocean, not only does it decrease the saturation state of CaCO3 making it harder for calcifying marine organisms to build their shells and skeletons, but also enhances the dissolution of CaCO3:

Although the surface ocean is predicted to remain supersaturated with respect to CaCO3 even under the worst case scenarios of the Intergovernmental Panel on Climate Change (IPCC 2007), significant decreases in marine calcification rates of 20-60% have been predicted (Gattuso et al. 1999; Kleypas et al. 1999; Feely et al. 2004; Hoegh-Guldberg et al. 2007; Guinotte, Fabry 2008; Doney et al. 2009; Hofmann et al. 2010; Kroeker et al.

2010; Chan, Connolly 2012; Andersson, Gledhill 2013). As reef building requires CaCO3 deposition in excess of physical, chemical, and biological erosion, present-day coral reefs may already experience deficits in net calcification (Kleypas et al. 1999).

1.2.3.1 The Effects of Ocean Acidification on Coral Calcification

Coral calcification, despite being strongly biologically controlled (see Allemand et al. 2011 for review), is affected by changes in seawater pH and carbonate chemistry.

Although the exact mechanisms are not fully understood, elevated pCO2 typically causes decreases in calcification (e.g. Marubini, Atkinson 1999; Leclercq et al. 2000; Langdon, Atkinson 2005; Renegar, Riegl 2005; Schneider, Erez 2006; Anthony et al. 2008; Jokiel et al. 2008; Marubini et al. 2008; Krief et al. 2010; Edmunds et al. 2012; Holcomb et al. 2012) but not always (Reynaud et al. 2003; Jury et al. 2010; Rodolfo-Metalpa et al. 2010; Edmunds 2011; Rodolfo-Metalpa et al. 2011; Edmunds et al. 2012; Houlbreque et al.

2012). When both temperature and pCO2 are elevated, decreases in calcification are often greater than due to one of these factors alone, suggesting an antagonistic interactive effect (Reynaud et al. 2003; Rodolfo-Metalpa et al. 2011). An interactive effect between high temperature and high pCO2 was not observed in the temperate coral Cladocora caespitosa (Rodolfo-Metalpa et al. 2010), or when the elevated temperature was within the normal, seasonal range (Langdon, Atkinson 2005). Further studies are required to gain a better understanding of the interactive effects of temperature and pCO2 on coral physiology and resilience and how that varies among species, since future conditions on reefs will include simultaneous increases in both OA and temperature.

1.2.3.2 The Effects of Ocean Acidification on Coral Energy Reserves

Almost nothing is known about how ocean acidification affects energy reserves in corals. Protein concentrations of Stylophora pistillata were unaffected by either elevated

15 pCO2 or elevated temperature or a combination of both (Reynaud et al. 2003). Similarly, protein content of the temperate coral Cladocora caespitosa was unaffected by any of these factors or their combination (Rodolfo-Metalpa et al. 2010). In contrast, protein concentrations of both massive Porites sp. and Stylophora pistillata seemed to increase at lower pH in another study (Krief et al. 2010). However, differences in experimental running time and pCO2 make comparisons between studies difficult. More studies specifically addressing all three energy reserve pools (i.e., lipids, protein, and carbohydrates) in more species are desperately needed to get a better understanding of the effects of ocean acidification on coral energy reserves, host physiology, and coral resilience.

1.3 DISSERTATION OUTLINE

The goal of this dissertation was to investigate the effects of multiple climate change stressors on the physiology and biogeochemistry of Caribbean and Pacific corals. Specifically, I investigated in two separate experiments how annually repeated coral bleaching and combined ocean acidification and warming affect the overall physiology of corals, and which aspects of their physiology and biogeochemistry appear to render them resilient or not. Further, I studied how coral bleaching and the associated physiological changes are recorded in coral skeletal and tissue isotopes. The results of this research are presented in three chapters:

Chapter 2: The effect of repeat bleaching on the physiology and biogeochemistry of three Caribbean coral species Chapter 3: Kinetic and metabolic isotope effects in coral skeletons: a re- evaluation using experimental coral bleaching as a case study

Chapter 4: Coral energy reserves and calcification in a high-CO2 world at two temperatures

Chapter 2: The effect of repeat bleaching on the physiology and biogeochemistry of three Caribbean coral species

Due to increasing atmospheric CO2 concentrations, coral bleaching has been predicted to increase in frequency and intensity over the coming decades (Hoegh- Guldberg 1999; Eakin et al. 2009). Despite this knowledge, it is currently unknown how repeat bleaching will impact coral physiology, biogeochemistry, and overall resilience to climate change. This is of particular concern for Caribbean coral reefs because they are in significant decline (Gardner et al. 2003), and have been predicted to experience coral bleaching biannually as early as 2030 (Donner et al. 2007). For the first time, three Caribbean coral species (Montastraea faveolata, Porites astreoides, Porites divaricata) were experimentally bleached in two consecutive summers (2009 and 2010) and then their short and long term recovery monitored over one full year. The impacts of repeat bleaching on their physiology and biogeochemistry were assessed in great detail by measuring bleaching intensity and mortality, calcification, tissue biomass, energy reserves (i.e., lipid, protein, and carbohydrate content), photosynthesis and respiration, feeding, and the stable carbon and nitrogen isotopes of the coral tissue. The physiological and biogeochemical responses to repeat bleaching were compared to the response of these coral species to single bleaching (Levas 2012). The physiological variables underlying resilience to repeat bleaching were identified and a working model of the impact of repeat bleaching stress on coral persistence developed.

Chapter 3: Kinetic and metabolic isotope effects in coral skeletons: a re- evaluation using experimental coral bleaching as a case study

Coral skeletal carbon isotopes are important paleoclimate proxies (e.g. Fairbanks, Dodge 1979; Gagan et al. 1994; Swart et al. 1996b; Grottoli 1999; Grottoli, Wellington 1999; Grottoli 2002) and have the potential to record coral bleaching events because

17 reduced photosynthesis rates result in lighter skeletal carbon isotopes (Porter et al. 1989; Grottoli et al. 2004; Rodrigues, Grottoli 2006). However, kinetic isotope effects due to compromised calcification rates in bleached corals often mask metabolic effects and thus the overall bleaching signal in coral skeletons (McConnaughey 1989a; Leder et al. 1991; McConnaughey et al. 1997; Rodrigues, Grottoli 2006; Hartmann et al. 2010; Levas 2012; Levas et al. 2013). Heikoop et al. (2000a) presented a data transformation to correct for the presence of kinetic isotope effects, but the correction as well as the assumptions of the underlying model (McConnaughey 1989a; McConnaughey et al. 1997) have never been tested using controlled, replicated experiments. Three Caribbean coral species (Montastraea faveolata, Porites astreoides, Porites divaricata) were experimentally bleached in two consecutive summers (2009 and 2010) and then their short and long term recovery monitored over one full year after each bleaching. Skeletal carbon isotopes were analyzed and assessed for the presence of kinetic and metabolic isotope effects in both healthy and bleached corals. Skeletal oxygen isotopes were used to correct for the presence of kinetic isotope effects (Heikoop et al. 2000a), and the transformed carbon isotope data were then compared to the measured physiological changes caused by coral bleaching. This enabled me to test experimentally if metabolic effects can be successfully de-convoluted from kinetic effects, and to re- evaluate their relative contributions to the carbon isotopic composition of coral skeletons.

Chapter 4: Coral energy reserves and calcification in a high-CO2 world at two temperatures

The combination of warming and acidifying oceans represents one of the biggest threats to coral reefs today (e.g. Hoegh-Guldberg et al. 2007; Frieler et al. 2012), and while we have begun to understand each of these factors alone, their combined impacts on coral physiology and resilience remain largely unknown. The majority of coral species decrease calcification when seawater pH decreases (Chan, Connolly 2012) but it is becoming increasingly clear that not all species respond to ocean acidification (OA) the

18 same way. Nevertheless, the mechanisms underlying resistance to ocean acidification are poorly understood. Coral energy reserves are important indicators of coral health, and might be catabolized under OA conditions to maintain calcification rates, yet to date have not been studied under OA conditions. Further, elevated temperature may exacerbate or mitigate the impacts of OA on coral physiology, and studies measuring calcification in combination with energy reserves are therefore desperately needed to understand coral resilience under combined ocean acidification and warming. Four Pacific coral species (Acropora millepora, Pocillopora damicornis, Montipora monasteriata, Turbinaria reniformis) were maintained under a total of six conditions for 3.5 weeks, representing three pCO2 levels (382, 607, 741 μatm), and two temperature regimes (26.5, 29.0°C) within each pCO2 level. Coral calcification was measured at the beginning, middle, and end of the experiment to see if calcification rates differ depending on exposure duration. For the first time, tissue biomass, lipid, protein, and carbohydrate content were measured in corals subjected to OA conditions combined with elevated temperature to assess if they are catabolized under OA and/or to maintain calcification under these conditions. Symbiodinium and chlorophyll a concentrations were measured to monitor potential effects of OA and OA with elevated temperature on the algal endosymbiont. The results were then compared to other studies addressing coral physiology under ocean acidification and warming, and mechanisms promoting resilience were assessed.

1.4 REFERENCES

Al-Horani FA, Al-Rousan SA, Manasrah RS, Rasheed MY (2005) Coral calcification: Use of radioactive isotopes and metabolic inhibitors to study the interactions with photosynthesis and respiration. Chemistry and Ecology 21:325-335

Allemand D, Tambutte E, Zoccola D, Tambutte S (2011) Coral calcification, cells to reefs. In: Dubinsky Z, Stambler N (eds) Coral Reefs: An Ecosystem in Transition. Springer, pp 119-150

Andersson AJ, Gledhill DK (2013) Ocean acidification and coral reefs: Effects on breakdown, dissolution, and net calcification. Annual Review of Marine Science 5:1.1-1.28

Anthony KRN (2000) Enhanced particle-feeding capacity of corals on turbid reefs (Great Barrier Reef, Australia). Coral Reefs 19:59-67

Anthony KRN, Connolly SR, Willis BL (2002) Comparative analysis of energy allocation to tissue and skeletal growth in corals. Limnology and Oceanography 47:1417-1429

Anthony KRN, Fabricius KE (2000) Shifting roles of heterotrophy and autotrophy in coral energetics under varying turbidity. Journal of Experimental Marine Biology and Ecology 252:221-253

Anthony KRN, Kline DI, Diaz-Pulido G, Dove S, Hoegh-Guldberg O (2008) Ocean acidification causes bleaching and productivity loss in coral reef builders. Proceedings of the National Academy of Sciences of the USA 105:17442-17446

Armoza-Zvuloni R, Segal R, Kramarsky-Winter E, Loya Y (2011) Repeated bleaching events may result in high tolerance and notable gametogenesis in stony corals: Oculina patagonica as a model. Marine Ecology Progress Series 426:149–159

Baird AH (1999) A large aggregation of Drupella rugosa following the mass bleaching of corals on the Great Barrier Reef. Reef Research 9:6-7

Bak RPM (1978) Lethal and sublethal effects of dredging reef corals. Marine Pollution Bulletin 9:14-16

Baker AC (2001) Reef corals bleach to survive change. Nature 411:765-766

Baker AC, Glynn PW, Riegl B (2008) Climate change and coral reef bleaching: An ecological assessment of long-term impacts, recovery trends and future outlook. Estuarine, Coastal and Shelf Science 80:435-471

Banaszak AT, Lesser MP (2009) Effects of ultraviolet radiation on coral reef organisms. Photochemical and Photobiological Sciences 8:1276-1294

Brown BE (1997a) Adaptations of reef corals to physical environmental stress. Advances in Marine Biology 31:222-299

Brown BE (1997b) Coral bleaching: causes and consequences. Coral Reefs 16 suppl:s129-s138

Brown BE, Dunne RP (2008) Solar radiation modulates bleaching and damage protection in a shallow water coral. Marine Ecology Progress Series 362:99-107

Brown BE, Dunne RP, Ambarsari I, Tissier MDAL, Satapoomin U (1999) Seasonal fluctuations in environmental factors and variations in symbiotic algae and chlorophyll pigments in four Indo-Pacific coral species. Marine Ecology Progress Series 191:53-69

Brown BE, Dunne RP, Goodson MS, Douglas AE (2002) Experience shapes the susceptibility of a reef coral to bleaching. Coral Reefs 21:119–126

Brown BE, Dunne RP, Scoffin TP, LeTissier MDA (1994) Solar damage in intertidal corals. Marine Ecology Progress Series 105:219-230

Bruno JF, Selig ER, Casey KS, Page CA, Willis BL, Harvell CD, Sweatman H, Melendy AM (2007) Thermal stress and coral cover as drivers of coral disease outbreaks Public Library of Science Biology 5:1220-1227

Caldeira K, Wickett ME (2003) Anthropogenic carbon and ocean pH. Nature 425:365

Carpenter KE, Abrar M, Aeby G, Aronson RB, Banks S, Bruckner A, Chiriboga A, Cortés J, Delbeek JC, DeVantier L, Edgar GJ, Edwards AJ, Fenner D, Guzmán HM, Hoeksema BW, Hodgson G, Johan O, Licuanan WY, Livingstone SR, Lovell ER, Moore JA, Obura DO, Ochavillo D, Polidoro BA, Precht WF, Quibilan MC, Reboton C, Richards ZT, Rogers AD, Sanciangco J, Sheppard A, Sheppard C, Smith J, Stuart S, Turak E, Veron JEN, Wallace C, Weil E, Wood E (2008) One-third of reef-building corals face elevated extinction risk from climate change and local impacts. Science 321:560-563

Castillo KD, Ries JB, Weiss JM, Lima FP (2012) Decline of forereef corals in response to recent warming linked to history of thermal exposure. Nature Climate Change:doi: 10.1038/nclimate1577

Chan NCS, Connolly SR (2012) Sensitivity of coral calcification to ocean acidification: a meta-analysis. Global Change Biology:doi: 10.1111/gcb.12011

Dollar SJ, Grigg RW (1981) Impact of a kaolin clay spill on a coral reef in Hawaii. Marine Biology 65:269-276

Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO2 problem. Annual Review of Marine Science 1:169-192

Donner SD (2009) Coping with commitment: Projected thermal stress on coral reefs under different future scenarios. PLoS ONE 4:e5712

Donner SD (2011) An evaluation of the effect of recent temperature variability on the prediction of coral bleaching events. Ecological Applications 21:1718–1730

Donner SD, Knutson TR, Oppenheimer M (2007) Model-based assessment of the role of human-induced climate change in the 2005 Caribbean coral bleaching event. Proceedings of the National Academy of Sciences of the USA 104:5483-5488

Druffel ERM (1997) Geochemistry of corals: Recorders of past ocean chemistry, ocean circulation and climate. Proceedings of the National Academy of Sciences of the USA 94:8354-8361

Dunne RP (2010) Synergy or antagonism - interactions between stressors on coral reefs. Coral Reefs 29:145-152

Eakin CM, Lough JM, Heron SF (2009) Climate variability and change: Monitoring data and evidence for increased coral bleaching stress. In: van Oppen MJH, Lough JM (eds) Coral Bleaching: Patterns, Processes, Causes and Consequences. Springer, Berlin Heidelberg, pp 41-67

Edmunds PJ (2011) Zooplanktivory ameliorates the effects of ocean acidification on the reef coral Porites spp. Limnology and Oceanography 56:2402-2410

Edmunds PJ, Brown D, Moriarty V (2012) Interactive effects of ocean acidification and temperature on two scleractinian corals from Moorea, French Polynesia. Global Change Biology 18:2173-2183

Edmunds PJ, Spencer Davies P (1986) An energy budget for Porites porites (Scleractinia). Marine Biology 92:339-347

Fairbanks RG, Dodge RE (1979) Annual periodicity of the 18O/16O and 13C/12C ratios in the coral Montastea annularis. Geochimica and Cosmochimica Acta 43:1009- 1020

Feely RA, Sabine CL, Lee K, Berelson W, Kleypas J, Fabry VJ, Millero FJ (2004) Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 305:362-366

Fitt WK, Brown BE, Warner ME, Dunne RP (2001) Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs 20:51-65

Fitt WK, Spero HJ, Halas J, White MW, Porter JW (1993) Recovery of the coral Montastraea annularis in the Florida Keys after the 1987 Caribbean "bleaching event". Coral Reefs 12:57-64

Fitt WK, Warner ME (1995) Bleaching patterns of four species of Caribbean reef corals. Biological Bulletin 189:298-307

Frieler K, Meinshausen M, Golly A, Mengel M, Lebek K, Donner SD, Hoegh-Guldberg O (2012) Limiting global warming to 2°C is unlikely to save most coral reefs. Nature Climate Change:doi: 10.1038/nclimate1674

Gagan MK, Chivas AR, Isdale PJ (1994) High-resolution isotopic records from corals using ocean temperature and mass-spawning chronometers. Earth and Planetary Science Letters 121:549-558

Gagan MK, Chivas AR, Isdale PJ (1996) Timing coral-based climatic histories using 13C enrichments driven by synchronized spawning. Geology 24:1009-1012

Gardner TA, Côté IM, Gill JA, Grant A, Watkinson AR (2003) Long-term regions-wide declines in Caribbean corals. Science 301:958-960

Gates RD, Baghdasarian G, Muscatine L (1992) Temperature stress causes host cell detachment in symbiotic cnidarians: implications for coral bleaching. Biological Bulletin 182:324-332

Gattuso J-P, Allemand D, Frankignoulle M (1999) Photosynthesis and calcification at cellular, organismal, and community levels in coral reefs: a review on interactions and control by carbonate chemistry. American Zoologist 39:160-183

Gleason DF, Wellington GM (1993) Ultraviolet radiation and coral bleaching. Nature 365:836-838

Glynn PW (1993) Coral reef bleaching: ecological perspectives. Coral Reefs 12:1-17

Glynn PW (1996) Coral reef bleaching: facts, hypotheses and implications. Global Change Biology 2:495-509

Glynn PW (1985) El Nino-associated disturbance to coral reefs and post disturbance mortality by Acanthaster planci. Marine Ecology Progress Series 26:295-300

Glynn PW (1988) El Nino warming, coral mortality, and reef framework destruction by echinoid bioerosion in the eastern Pacific. Galaxea 7:129-160 23

Glynn PW (1994) State of coral reefs in the Galapagos Islands: natural vs. anthropogenic impacts. Marine Pollution Bulletin 29:131-140

Glynn PW, Perez M, Gilchrist SL (1985) Lipid decline in stressed corals and their crustacean symbionts. Biological Bulletin 168:276-284

Goreau TF (1964) Mass expulsion of zooxanthellaefrom Jamaican reef communities after Hurricane Flora. Science 145:383-386

Grottoli AG (2002) Effect of light and brine shrimp levels on skeletal 13C values in the Hawaiian coral Porites compressa: A tank experiment. Geochimica and Cosmochimica Acta 66:1955-1967

Grottoli AG (2000) Stable carbon isotopes (13C) in coral skeletons. Oceanography 13:93-97

Grottoli AG (1999) Variability of stable isotopes and maximum linear extension in reef- coral skeletons at Kaneohe Bay, Hawaii. Marine Biology 135:437-449

Grottoli AG, Eakin CM (2007) A review of modern coral 18O and 14C proxy records. Earth-Science Reviews 81:67-91

Grottoli AG, Rodrigues LJ, Juarez C (2004) Lipids and stable carbon isotopes in two species of Hawaiian corals, Porites compressa and Montipora verrucosa, following a bleaching event. Marine Biology 145:621-631

Grottoli AG, Rodrigues LJ, Palardy JE (2006) Heterotrophic plasticity and resilience in bleached corals. Nature 440:1186-1189

Grottoli AG, Wellington GM (1999) Effect of light and zooplankton on skeletal C values in the Eastern Pacific corals Pavona clavus and Pavona gigantea. Coral Reefs 18:29-41

Guest JR, Baird AH, Maynard JF, Muttaqin E, Edwards AJ, Campbell SJ, Yewdall K, Affendi YA, Chou LM (2012) Contrasting patterns of coral bleaching susceptibility in 2010 suggest an adaptive response to thermal stress. PLoS ONE 7:e33353. doi:33310.31371/journal.pone.0033353

Guinotte JM, Fabry VJ (2008) Ocean acidification and its potential effects on marine ecosystems. Annals of the New York Academy of Sciences 1134:320-342

Guo L, Noriyuki T, Donald MS, Peter HS (2003) Nitrogen and carbon isotopic composition of high-molecular-weight dissolved organic matter in marine environments. Marine Ecology Progress Series 252:51-60

Hartmann AC, Carilli JE, Norris RD, Charles CD, Deheyn DD (2010) Stable isotopic records of bleaching and endolithic algae blooms in the skeleton of the boulder forming coral Montastraea faveolata. Coral Reefs 29:1079-1089

Harvey BP, Gwynn-Jones D, Moore PJ (2013) Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming. Ecology and Evolution:DOI: 10.1002/ece1003.1516

Haslun JA, Strychar KB, Buck G, Sammarco PW (2011) Coral bleaching susceptibility is decreased following short-term (1–3 year) prior temperature exposure and evolutionary history. Journal of Marine Biology 2011:doi:10.1155/2011/406812

Hayes RL, Bush PG (1990) Microscopic observations of recovery in the reef-building scleractinial coral, Montastraea annularis, after bleaching in a Cayman reef. Coral Reefs 8:203-209

Heikoop JM, Dunn JJ, Risk MJ, Schwarcz HP, McConnaughey TA, Sandeman IM (2000a) Separation of kinetic and metabolic isotope effects in carbon-13 records preserved in reef coral skeletons. Geochimica et Cosmochimica Acta 64:975-987

Heikoop JM, Risk MJ, Lazier AV, Edinger EN, Jompa J, Limmon GV, Dunn JJ, Browne DR, Schwarcz HP (2000b) Nitrogen-15 signals of anthropogenic nutrient loading in reef corals. Marine Pollution Bulletin 40:628-636

Hoegh-Guldberg O (1999) Climate change, coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research 50:839-866

Hoegh-Guldberg O, Mumby PJ, Hooten AJ, Steneck R, Greenfield P, Gomez E, Harvell CD, Sale PF, Edwards AJ, Caldeira K, Knowlton N, Eakin CM, Iglesias-Prieto R, Muthiga N, Bradbury RH, Dubi A, Hatziolos ME (2007) Coral reefs under rapid climate change and ocean acidification. Science 318:1737-1742

Hoegh-Guldberg O, Muscatine L, Goiran C, Siggard D, Marion G (2004) Nutrient- induced perturbations to 13C and 15N in symbiotic dinoflagellates and their coral hosts. Marine Ecology Progress Series 280:105-114

Hoegh-Guldberg O, Smith GJ (1989) The effect of sudden changes in temperature, light, and salinity on the population density and export of zooxanthellae from the reef coral Stylophora pistillata Esper and Seriatopora hystrix Dana Journal of Experimental Marine Biology and Ecology 129:279-303

Hofmann GE, Barry JP, Edmunds PJ, Gates RD, Hutchins DA, Klinger T, Sewell MA (2010) The effect of ocean acidification on calcifying organisms in marine ecosystems: an organism-to-ecosystem perspective. Annual Review of Ecology, Evolution, and Systematics 41:127-147 25

Holcomb M, Cohen AL, McCorkle DC (2012) An investigation of the calcification response of the sceractinian corals Astrangia poculata to elevated pCO2 and the effects of nutrients, zooxanthellae and gender. Biogeosciences 9:29-39

Hoogenboom MO, Campbell DA, Beraud E, DeZeeuw K, Ferrier-Pages C (2012) Effects of light, food availability and temperature stress on the function of photosystem II and photosystem I of coral symbionts. PLoS ONE 7:e30167. doi:30110.31371/journal.pone.0030167

Houlbreque F, Ferrier-Pages C (2009) Heterotrophy in tropical scleractinian corals. Biological Reviews 84:1-17

Houlbreque F, Rodolfo-Metalpa R, Jeffree R, Oberhaensli F, Teyssie J-L, Boisson F, Al- Trabeen K, Ferrier-Pages C (2012) Effects of increased pCO2 on zinc uptake and calcification in the tropical coral Stylophora pistillata. Coral Reefs 31:101-109

Hughes AD, Grottoli AG, Pease TK, Matsui Y (2010) Acquisition and assimilation of carbon in non-bleached and bleached corals. Marine Ecology Progress Series 420:91-101

Iglesias-Prieto R, Matta JL, Robins WA, Trench RK (1992 ) Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proceedings of the National Academy of Sciences of the USA 89:10302-10305

Iglesias-Prieto R, Reyes-Bonilla H, Riosmena-Rodriguez R (2003) Effects of the 1997- 1998 ENSO on coral reef communities in the Gulf of California, Mexico. Geofisica Internacional 42:467-471

IPCC (2007) Climate Change 2007: The physical science basis. Summary for policymakers. Contribution of working group I to the fourth assessment report of the Intergovernmental Panel on Climate Change. http://www.ipcc.ch website

Jokiel PL, Coles SL (1977) Effects of temperature on the mortality and growth of Hawaiian reef corals. Marine Biology 43:201-208

Jokiel PL, Coles SL (1990) Response of Hawaiian and other Indo-Pacific reef corals to elevated temperature. Coral Reefs 8:155-162

Jokiel PL, Rodgers KS, Kuffner IB, Andersson AJ, Cox EF, T. Mackenzie F (2008) Ocean acidification and calcifying reef organisms: a mesocosm investigation. Coral Reefs 27:473-483

Jones RJ (1997) Changes in zooxanthellar densities and chlorophyll concentrations in corals during and after a bleaching event. Marine Ecology Progress Series 158:51- 59

Jones RJ, Ward S, Amri AY, Hoegh-Guldberg O (2000) Changes in quantum efficiency of Photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event. Marine and Freshwater Research 51:63-71

Jury CP, Whitehead RF, Szmant AM (2010) Effects of variation in carbonate chemistry on the calcification rates of Madracis auretenra (Madracis mirabilis sensu Wells, 1973): bicarbonate concentrations best predict calcification rates. Global Change Biology 16:1632-1644

Kerswell AP, Jones RJ (2003) Effects of hypo-osmosis on the coral Stylophora pistillata: nature and cause of 'low-salinity bleaching'. Marine Ecology Progress Series 253:145-154

Kleypas J, Buddemeier RW, Archer D, Gattuso J-P, Langdon C, Opdyke BN (1999) Geochemical consequences of increased atmospheric carbon dioxide on coral reefs. Science 284:118-120

Krief S, Hendy EJ, Fine M, Yam R, Meibom A, Foster GL, Shemesh A (2010) Physiological and isotopic responses of scleractinian corals to ocean acidification. Geochimica and Cosmochimica Acta 74:4988-5001

Kroeker KJ, Kordas RL, Crim RN, Singh GG (2010) Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms. Ecology Letters 13:1419-1434

Kushmaro A, Loya Y, Fine M, Rosenberg E (1996) Bacterial infection and bleaching. Nature 380:396

Langdon C, Atkinson MJ (2005) Effect of elevated pCO2 on photosynthesis and calcification on corals and interactions with seasonal change in temperature/irradance and nutrient enrichment. Journal of Geophysical Research 110:C09S07, doi:10.1029/2004JC002576

Leclercq N, Gattuso J-P, Jaubert J (2000) CO2 partial pressure controls the calcification rate of a coral community. Global Change Biology 6:329-334

Leder JJ, Szmant AM, Swart P (1991) The effect of prolonged "bleaching" on skeletal banding and stable isotopic composition in Montastraea annularis. Coral Reefs 10:19-27

Lesser M, Mazel CH, Gorbunov MY, Falkowski PG (2004) Discovery of symbiotic nitrogen-fixing cyanobacteria in corals. Science 305:997-1000

Lesser MP (2011) Coral bleaching: Causes and mechanisms. In: Dubinsky Z, Stambler N (eds) Coral Reefs: An Ecosystem in Transition. Springer, pp 405-419

Lesser MP (2007) Coral reef bleaching and global climate change: Can coral survive the next century? Proceedings of the National Academy of Sciences of the USA 104:5259-5260

Lesser MP (1996) Elevated temperatures and ultraviolet radiation cause oxidative stress and inhibit photosynthesis in symbiotic dinoflagellates. Limnology and Oceanography 41:271-283

Lesser MP (2004) Experimental coral reef biology. Journal of Experimental Marine Biology and Ecology 300

Lesser MP (1997) Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16:187-192

Lesser MP (2006) Oxidative stress in marine environments: biochemistry and physiological ecology. Annual Review of Physiology 68:253-278

Levas SJ (2012) Biogeochemistry and Physiology of Bleached and Recovering Hawaiian and Caribbean Corals. PhD Thesis, The Ohio State University, p 238

Levas SJ, Grottoli AG, Hughes AD, Osburn CL, Matsui Y (2013) Physiological and biogeochemical traits of bleaching and recovery in the mounding species of coral Porites lobata: Implications for resilience in mounding corals. PLoS ONE 8:e63267. doi:63210.61371/journal.pone.0063267

Lüthi D, Le Floch M, Bereiter B, Blunier T, Barnola J-M, Siegenthaler U, Raynaud D, Jouzel J, Fischer H, Kawamura K, Stocker TF (2008) High-resolution carbon dioxide concentration record 650,000–800,000 years before present. Nature 453:379-382

Marubini F, Atkinson MJ (1999) Effects of lowered pH and elevated nitrate on coral calcification. Marine Ecology Progress Series 188:117-121

Marubini F, Ferrier-Pages C, Furla P, Allemand D (2008) Coral calcification responds to seawater acidification: a working hypothesis towards a physiological mechanism. Coral Reefs 27:491-499

Maynard JA, Anthony KRN, Marshall PA, Masiri I (2008) Major bleaching events can lead to increased thermal tolerance in corals. Marine Biology 155:173–182

McConnaughey T (1989) 13C and 18O isotopic disequilibrium in biological carbonates: I. patterns. Geochimica and Cosmochimica Acta 53:151-162

McConnaughey T (1989a) 13C and 18O isotopic disequilibrium in biological carbonates: I. Patterns. Geochimica et Cosmochimica Acta 53:151-162

McConnaughey T (1989b) 13C and 18O isotopic disequilibrium in biological carbonates: II. In vitro simulation of kinetic isotope effects. Geochimica et Cosmochimica Acta 53:163-171

McConnaughey TA, Burdett J, Whelan JF, Paull CK (1997) Carbon isotopes in biological carbonates: Respiration and photosynthesis. Geochimica et Cosmochimica Acta 61:611-622

Mendes JM, Woodley JD (2002) Effect of the 1995-1996 bleaching event on polyp tissue depth, growth, reproduction and skeletal band formation in Montastraea annularis. Marine Ecology Progress Series 235:93-102

Middlebrook R, Hoegh-Guldberg O, Leggat W (2008) The effect of thermal history on the susceptibility of reef-building corals to thermal stress. Journal of Experimental Biology 211:1050-1056

Muehllehner N, Edmunds PJ (2008) Effects of ocean acidification and increased temperature on skeletal growth of two scleractinian corals, Pocillopora meandrina and Porites rus. Proceedings of the 11th International Coral Reef Symposium, Ft. Lauderdale, Florida, 7-11 July 2008

Muscatine L, Goiran C, Land L, Jaubert J, Cuif J-P, Allemand D (2005) Stable isotopes (d13C and d15N) of organic matrix from coral skeleton. Proceedings of the National Academy of Sciences of the USA 102:1525-1530

Muscatine L, McCloskey LR, Marian RE (1981) Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration. Limnology and Oceanography 26:601-611

Muscatine L, Porter JW (1977) Reef corals: mutualistic symbioses adapted to nutrient- poor environments. BioScience 27:454-460

Muscatine L, Porter JW, Kaplan IR (1989) Resource partitioning by reef corals as determinded from stable isotope composition. I. 13C of zooxanthellae and animal tissue vs. depth. Marine Biology 100:185-193

Omata T, Suzuki A, Kawahat H, Okamoto M (2005) Annual fluctuation in the stable carbon isotope ratio of coral skeletons: The relative intensities of kinetic and metabolic isotope effects. Geochimica and Cosmochimica Acta 69:3007-3016

Palardy JE, Rodrigues LJ, Grottoli AG (2008) The importance of zooplankton to the daily metabolic carbon requirements of healthy and bleached corals at two depths. Journal of Experimental Marine Biology and Ecology 367:180-188

Pandolfi JM, Bradbury RH, Sala E, Hughes TP, Bjorndal KA, Cooke RG, McArdle D, McClenachan L, Newman MJH, Paredes G, Warner RR, Jackson JBC (2003) Global trajectories of the long-term decline of coral reef ecosystems. Science 301:955-958

Pandolfi JM, Connolly SR, Marshall DJ, Cohen AL (2011) Projecting coral reef futures under global warming and ocean acidification. Science 333:418-422

Pecheux M (2002) CO2 increase, a direct cause of coral reef mass bleaching? Marine Life 12:63-68

Penin L, Vidal-Dupiol J, Adjeroud M (2012) Response of coral assemblages to thermal stress: are bleaching intensity and spatial patterns consistent between events? Environmental Monitoring and Assessment:doi: 10.1007/s10661-10012-12923- 10663

Porter JW, Fitt WK, Spero HJ, Rogers AD, White MW (1989) Bleaching in reef corals: physiological and stable isotopic responses. Proceedings of the National Academy of Sciences of the USA 86:9342-9346

Rau GH, Takahashi T, Des Marais DJ (1989) Latitudinal variations in plankton 13C: implications for CO2 and prdocutivity in past oceans. Nature 341:516-518

Rau GH, Teyssie J-L, Rassoulzadegan F, Fowler SW (1990) 13C/12C and 15N/14N variations among size-fractionated marine particles: implications for their origin and trophic relationships. Marine Ecology Progress Series 59:33-38

Renegar DA, Riegl B (2005) Effect of nutrient enrichment and elevated CO2 partial pressure on growth rate of Atlantic scleractinian coral Acropora cervicornis. Marine Ecology Progress Series 293:69-76

Reynaud S, Leclercq N, Romaine-Lioud S, Ferrier-Pages C, Jaubert J, Gattuso J-P (2003) Interacting effects of CO2 partial pressure and temperature on photosynthesis and calcification in a scleractinian coral. Global Change Biology 9:1660-1668

Rodolfo-Metalpa R, Houlbreque F, Tambutte E, Boisson F, Baggini C, Patti FP, Jeffree R, Fine M, Foggo A, Gattuso J-P, Hall-Spencer JM (2011) Coral and mollusc resistance to ocean acidification adversely affected by warming. Nature Climate Change 1:308-312

Rodolfo-Metalpa R, Martin S, Ferrier-Pages C, Gattuso J-P (2010) Response of the temperate coral Cladocora caespistosa to mid- and long-term exposure to pCO2 and temperature levels projected for the year 2100 AD. Biogeosciences 7:289-300

Rodrigues LJ, Grottoli AG (2006) Calcification rate and the stable carbon, oxygen, and nitrogen isotopoes in the skeleton, host tissue, and zooxanthellae of bleached and recovering Hawaiian corals. Geochimica and Cosmochimica Acta 70:2781-2789

Rodrigues LJ, Grottoli AG (2007) Energy reserves and metabolism as indicators of coral recovery from bleaching. Limnology and Oceanography 52:1874-1882

Rodrigues LJ, Grottoli AG, Lesser MP (2008) Long-term changes in the chlorophyll fluorescence of bleached and recovering corals from Hawaii. The Journal of Experimental Biology 211:2502-2509

Rogers CS (1990) Responses of coral reefs and reef organisms to sedimentation. Marine Ecology Progress Series 62:185-202

Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL, Wanninkhof R, Wong CS, Wallace DWR, Tilbrook B, Millero FJ, Peng T-H, Kozyr A, Ono T, Rios AF (2004) The oceanic sink for anthropogenic CO2. Science 305:367-371

Schneider K, Erez J (2006) The effect of carbonate chemistry on calcification and photosynthesis in the hermatypic coral Acropora eurystoma. Limnology and Oceanography 51:1284-1293

Sheppard CRC (2003) Predicted recurrences of mass coral mortality in the Indian Ocean. Nature 425:294-297

Siegenthaler U, Stocker TF, Monnin E, Lüthi D, Schwander J, Stauffer B, Raynaud D, Barnola J-M, Fischer H, Masson-Delmotte V, Jouzel J (2005) Stable carbon cycle-climate relationship during the Late Pleistocene. Science 310:1313-1317

Sorokin Y (1973) On the feeding of some scleractinian corals with bacteria and dissolved organic matter. Limnology and Oceanography 18:380-385

Swart P, Dodge RE, Hudson HJ (1996a) A 240-year stable oxygen and carbon isotopic record in a coral from South Florida: Implications for the prediction of precipitation in Southern Florida. Palaios 11:362-373

Swart P, Leder JJ, Szmant AM, Dodge RE (1996b) The origin of variations in the isotopic record of scleractinian corals: II. Carbon. Geochimica and Cosmochimica Acta 60:2871-2885

Swart PK (1983) Carbon and oxygen isotope fractionation in scleractinian corals: A review. Earth Science Reviews 19:51-80 31

Szmant AM, Gassman NJ (1990) The effects of prolonged "bleaching" on the tissue biomass and reproduction of the reef coral Montastrea annularis. Coral Reefs 8:217-224

Thornhill DJ, Rotjan RD, Todd BD, Chilcoat GC, Iglesias-Prieto R, Kemp DW, LaJeunesse TC, McCabe Reynolds J, Schmidt GW, Shannon T, Warner ME, Fitt WK (2011) A connection between colony biomass and death in Caribbean reef- building corals. PLoS ONE 6:e29535. doi:29510.21371/journal.pone.0029535

Tolosa I, Treignier C, Grover R, Ferrier-Pages C (2011) Impact of feeding and short-term temperature stress on the content and isotopic signature of fatty acids, sterols, and alcohols in the scleractinian coral Turbinaria reniformis. Coral Reefs 30:763–774

Ward S, Harrison PJ, Hoegh-Guldberg O (2000) Coral bleaching reduces reproduction of scleractinian corals and increases susceptibility to future stress. Proceedings of the 9th International Coral Reef Symposium 2:1123-1128

Wild C, Hoegh-Guldberg O, Naumann MS, Colombo-Pallotta MF, Ateweberhan M, Fitt WK, Iglesias-Prieto R, Palmer C, Bythell JC, Ortiz J-C, Loya Y, van Woesik R (2011) Climate change impedes scleractinian corals as primary reef ecosystem engineers. Marine and Freshwater Research 62:205-215

Wilkinson C (2008) Status of coral reefs of the world: 2008. Global Coral Reef Monitoring Network and Reef and Rainforest Research Centre:296p

Yamamuro M, Kayanne H, Minagawa H (1995) Carbon and nitrogen stable isotopes of primary producers in coral reef ecosystems. Limnology and Oceanography 40:617-621

2. THE EFFECT OF REPEAT BLEACHING ON THE PHYSIOLOGY AND BIOGEOCHEMISTRY OF THREE CARIBBEAN CORAL SPECIES

Verena Schoepf1, Andréa G. Grottoli1, Stephen J. Levas1, Matthew D. Aschaffenburg2, Justin H. Baumann1, Mark E. Warner2

1 School of Earth Sciences, The Ohio State University, Columbus, OH, USA 2 School of Marine Science and Policy, University of Delaware, Lewes, DE, USA.

2.1 ABSTRACT

Mass coral bleaching events where coral communities worldwide lose their vital algal endosymbionts and/or photosynthetic pigments have increased in frequency and intensity over the past decades. As atmospheric CO2 concentrations keep increasing, bleaching events have been predicted to occur annually worldwide later this century, and even sooner in the Caribbean. However, despite this knowledge, the impacts of annually recurring coral bleaching on physiology, biogeochemistry, and overall resilience remain largely unknown. Using replicated, controlled tank experiments, we simulated annually recurring coral bleaching by subjecting three Caribbean coral species (Orbicella faveolata, Porites astreoides, and Porites divaricata) to elevated temperature for 2.5 weeks in two consecutive years. The impacts of repeat bleaching stress on coral physiology (i.e., metabolism, heterotrophy, chlorophyll a, energy reserves, tissue biomass, calcification) as well as biogeochemistry (i.e., tissue and skeletal stable isotopes) were assessed immediately after repeat bleaching, and after both short and long term recovery (1.5 and 11 months, respectively). By comparing the response of these coral species to single versus repeat bleaching, we show that repeat bleaching can dramatically alter coral thermal tolerance with the potential for both rapid acclimation (P. divaricata) and increased bleaching susceptibility (P. astreoides) to repeat bleaching stress. Thus, susceptibility or resistance to single bleaching is a poor predictor for a coral’s response to repeat bleaching. While two out of the three species studied here were able to fully recover from repeat bleaching within one year, P. astreoides was still impacted 11 months later, suggesting that this species might face significant demise in a future of frequent thermal stress. Combined with other stressors such as ocean acidification, it is therefore likely that over the coming decades, Caribbean coral reefs – and potentially reefs worldwide – will experience significant shifts in coral community composition and diversity, and potential extinction of temperature- and pH- sensitive species.

2.2 INTRODUCTION

In the present world of global climate change, the future of coral reefs is at risk (Hughes et al. 2003; Pandolfi et al. 2003; Sheppard 2003; Bellwood et al. 2004; Hoegh- Guldberg et al. 2007; Pandolfi et al. 2011; Wild et al. 2011; Frieler et al. 2012). A combination of both anthropogenic and natural threats has caused an effective loss of 19% of the world’s reefs, threatens another 35% over the coming decades (Wilkinson 2008), and has put one third of reef corals at elevated risk of extinction (Carpenter et al.

2008). Rising levels of atmospheric CO2 cause increases in sea surface temperatures (Levitus et al. 2009), which in turn are responsible for mass bleaching events where coral communities region- or worldwide lose a significant portion of their vital algal endosymbionts and/or photosynthetic pigments (Hoegh-Guldberg, Smith 1989; Jokiel, Coles 1990; Glynn 1993, 1996; Brown 1997b; Hoegh-Guldberg 1999; Fitt et al. 2001; Baker et al. 2008). As healthy corals typically meet most of their daily energy requirements via photosynthesis by their endosymbionts (e.g. Muscatine et al. 1981), bleaching threatens their survival and significantly contributes to their worldwide decline. Mass bleaching events have increased in frequency over the past decades (Hoegh- Guldberg 1999; Eakin et al. 2009) and, at the current rate of global warming (~0.2°C per decade), are predicted to occur every 5 years for the majority of reefs worldwide by 2030 (Sheppard 2003; Donner 2009) or when global mean temperatures increase 1.5°C relative to pre-industrial times (Frieler et al. 2012). Once they exceed 2.0°C warming relative to pre-industrial times, coral reefs worldwide are likely to experience severe bleaching annually, and more than 90% will be at risk of long term degradation (Frieler et al. 2012). However, despite this knowledge, the effects of annual bleaching on coral physiology, recovery capacity, and resilience remain poorly understood. Grottoli et al. (in review) showed for the first time that some species of Caribbean coral will be able to acclimate to annual bleaching stress, while others may become more susceptible to bleaching. This is consistent with mounting evidence that the thermal history of corals influences bleaching susceptibility, and that prior exposure to thermal

35 stress can result in acclimation and/or potentially adaption of the coral holobiont. For example, when bleaching response of coral communities was compared across natural bleaching events (4-5 years apart), they were found to be more resistant in later bleaching events (Maynard et al. 2008; Guest et al. 2012; Penin et al. 2013), even when temperature stress was more severe (Maynard et al. 2008; Penin et al. 2013). Further, when some species of corals were pre-exposed to thermal stress 1-2 weeks (Middlebrook et al. 2008) or 1-3 years (Haslun et al. 2011) prior to a simulated bleaching event, they were found to be more tolerant to bleaching stress (Middlebrook et al. 2008; Haslun et al. 2011) and to have more effective photoprotective mechanisms (Middlebrook et al. 2008). Siderastrea siderea corals experiencing high diurnal and seasonal thermal variability were found to maintain calcification rates despite decades of ocean warming compared to conspecifics from a more stable thermal environment (Castillo et al. 2012). Further, the importance of thermal history in determining bleaching susceptibility was also confirmed by a study that showed that estimating bleaching thresholds from local historical sea surface temperature variability delivered the highest predictive power (Donner 2011). However, no studies to date have experimentally evaluated the effect of annual bleaching on coral physiology and biogeochemistry in detail, nor the impact of recurring bleaching on long- term recovery. Coral bleaching represents a form of environmental stress that results in severe resource limitation accompanied by dramatic changes in physiology. Both partners of the endosymbiosis have to employ a variety of strategies to ensure the survival and recovery of the coral holobiont. Although the algal endosymbiont (Symbiodinium sp.) plays a crucial role in determining bleaching susceptibility (e.g. Buddemeier, Fautin 1993; Baker 2001; Baker et al. 2004; Berkelmans, van Oppen 2006; Jones et al. 2008), recent studies have shown that the animal host is also essential in determining tolerance to bleaching and promoting recovery (Grottoli et al. 2006; Baird et al. 2008; Anthony et al. 2009; Fitt et al. 2009). Healthy corals typically acquire most of their carbon photosynthetically via their endosymbionts (e.g. Muscatine et al. 1981; Falkowski et al. 1984; Edmunds, Spencer Davies 1986; Grottoli et al. 2006; Tremblay et al. 2012a). In addition, they

36 acquire heterotrophic carbon from feeding on zooplankton, dissolved and particulate organic matter, and bacteria, (e.g. Anthony 2000; Grottoli et al. 2006; Palardy et al. 2008; Houlbreque, Ferrier-Pages 2009; Levas 2012; Tremblay et al. 2012b). However, when bleached, photosynthesis rates typically decline significantly (Porter et al. 1989; Fitt, Warner 1995; Jones 1997; Lesser 1997; Grottoli et al. 2006; Rodrigues, Grottoli 2007; Rodrigues et al. 2008; Levas 2012), resulting in dramatic decreases in the amount of carbon being translocated to the host (Hughes et al. 2010). As a consequence, the animal host has to employ one or more of the following strategies to sustain itself and facilitate recovery: (1) decrease calcification (Jokiel, Coles 1977; Leder et al. 1991; Mendes, Woodley 2002; Rodrigues, Grottoli 2006; Levas 2012; Levas et al. 2013; Grottoli et al. in review), (2) catabolize energy reserves (Porter et al. 1989; Fitt et al. 1993; Grottoli et al. 2004; Grottoli et al. 2006; Rodrigues, Grottoli 2007; Thornhill et al. 2011; Levas 2012; Grottoli et al. in review), (3) increase feeding (Grottoli et al. 2006; Palardy et al. 2008; Anthony et al. 2009; Levas 2012; Grottoli et al. in review), (4) decrease respiration (Rodrigues, Grottoli 2007; Levas 2012) and/or (5) translocate carbon to the endosymbiont to facilitate their recovery (Hughes et al. 2010). Not surprisingly, large amounts of energy reserves as well as high feeding rates have been found to promote bleaching resilience and recovery (Grottoli et al. 2006; Anthony et al. 2009). Biogeochemical studies have further advanced our understanding of the physiological changes occurring during bleaching and recovery from bleaching. The 13 13 stable carbon isotopic composition of the animal host (δ Ch) and endosymbiont (δ Ce) track changes in photosynthesis and feeding rates as well as their relative contribution to 13 the internal carbon pool (δ Ch- e), whereas their stable nitrogen isotopic composition 15 15 (δ Nh and δ Ne) record the inorganic and organic sources of nitrogen to the coral holobiont (Heikoop et al. 2000b; Hoegh-Guldberg et al. 2004; Rodrigues, Grottoli 2006; Levas et al. 2013). Further, skeletal stable carbon isotopes are sensitive to changes in the sources of carbon to the animal host (Fairbanks, Dodge 1979; Muscatine et al. 1981; Porter et al. 1989; McConnaughey 1989a; Swart et al. 1996b; McConnaughey et al. 1997; Grottoli, Wellington 1999; Grottoli 2000, 2002; Rodrigues, Grottoli 2006), while skeletal

37 oxygen isotopes (δ18O) record changes in seawater temperature when salinity is relatively constant (e.g. Fairbanks, Dodge 1979; Porter et al. 1989; Gagan et al. 1994; Swart et al. 1996a; Druffel 1997; Grottoli, Eakin 2007). However, in cases of severe bleaching associated with compromised calcification, skeletal isotopic signatures can become unclear and sometimes be difficult to interpret (Leder et al. 1991; Rodrigues, Grottoli 2006; Hartmann et al. 2010; Levas 2012). Detailed physiological measurements coupled with isotopic analyses are therefore powerful tools to study the impact of repeat bleaching on the coral holobiont and to identify the underlying factors that promote bleaching tolerance, recovery, and overall resilience to frequent thermal stress. As bleaching events are expected to increase in frequency and intensity over the coming decades (Hoegh-Guldberg 1999; Hoegh- Guldberg et al. 2007; Donner 2009; Eakin et al. 2009; Frieler et al. 2012), it is critical to understand if coral species respond to repeat bleaching the same way they do to single bleaching, and if repeat bleaching will overwhelm their capacity to recover in between bleaching events. For example, Porites astreoides has a high tolerance to single bleaching but was extremely sensitive to annual temperature stress (Aschaffenburg 2012; Levas 2012; Grottoli et al. in review). Further, bleached P. compressa required up to 8 months to replenish energy reserves and tissue biomass, even though photosynthesis rates and chlorophyll a concentrations had already recovered 1.5 and 4 months after the bleaching event, respectively (Rodrigues, Grottoli 2007; Hughes, Grottoli in review). Understanding coral resilience in a future of frequent thermal stress is of particular importance in the Caribbean, where coral reefs are in serious decline (Gardner et al. 2003; Eakin et al. 2010; Perry et al. 2013), heavily affected by human impacts (Pandolfi, Jackson 2006; Mora 2008), and recovery of bleached reefs is slow to non-existent (Baker et al. 2008). Further, mass bleaching events have been predicted to occur biannually in the Eastern Caribbean by 2030 (Donner et al. 2007). Existing studies assessing coral bleaching in the Caribbean have provided important knowledge about the physiology of singly bleached Caribbean corals or bleaching events separated by 3-5 years (Porter et al. 1989; Fitt et al. 1993; e.g. Warner et

38 al. 1999; Fitt et al. 2000). Grottoli et al. (in review) showed for the first time the cumulative impact of annual bleaching stress on three Caribbean species (measured over two consecutive years) but did not assess long term recovery and potential prolonged impacts on coral physiology. Further, impacts of recurring annual bleaching on coral biogeochemistry have not been studied to date and could significantly enhance our understanding of the mechanisms driving resilience to and recovery from annual bleaching events. The present study is thus the first to assess the impact of annually repeated coral bleaching on both physiology and biogeochemistry of Orbicella faveolata (formerly Montastraea faveolata (Budd et al. 2012)), P. astreoides, and P. divaricata with an emphasis on long term recovery and the response of the animal host. We conducted a controlled experiment simulating annually repeated bleaching on ecologically relevant timescales to determine (1) how annually recurring thermal stress will impact the physiology and resilience of both endosymbiont and animal host, (2) if their response and recovery differs from that to single bleaching, and (3) which underlying mechanisms promote acclimation, increased temperature tolerance, and rapid recovery - or conversely are associated with reduced tolerance to elevated temperature and prolonged recovery from bleaching.

2.3 MATERIAL AND METHODS

2.3.1 Coral Collection

Coral fragments of Orbicella faveolata, Porites astreoides, and Porites divaricata were collected in July 2009 from reefs near Puerto Morelos, Mexico, from shallow depth (3-8 m) (see Table 2.1 for details on collection dates, sites, and depths). Eight fragments were collected from nine parent colonies (a total of 72 fragments per species), which were separated by at least 10 m to increase the likelihood of sampling distinct genotypes. All fragments were mounted onto pre-labeled PVC tiles using marine epoxy, and maintained in outdoor flow-through seawater tanks shaded with two layers of neutral 39 density mesh to mimic light levels at collection depth (~600 μmol photons m-2 s-1, measured with a 2-pi quantum sensor (Licor Inc.)). Incoming seawater was pumped from the back-reef lagoon and passed through coarse filters to remove debris. Corals were allowed to recover for 5 days until visible tissue growth was observed, and then buoyantly weighed prior to the start of the experiment.

2.3.2 Repeat Bleaching Experiment

Single Bleaching Treatment. On 14 July 2009, half of the coral fragments from each parent colony were randomly assigned to each treatment: (1) ambient control fragments (later referred to as non-bleached corals) were maintained in tanks with ambient seawater temperature (30.66 ± 0.24°C), whereas (2) treatment fragments (later referred to as singly bleached corals) were placed in tanks with elevated seawater temperature (increased from 30.66 to 31.48 ± 0.20°Cover seven days and then held at that temperature for another eight days) (Fig. 2.1). Seawater temperature in the treatment tanks was gradually elevated using industrial heaters to mimic warming rates at the onset of a natural bleaching event and to prevent heat shock. Fragments were rotated daily within and among tanks of the same treatment to minimize any position or tank-specific effects. After a total of 15 days (29 July 2009 all coral fragments were buoyantly weighed and then placed on the back reef (3.5 m, 600-700 μmol photons m-2 s-1) to recover in situ for a full year.

Repeat Bleaching Treatment. On 9 July 2010, all coral fragments were recollected, thoroughly cleaned, and then buoyantly weighed. On 20 July 2010, all corals that had served as ambient control fragments the previous summer were placed in tanks with ambient seawater (30.40 ± 0.23°C) (later referred to as non-bleached corals), and all corals that had been used as treatment fragments were placed in tanks at elevated temperature once again (31.60 ± 0.24°C) (later referred to as repeat bleached corals) for 17 days (Fig. 2.1). The seawater temperature in the treatment tanks was gradually

40 elevated over the course of 7 days. Although the repeat bleaching treatment was designed to be as similar as possible to the single bleaching treatment, an additional 2 days were deemed necessary to achieve a similar amount of temperature stress due to a bad weather episode, which resulted in unusually low temperatures in both treatments for 2 days. During the last days of the repeat bleaching treatment, photosynthesis and respiration rates were measured on both ambient control and treatment corals (6-7 August 2010). On 6 August 2010, all tanks were returned to ambient temperature levels. Corals were visually assessed for their bleaching status, and then buoyantly weighed. Nine fragments per treatment (i.e., one fragment per parent colony) were then frozen for the physiological measurements (see below). The remaining fragments were placed back in the back-reef lagoon (3.5 m, 600-700 μmol photons m-2 s-1) for either feeding experiments (another nine fragments per treatment; see below) or to recover in situ (the remaining 50% of the fragments)

Recovery from Repeat Bleaching Treatment. To assess short- and long-term recovery from repeat bleaching, nine fragments per treatment (i.e., one fragment per parent colony) were collected 1.5 and 11 months after the repeat bleaching treatment (22 September 2010 and 14 June 2011, respectively) (Fig. 2.1). They were visually assessed for their bleaching status and mortality, thoroughly cleaned and buoyantly weighed, and then frozen for the following physiological measurements (see below): chlorophyll a, lipid, protein, carbohydrate content, tissue biomass, tissue and skeletal isotopes. Feeding, photosynthesis, and respiration rates were not measured at these recovery intervals.

A previous study (Levas 2012) assessed the physiological and biogeochemical responses of all three coral species to single bleaching at the same recovery intervals that were used to assess recovery from repeat bleaching. This allowed for the comparison of their physiological and biogeochemical responses to single bleaching to their responses to repeat bleaching.

2.3.3 Physiological Analyses

Bleaching and Mortality. Bleaching status and (partial) mortality of each fragment were visually assessed after each bleaching treatment and at each recovery interval prior to freezing according to metrics in Rodrigues, Grottoli (2007) and Levas (2012). In brief, corals were assigned one of the following designations based on their appearance: 1- non-bleached fragments were dark brown in color and completely covered by living tissue, 2- partially bleached fragments were either entirely pale but not white or some of the tissue was bleached and some healthy, and they were completely covered by live tissue, 3- bleached fragments were either 100% white in color or >50% white and the rest pale, and they were completely covered by living tissue, 4- partially dead fragments were partially covered by filamentous or encrusting algae (or both) and partially covered by patches of living tissue that varied in color from pale to dark brown, 5- partially dead bleached fragments were partially covered by filamentous or encrusting algae (or both) and partially covered by patches of living tissue that was white in color, and 6- dead fragments were completely covered by filamentous or encrusting algae (or both), with no living tissue remaining.

Metabolism and CZAR. Photosynthesis (P) and respiration (R) rates were measured to quantify the impacts of repeat bleaching on coral metabolism, and to calculate the amount of photoautotrophically carbon available to the animal host to meet metabolic demands (CZAR). On 6 and 7 August 2010, ambient control and treatment corals were incubated to measure gross P and day and night R. They were individually placed in 500 ml sealed UV-transparent Plexiglas chambers filled with seawater and a stir bar, and all gaseous oxygen was purged by bubbling in nitrogen. Stir bars ensured adequate mixing within the chambers. Chambers were then placed into a 20 L Plexiglas tank filled with freshwater, which was temperature controlled using a heater/chiller and set to 30°C or 32°C for ambient control and treatment fragments, respectively. P and R rates were determined from the change in dissolved O2 concentration in the chambers

42 using D901 Miniature Galvanic DO2 probes (Qubit Systems) connected to a laptop, which ran LoggerPro software to record and analyze data. P and R rates were determined by taking the slopes of the LoggerPro graphs of dissolved O2 concentration versus time. They were allowed to stabilize for the first 1-5 minutes of each run; only data collected after this stabilization period were used to calculate the P and R rates. Daytime R was determined by covering the chambers with black plastic and allowing for the fragments to establish a constant respiration rate (usually 10 minutes). Shading was removed and an array of LED lights was turned on exposing the corals to 415 μmol photons m-2 s-1of light to induce maximal photosynthesis (usually 10-15 minutes). After a photosynthetic rate was established, corals were removed from the chamber and placed in their respective tanks. At night, evening respiration rates were determined for the same corals. The same procedure as above was carried out, but only for respiration. The percent contribution of photoautotrophic carbon to daily animal respiration (CZAR) (Muscatine et al. 1981) was then calculated for each fragment using P and R rates assuming a 12:12 diurnal cycle.

Feeding Experiments and CHAR. Feeding rates were measured after repeat bleaching to calculate the amount of heterotrophically carbon available to the animal host to meet metabolic demands (CHAR). After the repeat bleaching treatment, a quarter of all ambient control and treatment corals were placed on the reef to acclimate for one week. Feeding experiments were performed on two nights (August 13 and 15, 2010). Coral feeding was performed according to methods outlined in Palardy et al (2005). In brief, coral fragments were starved for 12 hours prior to feeding by covering them with clear 50 L plastic chambers. Chambers had windows covered with 50 μm Nitex screen to allow for sufficient flow but prevent zooplankton from entering (Palardy et al. 2005), thus enabling the corals to fully empty their guts. One hour after dusk, the chambers were removed and the coral fragments were allowed to feed for one hour. Then, fragments were collected and fixed in 4% formalin to prevent digestion of ingested zooplankton. Within 48 hours of feeding, 150 polyps or all polyps on the coral fragments (whichever came first) were dissected as per Palardy et al. (2005). The number of zooplankton eaten

43 per polyp, as well as the prey type and size were recorded. Feeding rates were standardized to grams ash-free-dry-weight (gdw) of each coral fragment (plankton captured/hour/gdw). For ash-free dry weights, the entire coral fragment was ground, dried at 60°C to a constant dry weight, and then combusted at 450°C for 6 hours. Feeding rates were then used to calculate the percent contribution of zooplankton- acquired heterotrophic carbon (C) to daily animal respiration (CHAR) as per (Grottoli et al. 2006; Palardy et al. 2008). CHAR is assumed to be a conservative estimate of heterotrophic carbon input because it does not take into account heterotrophic carbon acquired from nano- and picoplankton (e.g. Tremblay et al. 2012b), dissolved organic carbon (e.g. Levas 2012), or small particulate organic carbon (e.g. Anthony 2000; see Houlbreque, Ferrier-Pages 2009 for review). In addition, an assumed 8 hour feeding period is also likely to be conservative as all three species often have their tentacles extended throughout the day and can potentially acquire heterotrophic carbon during all 24 hours of the day. The sum of CZAR and CHAR was also calculated as CTAR (Grottoli et al. in review), which represents the total contribution of acquired carbon to daily animal respiration.

Chlorophyll a. Chlorophyll a content was analyzed to quantify the loss of photosynthetic pigment in the algal endosymbionts due to repeat bleaching. Coral tissue was removed with a WaterPik® and the total extraction volume measured. The blastate was homogenized and the resulting slurry centrifuged for 5 min at 6500 g. The supernatant was then removed and the algal pellet resuspended in artificial seawater (Instant Ocean). A subsample of the resuspended algal pellet was broken using glass beads in 100% acetone and subsequently stored at -20°C overnight. Samples were then centrifuged and chlorophyll a was determined using a Shimadzu UV-VIS spectrophotometer using the equations of Jeffrey and Humphrey (1975). Chlorophyll a content was standardized to surface area which was determined using the aluminum foil method (Marsh 1970).

Energy Reserves and Tissue Biomass. Energy reserves and tissue biomass were measured to quantify if lipid, protein, and carbohydrate were catabolized to compensate for a loss of photoautotrophic carbon due to repeat bleaching. Soluble lipids (referred to hereafter simply as lipids) were extracted from a whole, ground coral sample (skeleton + animal tissue + algal endosymbiont) in a 2:1 chloroform:methanol solution for 1 hour (Grottoli et al. 2004; Rodrigues, Grottoli 2007), washed in 0.88% KCl followed by 100% chloroform and another wash with 0.88% KCl. The extract was dried to constant weight under a stream of pure nitrogen (UPH grade 5.0) and standardized to the ash-free dry weight. Animal soluble protein and carbohydrate (referred to hereafter simply as protein and carbohydrate, respectively) were extracted from a second whole, ground coral sample taken from the same fragment as the lipid sample (Rodrigues, Grottoli 2007). Briefly, Milli-Q water was added to the ground coral sample and the resulting slurry was homogenized and then centrifuged twice (5000 rpm, 10 min) to separate the animal tissue from the skeleton and endosymbiotic algae. Protein and carbohydrate was extracted from the animal tissue only. One subsample of this animal tissue slurry was used for protein extraction using the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as a standard (Pierce BCA Protein Assay Kit). A second subsample was used for carbohydrate quantification using the phenol-sulfuric acid method (Dubois et al. 1956) with glucose as a standard. Soluble animal protein and carbohydrate concentrations were standardized to ash-free dry weight. Tissue biomass was determined by adding the dry lipid, protein, and carbohydrate content to the ash-free dry weights of the extracted coral samples, and was then standardized to surface area.

Calcification. Net calcification was determined using the buoyant weight technique (Jokiel et al. 1978) to see if corals were able to maintain skeletal growth after repeat bleaching. Each coral fragment was buoyantly weighed at the beginning and end of each bleaching treatment as well as each recovery interval. Daily calcification rates

45 were calculated as the difference between weights at different time points, divided by the respective number of days elapsed, and standardized to surface area. Surface area was determined using the aluminum foil technique (Marsh 1970).

2.3.4 Isotopic Analyses

Tissue Isotopes. Stable C and N isotopes were analyzed to assess changes in photosynthesis, feeding, and nitrogen uptake in the animal host and algal endosymbiont. Coral tissue was removed from half of each fragment using an airbrush, separated into animal host and endosymbiotic algal fraction by centrifugation, and then individually loaded onto pre-burned glass fiber filters (Rodrigues, Grottoli 2006; Hughes et al. 2010; Levas et al. 2013). Filters were gently shaved with razor blades, and the shaved material containing either the animal host or endosymbiont fraction was then packed into tin capsules. The tin capsules were combusted in a Costech Elemental Analyzer, and the resulting CO2 and N2 gas was automatically analyzed with a Finnigan Delta Plus Advantage stable isotope ratio mass spectrometer (SIRMS) via a Conflo III open split interface in Grottoli’s Stable Isotope Biogeochemistry Lab at The Ohio State University. 13 The carbon isotopic composition of the animal host (δ Ch) and algal endosymbiont 13 13 12 (δ Ce) were reported as the per mil deviation of the stable isotopes C: C relative to 13 Vienna-Peedee Belemnite Limestone standard (v-PDB). The difference between δ Ch 13 and δ Ce was calculated to determine the relative contribution of photoautotrophic versus heterotrophic carbon to the internal DIC pool (Muscatine et al. 1989b). The nitrogen 15 15 isotopic composition of the animal host (δ Nh) and endosymbiont (δ Ne) was reported as the per mil deviation of the ratio of stable isotopes 15N:14N relative to air. Repeated measurements of the commercial standard USGS-24 (n=55) had a SD of ± 0.04‰ for δ13C. Repeated measurements of the commercial standard IAEA-N2 (n=51) had a SD of ± 0.11‰ for δ15N.

Skeletal Isotopes. Stable C and O isotopes were analyzed to assess if changes in photosynthesis, feeding, and seawater temperature due to repeat bleaching are recorded in the coral skeleton. The exposed skeleton from samples processed for tissue isotopes was dried at 60°C for several days. Then, the uppermost layer of the skeleton was gently shaved with a Dremel tool fitted with a diamond-tipped drill bit and ground to fine powder with a mortar and pestle. About 80-100 μg of the skeletal powder were analyzed for δ13C and δ18O using an automated Kiel Carbonate Device coupled to a Finnigan Delta Plus Advantage SIRMS. Samples were acidified under vacuum with 100% ortho- phosphoric acid, and the resulting CO2 gas was cryogenically purified and delivered to 13 the mass spectrometer. Skeletal carbon isotopes (δ Cs) were reported as the per mil deviation of the stable isotopes 13C:12C relative to Vienna-Peedee Belemnite Limestone 18 standard (v-PDB), whereas skeletal oxygen isotopes (δ Os) were reported as the per mil deviation of the ratio of stable isotopes 18O:16O relative to v-PDB. Repeated measurements (n=55) of an internal standard had a SD of ± 0.03‰ for δ13C and ± 0.07‰ for δ18O, respectively. For both organic and skeletal isotopes, approximately 10% of all samples were run in duplicate.

2.3.5 Statistical Analyses

Two-way mixed model analyses of variance were used to test the effect of temperature and genotype on gross photosynthesis, day respiration, night respiration, CZAR, CHAR, CTAR, and feeding rate for each species separately. Temperature was fixed with two levels (non-bleached and repeat bleached), and genotype was a random factor. Three-way mixed model analyses of variance (ANOVA) were used to test the effect of temperature, recovery, and genotype on chlorophyll a, lipid, protein, 13 18 13 15 13 15 carbohydrate, tissue biomass, calcification, δ Cs, δ Os, δ Ch, δ Nh, δ Ce, δ Ne, and 13 13 δ Ch- δ Ce values for each species separately. Temperature was fixed with two levels (non-bleached and repeat bleached), recovery was fixed with three levels (0, 1.5, and 11

47 months), and genotype was a random factor. As genotype was a random factor in these ANOVA analyses, interaction terms with genotype were not included in the model. To determine if zooplankton captures were affected by bleaching status and zooplankton size, all zooplankton captured were converted into proportions by taxa and size class (Palardy et al. 2008; Levas 2012). These proportional assemblages were tested for differences across species and bleaching status using a factorial MANOVA. Since no differences in the composition of zooplankton taxa and size were found among species or bleaching status, data were pooled among experimental treatments and species. All data were tested for normality using Shapiro-Wilk’s test and the residuals of each variable. Non-normal data sets were log or square root transformed with the addition of a constant to make all data points positive. A total of four outliers defined after Hoaglin et al. (1983) were removed from the entire data set because transformations alone did not achieve normality. P-values ≤0.05 were considered significant. Post hoc Tukey tests were performed when main effects were significant. When significant genotype effects were detected, Tukey tests revealed that all average values overlapped among genotypes. As such, we concluded that the selected colonies represented the natural variation in the population well. A posteriori slice tests (e.g., tests of simple effects, Winer 1971) determined if the non-bleached and repeat bleached treatment averages significantly differed within each recovery interval. Corals were considered to be fully recovered once the average value of repeat bleached corals no longer significantly differed from the average of non-bleached controls. Since all fragments were exposed to identical conditions except temperature during the single and repeat bleaching treatment, any differences in the observed responses were due to temperature effects alone and independent of seasonal variation. Bonferroni corrections were not applied due to increased likelihood of false negatives (Quinn, Keough 2002; Moran 2003). Statistical analyses were performed using SAS software, Version 9.2 of the SAS System for Windows.

2.4 RESULTS

2.4.1 Bleaching and Mortality Status

Prior to the start of the repeat bleaching treatment, all coral fragments of all species appeared healthy and non-bleached (i.e., dark brown or yellow in color). After 17 days of exposure to elevated seawater temperature (= 0 month recovery), 44% and 100% of repeat bleached O. faveolata and P. astreoides were visibly bleached, respectively (Fig. 2.3). However, P. divaricata were only slightly pale in 43% of cases (Fig. 2.2, 2.3). Only one fragments of O. faveolata died during the repeat bleaching treatment (Fig. 2.3A). After 1.5 month of recovery on the back reef, the percentage of visibly bleached corals decreased to 6%, 84%, and 22% in O. faveolata, P. astreoides, and P. divaricata, respectively (Fig. 2.2, 2.3), and the percentage of dead fragments and/or fragments with partial mortality increased in both O. faveolata and P. astreoides. Except for P. astreoides, no coral fragments remained visibly bleached after 11 months of recovery (Fig. 2.2), but 63%, 33%, and 14% of all O. faveolata, P. astreoides, and P. divaricata, respectively, showed partial mortality (Fig. 2.3).

2.4.2 Physiology

Photosynthesis and Respiration. Average gross photosynthesis of repeat bleached corals did not decrease significantly compared to non-bleached controls in any of the species studied (Fig. 2.4, Table 2.2), although they tended to be lower in repeat bleached corals. Further, average day and night respiration did not differ significantly between non-bleached and repeat bleached corals in any of the three species (Fig. 2.4, Table 2.2), though night respiration tended to be greater in repeat bleached than in non- bleached corals across all species (Fig. 2.4).

Feeding. The size and type of zooplankton captured did not differ significantly among coral species, nor did it differ between non-bleached and repeat bleached corals (Table 2.3). Zooplankton captures were strongly dominated by zooplankton ranging from 400 to 1000 μm in size, followed by zooplankton 200-400 and >1000 μm in size (Fig. 2.5A). More than 50% of all captured zooplankton were copepods, followed by crab zoeae (16%), and snails (9%) (Fig. 2.5B). Average feeding rates did not significantly differ between non-bleached and repeat bleached corals in any of the three species (Fig. 2.5C, Table 2.4).

CZAR, CHAR, and CTAR. Average CZAR did not differ significantly between repeat bleached corals and non-bleached controls, although it tended to be lower in repeat bleached O. faveolata and P. divaricata (Fig. 2.6A, Table 2.5). Further, CZAR was less than 100% in repeat bleached O. faveolata and in both repeat bleached and non-bleached P. divaricata (Fig. 2.6A). Average CHAR of repeat bleached corals and non-bleached controls were the same, and it was highly variable among species and treatments (Fig. 2.6B, Table 2.5). Overall, heterotrophy contributed 0.6 - 14% of the daily metabolic demand. Both non-bleached O. faveolata and P. astreoides met more than 100% of their daily metabolic demand (i.e., CTAR), whereas non-bleached P. divaricata did not (Fig. 2.6C, Table 2.5). When repeat bleached, P. astreoides was the only species that was able to meet 100% of its metabolic demand from a combination of auto- and heterotrophy, whereas repeat-bleached O. faveolata and P. divaricata only met 90% and 56%, respectively. That said, average CTAR of repeat-bleached corals was not significantly different from non-bleached controls in any of the three species (Table 2.5).

Chlorophyll a, Energy Reserves, Tissue Biomass, and Calcification. In O. faveolata, average chlorophyll a concentrations were 52% and 28% lower in repeat bleached corals compared to non-bleached controls at 0 and 1.5 month of recovery, respectively, but were fully recovered after 11 months (Fig. 2.7A, Table 2.6). Average

50 lipid and protein concentrations of repeat bleached corals did not differ from non- bleached controls at any point during the study (Fig. 2.7B, D, Table 2.6). After 11 months of recovery, average carbohydrate concentrations of repeat bleached corals were 28% higher than in non-bleached controls (Fig. 2.7D). Average tissue biomass was the same for both non-bleached and repeat bleached corals throughout the study (Fig. 2.7E, Table 2.6). In contrast, average calcification rates of repeat bleached corals were significantly compromised (-212%) immediately after repeat bleaching; skeletal growth stopped which resulted in net dissolution of calcium carbonate (Fig. 2.7F, Table 2.6). Seasonal effects were detected in both chlorophyll a concentration and calcification rates, where highest concentrations/rates were observed in fall (1.5 month of recovery) and early summer (11 month of recovery), respectively (Fig. 2.7A, F, Table 2.6). In P. astreoides, average chlorophyll a concentrations of repeat bleached corals were 75% and 76% lower than in non-bleached controls at both 0 and 1.5 month of recovery, respectively, but were fully recovered after 11 months (Fig. 2.7G, Table 2.6). At the same time, average lipid concentrations were the same for both non-bleached and repeat bleached corals throughout the study (Fig. 2.7H, Table 2.6). In contrast, average protein concentrations of repeat bleached corals were 27% higher and 32% and 35% lower than in non-bleached corals at 0, 1.5, and 11 months of recovery, respectively. Importantly, protein concentrations of repeat bleached corals were not fully recovered 11 months after repeat bleaching (Fig. 2.7I). Repeat bleached corals had 37% more carbohydrate that non-bleached corals immediately after repeat bleaching (Fig. 2.7J, Table 2.6). Average tissue biomass was the same for both repeat bleached corals and non- bleached controls throughout the study, though biomass values showed seasonal variability with lowest values observed in fall at 1.5 month of recovery (Fig. 2.7K, Table 2.6). Average calcification rates were 69% lower in repeat bleached corals compared to non-bleached controls at 1.5 month of recovery, and still lower by 46% 11 months after repeat bleaching although this was not statistically significant (Fig. 2.7L, Table 2.6). In P. divaricata, average chlorophyll a concentrations of repeat bleached corals were 42% lower , 46% higher, and no different after 0, 1.5, and 11 months of recovery,

51 respectively (Fig. 2.7M, Table 2.6). Average lipid concentrations of repeat bleached corals were 30% lower than in non-bleached controls after 1.5 months of recovery but fully recovered after 11 months (Fig. 2.7N, Table 2.6). Average protein concentrations were the same for both repeat bleached corals and non-bleached controls throughout the study (Fig. 2.7O, Table 2.6). Average carbohydrate concentrations of repeat bleached corals were 18% higher than in non-bleached controls immediately after repeat bleaching but fully recovered after 1.5 months of recovery (Fig. 2.7P, Table 2.6). Carbohydrate concentrations also increased significantly throughout the study. Average tissue biomass was 24% higher in repeat bleached corals compared to non-bleached controls after 11 months of recovery (Fig. 2.7Q, Table 2.6). Average calcification rates were the same for both repeat bleached corals and non-bleached controls throughout the study (Fig. 2.7R, Table 2.6).

2.4.3 Tissue and Skeletal Isotopes

Tissue Isotopes. Immediately after bleaching, average stable carbon isotopes of 13 the animal host (δ Ch) of repeat bleached O. faveolata did not differ significantly from that of non-bleached controls (Fig. 2.8A, Table 2.7). After 1.5 month of recovery, 13 average δ Ch was significantly lighter in repeat bleached O. faveolata compared to non- bleached controls but had fully recovered at 11 months of recovery (Fig. 2.7A). In the 13 endosymbiont, average δ Ce of repeat bleached O. faveolata was overall lighter than non-bleached controls throughout the study, and was significantly lighter than non- bleached controls after 1.5 month of recovery (Fig. 2.8B, Table 2.7). A significant seasonal trend was also observed with heavier values in early summer compared to late 13 13 summer and fall (Table 2.7). Overall, average δ Ch - δ Ce was significantly greater in repeat bleached than non-bleached controls, which was largely driven by the significant difference immediately after bleaching (i.e., at 0 months recovery) (Fig. 2.8C, Table 2.7). 15 Average stable nitrogen isotopes of the animal host (δ Nh) were overall significantly heavier in repeat-bleached corals than in non-bleached controls, but did not significantly

52 differ between treatments at any point during the study (Fig. 2.8D, Table 2.7). Further, a significant seasonal trend was observed with lighter values in early summer (=11 month of recovery) compared to late summer and fall (=0 and 1.5 month of recovery, 15 respectively) (Fig. 2.8D, Table 2.7). Average δ Ne did not differ between repeat bleached and non-bleached controls at any point during the study, but was lighter in early summer compared to late summer and fall (Fig. 2.8E, Table 2.7). 13 In P. astreoides, average δ Ch of repeat bleached corals was significantly heavier than in non-bleached controls after 1.5 month of recovery and overall significantly heavier in early summer (=11 month of recovery) compared to late summer and fall (Fig. 13 2.8F, Table 2.7). Similarly, average δ Ce did not differ between repeat bleached and non- bleached corals throughout the study, but values were significantly heavier in early summer (=11 month of recovery) compared to late summer and fall (Fig. 2.8G, Table 13 13 2.7). The average difference between δ Ch and δ Ce was significantly lower in bleached corals after 11 months of recovery but did not differ between repeat bleached and non- 15 bleached corals at 0 and 1.5 months of recovery (Fig. 2.8H, Table 2.7). Average δ Nh of repeat bleached corals was significantly heavier than in non-bleached controls after 1.5 15 month of recovery (Fig. 2.8I, Table 2.7). Average δ Ne of repeat bleached corals was significantly heavier than non-bleached controls at both 0 and 1.5 month of recovery (Fig. 2.8J, Table 2.7). 13 13 In P. divaricata, average δ Ch and δ Ce did not significantly differ between repeat bleached and non-bleached controls throughout the study (Fig. 2.8K, L; Table 2.7). 13 13 Average δ Ch - δ Ce was significantly higher in repeat bleached than in non-bleached 15 control at 0 months recovery (Fig. 2.8M, Table 2.7). Similarly, average δ Nh did not significantly differ between repeat bleached and non-bleached controls throughout the study (Fig. 2.8N, Table 2.7). However, a significant seasonal trend was observed with lighter values in early summer (=11 month of recovery) compared to late fall (=1.5 15 months recovery) (Fig. 2.8N, Table 2.7). Average δ Ne of repeat bleached corals was significantly heavier than non-bleached controls immediately after repeat bleaching, but had recovered to non-bleached control values by 1.5 months of recovery (Fig. 2.8O,

15 Table 2.7). Overall, average δ Ne was lighter in early summer (11 months of recovery) compared to late summer and fall (Fig. 2.8O, Table 2.7).

13 18 Skeletal Isotopes. Average δ Cs and δ Os of repeat bleached O. faveolata were significantly heavier immediately after repeat bleaching compared to non-bleached controls but did not differ at any other time in the study (Fig. 2.9A, B, Table 2.8). In P. 13 18 astreoides, average δ Cs and δ Os of repeat bleached corals were significantly heavier than in non-bleached controls at 1.5 month of recovery, but did not differ at any other 13 18 time (Fig. 2.9C, D, Table 2.8). In P. divaricata, average δ Cs and δ Os of repeat bleached corals and non-bleached controls were the same throughout the study (Fig. 13 18 2.9E, F, Table 2.8). Seasonal trends were detected for both δ Cs and δ Os with heaviest 13 values observed at 0 and 11 months (late and early summer) of recovery for δ Cs, and at 18 11 months of recovery (early summer) for δ Os (Table 2.8).

2.5 DISCUSSION

Coral bleaching events are expected to increase in frequency and intensity over the coming decades (Hoegh-Guldberg 1999; Hoegh-Guldberg et al. 2007; Donner 2009; Eakin et al. 2009; Frieler et al. 2012; Grottoli et al. in review). Yet, the impacts of repeat bleaching on coral physiology and resilience remain largely unknown. Grottoli et al. (in review) showed for the first time that repeat bleaching can both lower or increase the thermal tolerance and overall resilience of the Caribbean coral species O. faveolata, P. astreoides, and P. divaricata. Here, we go beyond the findings of Grottoli et al (in review) and report a very detailed analysis of impacts of repeat bleaching on coral metabolism, heterotrophy, chlorophyll a, lipid, protein, carbohydrates, tissue biomass, calcification, and tissue and skeletal isotopes of these coral species. This was assessed immediately after repeat bleaching, and after both short and long term recovery. We show for the first time that when bleaching events are relatively mild (i.e., +1.2°C for 17

54 days), some corals can fully recover within one year after repeat bleaching while others will not be able to acclimate to annual bleaching stress.

2.5.1 Orbicella faveolata

Physiology. Mounding O. faveolata is a major reef builder in the Caribbean that is generally considered to be relatively susceptible to coral bleaching (Warner et al. 1999; Warner et al. 2006; Hennige et al. 2011). This is evident in their response to single bleaching, where 66% of all treatment corals were visibly bleached immediately after the elevated temperature stress, the overall performance of the algal endosymbiont was severely impacted (Aschaffenburg 2012), and some fragments had not visually recovered even after 11 months of recovery (Levas 2012). However after repeat bleaching, only 44% of all treatment corals were visibly bleached (Fig. 2.2, 2.3A), endosymbiont performance was improved (Aschaffenburg 2012), and photosynthesis rates were maintained (Fig. 2.3A). After 11 months on the reefs, these corals appeared visually recovered. This increased resistance to repeat bleaching stress was likely due to increased abundance of thermally resistant endosymbiont type D1a (McGinley 2012; Grottoli et al. in review). Nevertheless, CTAR values of repeat bleached O. faveolata averaged 90% indicating that they did not meet 100% of their daily metabolic demand via photoautotrophic carbon from their endosymbionts (Fig. 2.6A). This is not surprising considering that CZAR was below 100% and chlorophyll a concentrations were much lower than their non-bleached controls (Fig. 2.4A, 2.7A). Further, O. faveolata was not able to compensate for the loss of photoautotrophic carbon with increased zooplankton feeding because it does not display a large potential for heterotrophic plasticity when singly (Levas 2012) or repeat bleached (Fig. 2.5C). However, it is possible that repeat bleached O. faveolata could have obtained additional fixed carbon by grazing on pico- and nanoplankton, which can add up to 11% and 7% to their daily carbon budget, respectively (Tremblay et al. 2012b), or taking up dissolved organic carbon (DOC) from

55 seawater (Levas 2012; Levas et al. 2013). In fact, singly bleached O. faveolata met 16% of their daily metabolic demand this way (Levas 2012). It is therefore possible that repeat bleached O. faveolata met more that 100% of their daily metabolic demand, though additional study is needed to test this hypothesis. This would be in stark contrast to single bleaching, where O. faveolata only met a maximum of 68% of their respiratory needs even when all possible heterotrophic carbon sources were considered (Levas 2012). Even though O. faveolata maintained lipid, protein, carbohydrate content, and tissue biomass throughout recovery (Fig. 2.7B-E), the energetic value (Joules per mg coral tissue) of the three energy reserve pools (Gnaiger, Bitterlich 1984) in repeat bleached O. faveolata was lower than in controls corals immediately after repeat bleaching (Grottoli et al. in review). This indicates that there was indeed some deficit in the overall energy budget, which is consistent with skeletal dissolution at the same time point (Fig. 2.7F). Dramatically decreasing calcification rates can be interpreted as a strategy to save energy because calcification is thought to be energetically expensive (Allemand et al. 2011) and therefore often decreases in bleached corals (Jokiel, Coles 1977; Leder et al. 1991; Mendes, Woodley 2002; Rodrigues, Grottoli 2006; Levas 2012; Levas et al. 2013).

13 Isotopes. Stable carbon isotopes of the animal host (δ Ch) and endosymbiont 13 (δ Ce) track changes in the relative contribution of photosynthesis and heterotrophy to 13 13 the internal carbon pool. In repeat bleached O. faveolata, both δ Ch and δ Ce showed a lag in their response to repeat temperature stress. This is likely due to a dilution effect associated with the short duration of the temperature stress (i.e., 17 days), because sufficient cell turnover has to occur before the effect of repeat bleaching can be detected. 13 13 As a consequence, δ Ch and δ Ce were not significantly different from controls until 1.5 month after the temperature stress (Fig. 2.8A, C), even though chlorophyll a concentrations were already dramatically lower immediately after repeat bleaching. 13 δ Ch-e indicated that the relative contribution of photosynthetically versus heterotrophically acquired carbon to the internal carbon pool was greater in bleached than

56 in control corals immediately after repeat bleaching (Fig. 2.8C). Thus, even though the rate of photosynthesis was dramatically lower in bleached corals, the little carbon that was assimilated into coral tissues was predominantly photosynthetic in origin (Fig. 2.7E). Overall, the isotopic responses observed here are in contrast to O. faveolata’s response to 13 13 single bleaching where both δ Ch and δ Ce were unaffected despite significant declines in photoautotrophic carbon contribution to the internal carbon pool (Levas 2012). Coral nitrogen isotopes track changes in the source of dissolved inorganic 15 15 nitrogen (DIN) to the animal host (δ Nh) and endosymbiont (δ Ne). Bleached corals typically increase uptake of DIN to facilitate chlorophyll a recovery (Rodrigues, Grottoli 2006; Levas 2012; Levas et al. 2013). As uptake rates increase, this results in less 15 15 discrimination against the heavier N isotope, which in turn causes enriched δ Ne 15 15 values. Surprisingly, both δ Nh and δ Ne did not differ between repeat bleached and non-bleached O. faveolata at any point during recovery (Fig. 2.8D, E). Thus, repeat bleached O. faveolata did not appear to take up more DIN than the non-bleached controls, which could explain why chlorophyll a concentrations were still significantly lower after 1.5 months of recovery. 13 Coral skeletal stable carbon isotopes (δ Cs) failed to record decreases in CZAR of repeat bleached O. faveolata (Fig. 2.6A, 2.9A). Similarly, skeletal oxygen isotopes 18 (δ Os) did not track the elevated water temperature during the repeat bleaching treatment 13 (Fig. 2.9B). Typically, δ Cs decreases in bleached corals because fractionation increases as the rate of photosynthesis decreases so long as modest calcification rates are maintained (e.g. Porter et al. 1989; Grottoli, Wellington 1999; Grottoli 2002; Rodrigues, 18 Grottoli 2006; Levas et al. 2013). On the other hand, δ Os decreases as seawater temperature increases so long as salinity is relatively constant (e.g. Fairbanks, Dodge 1979; Porter et al. 1989; Gagan et al. 1994; Swart et al. 1996a; Druffel 1997; Grottoli, Eakin 2007). However, this is not always the case (e.g. Leder et al. 1991; Rodrigues, 13 18 Grottoli 2006; Hartmann et al. 2010; Levas 2012), and both δ Cs and δ Os of bleached corals can start approaching isotopic equilibrium with seawater (i.e., become more enriched than non-bleached controls) when calcification is severely compromised

(McConnaughey 1989a; Rodrigues, Grottoli 2006). It is therefore likely that in repeat 13 18 bleached O. faveolata, both δ Cs and δ Os failed to record changes in photosynthesis and seawater temperature due to impaired skeletal growth, but approached the values of non- bleached controls when calcification rates recovered.

Summary. Overall, O. faveolata was still susceptible to repeat bleaching but fully recovered within 1.5 – 11 months. Symbiont shuffling with increases in thermally resistant symbiont type D1a resulted in increased photochemical resilience and better overall endosymbiont performance during repeat bleaching (Aschaffenburg 2012; McGinley 2012), but appears to incur significant short-term costs to the animal host, such as impaired calcification. The ability to uptake significant amounts of DOC when bleached (Levas 2012) and potentially other sources of heterotrophic carbon (Tremblay et al. 2012b) may allow O. faveolata to compensate for some of these costs. This suggests overall that this species may persist on future Caribbean reefs but might not be able to maintain its role as a key reef builder in the long term.

2.5.2 Porites astreoides

Physiology. Mounding P. astreoides is a Caribbean coral species that has recently increased in abundance on Caribbean coral reefs due to its ability to survive disturbance better than other species (Green et al. 2008; Edmunds 2010). As expected, it was relatively resistant to single bleaching, with only 22% of all treatment corals showing visible signs of bleaching (Levas 2012) and only small decreases in endosymbiont performance (Aschaffenburg 2012). In contrast, after repeat bleaching 100% of all treatment P. astreoides were visibly bleached, and 1.5 and 11 months later some mortality had occurred and more than 80% and 10% had not visually recovered, respectively (Fig. 2.3B). This is consistent with dramatically lower chlorophyll a concentrations immediately after repeat bleaching and even 1.5 months later (Fig. 2.7G). Further, endosymbiont performance and densities suffered massive declines

(Aschaffenburg 2012; Grottoli et al. in review). It is therefore surprising that photosynthesis rates and CZAR of repeat bleached P. astreoides (Fig. 2.4A, 2.6A this study, Grottoli et al. in review) were not different from non-bleached controls. As photosynthesis rates were maintained, repeat bleached P. astreoides were able to meet 100% of their daily metabolic energy demand from photoautotrophic carbon alone. Zooplankton feeding contributed another 9% (Fig. 2.6). While meeting 100% of daily metabolic demand is unusual for bleached corals (e.g. Porter et al. 1989; Grottoli et al. 2006; Levas 2012), it is consistent with P. astreoides’ response to single bleaching (Levas 2012; Grottoli et al. in review). However, singly bleached P. astreoides were only able to do so by dramatically increasing zooplankton feeding rates so that CHAR alone contributed more than 100% to their total carbon budget (Levas 2012; Grottoli et al. in review). In contrast, feeding rates of repeat bleached P. astreoides were not higher than those of non-bleached controls (Fig. 2.5C, 2.6B), suggesting that heterotrophic plasticity is lost when thermal stress becomes more frequent. Although repeat bleached P. astreoides met 100% of their metabolic needs immediately after repeat bleaching, the full impacts on its physiology were not evident until later. Repeat bleached corals suffered dramatic decreases in protein concentrations (-32%) and calcification rates (-69%) after 1.5 months of recovery (Fig. 2.7I, L). Further, they stored 28% less energy in their energy reserves than non-bleached controls (Grottoli et al. in review), which was likely driven by significant catabolism of protein as both lipid and carbohydrate content were maintained (Fig. 2.7H, J, K). Thus, it seems that in this species protein is the preferred respiratory fuel during times of stress rather than lipids as is often assumed (see Lesser 2013 for discussion). Alternatively, this could also indicate that this species experienced extensive cellular damage. Most strikingly however, protein concentrations had not recovered 11 months after repeat bleaching and calcification rates were still ~50% lower (though not statistically significant) than in controls. P. astreoides was therefore not able to fully recover from repeat bleaching stress within one year, which calls into question if this species will survive in a future of annually recurring coral bleaching.

Isotopes. Equal stable carbon isotopic values in repeat bleached and control P. 13 13 astreoids coral animal host (δ Ch) and endosymbiont (δ Ce) is consistent with the fact that repeat bleached P. astreoides maintained photosynthesis rates and CZAR. This is further supported by significant enrichment of the stable nitrogen isotopes of both animal 15 15 host (δ Nh) and endosymbiont (δ Ne) at 0 and/or 1.5 month of recovery (Fig. 2.8i, J), which indicates DIN uptake from seawater to promote endosymbiont recovery (Rodrigues, Grottoli 2006; Levas 2012; Levas et al. 2013). 13 13 The strong negative difference between δ Ch and δ Ce after 11 months of recovery suggests that repeat bleached P. astreoides assimilated significant amounts of heterotrophic carbon at this time point (Fig. 2.8H), even though zooplankton feeding rates were low immediately after repeat bleaching (i.e., CHAR was <10%) (Fig. 2.5C). Irrespective of feeding rates, these results indicate that heterotrophic carbon is disproportionately assimilated into the tissues compared to photoautotrophically acquired carbon after 11 months recovery. This is consistent with findings on bleached Porites compressa corals in Hawaii that assimilate dramatically higher amounts of heterotrophically derived carbon 11 months after recovery compared to controls (Hughes, Grottoli in review) into their lipid energy reserves (Baumann 2013). Considering that protein concentrations were not fully recovered 11 months after repeat bleaching (this study), it would appear that P. astreoides could be heavily sequestering heterotrophic carbon as a viable strategy to promote recovery. 13 The lack of any decline in coral skeletal stable carbon isotopes (δ Cs) of repeat bleached P. astreoides (Fig. 2.9C) immediately after repeat bleaching is to be expected given the maintenance of CZAR (Fig. 2.6A) and calcification rates (Fig. 2.7L). However, by 1.5 months of recovery, calcification rates were severely compromised so that 13 18 metabolic isotope fractionation effects on δ Cs were reduced. δ Os also failed to record elevated seawater temperatures during the repeat bleaching treatment which is likely due to decreased calcification rates and thus reduced impact of temperature-dependent kinetic fractionation (Fig. 2.9D). This is consistent with other isotopic studies of singly bleached

60 corals where the bleaching event is not reliably recorded in the skeletal stable isotopic signature (Leder et al. 1991; Rodrigues, Grottoli 2006; Hartmann et al. 2010; Levas 2012).

Summary. After 11 months of recovery, P. astreoides corals had not fully recovered (the amount of protein and the calcification rate were much smaller in repeat bleached corals than control corals). Thus, annually repeated bleaching appears to have a cumulative stress effect on P. astreoides rendering it highly susceptible to frequent bleaching. Loss of heterotrophic plasticity (this study) coupled with a lack of flexibility in its association with different endosymbiont types (Grottoli et al. in review) appear to be the underlying factors driving the loss of bleaching tolerance in this species. Considering its increased bleaching susceptibility and long recovery time, P. astreoides is unlikely to acclimate to frequent thermal stress (this study, Aschaffenburg 2012; Levas 2012; McGinley 2012; Grottoli et al. in review). It may therefore lose its ecological advantage of being more stress-resistant than other species, and, given its sensitivity to ocean acidification (Albright et al. 2008; Albright, Langdon 2011; Crook et al. 2013), ultimately cease to be a prominent species on future Caribbean coral reefs.

2.5.3 Porites divaricata

Physiology. Porites divaricata is a branching coral species characterized by high growth rates and low bleaching susceptibility (Green et al. 2008; Edmunds 2010). After single bleaching, only 11% of all treatment corals showed visible signs of bleaching (Levas 2012), whereas almost 60% were paler than the non-bleached control corals after repeat bleaching (Fig. 2.3C). Chlorophyll a concentrations were initially lower in repeat bleached P. divaricata but increased by 169% - and thus surpassed those of non-bleached controls - at 1.5 month of recovery (Fig. 2.7M). However, endosymbiont concentrations (Aschaffenburg 2012; Grottoli et al. in review), photosynthesis rates (Fig 2.4A), and

CZAR (Fig 2.6A) were the same in non-bleached and repeat bleached corals. Thus, photosynthetic function and CZAR were maintained in repeat bleached P. divaricata. Surprisingly though, P. divaricata was not able to meet 100% of daily metabolic demand from a combination of photosynthesis and zooplankton feeding irrespective of bleaching status (Fig. 2.6C). Other sources of heterotrophic carbon such as DOC (Levas 2012), pico- and nanoplankton (Tremblay et al. 2012b), or particulate organic carbon (Anthony 2000) are unaccounted for in this study and may play a significant role in the total carbon budget of this species. The fact that this species maintains energy reserves (Fig. 2.7N-P), tissue biomass (Fig. 2.7Q), and calcification (Fig. 2.7R) when repeat bleached, strongly suggests that additional heterotrophic carbon sources are being accessed. Additional study is needed to test this hypothesis. Overall, P. divaricata had higher levels of energy reserves than either O. faveolata or P. astreoides (this study). Protein concentrations in particular were higher than in any of the other species throughout the study. Further, repeat bleached corals had significantly more carbohydrate than control corals immediately after repeat bleaching. As high levels of energy reserves are known to promote bleaching resistance and recovery from bleaching (Rodrigues, Grottoli 2007; Anthony et al. 2009), it is likely that this enabled P. divaricata to increase its bleaching resistance and to rapidly acclimate to repeat temperature stress.

Isotopes. Consistent with no changes in CZAR (Fig. 2-4A, 2-6A, Aschaffenburg 2012; Grottoli et al. in review), photosynthetic efficiency (Aschaffenburg 2012), and 13 CHAR (Fig. 2.6B), the stable carbon isotopes of the animal host (δ Ch) and 13 endosymbiont (δ Ce) of repeat bleached P. divaricata did not differ from their controls at any time during the study (Fig. 2.8K, L). Significant enrichment of the stable nitrogen 15 isotopes of the endosymbiont (δ Ne) at 0 month of recovery (Fig. 2.8O) indicates that the endosymbiont increased DIN uptake to maintain/and or recover chlorophyll a concentrations.

13 13 The strong negative difference between δ Ch and δ Ce of non-bleached controls at 0 month of recovery indicates that the contribution of heterotrophic carbon to the total pool of assimilated carbon in the tissues was greater than that of photoautotrophic carbon whereas the relative contribution of both sources of carbon was equal in repeat bleached 13 13 P. divaricata (Fig. 2.8M). Further, the decline in average δ Ch - δ Ce throughout recovery in both repeat bleached and non-bleached corals indicates that heterotrophy plays an increasingly important role in the carbon budget of this species over the course of the 11 month study. This contrasts the isotopic response of P. divaricata to single bleaching, where photosynthetic carbon contributions dominated the isotopic signal throughout recovery (Levas 2012). Skeletal stable carbon and oxygen isotopes of repeat bleached P. divaricata did not record any physiological or environmental changes (Fig. 2.9E, F). This is consistent with their overall response to single bleaching (Levas 2012), and the lack of dramatic changes in endosymbiont performance and/or calcification immediately after repeat bleaching. Apparently, small increases in seawater temperature – such as 1°C for 15-17 18 days - are not enough to be recorded in coral bulk δ Os because none of the three species 18 studied here showed the expected decrease in δ Os after single or repeat bleaching (Levas 2012, this study).

Summary. Overall, P. divaricata was moderately resilient to single bleaching, yet was able to acclimate to annual bleaching stress and dramatically increase its thermal tolerance. No mortality occurred at any point during the study. This improved resilience was likely driven by physiological acclimation of both endosymbiont and animal host (Grottoli et al. in review), as (1) thermally resistant symbiont type A4 gradually replaced C47 within treatment corals during repeat bleaching (McGinley 2012; Grottoli et al. in review), and (2) high levels of energy reserves are known to promote bleaching tolerance and recovery from bleaching (this study, Anthony et al. 2009; Grottoli et al. in review). Considering its rapid acclimation and high bleaching tolerance (this study, Grottoli et al.

63 in review) as well as relatively low sensitivity to ocean acidification (Crook et al. 2012), P. divaricata is likely to become a prominent species on future Caribbean coral reefs.

2.5.4 Implications for the Future of Coral Reefs

Caribbean coral reefs have undergone dramatic change due to human impacts over the past centuries despite marked ecological persistence of coral communities over the past 220,000 years (Pandolfi, Jackson 2006). Coral cover has declined by 80% (Gardner et al. 2003) due to a combination of overfishing (Jackson et al. 2001), coral and herbivore disease (Lessios 1988; Patterson et al. 2002), and climate change (Hughes et al. 2003). Further, mass bleaching events associated with slow to non-existent recovery have become more frequent (Donner et al. 2007; Baker et al. 2008; Eakin et al. 2010), and the once-dominant key reef builder A. palmata and A. cercivornis have mostly disappeared (Aronson, Precht 2001; Patterson et al. 2002). As a consequence, disturbance-resistant species such as P. astreoides have increased in abundance (Green et al. 2008; Edmunds 2010), yet it is unclear how and if today’s coral communities will persist in a future of repeat coral bleaching. The present study clearly demonstrates that susceptibility or resistance to single bleaching is a poor predictor for a coral’s response to repeat bleaching (see also Aschaffenburg 2012; McGinley 2012; Grottoli et al. in review), and that while some species (like P. divaricata) may be able to rapidly acclimate to annual bleaching stress, others (like P. astreoides) are likely to face significant demise. It is further evident that coral morphology (e.g. Loya et al. 2001) may not be an important predictive factor when it comes to repeat bleaching. Based on current model predictions, +1-1.5°C acclimation and/or adaptation would be required for Caribbean reef corals to keep up with rising temperatures (Teneva et al. 2012), and evidence for +1°C acclimation in Caribbean corals (this study, Aschaffenburg 2012; McGinley 2012; Grottoli et al. in review) and up to 1.5°C for Pacific coral species (Berkelmans, van Oppen 2006; Middlebrook et al. 2008) is therefore encouraging. Such acclimation and/or adaptation would also significantly delay the onset

64 of harmfully frequent bleaching events (Donner 2009), and reduce the percentage of reefs at risk of long term degradation worldwide (Frieler et al. 2012). Critically though, no modeling studies to date have considered the effect of increasing bleaching susceptibility of species such as P. astreoides on predicted future coral mortality, reef degradation, and frequency of mass bleaching events. It also has to be emphasized that temperature increases of +1-1.2°C for 2 weeks represent relatively mild bleaching events, suggesting that both the potential for acclimation and increased bleaching susceptibility are rather conservative estimates. Ocean acidification will compromise coral reef persistence in addition to coral bleaching (Kleypas et al. 1999; Doney et al. 2009), and has also been shown to increase bleaching susceptibility (Pecheux 2002; Anthony et al. 2008), thus indicating bleak prospects for future reefs. It is therefore likely that over the coming decades, Caribbean coral reefs – and potentially reefs worldwide – will experience yet another dramatic change associated with significant shifts in coral community composition and diversity, and potential extinction of temperature- and pH- sensitive species.

Acknowledgements. We thank Roberto Iglesias-Prieto, Ania Banaszak, Susana Enriquez, Robin Smith, and the staff of the Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, in Puerto Morelos for their generous time and logisitical support. We also thank Yohei Matsui, Teresa Huey, Dana Borg, Elizabeth Zebrowski, Jordan Scheuermann, and Michael McBride for help in the field and laboratory. This work was funded by the National Science Foundation (OCE#0825490 to AGG and OCE#0825413 to MEW). All work undertaken in this study complied with the current laws of Mexico and the United States of America.

2.6 REFERENCES

Albright R, Langdon C (2011) Ocean acidification impacts multiple early life history processes of the Caribbean coral Porites astreoides. Global Change Biology 17:2478-2487

Albright R, Mason B, Langdon C (2008) Effect of aragonite saturation state on settlement and post-settlement growth of Porites astreoides larvae. Coral Reefs 27:485-490

Allemand D, Tambutte E, Zoccola D, Tambutte S (2011) Coral calcification, cells to reefs. In: Dubinsky Z, Stambler N (eds) Coral Reefs: An Ecosystem in Transition. Springer, pp 119-150

Anthony KRN (2000) Enhanced particle-feeding capacity of corals on turbid reefs (Great Barrier Reef, Australia). Coral Reefs 19:59-67

Anthony KRN, Hoogenboom MO, Maynard JF, Grottoli AG, Middlebrook R (2009) Energetics approach to predicting mortality risk from environmental stress: a case study of coral bleaching. Functional Ecology 23:539-550

Aronson R, Precht W (2001) White-band disease and the changing face of Caribbean coral reefs. Hydrobiologia 460:25-38

Aschaffenburg M (2012) The physiological response of Symbiodinium spp. to thermal and light stress: A comparison of different phylotypes and implications for coral reef bleaching. PhD thesis, University of Delaware, p 155

Baird AH, Bhagooli R, Ralph PJ, Takahashi S (2008) Coral bleaching: the role of the host. Trends in Ecology and Evolution 24:16-20

Baker AC (2001) Reef corals bleach to survive change. Nature 411:765-766

Baker AC, Starger CJ, McClanahan TR, Glynn PW (2004) Corals' adaptive response to climate change. Nature 430:741

Baumann J (2013) The effects of elevated temperature stress on the acquisition and allocation of carbon to lipids in Hawaiian corals. MSc thesis, The Ohio State University, p

Bellwood DR, Hughes TP, Folke C, Nyström M (2004) Confronting the coral reef crisis. Nature 429:827-833

Berkelmans R, van Oppen MJH (2006) The role of zooxanthellae in the thermal tolerance of corals: a "nugget of hope" for coral reefs in an era of climate change. Proceedings of the Royal Society B: Biological Sciences 273:2305-2312

Brown BE (1997) Coral bleaching: causes and consequences. Coral Reefs 16 suppl:s129- s138

Budd AF, Fukami H, Smith ND, Knowlton N (2012) Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia). Zoological Journal of the Linnean Society 166:465-529

Buddemeier RW, Fautin DG (1993) Coral bleaching as an adaptive mechanism. BioScience 43:320-326

Castillo KD, Ries JB, Weiss JM, Lima FP (2012) Decline of forereef corals in response to recent warming linked to history of thermal exposure. Nature Climate Change:doi: 10.1038/nclimate1577

Crook ED, Cohen AL, Rebolledo-Vieyra M, Hernandez L, Paytan A (2013) Reduced calcification and lack of acclimatization by coral colonies growing in areas of persistent natural acidification. Proceedings of the National Academy of Sciences:doi:10.1073/pnas.1301589110

Crook ED, Potts D, Rebolledo-Vieyra M, Hernandez L, Paytan A (2012) Calcifying coral abundance near low-pH springs: implications for future ocean acidification. Coral Reefs 31:239-245

Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO2 problem. Annual Review of Marine Science 1:169-192

Donner SD (2009) Coping with commitment: Projected thermal stress on coral reefs under different future scenarios. PLoS ONE 4:e5712

Donner SD (2011) An evaluation of the effect of recent temperature variability on the prediction of coral bleaching events. Ecological Applications 21:1718–1730

Druffel ERM (1997) Geochemistry of corals: Recorders of past ocean chemistry, ocean circulation and climate. Proceedings of the National Academy of Sciences of the USA 94:8354-8361

Dubois M, Giles KA, Hamilton JK, Pebers PA, Smith F (1956) Colorimetric method for determination of sugar and related substances. Analytical Chemistry 28:350-356

Eakin CM, Morgan JA, Heron SF, Smith TB, Liu G, Alvarez-Filip L, Baca B, Bartels E, Bastidas C, Bouchon C, Brandt M, Bruckner AW, Bunkley-Williams L, Cameron A, Causey BD, Chiappone M, Christensen TRL, Crabbe MJC, Day O, de la Guardia E, Diaz-Pulido G, DiResta D, Gil-Agudelo DL, Gilliam DS, Ginsburg RN, Gore S, Guzman HM, Hendee JC, Hernandez-Delgado EA, Husain E, Jeffrey CFG, Jones RJ, Jordan-Dahlgren E, Kaufman LS, Kline DI, Kramer PA, Lang JC, Lirman D, Mallela J, Manfrino C, Marechal J-P, Marks K, Mihaly J, Miller WJ, Mueller EM, Muller EM, Orozco Toro CA, Oxenford HA, Ponce-Taylor D, Quinn N, Ritchie KB, Rodriguez S, Ramirez AR, Romano S, Samhouri JF, Sanchez JA, Schmahl GP, Shank BV, Skirving WJ, Steiner SCC, Villamizar E, Walsh SM, Walter C, Weil E, Williams EH, Roberson KW, Yusuf Y (2010) Caribbean corals in crisis: Record thermal stress, bleaching, and mortality in 2005. PLoS ONE 5:e13969

Edmunds PJ (2010) Population biology of Porites astreoides and Diploria strigosa on a shallow Caribbean reef. Marine Ecology Progress Series 418:87-104

Edmunds PJ, Spencer Davies P (1986) An energy budget for Porites porites (Scleractinia). Marine Biology 92:339-347

Fairbanks RG, Dodge RE (1979) Annual periodicity of the 18O/16O and 13C/12C ratios in the coral Montastea annularis. Geochimica and Cosmochimica Acta 43:1009- 1020

Falkowski PG, Dubinsky Z, Muscatine L, Porter JW (1984) Light and the bioenergetics of a symbiotic coral. BioScience 34:705-709

Fitt WK, Brown BE, Warner ME, Dunne RP (2001) Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs 20:51-65

Fitt WK, Gates RD, Hoegh-Guldberg O, Bythell JC, Jatkar A, Grottoli AG, Gomez M, Fisher P, Lajuenesse TC, Pantos O, Iglesias-Prieto R, Franklin DJ, Rodrigues LJ, Torregiani JM, van Woesik R, Lesser MP (2009) Response of two species of Indo-Pacific corals, Porites cylindrica and Stylophora pistillata, to short-term thermal stress: The host does matter in determining the tolerance of corals to bleaching. Journal of Experimental Marine Biology and Ecology 373:102-110

Fitt WK, McFarland FK, Warner ME, Chilcoat GC (2000) Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching. Limnology and Oceanography 45:677-685

Fitt WK, Spero HJ, Halas J, White MW, Porter JW (1993) Recovery of the coral Montastraea annularis in the Florida Keys after the 1987 Caribbean "bleaching event". Coral Reefs 12:57-64

Fitt WK, Warner ME (1995) Bleaching patterns of four species of Caribbean reef corals. Biological Bulletin 189:298-307

Gagan MK, Chivas AR, Isdale PJ (1994) High-resolution isotopic records from corals using ocean temperature and mass-spawning chronometers. Earth and Planetary Science Letters 121:549-558

Gardner TA, Côté IM, Gill JA, Grant A, Watkinson AR (2003) Long-term regions-wide declines in Caribbean corals. Science 301:958-960

Glynn PW (1993) Coral reef bleaching: ecological perspectives. Coral Reefs 12:1-17

Glynn PW (1996) Coral reef bleaching: facts, hypotheses and implications. Global Change Biology 2:495-509

Gnaiger E, Bitterlich G (1984) Proximate biochemical composition and caloric content calculated from elemental CHN analysis: a stoichiometric concept. Oecologia 62:289-298

Green DH, Edmunds PJ, Carpenter RC (2008) Increasing relative abundance of Porites astreoides on Caribbean reefs mediated by an overall decline in coral cover. Marine Ecology Progress Series 359:1-10

Grottoli AG (2002) Effect of light and brine shrimp levels on skeletal 13C values in the Hawaiian coral Porites compressa: A tank experiment. Geochimica and Cosmochimica Acta 66:1955-1967

Grottoli AG (2000) Stable carbon isotopes (13C) in coral skeletons. Oceanography 13:93-97

Grottoli AG, Eakin CM (2007) A review of modern coral 18O and 14C proxy records. Earth-Science Reviews 81:67-91

Grottoli AG, Rodrigues LJ, Palardy JE (2006) Heterotrophic plasticity and resilience in bleached corals. Nature 440:1186-1189

Grottoli AG, Warner M, Levas SJ, Aschaffenburg M, Schoepf V, McGinley M, Baumann J, Matsui Y (in review) Acclimation to annually recurring coral bleaching determined by coral phenotypic plasticity. Submitted to Science

Grottoli AG, Wellington GM (1999) Effect of light and zooplankton on skeletal C values in the Eastern Pacific corals Pavona clavus and Pavona gigantea. Coral Reefs 18:29-41

Heikoop JM, Risk MJ, Lazier AV, Edinger EN, Jompa J, Limmon GV, Dunn JJ, Browne DR, Schwarcz HP (2000) Nitrogen-15 signals of anthropogenic nutrient loading in reef corals. Marine Pollution Bulletin 40:628-636

Hennige SJ, McGinley MP, Grottoli AG, Warner ME (2011) Photoinhibition of Symbiodinium spp. within the reef corals Montastraea faveolata and Porites astreoides: implications for coral bleaching. Marine Biology:doi 10.1007/s00227- 00011-01752-00221

Hoaglin E, Musteller F, Tukey J (1983) Understanding Robust and Exploratory Data Analysis. John Wiley & Sons, New York

Hoegh-Guldberg O (1999) Climate change, coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research 50:839-866

Houlbreque F, Ferrier-Pages C (2009) Heterotrophy in tropical scleractinian corals. Biological Reviews 84:1-17

Hughes AD, Grottoli AG (in review) Heterotrophic compensation: A possible mechanism for resilience of coral reefs to global warming, or a sign of prolonged stress? Submitted to PLoS ONE

Hughes AD, Grottoli AG, Pease TK, Matsui Y (2010) Acquisition and assimilation of carbon in non-bleached and bleached corals. Marine Ecology Progress Series 420:91-101

Hughes TP, Baird AH, Bellwood DR, Card M, Connolly SR, Folke C, Grosberg R, Hoegh-Guldberg O, Jackson JBC, Kleypas J, Lough JM, Marshall P, Nyström M, Palumbi SR, Pandolfi JM, Rosen B, Roughgarden J (2003) Climate change, human impacts, and the resilience of coral reefs. Science 301:929-933

Jackson JBC, Kirby MX, Berger WH, Bjorndal KA, Botsford LW, Bourque BJ, Bradbury RH, Cooke R, Erlandson J, Estes JA, Hughes TP, Kidwell S, Lange CB, Lenihan HS, Pandolfi JM, Peterson CH, Steneck RS, Tegner MJ, Warner RR (2001) Historical overfishing and the recent collapse of coastal ecosystems. Science 293:629-637

Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1, and c2 in higher plants, algae, and natural phytoplankton. Biochem Physiol Pflanzen 167:191-194

Jokiel PL, Coles SL (1977) Effects of temperature on the mortality and growth of Hawaiian reef corals. Marine Biology 43:201-208

Jokiel PL, Coles SL (1990) Response of Hawaiian and other Indo-Pacific reef corals to elevated temperature. Coral Reefs 8:155-162

Jokiel PL, Maragos JE, Franzisket L (1978) Coral growth: buoyant weight technique. In: Stoddart DR, Johannes RE (eds) Coral Reefs: Resesarch Methods. UNESCO, Paris, pp 529-541

Jones A, Berkelmans R (2010) Potential costs of acclimatization to a warmer climate: Growth of a reef coral with heat tolerant vs. sensitive symbiont types. PLoS ONE 5:e10437

Jones AM, Berkelmans R, van Oppen MJH, Mieog JC, Sinclair W (2008) A community change in the algal endosymbionts of a scleractinian coral following a natural bleaching event: field evidence of acclimatization. Proceedings of the Royal Society B: Biological Sciences 275:1359-1365

Jones RJ (1997) Changes in zooxanthellar densities and chlorophyll concentrations in corals during and after a bleaching event. Marine Ecology Progress Series 158:51- 59

Kleypas J, Buddemeier RW, Archer D, Gattuso J-P, Langdon C, Opdyke BN (1999) Geochemical consequences of increased atmospheric carbon dioxide on coral reefs. Science 284:118-120

Leder JJ, Szmant AM, Swart P (1991) The effect of prolonged "bleaching" on skeletal banding and stable isotopic composition in Montastraea annularis. Coral Reefs 10:19-27

Lesser MP (1997) Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16:187-192

Lesser MP (2013) Using energetic budgets to assess the effects of environmental stress on corals: are we measuring the right things? Coral Reefs 32:25-33

Lessios HA (1988) Mass mortality of Diadema antillarum in the Caribbean: what have we learned? Annual Review of Ecology and Systematics 19:371-393

Levas SJ (2012) Biogeochemistry and Physiology of Bleached and Recovering Hawaiian and Caribbean Corals. PhD Thesis, The Ohio State University, p 238

Levitus S, Antonov JI, Boyer TP, Locarnini RA, Garcia HE, Mishonov AV (2009) Global ocean heat content 1955–2008 in light of recently revealed instrumentation problems. Geophys. Res. Lett. 36:L07068.

Loya Y, Sakai K, Yamazato K, Nakan Y, Sambali H, van Woesik R (2001) Coral bleaching: the winners and the losers. Ecology Letters 4:122-131

Marsh JA (1970) Primary productivity of reef-building calcareous red algae. Ecology 51:255-263

Maynard JA, Anthony KRN, Marshall PA, Masiri I (2008) Major bleaching events can lead to increased thermal tolerance in corals. Marine Biology 155:173–182

McConnaughey T (1989a) 13C and 18O isotopic disequilibrium in biological carbonates: I. Patterns. Geochimica et Cosmochimica Acta 53:151-162

McConnaughey TA, Burdett J, Whelan JF, Paull CK (1997) Carbon isotopes in biological carbonates: Respiration and photosynthesis. Geochimica et Cosmochimica Acta 61:611-622

McGinley M (2012) The impact of environmental stress on Symbiodinium spp.: A molecular and community-scale investigation. PhD thesis, University of Delaware, p 173

Middlebrook R, Hoegh-Guldberg O, Leggat W (2008) The effect of thermal history on the susceptibility of reef-building corals to thermal stress. Journal of Experimental Biology 211:1050-1056

Mora C (2008) A clear human footprint in the coral reefs of the Caribbean. Proceedings of the Royal Society B: Biological Sciences 275:767-773

Moran MD (2003) Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos 100:403-405

Muscatine L, McCloskey LR, Marian RE (1981) Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration. Limnology and Oceanography 26:601-611

Muscatine L, Porter JW, Kaplan IR (1989) Resource partitioning by reef corals as determined from stable isotope composition. I. 13C of zooxanthellae and animal tissue vs. depth. Marine Biology 100:185-193

Palardy JE, Grottoli AG, Matthews KA (2005) Effects of upwelling, depth, morphology and polyp size on feeding of three species of Panamanian corals. Marine Ecology Progress Series 300:79-89

Pandolfi JM, Connolly SR, Marshall DJ, Cohen AL (2011) Projecting coral reef futures under global warming and ocean acidification. Science 333:418-422

Pandolfi JM, Jackson JBC (2006) Ecological persistence interrupted in Caribbean coral reefs. Ecology Letters 9:818-826

Patterson KL, Porter JW, Ritchie KB, Polson SW, Mueller E, Peters EC, Santavy DL, Smith GW (2002) The etiology of white pox, a lethal disease of the Caribbean elkhorn coral, Acropora palmata. Proceedings of the National Academy of Sciences 99:8725-8730

Pecheux M (2002) CO2 increase, a direct cause of coral reef mass bleaching? Marine Life 12:63-68

Perry CT, Murphy GN, Kench PS, Smithers SG, Edinger EN, Steneck RS, Mumby PJ (2013) Caribbean-wide decline in carbonate production threatens coral reef growth. Nature Communications 4: doi:10.1038/ncomms2409

Porter JW, Fitt WK, Spero HJ, Rogers AD, White MW (1989) Bleaching in reef corals: physiological and stable isotopic responses. Proceedings of the National Academy of Sciences of the USA 86:9342-9346

Quinn GP, Keough MJ (2002) Experimental Design and Data Analysis for Biologists. Cambridge University Press, 557 p, New York

Rodrigues LJ, Grottoli AG (2007) Energy reserves and metabolism as indicators of coral recovery from bleaching. Limnology and Oceanography 52:1874-1882

Rodrigues LJ, Grottoli AG, Lesser MP (2008) Long-term changes in the chlorophyll fluorescence of bleached and recovering corals from Hawaii. The Journal of Experimental Biology 211:2502-2509

Sheppard CRC (2003) Predicted recurrences of mass coral mortality in the Indian Ocean. Nature 425:294-297

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150:76-85

Swart P, Leder JJ, Szmant AM, Dodge RE (1996b) The origin of variations in the isotopic record of scleractinian corals: II. Carbon. Geochimica and Cosmochimica Acta 60:2871-2885

Teneva L, Karnauskas M, Logan C, Bianucci L, Currie J, Kleypas J (2012) Predicting coral bleaching hotspots: the role of regional variability in thermal stress and potential adaptation rates. Coral Reefs 31:1-12

Tremblay P, Grover R, Maguer JF, Legendre L, Ferrier-Pages C (2012a) Autotrophic carbon budget in coral tissue: a new 13C-based model of photosynthate translocation. The Journal of Experimental Biology 215:1384-1393

Tremblay P, Naumann MS, Sikorski S, Grover R, Ferrier-Pagès C (2012b) Experimental assessment of organic carbon fluxes in the scleractinian coral Stylophora pistillata during a thermal and photo stress event. Marine Ecology Progress Series 453:63- 77

Warner ME, Fitt WK, Schmidt GW (1999) Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching. Proceedings of the National Academy of Sciences of the USA 96:8007-8012

Warner ME, LaJeunesse TC, Robison JD, Thur RM (2006) The ecological distribution and comparative photobiology of symbiotic dinoflagellates from reef corals in Belize: potential implications for coral bleaching. Limnology and Oceanography 51:1887-1897

Wilkinson C (2008) Status of coral reefs of the world: 2008. Global Coral Reef Monitoring Network and Reef and Rainforest Research Centre:296p

Winer BJ (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York

2.7 TABLES

Table 2.1. Parent colony (=genotype) collection dates, depths, and locations.

Species Genotype Date Depth Location Coordinates O. faveolata 5, 9 6/18/09 5 m The Wall 20°49.432’N, 86°52.664’W O. faveolata 8 6/18/09 5 m Jardines 20°50.045’N, 86°52.694’W O. faveolata 1-4, 6, 7 6/18/09 5 m Radio Pirata 20°51.260’N, 86°51.909’W P. astreoides 1-9 6/18/09 3 m El Islote 20°55.607’N, 86°49.882’W P. divaricata 1-9 6/18/09 3 m El Islote 20°55.607’N, 86°49.882’W

Variable Effect df SS F-statistic p-value O. faveolata

Gross P Model 9, 15 0.4358 0.83 0.6164 T 1 0.1063 1.82 0.2261 G 8 0.3546 0.76 0.6511 Day R Model 9, 15 0.2839 0.75 0.6652 T 1 0.0033 0.08 0.7875 G 8 0.2794 0.83 0.6076 Night R Model 9, 16 0.2000 0.49 0.8451 T 1 0.0519 1.14 0.3221 G 8 0.1353 0.37 0.9065 P. astreoides

Gross P Model 9, 17 0.2899 0.87 0.5848 T 1 0.0288 0.78 0.4040 G 8 0.2611 0.88 0.5699 Day R Model 9, 17 0.1314 0.65 0.7334 T 1 0.0051 0.23 0.6473 G 8 0.1263 0.70 0.6852 Night R Model 9, 17 0.5938 0.83 0.6073 T 1 0.1399 1.77 0.2205 G 8 0.4539 0.72 0.6759 P. divaricata

Gross P Model 9, 15 0.4046 0.58 0.7808 T 1 0.0758 0.97 0.3625 G 8 0.3517 0.56 0.7785 Day R Model 9, 15 0.0869 0.65 0.7286 T 1 0.0059 0.40 0.5503 G 8 0.0810 0.68 0.6978 Night R Model 9, 15 0.1105 0.45 0.8661 T 1 0.0163 0.60 0.4697 G 8 0.1013 0.46 0.8461 df, degrees of freedom; SS, sum of squares of the effect;

Variable Effect df F-statistic V-statistic p-value Zoopl. size Model S 6, 30 0.52 0.19 0.7863 T 3, 14 1.79 0.28 0.1945 S x T 6, 30 0.16 0.06 0.9851 Zoopl. taxa Model S 16, 20 0.89 0.83 0.5873 T 8, 9 0.97 0.46 0.5134 S x T 16, 20 0.54 0.61 0.8909 df, degrees of freedom; V-statistic, value of Pillai’s trace statistic;

Species Effect df SS F-statistic p-value O. faveolata Model 9, 16 157.75 1.91 0.2033 T 1 4.3114 0.47 0.5154 G 8 156.40 2.13 0.1679 P. astreoides Model 9, 17 8.6992 0.62 0.7579 T 1 0.9663 0.62 0.4554 G 8 7.7329 0.62 0.7461 P. divaricata Model 9, 15 115.523 1.42 0.3445 T 1 19.5302 2.16 0.1917 G 8 97.9932 1.36 0.3649 df, degrees of freedom; SS, sum of squares of the effect;

Table 2.5. Results of two-way ANOVAs for CZAR, CHAR, and CTAR of O. faveolata, P. astreoides, and P. divaricata at 0 month recovery. The effect of temperature (T) was fixed and fully crossed with two levels (NB = non-bleached 30.4°C, BL = repeat bleached 31.6°C). Genotype (G) was a random factor with 9 levels (1-9). Significant p- values (p≤0.05) are highlighted in bold.

Variable Effect df SS F-statistic p-value O. faveolata

CZAR Model 9, 16 52009.90 0.89 0.5749 T 1 20065.43 3.09 0.1222 G 8 31871.47 0.61 0.7461 CHAR Model 9, 16 1745.46 1.75 0.2372 T 1 305.20 2.75 0.1413 G 8 1374.90 1.55 0.2891 CTAR Model 9, 14 13827.86 1.09 0.4892 T 1 3472.76 2.46 0.1775 G 8 8301.36 0.74 0.6676 P. astreoides

CZAR Model 9, 17 4.6125 2.30 0.1276 T 1 0.8442 3.79 0.0875 G 8 3.7682 2.11 0.1552 CHAR Model 9, 17 14.9978 0.60 0.7671 T 1 1.3317 0.48 0.5074 G 8 13.6661 0.62 0.7445 CTAR Model 9, 17 61837.60 1.40 0.3238 T 1 1185.36 0.24 0.6365 G 8 60652.24 1.54 0.2770 P. divaricata

CZAR Model 9, 15 5.2488 1.84 0.2365 T 1 1.4619 4.60 0.0755 G 8 4.3974 1.73 0.2599 CHAR Model 9, 15 187.361 2.02 0.2019 T 1 27.6083 2.68 0.1526 G 8 162.336 1.97 0.2118 CTAR Model 9, 15 9029.52 2.53 0.1354 T 1 3184.90 8.03 0.0298 G 8 6882.82 2.17 0.1804 df, degrees of freedom; SS, sum of squares of the effect;

Variable Effect df SS F- p-value Tukey statistic O. faveolata

Chl a Model 13, 47 235.28 3.56 0.0015 T 1 90.146 17.72 0.0002 NB > BL R 2 106.01 10.42 0.0003 1.5 > 11=0 G 8 12.337 0.30 0.9597 T x R 2 16.545 1.63 0.2117 Lipid Model 13, 45 0.0691 1.37 0.2285 T 1 0.0048 1.25 0.2727 R 2 0.0132 1.70 0.1986 G 8 0.0392 1.26 0.2979 T x R 2 0.0131 1.68 0.2022 Protein Model 13, 46 0.2092 2.83 0.0078 T 1 0.0026 0.46 0.5022 R 2 0.0108 0.95 0.3958 G 8 0.1763 3.88 0.0026 2=3=4=1=7=6=8 > 3=4=1=7=6=8=9=5 T x R 2 0.0106 0.93 0.4038 Carbs Model 13, 46 0.0016 15.23 <0.0001 T 1 0.0000 1.95 0.1722 R 2 0.0013 83.28 <0.0001 G 8 0.0001 0.76 0.6379 T x R 2 0.0001 3.79 0.0329 Biomass Model 13, 45 1840.47 1.44 0.1965 T 1 233.58 2.37 0.1335 R 2 388.91 1.97 0.1555 G 8 1146.28 1.45 0.2128 T x R 2 5.9316 0.03 0.9704

continued

Table 2.6 continued

Calc. Model 13, 46 4.8861 3.49 0.0018 T 1 0.3245 3.02 0.0917 R 2 2.7474 12.76 <0.0001 11=1.5 > 0 G 8 1.4813 1.73 0.1279 T x R 2 0.4561 2.12 0.1361 P. astreoides

Chl a Model 13, 50 509.98 14.59 <0.0001 T 1 278.02 103.43 <0.0001 R 2 7.9396 1.48 0.2415 G 8 35.119 1.63 0.1488 T x R 2 176.36 32.80 <0.0001 Lipid Model 13, 51 0.0491 1.51 0.1578 T 1 0.0009 0.34 0.5627 R 2 0.0029 0.58 0.5659 G 8 0.0351 1.76 0.1166 T x R 2 0.0091 1.83 0.1745 Protein Model 13, 52 0.5846 6.69 <0.0001 T 1 0.1208 17.97 0.0001 R 2 0.14237 10.59 0.0002 G 8 0.0978 1.82 0.1029 T x R 2 0.2038 15.16 <0.0001 Carbs Model 13, 52 0.0019 2.41 0.0171 T 1 0.0002 2.66 0.1112 R 2 0.0002 1.61 0.2138 G 8 0.0011 2.27 0.0421 all the same T x R 2 0.0005 3.87 0.0293 Biomass Model 13, 52 3814.24 3.04 0.0036 T 1 262.67 2.72 0.1072 R 2 2998.66 15.52 <0.0001 11=0 > 1.5 G 8 483.90 0.63 0.7507 T x R 2 67.80 0.35 0.7062 Calc. Model 13, 47 1.3173 5.76 <0.0001 T 1 0.3843 21.83 <0.0001 R 2 0.4149 11.79 0.0001 G 8 0.1731 1.23 0.3125 T x R 2 0.2798 7.95 0.0015

continued

Table 2.6 continued

P. divaricata

Chl a Model 13, 40 199.478 3.63 0.0023 T 1 0.2038 0.05 0.8280 R 2 98.1069 11.59 0.0002 G 8 30.5531 0.90 0.5286 T x R 2 52.6506 6.22 0.0060 Lipid Model 13, 39 0.1994 5.43 0.0001 T 1 0.0006 0.20 0.6551 R 2 0.1057 18.71 <0.0001 G 8 0.0296 1.31 0.2820 T x R 2 0.0321 5.68 0.0090 Protein Model 13, 39 0.0279 0.61 0.8202 T 1 0.0003 0.08 0.7781 R 2 0.0170 2.43 0.1080 G 8 0.0104 0.37 0.9265 T x R 2 0.0003 0.04 0.9604 Carbs Model 13, 39 0.0035 6.85 <0.0001 T 1 0.0001 2.87 0.1020 R 2 0.0025 32.03 <0.0001 11 > 1.5 > 0 G 8 0.0004 1.36 0.2615 T x R 1 0.0001 1.59 0.2222 Biomass Model 13, 39 1.2552 3.84 0.0017 T 1 0.0016 0.06 0.8019 R 2 0.4884 9.71 0.0007 G 8 0.5496 2.73 0.0249 all the same T x R 2 0.2042 4.06 0.0292 Calc. Model 13, 39 0.5093 1.12 0.3863 T 1 0.0393 1.12 0.2988 R 2 0.0840 1.20 0.3169 G 8 0.4242 1.52 0.1996 T x R 2 0.0082 0.12 0.8896 df, degrees of freedom; SS, sum of squares of the effect;

Variable Effect df SS F- p-value Tukey statistic O. faveolata

13 δ Ch Model 13, 47 19.9135 6.38 <0.0001 T 1 0.3334 1.39 0.2467 R 2 3.4247 7.14 0.0026 G 8 13.9428 7.26 <0.0001 9=2=3=6 > 2=3=6=8=7=5 > 3=6=8=7=5=14 T x R 2 2.2763 4.74 0.0153 13 δ Ce Model 13, 47 20.7655 5.97 <0.0001 T 1 4.3534 16.28 0.0003 NB > BL R 2 3.2393 6.06 0.0056 11 > 0=1.5 G 8 12.3950 5.80 0.0001 9=3=2=8 > 3=2=8=6=4=5=1=7 T x R 2 0.4332 0.81 0.4532 13 δ Ch - Model 13, 47 9.0652 4.54 0.0002 13 δ Ce T 1 2.2774 14.82 0.0005 R 2 1.9827 6.45 0.0042 G 8 3.7909 3.08 0.0100 7=6=2=9=5=3=1=8 > 6=2=9=5=3=1=8=4 T x R 2 1.0378 3.38 0.0459 15 δ Nh Model 13, 46 0.1744 6.70 <0.0001 T 1 0.0156 7.80 0.0086 BL > NB R 2 0.1061 26.48 <0.0001 0=1.5 > 11 G 8 0.0373 2.33 0.0418 5=8=6=1=9=4=7 > 8=6=1=9=4=7=2 > 6=1=9=4=7=2=3 T x R 2 0.0005 0.13 0.8779

continued

Table 2.7 continued

15 δ Ne Model 13, 47 11.7354 8.67 <0.0001 T 1 0.0948 0.91 0.3466 R 2 7.1388 34.29 <0.0001 1.5=0 > 11 G 8 3.3887 4.07 0.0018 6=5=9=8=4=1=7 > 5=9=8=4=1=7=3 > 4=1=7=3=2 T x R 2 0.1639 0.79 0.4631 P. astreoides

13 δ Ch Model 13, 50 7.2590 2.09 0.0392 T 1 0.0342 0.13 0.7222 R 2 2.1075 3.95 0.0278 11 > 0=1.5 G 8 3.4347 1.61 0.1552 T x R 2 1.2661 2.37 0.1071 13 δ Ce Model 13, 50 35.3205 2.56 0.0125 T 1 0.7803 0.74 0.3966 R 2 20.7324 9.77 0.0004 11 > 1.5=0 G 8 14.1237 1.66 0.1403 T x R 2 2.0659 0.97 0.3871 13 δ Ch - Model 13, 50 16.4006 4.06 0.0004 13 δ Ce T 1 1.6308 5.24 0.0278 R 2 9.1049 14.64 <0.0001 G 8 3.8810 1.56 0.1705 T x R 2 4.8776 7.84 0.0014 15 δ Nh Model 13, 49 22.0190 6.07 <0.0001 T 1 3.4501 12.37 0.0012 R 2 13.5470 24.29 <0.0001 G 8 3.0233 1.36 0.2492 T x R 2 2.9674 5.32 0.0095 15 δ Ne Model 13, 50 12.5850 6.77 <0.0001 T 1 4.5010 31.55 <0.0001 R 2 2.2163 7.75 0.0015 G 8 3.2946 2.88 0.0134 5=6=1=4=7 > 6=1=4=7=8=2=3=9 T x R 2 1.6751 5.86 0.0062

continued

Table 2.7 continued

P. divaricata

13 δ Ch Model 13, 31 8.8066 1.69 0.1492 T 1 0.8290 2.07 0.1675 R 2 0.0286 0.04 0.9651 G 8 7.0898 2.21 0.0773 T x R 2 0.4791 0.60 0.5606 13 δ Ce Model 13, 31 12.1400 2.87 0.0199 T 1 0.1249 0.38 0.5433 R 2 0.5134 0.79 0.4694 G 8 9.8585 3.79 0.0090 6=9=5=2=7=8=4=3 > 9=5=2=7=8=4=3=1 T x R 2 1.7399 2.67 0.0962 13 δ Ch - Model 13, 31 4.0121 2.47 0.0385 13 δ Ce T 1 0.3104 2.48 0.1325 R 2 0.4782 1.91 0.1765 G 8 0.7805 0.78 0.6252 T x R 2 1.3400 5.36 0.0149 15 δ Nh Model 13, 31 8.4067 2.62 0.0300 T 1 0.4368 1.77 0.2000 R 2 2.3564 4.77 0.0217 1.5=0 > 0=11 G 8 3.3117 1.68 0.1724 T x R 2 0.3331 0.67 0.5217 15 δ Ne Model 13, 31 4.2960 4.44 0.0021 T 1 0.3398 4.57 0.0466 BL > NB R 2 2.4058 16.17 <0.0001 0=1.5 > 11 G 8 0.3257 0.55 0.8060 T x R 2 0.1292 0.87 0.4365 df, degrees of freedom; SS, sum of squares of the effect;

Variable Effect df SS F- p-value Tukey statistic O. faveolata

13 δ Cs Model 13, 47 27.7784 3.99 0.0006 T 1 0.4855 0.91 0.3479 R 2 2.4899 2.32 0.1134 G 8 18.1048 4.22 0.0014 3=8=5=2=6=4=9=7 > 6=4=9=7=1 T x R 2 7.3880 6.89 0.0031 18 δ Os Model 13, 47 12.2674 6.02 <0.0001 T 1 0.6775 4.32 0.0453 R 2 0.9091 2.90 0.0689 G 8 9.5328 7.60 <0.0001 8=5=3=2=4=6=9 > 4=6=9=1 > 6=9=1=7 T x R 2 1.5541 4.95 0.0129 P. astreoides

13 δ Cs Model 13, 50 12.9824 5.99 <0.0001 T 1 3.2157 19.28 <0.0001 R 2 1.8886 5.66 0.0072 G 8 4.1297 3.09 0.0089 8=4=1=7=2=3=6=5 > 4=1=7=2=3=6=5=9 T x R 2 3.1606 9.47 0.0005 18 δ Os Model 13, 50 1.5323 4.14 0.0003 T 1 0.5992 21.06 <0.0001 R 2 0.0472 0.83 0.4442 G 8 0.3197 1.40 0.2269 T x R 2 0.5086 8.94 0.0007

continued

Table 2.8 continued

P. divaricata

13 δ Cs Model 13, 31 10.8216 2.52 0.0355 T 1 0.4353 1.32 0.2662 R 2 2.3805 3.60 0.0484 11=0 > 1.5 G 8 6.4728 2.45 0.0547 T x R 2 0.2173 0.33 0.7241 18 δ Os Model 13, 31 1.7622 3.31 0.0101 T 1 0.1101 2.69 0.1185 R 2 0.5925 7.23 0.0050 11 > 1.5=0 G 8 0.8720 2.66 0.0404 9=2=6=1=4=3=7 > 2=6=1=4=3=7=5=8 T x R 2 0.0547 0.67 0.5256 df, degrees of freedom; SS, sum of squares of the effect;

2.8 FIGURES

O. faveolata P. astreoides P. divaricata 100 A B C 80 60

corals 40 20

% of treatment treatment of %

0 1.5 11 0 1.5 11 0 1.5 11 Months of recovery after repeat bleaching

non-bleached partially bleached bleached partially dead non-bleached partially dead bleached dead

Figure 2.3. Bleaching and mortality status for repeat bleached (A) Orbicella faveolata, (B) Porites astreoides, and (C) Porites divaricata at 0, 1.5, and 11 months of recovery. Corals designated as non-bleached were dark brown in color and completely covered by living tissue. Partially bleached fragments were either entirely pale (but not white) or some of the tissue was bleached and some healthy, and they were completely covered by live tissue. Bleached fragments were either 100% white in color or >50% white and the rest pale, and they were completely covered by living tissue. Partially dead fragments were partially covered by filamentous or encrusting algae (or both), and partially covered by patches of living tissue that varied in color from pale to dark brown. Partially dead bleached fragments were partially covered by filamentous or encrusting algae (or both), and partially covered by patches of living tissue that was white in color. Dead fragments were completely covered by filamentous or encrusting algae (or both), with no living tissue remaining.

)

2 -1 A non-bleached 1.2 repeat bleached

gdw -1 0.8

Gross P 0.4

mol min mol

Production of O



(

)

-1 0.6 B

gdw

-1 0.4

Day R 0.2

mol min mol



(

Consumption ofConsumption O

)

-1 0.6 C

gdw

-1 0.4

Night R 0.2

mol min mol



(

Consumption ofConsumption O OF PA PD Species

0.6 A B 0.5 0.4 0.3

Proportion Proportion 0.2

of assemblage of 0.1

CO CZ SN PO UN AM IS OS CU 50-100 >1000 101-200201-400401-1000 Size class (m) Zooplankton Group

) C non-bleached

-1 6 repeat bleached

gdw

-1 4

Feeding rate Feeding 2

(captures hr (captures

OF PA PD Species

Figure 2.5. Proportion of zooplankton capture assemblage by (A) size class and (B) zooplankton group for all species combined immediately after the repeat bleaching treatment (=0 month recovery). Proportions were calculated from a total of 84 zooplankton captures. (C) Average feeding rate of Orbicella faveolata (OF), Porites astreoides (PA), and Porites divaricata (PD) at 0 month recovery. Averages are shown ± 1 SE. Sample size ranges from 8-9. CO=copepod, CZ=carb zoeae, SN=snail, PO=polychaete, UN=unidentified, AM=amphipod, IS=isopod, OS=ostracod, CU=cumaceae.

250 A non-bleached repeat bleached 200 150 100 CZAR (%) CZAR 50

B 20 15 10

CHAR (%) CHAR 5

C 200 NB-mean BL-mean 150 100

CTAR (%) CTAR 50

OF PA PD Species

O. faveolata P. astreoides P. divaricata A G M non-bleached

) 15 * * repeat bleached

-2 * * a * * 10

g cm

Chl Chl

 ( 5

0.4 B H N

) * -1 0.3 0.2

Lipid Lipid (g gdw 0.1

0.6 C I * * * O

) -1 0.4

Protein

(g gdw 0.2

D J P

) 0.06 * -1 * 0.04 *

(g gdw 0.02

Carbohydrate

60 E K Q

)

-2 40 *

(mg cm (mg 20

Tissue biomass Tissue

) F L R -2 1.5 *

-1 1.0 * 0.5

Calcification 0.0

(mg day (mg

0 1.5 11 0 1.5 11 0 1.5 11 Months of recovery

Figure 2.7. Average chlorophyll a, lipid, protein, carbohydrate, tissue biomass, and calcification rates of (A-F) Orbicella faveolata, (G-L) Porites astreoides, and (M-R) Porites divaricata at 0, 1.5, and 11 months of recovery. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a recovery interval. Sample size ranges from 5-9. 96

O. faveolata P. astreoides P. divaricata -12 A F K non-bleached * * repeat bleached h -13

 -14

(‰, v-PDB) (‰, -15

-12 B G L * e -13

 -14

(‰, v-PDB) (‰, -15 C H M 1 * *

h-e

C 0

13  * -1

(‰, v-PDB) (‰,

6 D I N

h 5





 4 (‰, air) (‰, * 3 E J O 5

 4



 * (‰, air) (‰, 3 * *

0 1.5 11 0 1.5 11 0 1.5 11 Months of recovery

13 Figure 2.8. Average stable carbon isotopes of the animal host (δ Ch) and endosymbiont 13 (δ Ce), the difference between the stable carbon isotopes of animal host and 13 15 endosymbiont (δ Ch-e) , and the stable nitrogen isotopes of the animal host (δ Nh) and 15 endosymbiont (δ Ne) of (A-E) Orbicella faveolata, (F-J) Porites astreoides, and (K-O) 13 Porites divaricata at 0, 1.5, and 11 months of recovery. For δ Ch-e, heterotrophy contributes more to the fixed carbon pool when the difference is <0, whereas photosynthesis contributes more when the difference is ≥0. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a specific recovery interval. Sample size ranges from 3-9.

O. faveolata P. astreoides P. divaricata 0 A C E

s -2

C *



± 1 SE ± -4 * (‰, VPDB) VPDB) (‰, -6 B D F -5 *

 -4



 ± 1 SE ± * -3 non-bleached (‰, VPDB) VPDB) (‰, repeat bleached

0 1.5 11 0 1.5 11 0 1.5 11 Months of recovery

13 Figure 2.9. Average stable carbon isotopes of the skeleton (δ Cs) and stable oxygen 18 isotopes of the skeleton (δ Os) of (A-B) Orbicella faveolata, (C-D) Porites astreoides, and (E-F) Porites divaricata at 0, 1.5, and 11 months of recovery. Averages are shown ± 1 SE. Asterisks indicate significant differences between non-bleached and repeat bleached corals within a specific recovery interval. Sample size ranges from 3-9. Note 18 that the axis for δ Os is reversed.

3. KINETIC AND METABOLIC ISOTOPE EFFECTS IN CORAL SKELETONS: A RE-EVALUATION USING EXPERIMENTAL CORAL BLEACHING AS A CASE STUDY

Verena Schoepf1, Stephen J. Levas1, Lisa J. Rodrigues2, Michael O. McBride1, Matthew D. Aschaffenburg3, Mark E. Warner3, and Andréa G. Grottoli1

1. School of Earth Sciences, The Ohio State University, Columbus, OH, USA 2. Department of Geography and Environment, Villanova University, Villanova, PA, USA 3. School of Marine Science and Policy, University of Delaware, Lewes, DE, USA

3.1 ABSTRACT

Coral skeletal δ18O and δ13C are important paleo-climate proxies that are both influenced by kinetic fractionation effects and in the case of δ13C, metabolic fractionation effects as well. However, kinetic isotope effects can overpower metabolic isotope effects and thus compromise the use of coral skeletal δ13C as a proxy of metabolism. Heikoop et al. (2000) proposed a simple data correction to remove kinetic isotope effects from coral skeletal δ13C, as well as an equation to calculate P/R ratios from coral isotopes. However, these data corrections have never been directly tested. Here, we tested the δ13C correction and the P/R calculation using six species of corals from controlled bleaching experiments where both the stable isotopes and the physiological variables that cause isotopic fractionation (i.e., photosynthesis, respiration, and calcification) have been simultaneously measured. Experimental coral bleaching represents a unique environmental scenario to test this because bleaching is caused by elevated temperature (which drives kinetic effects), and produces large physiological responses in corals (which drives metabolic effects). We show for the first time that the data correction proposed by Heikoop et al. (2000) does not effectively remove kinetic effects in the coral species studied here, and did not improve the metabolic signal of bleached and non- bleached corals. In addition, isotope-based P/R ratios were in poor agreement with measured P/R ratios, even when the data correction was applied. We therefore recommend that the data correction not be routinely applied for paleo-climate reconstruction, and that P/R ratios should only be obtained by direct measurement of P and R by respirometry.

100

3.2 INTRODUCTION

The stable carbon and oxygen isotopes of coral skeletal carbonate are powerful tools that have significantly advanced our understanding of past climate. Coral skeletal δ13C has been established as a proxy for light levels or cloud cover, seasonality, and nutrient/zooplankton levels (e.g. Fairbanks, Dodge 1979; Gagan et al. 1994; Swart et al. 1996b; Grottoli 1999; Grottoli, Wellington 1999; Grottoli 2002), whereas coral skeletal δ18O records sea surface temperature (SST) and salinity (SSS) (e.g. Fairbanks, Dodge 1979; Gagan et al. 1994; Swart et al. 1996a; Druffel 1997; Grottoli, Eakin 2007). However, coral aragonite does not precipitate in equilibrium with the isotopic composition of seawater, and is typically depleted in both δ13C and δ18O relative to seawater (Swart 1983b; McConnaughey 1989a) (Fig. 3.1), which has important implications for paleo-climate reconstruction. Two patterns of isotopic disequilibrium are particularly common in biological carbonates: (1) metabolic isotope effects related to photosynthesis and respiration, which modulate the isotopic composition of the internal dissolved inorganic carbon (DIC) pool from which carbonate is precipitated (e.g. Swart 1983b; McConnaughey 1989a; McConnaughey et al. 1997), and (2) kinetic isotope effects related to CO2 hydration and hydroxylation during calcification (McConnaughey 1989a, 1989b; Allison et al. 1996; Cohen, Hart 1997). In tropical scleractinian corals, metabolic isotope effects are thought to dominate coral skeletal δ13C, which allows for their use as powerful proxies for light levels or cloud cover, seasonality, and nutrient/zooplankton levels (e.g. Fairbanks, Dodge 1979; Gagan et al. 1994; Swart et al. 1996b; Grottoli 1999; Grottoli, Wellington 1999; Grottoli 2002; Allison et al. 2012) . However, their fast calcification rates related to the symbiosis with single-celled dinoflagellates (Symbiodinium spp.) (Goreau, Goreau 1959; Gattuso et al. 1999) also favor strong kinetic effects (McConnaughey 1989a, 1989b; Allison et al. 1996; Cohen, Hart 1997; Heikoop et al. 2000a) that can overpower and mask the presence of metabolic effects (McConnaughey 1989a; Allison et al. 1996; Cohen, Hart

101

1997; McConnaughey et al. 1997; Heikoop et al. 2000a). Thus, coral skeletal δ13C is challenging to use as climate proxy. While metabolic isotope effects influence only carbon isotopic composition, kinetic isotope effects result in the simultaneous depletion of δ13C and δ18O in an approximate ratio of 3:1 (McConnaughey 1989a). As a consequence, a strong correlation between δ13C and δ18O (due to their simultaneous depletion) can be used as a diagnostic tool to detect the presence of strong kinetic effects (McConnaughey 1989a). In contrast, the presence of metabolic isotope effects is indicated by the lack of a strong correlation of δ13C and δ18O (McConnaughey 1989a). These characteristic relationships between δ13C and δ18O have been used to propose a simple data correction that removes kinetic isotope effects and reveals potentially hidden metabolic effects in the δ13C signature (Heikoop et al. 2000a; Suzuki et al. 2005; Ourbak et al. 2008). Kinetic and metabolic isotope effects are best assessed when visualized in δ18O vs. δ13C space (Fig. 3.1). Carbonate precipitated in isotopic equilibrium with seawater is expected to plot close to isotopic equilibrium composition (Fig. 3.1). Both metabolic and kinetic isotope effects (KIE) cause significant offsets from isotopic equilibrium composition. As KIE result in the simultaneous depletion of δ13C and δ18O in an approximate ratio of 3:1 (McConnaughey 1989a), corals typically plot along or parallel to the so-called KIE line when kinetic isotope effects dominate (Fig. 3.1). Faster growing corals are expected to plot further away from isotopic equilibrium composition than slower growing corals due to more pronounced KIE effects (Fig.3.1) (McConnaughey 1989a; Allison et al. 1996). In contrast, metabolic isotope effects cause offsets from the KIE line towards both more enriched and more depleted δ13C (Fig. 3.1). This is because photosynthesis enriches the internal DIC pool from which the skeleton is precipitated as photosynthesis preferentially removes 12C, whereas respiration leads to the incorporation of isotopically depleted metabolic C. Generally, photosynthesis affects skeletal δ13C more strongly (up to 11‰) than respiration (about 1.5‰) because symbiotic corals calcify mainly during the day when photosynthetic CO2 uptake is several times faster than respiratory CO2 release (McConnaughey 1989a; McConnaughey et al. 1997). Since high

102 photosynthesis rates are generally related to high calcification rates, fast growing healthy corals are expected to plot towards more enriched δ13C and more depleted δ18O values, respectively (i.e, in the lower right quadrant in Fig. 3.1). Beyond its implications for paleoclimate reconstruction, the concept of metabolic and kinetic isotope effects as indicated by plots of skeletal δ18O vs. δ13C (Fig. 3.1) is also a valuable tool to detect changes in coral metabolism, the degree of auto- vs. heterotrophy, and changes in calcification rates. They can thus be used to infer ecological and physiological plasticity in corals (Maier et al. 2003), and to trace physiological changes during a variety of environmental scenarios including coral bleaching (Suzuki et al. 2003), haze events (Risk et al. 2003), and different water flow conditions (Suzuki et al. 2008). Further, they can be used to estimate the ratio of photosynthesis to respiration (P/R ratio) based on where corals plot in the space of skeletal δ18O vs. δ13C (Maier et al. 2003) or based on calculations using coral skeletal and tissue isotopes (Heikoop et al. 2000a; Maier 2004; Kaandorp et al. 2005; Lesser et al. 2010). Although many studies support this theory of metabolic and kinetic isotope fractionation effects (e.g. McConnaughey 1989b; Allison et al. 1996; Cohen, Hart 1997; Heikoop et al. 2000a), it has never been tested using controlled, replicated experiments where the extent of both isotope fractionation effects is directly quantified by simultaneously measuring the physiological variables that cause fractionation (i.e., photosynthesis, respiration, and calcification) and the paired skeletal stable isotopes values. More specifically, such a direct test is needed to determine if (1) the data correction proposed by Heikoop et al. (2000a) effectively removes kinetic isotope effects, (2) if isotope-based P/R ratios are reliable proxies for P/R ratios measured by respirometry, and (3) if the data correction can be applied to a wide range of coral species under a range of environmental scenarios that influence both metabolic and kinetic isotope effects. One such environmental scenario that affects both metabolic and kinetic isotope effects is coral bleaching, which is largely caused by periods of elevated seawater temperature (Glynn 1993; Brown 1997b, 1997a; Hoegh-Guldberg 1999; Lesser 2004;

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Baker et al. 2008). During bleaching, corals lose significant amounts of their algal endosymbionts and/or photosynthetic pigments within the endosymbionts (Hoegh- Guldberg, Smith 1989; Jokiel, Coles 1990; Glynn 1993, 1996; Brown 1997b; Hoegh- Guldberg 1999; Fitt et al. 2001; Baker et al. 2008), which renders them pale or “bleached” in appearance. This can result in dramatic declines in photosynthesis (Porter et al. 1989; Fitt, Warner 1995; Jones 1997; Lesser 1997; Grottoli et al. 2006; Rodrigues, Grottoli 2007; Rodrigues et al. 2008; Levas 2012), which means that significantly less 12C is removed from the internal DIC pool from which the skeleton is precipitated. As a consequence, the δ13C of bleached corals that maintain modest rates of calcification is typically (though not always) more depleted compared to healthy, non-bleached corals (Porter et al. 1989; Grottoli et al. 2004; Rodrigues, Grottoli 2006; Levas et al. 2013). When corals recover from bleaching, skeletal δ13C is expected to increase until it is no longer different from non-bleached controls. In δ18O vs. δ13C space, bleached corals are expected to plot closer to the KIE line than healthy corals due to reduced photosynthesis and also closer to equilibrium as calcification rates are often compromised (Fig. 3.1). However, bleached corals that are 13 still modestly calcifying do not always show this expected decrease in δ Cs (Leder et al. 1991; Rodrigues, Grottoli 2006; Hartmann et al. 2010; Levas 2012), which is potentially due to strong kinetic effects masking changes in photosynthesis and/or respiration. The application of a data correction to remove kinetic isotope effects (Heikoop et al. 2000a) might therefore reveal the masked metabolic isotope effects, and thus improve accuracy and interpretation of skeletal isotopes in bleached corals. Corals that were bleached under controlled experimental conditions present a unique case study for directly testing the data correction to remove kinetic isotope effects proposed by Heikoop et al. (2000a), and if coral skeletal isotopes are reliable proxies for P/R ratios. This is because bleaching is caused by elevated temperature (which drives kinetic effects) and produces large physiological responses in corals (which drives metabolic effects). Here, we reanalyzed previously published data from the controlled bleaching experiments of Rodrigues and Grottoli (2006), Levas et al. (2013), and Chapter

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1 that meet these criteria. In these studies, three Hawaiian and three Mexican coral species were either bleached by subjecting them to elevated seawater temperature for 2.5 – 4 weeks or were kept at ambient temperatures as controls. Hawaiian corals were exposed to elevated temperature once, whereas Mexican corals were repeat bleached by exposing them to elevated temperature in two consecutive summers. This was done to assess any potential effects of frequent thermal stress on coral isotopes, which could have implications for the reconstruction of past bleaching events from coral skeletons. Short and long term recovery was assessed over 8-11 months, and P/R ratios were measured in addition to their tissue and skeletal isotopes at relevant time points. A suite of other physiological measurements were performed in these coral studies (Chapter 1, Grottoli et al. 2006; Rodrigues, Grottoli 2006, 2007; Rodrigues et al. 2008; Aschaffenburg 2012; Levas et al. 2013; Grottoli et al. in review), providing a rich background of physiological information within which to interpret our findings. 13 13 13 We hypothesize that (1) the correlation between skeletal δ C (δ Cs) and tissue δ C 13 13 13 (δ Ch) improves when δ Cs values are corrected (δ Cscorr ) according to Heikoop et al. 13 (2000a), and (2) that as photosynthesis and calcification decline with bleaching, δ Cscorr values move towards the upper left corner in δ18O vs. δ13C space. Further, we hypothesize that isotope-based calculated P/R ratios (Kaandorp et al. 2005) are significantly correlated with P/R ratios measured by respirometry. If these hypotheses are confirmed, the data correction could be routinely applied to paleo-climate reconstructions to improve accuracy of coral proxy records. In addition, improved metabolic signals in bleached corals might allow for the reconstruction of past bleaching events from coral skeletons. Finally, isotope-based P/R ratios could be used to infer coral metabolism in mesophotic or deep sea environments, where respirometry is either not practical or cannot be easily performed. Overall, this approach of combined physiological and isotopic analyses should significantly promote the understanding of the functional processes underlying isotopic proxy signals in coral skeletons.

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3.3 MATERIAL AND METHODS

3.3.1 Hawaii Bleaching Experiments

A detailed description of the bleaching experiment in Hawaii can be found in Rodrigues and Grottoli (2006). Briefly, coral fragments from Porites compressa and Montipora capitata were collected from Point Reef, Kaneohe Bay, Hawaii (21°26.18’N; 157°47.56’W) in late August 2003 from 2 m depth. On 4 September 2003, half of all fragments were placed in shaded outdoor tanks with ambient seawater (26.8°C ± 0.04 SE) (non-bleached controls), whereas the other half was placed in tanks with elevated temperature (30.1°C ± 0.05 SE) (bleached corals). Temperature was gradually elevated over the course of three days. Corals were not fed during the experiment, and inflow pipes were fitted with a 50 μm-filter. To minimize positional effects, corals were rotated within and among tanks of the same treatment daily. After one month, on 4 October 2003, 25% of all treatment and control fragments were collected and frozen for isotopic analyses (= 0 month recovery), whereas the remaining corals were placed back on the reef to recover in situ. To assess short and long term recovery, a third of all remaining treatment and control corals were collected after 1.5, 4, and 8 months on 16 November 2003, 2 February 2004, and 4 June 2004, respectively. Photosynthesis and respiration rates were measured at each recovery interval before corals were frozen for isotopic analyses. A similar bleaching experiment was performed in summer 2006 to assess bleaching impacts on Porites lobata. A detailed description of the experimental design can be found in Levas et al. (2013). Briefly, coral fragments were collected from Sanpan Channel, Kaneohe Bay, Hawaii (21°26.18’N; 157°47.56’W) in August 2006 from 10-12 m depth. On 18 August 2006, half of all fragments were placed in shaded outdoor tanks with ambient seawater (27.5°C ± 0.08 SE) (non-bleached controls), whereas the other half was placed in tanks with elevated temperature (30.2°C ± 0.20 SE) (bleached corals). Temperature was gradually elevated over the course of seven days. Corals were fed

106 freshly caught zooplankton for 1 h at dusk every other night. To minimize positional effects, corals were rotated within and among tanks of the same treatment daily. After 23 days, on 9 September 2006, 25% of all treatment and control fragments were collected and frozen for isotopic analyses (= 0 month recovery), whereas the remaining corals were placed back on the reef to recover in situ. To assess short and long term recovery, a third of all remaining treatment and control corals were collected after 1, 5, and 11 months on 7 October 2006, 4 February 2007, and 20 August 2007, respectively. Photosynthesis and respiration rates were measured at each recovery interval before corals were frozen for isotopic analyses.

3.3.2 Mexico Bleaching Experiment

A detailed description of the experimental design can be found in Chapter 1 of this dissertation (Fig. 2.1). Briefly, coral fragments of Orbicella faveolata (formerly Montastraea faveolata (Budd et al. 2012)), Porites astreoides, and Porites divaricata were collected in July 2009 from reefs near Puerto Morelos, Mexico, from shallow depth (3-8 m) (see Table 2.1 for details on collection dates, sites, and depths). On 14 July 2009, half of the coral fragments from each parent colony were randomly assigned to each treatment: (1) ambient control fragments (non-bleached corals) were maintained in tanks with ambient seawater temperature (30.66 ± 0.24°C), whereas (2) treatment fragments (singly bleached corals) were placed in tanks with elevated seawater temperature (31.48 ± 0.20°C). Seawater temperature in the treatment tanks was gradually elevated over the course of a week. Corals were not fed during the experiment but had access to unfiltered seawater. Fragments were rotated daily within and among tanks of the same treatment to minimize any position or tank-specific effects. After a total of 15 days on 29 July 2009, temperature in all tanks was returned to ambient levels, and all coral fragments were placed on the back reef to recover in situ. On 9 July 2010, all coral fragments that had recovered on the back reef for 1 year were recollected and thoroughly cleaned. On 20 July 2010, all corals that had served as

107 ambient control fragments the previous summer were placed in tanks with ambient seawater (30.40 ± 0.23°C) (non-bleached corals), whereas all corals that had been used as treatment fragments were maintained in tanks with elevated temperature (gradually elevated to 31.6 ° over 7 days and then held at 31.60 ± 0.24°C for 10 days) (repeat bleached corals). Corals were not fed during the experiment but had access to unfiltered seawater. Fragments were rotated daily within and among tanks of the same treatment to minimize any position or tank-specific effects. After 17 days on 6 August 2010, all tanks were returned to ambient temperature levels. During the last days of the repeat bleaching treatment, photosynthesis and respiration rates were measured on one ambient control and one treatment coral fragment of each colony and species (i.e., n=9 fragments per species and treatment) (6-7 August 2010), and were then frozen for additional physiological and isotopic analyses. All remaining fragments were placed on the back reef to recover in situ. To assess short- and long-term recovery from repeat bleaching, one fragment from each colony and treatment was recollected from the reef after 1.5 and 11 months of recovery (22 September 2010 and 14 June 2011), and then frozen for isotopic analyses. Photosynthesis and respiration rates were not measured at these recovery intervals.

3.3.3 Physiological Analyses

Photosynthesis and Respiration. Photosynthesis (P) and respiration (R) rates were measured by incubating non-bleached and bleached corals in plastic chambers and measuring changes in dissolved oxygen during light and dark conditions. P and R were standardized to coral ash-free dry tissue biomass (O. faveolata, P. astreoides, P. divaricata, P. compressa, M. capitata) or surface area (P. lobata). P/R ratios were then calculated by dividing gross P by day R. A detailed description of the P and R measurement methods for the Mexican corals can be found in Chapter 1 (section 2.3.3), and method details for the Hawaiian corals are given in Rodrigues and Grottoli (2007) and Levas et al. (2013). P/R ratios derived from measured P and R values were then

108 compared to P/R ratios calculated from coral skeletal and tissue isotopes (see methods below).

3.3.4 Isotopic Analyses

Seawater DIC Isotopes. A total of nine filtered seawater samples from Kaneohe 13 Bay, Hawaii, were collected throughout 2006/07 for δ CDIC analyses. They were preserved with anhydrous MgCl according to methods by Raymond and Bauer (Raymond, Bauer 2001). In the laboratory, each sample was acidified on the vacuum extraction line under high-purity helium flow, with the resulting CO2 gas cryogenically isolated under vacuum, and the DIC concentration was determined (McNichol et al.

1994). The CO2 from each DIC sample was sealed in Pyrex ampoules and introduced into a Finnigan Delta IV SIRMS via an automated 10-port inlet. All δ13C values were reported 13 as per mil values relative to v-PDB. δ CDIC analyses were not performed for seawater from Puerto Morelos, Mexico. The standard deviation of repeated measurements of an internal standard was ± 0.03‰ (n = 37).

Tissue and Skeletal Isotopes. A detailed description of the isotopic analyses for the Mexican corals can be found in Chapter 1 (section 2.3.4), and for the Hawaiian corals in Rodrigues and Grottoli (2006) and Levas et al. (2013). Briefly, coral tissue was removed from the skeleton using an artist’s airbrush, homogenized, and separated into animal host and algal endosymbiont by centrifugation. The two fractions were then individually transferred onto pre-burned glass fiber filters and combusted in an Elemental Analyzer coupled to a stable isotope ratio mass spectrometer (SIRMS). For skeletal isotopes, the uppermost layer of the dried skeleton was gently shaved with a diamond- tipped Dremel tool and ground to fine powder. About 80-100 μg of the skeletal powder were analyzed for δ13C and δ18O using an automated Kiel Carbonate Device coupled to a SIRMS. Samples were acidified under vacuum with 100% ortho-phosphoric acid. The 13 13 carbon isotopic composition of the animal host (δ Ch), algal endosymbiont (δ Ce), and

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13 13 12 skeleton (δ Cs) were reported as the per mil deviation of the stable isotopes C: C relative to Vienna-Peedee Belemnite Limestone standard (v-PDB). Skeletal oxygen 18 isotopes (δ Os) were reported as the per mil deviation of the ratio of stable isotopes 18O:16O relative to v-PDB. For both organic and skeletal isotopes, approximately 10% of all samples were run in duplicate. For Porites compressa and Montipora capitata, the 13 standard deviation of repeated measurements of internal standards was ± 0.16‰ for δ Ch 13 13 18 and δ Ce (n = 140), ± 0.05‰ for δ Cs, and ± 0.08‰ for δ Os (n = 96) (Rodrigues, Grottoli 2006). For Porites lobata, the standard deviation of repeated measurements of 13 13 13 internal standards was ± 0.08‰ for δ Ch and δ Ce (n = 24), ± 0.03‰ for δ Cs, and ± 18 0.07‰ for δ Os (n = 50) (Levas et al. 2013). For the Mexican coral species, the standard 13 deviation of repeated measurements of internal standards was ± 0.04‰ for δ Ch and 13 13 18 δ Ce (n = 55), ± 0.03‰ for δ Cs, and ± 0.07‰ for δ Os (n = 55) (Chapter 1).

13 Data Correction. Coral skeletal carbon isotopes (δ Cs) were corrected using 18 skeletal oxygen isotopes (δ Os) to remove kinetic effects according to the following equation (Heikoop et al. 2000a):

13 13 18 18 δ Cs corrected = δ Cs original –(3*( δ Os original – δ Os average)) (1)

18 Here, δ Os average was calculated individually for each treatment and recovery interval in each species.

13 Carbon Isotopic Equilibrium. Carbon isotopic equilibrium (δ Ceq) was calculated following McConnaughey et al. (1997) and Heikoop et al. (2000a) using the equation of Romanek et al. (1992):

13 13 δ Caragonite = δ CDIC + 2.7 (2)

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13 For Hawaiian corals, an average δ CDIC of +0.12 ‰ ± 0.44 SD (n=9) was measured in 13 Kaneohe Bay in 2006 and 2007 (Table 3.1), and δ Ceq was therefore estimated to be 13 +2.82‰. For Mexican corals, δ CDIC is unknown for Puerto Morelos and was therefore estimated to be +1.15‰ based on literature values from other locations in the Caribbean (Grossman, Ku 1986; Swart et al. 1996b; Watanabe et al. 2002; Maier 2004; Maier et al. 13 2010; Moyer, Grottoli 2011). Thus, an average δ Ceq of +3.85‰ was used.

Oxygen Isotopic Equilibrium. Two different methods exist in the literature to 18 calculate oxygen isotopic equilibrium composition for the coral skeleton (δ Oeq), only one of which incorporates temperature-dependent fractionation. To facilitate comparisons 18 across studies, both methods were used in this study. First, δ Oeq was calculated using the equation by Grossman and Ku (1986):

(dc – dw) = 4.75 – 0.23*T (°C) (3)

18 18 where dc is δ Oeq and dw is δ Oseawater expressed on the same isotopic scale. For 18 Hawaiian corals, δ Oseawater of Kaneohe Bay is unknown and was estimated to be +0.4‰ (SMOW) or +0.13‰ (v-PDB) based on values from the global 18O database (Epstein, Mayeda 1953; Ostlund et al. 1987; Schmidt et al. 1999). Seawater temperatures range from 23.0 – 28.0°C in Kaneohe Bay (Rodrigues, Grottoli 2006; Levas 2012), which 18 resulted in an average δ Oeq of –1.24‰ when the temperature of the bleaching treatments (30.1°C and 30.2°C) was included. 18 For Mexican corals, δ Oseawater is unknown for Puerto Morelos and was therefore estimated to be +0.85‰ (SMOW) based on literature values from the Caribbean (Leder et al. 1996; Watanabe et al. 2002; Maier 2004). This value was then converted to v-PDB 18 scale by subtracting 0.27‰ (Schmidt 1999), resulting in an average δ Oseawater of +0.58‰ (v-PDB). Seawater temperature was monitored using HOBO temperature loggers throughout the study, with an annual range of 26.3 – 30.7°C during 2009/2010, 18 resulting in an average δ Oeq of -1.32‰ for singly bleached corals when the temperature

111 of the bleaching treatment (31.5°C) was included. Similarly, during 2010/2011 seawater 18 temperatures ranged from 25.5 – 30.4°C, resulting in an average δ Oeq of -1.24‰ for repeat bleached corals when the temperature of the bleaching treatment (31.6°C) was included. 18 18 Second, δ Oeq was calculated according to Maier (2004) where δ Oeq equals 18 δ Oseawater after conversion to v-PDB isotopic scale:

18 18 δ Oeq (v-PDB) = δ Oseawater (SMOW) - 0.27 (4)

This equilibrium value is independent of seawater temperature. For Hawaiian and 18 Mexican corals, δ Oeq was therefore +0.13‰ and +0.58‰ (v-PDB), respectively.

Isotope-based P/R Ratios. P/R ratios were calculated from skeletal and tissue isotopes according to the following equations (Maier 2004; Kaandorp et al. 2005):

13 18 18 13 Moffset = (δ Cs original – α (δ Os original - δ Oeq) - δ Ceq) (5)

13 13 P/R = ((Moffset – r)/r) / (δ Ce/ δ Ch) (6)

where Moffset is the metabolic offset from the kinetic isotope effect (KIE) line, α is the 18 13 slope of relation of δ Os to δ Cs which is estimated to be 0.33 based on the simultaneous 13 18 depletion of δ Cs and δ Os in an approximate ratio of 3:1 due to KIE effects 13 (McConnaughey 1989a; Heikoop et al. 2000a), r is the offset of δ Cs from the KIE line due to respiration which is estimated to be -1.5‰ (McConnaughey et al. 1997; Heikoop 13 et al. 2000a), δ Ce is the carbon isotopic composition of the algal endosymbiont, and 13 δ Ch is the carbon isotopic composition of the animal host. Absolute values of r were 18 used for calculations. Isotope-based P/R ratios were calculated using δ Oeq calculated 18 after both Grossman and Ku (1986) and Maier (2004). When δ Oeq calculated after Grossman and Ku (1986) was used, it was calculated individually for each treatment and

112 recovery interval, thus taking into account temperature differences due to treatment or season.

3.3.5 Statistical Analyses

To determine the presence of kinetic isotope effects, correlations between 13 13 18 uncorrected original δ Cs (i.e., δ Csorig) and δ Os were calculated using Spearman’s 13 rank correlation coefficient r. Following Heikoop et al. (2000a), δ Csorig was considered to be dominated by kinetic isotope effects when this correlation was statistically significant (p-values≤0.05). To test the effectiveness of the data transformation in removing kinetic isotope effects, two methods were used. First, following (Heikoop et al. 2000a), correlations were 13 13 13 computed for δ Csorig vs. δ C of the animal host tissue (i.e., δ Ch), and for corrected 13 13 13 δ Cs (i.e., δ Cscorr) vs δ Ch. Since coral tissue isotopes are not affected by kinetic isotope effects related to calcification, they can be used to assess if the data correction was effective. Thus, good correlation between the skeleton and tissue δ13C should indicate that metabolic isotope effects dominate the skeletal isotope signal, and that kinetic isotope effects were successfully removed. While Heikoop et al. (2000) used whole tissue (i.e., animal host + algal endosymbiont) isotopes for this comparison, we chose to use animal host isotopes as the coral tissue is made up of much more animal host 13 13 13 cells compared to algal cells. Nevertheless, both δ Ch and δ Ce versus δ Csorig and 13 13 δ Cscorr were evaluated (Table 3.8). However, correlations with δ Ce were generally 13 even weaker than those with δ Ch and so were not evaluated any further. Correlation analyses were computed (1) for all Hawaiian and Mexican coral species, respectively, (2) for each species and treatment pooled across all recovery intervals, and (3) individually for each species, treatment, and recovery interval as long as sample size was at least four. The data correction was considered to be effective in removing kinetic isotope effects when (1) the correlation was statistically significant and (2) Spearman’s r was higher for 13 13 13 13 δ Cscorr vs. δ Ch compared to δ Csorig vs. δ Ch (Heikoop et al. 2000a).

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13 Second, δ Cscorr was plotted for each species, treatment, and recovery interval in δ18O vs. δ13C space. It was then assessed if bleached corals plotted more towards lower photosynthesis and lower calcification rates (i.e., towards the upper left in δ18O vs. δ13C space – see Fig. 3.1) compared to non-bleached corals, as would be expected based on measured changes in physiology and calcification (Chapter 1, Rodrigues, Grottoli 2006, 2007; Aschaffenburg 2012; Levas et al. 2013; Grottoli et al. in review). To compare measured and isotope-based P/R ratios, correlations were calculated using Spearman’s rank correlation coefficient r for the following comparisons: (1) 13 18 measured P/R vs. isotope based P/R using δ Csorig and δ Oeq after Grossman and Ku 13 18 (1986), (2) measured P/R vs. isotope based P/R using δ Cscorr and δ Oeq after Grossman 13 18 and Ku (1986), (3) measured P/R vs. isotope based P/R using δ Csorig and δ Oeq after 13 18 Maier (2004), and (4) measured P/R vs. isotope based P/R using δ Cscorr and δ Oeq after Maier (2004). In addition, paired t-tests were computed for the same four comparisons to test if the averages of the measured and isotope-based P/R ratios differed significantly from one another. For t-test analyses, data were first tested for normality using residual plots and Shapiro-Wilk’s test and when necessary, data were transformed to meet the assumption of normality. Bonferroni corrections were not applied because they increase the risk of false negatives (Quinn, Keough 2002; Moran 2003). A total of three outliers (two from P. compressa, one from M. capitata) were excluded from all statistical analyses but are clearly indicated in the figures. Statistical analyses were performed using SAS software, Version 9.2 of the SAS System for Windows.

3.4 RESULTS

13 18 The skeletal carbon and oxygen isotopes (δ Cs and δ Os, respectively) of all Mexican and Hawaiian coral species plotted roughly parallel to the KIE line, but were slightly offset towards more depleted δ18O values (Fig. 3.1). They were much closer to

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18 the KIE line that used oxygen isotopic equilibrium (δ Oeq) calculated using Grossman 18 and Ku (1986) compared to that using δ Oeq calculated using Maier (2004).

3.4.1 Isotope correlations

13 18 Hawaiian Corals. δ Csorig and δ Os were highly correlated in most Hawaiian corals, even when the data set was analyzed individually for each species and treatment (Table 3.2A), with the exception of the P. compressa (all corals) and control M. capitata 13 13 (Table 3.2A). δ Csorig and δ Ch were significantly correlated in five cases, but none of 13 them showed improved correlations when computed with δ Cscorr (Table 3.2A). When correlations were calculated for each individual treatment and recovery 13 18 interval in each species, significant correlations between δ Csorig and δ Os were only observed in the following treatments: bleached P. compressa at 4 months of recovery, non-bleached M. capitata at 0 months of recovery, bleached M. capitata at 1.5 months of recovery, and bleached P. lobata at 0 month of recovery (Table 3.2B). Furthermore, only 13 13 three of the 16 cases showed significant correlations between δ Csorig and δ Ch (Table 13 3.2B). Of these, none of the correlation coefficients improved with δ Cscorr. However, in 13 one case the correlation with δ Cscorr was significant even though it had not been 13 significant for δ Csorig (Table 3.2B). 13 18 13 In δ C vs. δ O space, bleached δ Cscorr at 0 and 1.5 months of recovery were 13 somewhat more depleted (i.e., had lower photosynthesis) compared to bleached δ Cscorr at 4 and 8 months of recovery (Fig. 3-2H, J, L), but did not differ from non-bleached 13 control δ Cscorr values within any recovery interval (Fig. 3.2G, I, K). Further, no changes 13 in δ Cscorr were observed to indicate decreases in calcification at 0 and 1.5 months of recovery compared to non-bleached controls (Fig. 3.2).

Mexican Corals. Most Mexican corals showed highly significant correlations 13 18 between δ Csorig and δ Os, even when the data set was analyzed individually for each species and treatment (Table 3.3A). The only exception was non-bleached P. divaricata

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13 13 corals (Table 3.3A). In addition, of the 8 cases where δ Csorig and δ Ch were 13 significantly correlated, only two had stronger correlations with δ Cscorr, (Table 3.3A). When correlations were calculated for each treatment and recovery interval 13 18 individually, δ Csorig and δ Os were highly correlated for both non-bleached and repeat bleached O. faveolata at 0 and 1.5 months of recovery, but not at 11 months of recovery (Table 3.3B). For P. astreoides, only non-bleached corals at both 0 and 1.5 months of 13 18 recovery showed significantly correlated δ Csorig and δ Os (Table 3.3). In P. divaricata, 13 18 δ Csorig and δ Os of both non-bleached and repeat bleached corals were highly correlated at 0 months of recovery, but not at any other recovery interval (Table 3.3B). 13 Furthermore, only three of the 17 cases showed significant correlations between δ Csorig 13 13 and δ Ch (Table 3.3B). Of these, the correlation improved in only one case when δ Cscorr was used (Table 3.3B). 13 18 13 In δ C vs. δ O space, O. faveolata, bleached δ Cscorr at 1.5 months of recovery 13 13 was more depleted in δ C (i.e., had lower photosynthesis) compared to bleached δ Cscorr at 0 and 11 months of recovery (Fig. 3.3H), and compared to non-bleached controls at 1.5 13 (but not 0) months of recovery (Fig. 3.3G). In P. astreoides, bleached δ Cscorr at 0 month 13 13 of recovery was more depleted in δ C compared to bleached δ Cscorr at 1.5 and 11 13 months of recovery (Fig. 3.3H, J, L), but not compared to non-bleached δ Cscorr at 0 13 month of recovery (Fig. 3.2I). In P. divaricata, bleached δ Cscorr at 0 and 1.5 months of 13 13 recovery was more depleted in δ C compared to bleached δ Cscorr at 11 months of 13 recovery (Fig. 3.3L), but not compared to non-bleached control δ Cscorr (Fig. 3.2K). Only 13 18 in P. astreoides was bleached δ Cscorr more enriched in δ O (i.e., had lower calcification) at 1.5 months of recovery compared to non-bleached controls (Fig. 3.3I, J).

3.4.2 Measured and isotope-based P/R ratios

Hawaiian Corals. In almost all cases, measured P/R ratios of Hawaiian corals were not significantly correlated with any isotope-based P/R ratios (Table 3.4, Fig. 3.4). However, in P. compressa, measured P/R ratios were negatively correlated with isotope-

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18 based P/R ratios using δ Oeq calculated after Maier (2004), independent of whether 13 original or corrected δ Cs was used (Table 3.4, Fig. 3.4). In P. lobata, measured P/R 18 ratios were positively correlated with isotope-based P/R ratios using δ Oeq calculated 13 after Grossman and Ku (1986), independent of whether original or corrected δ Cs was used (Table 3.4, Fig. 3.4). Paired t-tests indicated that typically isotope-based P/R ratios were significantly different from measured P/R ratios in Hawaiian corals (Table 3.5). Despite the significant 18 correlations of measured and isotope-based P/R ratios using δ Oeq calculated after Maier (2004) in P. compressa, t-tests indicated that they were significantly different (Table 3.5). 18 In contrast, t-tests confirmed that isotope-based P/R ratios using δ Oeq calculated after Grossman and Ku (1986) in P. lobata were not significantly different from measured P/R 13 18 ratios (Table 3.5). Further, isotope-based P/R ratios using corrected δ Cs and δ Oeq calculated after Grossman and Ku (1986) in M. capitata were not significantly different from measured P/R ratios (Table 3.5), even though they were not significantly correlated (Table 3.4).

Mexican Corals. Measured and isotope-based P/R ratios were not significantly 18 correlated in any Mexican coral species, independent of the type of δ Oeq and whether 13 original or corrected δ Cs was used (Fig. 3.5, Table 3.6). Similarly, isotope-based P/R ratios differed significantly from measured P/R ratios in all cases (Table 3.7).

18 Generally, isotope-based P/R ratios calculated using δ Oeq after Grossman and Ku (1986) tended to underestimate P/R ratios in both Hawaiian and Mexican coral 18 species, whereas isotope-based P/R ratios calculated using δ Oeq after Maier (2004) were typically higher than measured P/R ratios (Fig. 3.4, 3.5). Further, isotope-based P/R ratios 18 calculated using δ Oeq after Grossman and Ku (1986) sometimes resulted in negative P/R ratios, but measured P/R ratios were always greater than zero (Fig. 3.4, 3.5).

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3.5 DISCUSSION

In the present study, we re-evaluated metabolic and kinetic isotope effects in coral skeletons of non-bleached and bleached Mexican and Hawaiian coral species of different growth morphologies. We show for the first time that despite the fact that all coral species showed significant kinetic isotope effects, the data correction proposed by Heikoop et al. (2000a) did not improve the metabolic signal in these corals. Further, isotope-based P/R ratios differed significantly from P/R ratios measured by respirometry, independent of whether the data correction was used or not.

3.5.1 Presence of kinetic isotope effects

Both Mexican and Hawaiian coral species showed highly significant correlations 13 18 between δ Csorig and δ Os (Fig. 3.1, Tables 3.2, 3.3). While this was independent of colony morphology and origin, correlation coefficients were generally higher in Mexican 13 compared to Hawaiian corals. The presence of significant correlations between δ Csorig 18 and δ Os is consistent with other studies for a wide range of both Pacific and Caribbean corals (McConnaughey 1989a; McConnaughey et al. 1997; Heikoop et al. 2000a; Maier et al. 2003; Risk et al. 2003; Suzuki et al. 2003; Omata et al. 2005; Suzuki et al. 2008). Overall, this indicates that the isotopic signal of the corals studied here has a strong kinetic fractionation component (McConnaughey 1989a; McConnaughey et al. 1997; Heikoop et al. 2000a). 18 Theoretically, the observed variability in δ Os could have been caused by factors other than kinetic isotope effects, such as variations in seawater δ18O. Considering that non-bleached and bleached corals of each species were exposed to identical environmental conditions except for temperature during the experimental bleaching 18 treatment in tanks, it is very unlikely that the observed variability in δ Os was caused by differences in salinity. Further, corals were exposed to identical environmental conditions throughout the 8-11 months of recovery (i.e., temperature, salinity, light, flow, plankton

118 abundance, etc.) within their respective locations (i.e., Mexico or Hawaii), thus 18 eliminating the possibility that the observed δ Os within each location was caused by differences in environmental conditions during recovery. However, it is possible that the shaving method used to sample the skeleton could have produced some isotopic variability among samples. Despite efforts to sample an identical time period when shaving the coral skeleton for isotopic analyses, it is possible that small differences in time periods were sampled. Thus, some of the observed 13 18 variability in δ Csorig and δ Os may be due to slightly different time periods being 13 18 represented with each sample. Nevertheless, the majority of δ Csorig and δ Os variability is likely due to kinetic isotope effects given their significant correlations and the high degree of agreement with other studies.

13 3.5.2 Evaluation of the δ Cs data correction

13 Two methods were used to evaluate the effectiveness of the δ Cs correction. 13 13 13 13 First, δ Csorig versus δ Ch correlations were compared to δ Cscorr versus δ Ch 18 13 correlations. Second, δ Os versus δ Cscorr plots of non-bleached and bleached corals of the same species were compared to determine if measured changes in photosynthesis, respiration, and calcification due to bleaching were reflected in the theoretically expected changes in the isotopic composition of the skeleton. First, the data correction proposed by Heikoop et al. (2000a) to remove kinetic isotope effects was generally not effective in any of the six coral species studied here (Tables 3.2, 3.3), despite the fact that two of the species were the same as those studied by Heikoop et al. (2000a), and a third one was the same genus. Heikoop et al. (2000a) provided evidence for the efficiency of the data correction by correlating both original 13 and corrected δ Cs with whole tissue isotopes as tissue isotopes are not affected by 13 13 kinetic isotope effects related to calcification.. They showed that δ Cscorr versus δ Ctissue correlations were stronger in both Pacific and Caribbean corals collected over depth and 13 light gradients compared to the same correlations using original δ Cs. Overall, our data

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13 overwhelmingly showed that for 49 of 53 cases evaluated, δ Cscorr resulted in no change 13 13 or weaker correlations with δ Ch than with δ Corig for all species and all cases within each species (Tables 3.2, 3.3). 18 13 Second, plots of δ Os vs. δ Cscorr did not produce results consistent with the measured changes in photosynthesis, respiration, and calcification in either Hawaiian or 18 13 Mexican corals (Fig. 3.2, 3.3). In δ Os vs. δ Cs space, bleached corals were expected to 13 18 plot towards more depleted δ Cs and more enriched δ Os values (i.e., towards the upper left ) compared to non-bleached corals as both photosynthesis and calcification rates are typically compromised during the first two months after bleaching (Chapter 1, Rodrigues, Grottoli 2006, 2007; Levas et al. 2013). Photosynthesis and calcification rates were fully recovered in most species after 8-11 months of recovery (Chapter 1, Rodrigues, Grottoli 2006, 2007; Levas et al. 2013). Therefore, bleached corals were expected to plot in similar areas as the non-bleached control corals later during recovery. However, this was not generally the case (Fig. 3.2, 3.3). It is also worth noting that calcification of O. faveolata had essentially stopped at 0 month of recovery (Chapter 1 Fig. 2.7) yet displayed strong kinetic effects (Table 3.3), while at 11 months of recovery when calcification rates were the highest, kinetic effects were not detected (Chapter 1 Fig. 2.7, this chapter Table 3.3). Further, calcification rates of P. compressa were significantly lower in bleached compared to non-bleached corals 13 throughout 8 months of recovery (Rodrigues, Grottoli 2006), yet corrected δ Cs of 18 bleached corals did not plot towards more enriched δ Os (i.e., lower calcification) 13 compared to non-bleached δ Cscorr (Fig. 3.2G, H). Thus, both methods used to test the effectiveness of the data correction proposed by Heikoop et al. (2000a) demonstrated convincingly that (1) the data correction does not effectively remove kinetic isotope effects, and that (2) it does not improve the metabolic signal in bleached corals. Several factors may have contributed to the observed differences between the Heikoop et al. (2000) study and the present study. First, Heikoop et al. (2000a) used 13 13 whole tissue (animal host + algal endosymbiont) δ C for the correlations with δ Csorig 13 13 13 and δ Cscorr, while δ Ch was used in the present study because whole tissue δ C was not

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13 13 available. Correlations were evaluated for both δ Ch and algal endosymbiont δ C, but 13 13 the latter correlations were generally even weaker than those with δ Ch for both δ Csorig 13 and δ Cscorr (Table 3.8). Second, Heikoop et al. (2000a) pretreated samples with 6% sodium hypochlorite to remove organic matter, whereas the samples in this study were not pre-treated. It has been shown that pre-treatment with sodium hypochlorite can have 13 18 significant and non-correctable effects on both skeletal δ Cs and δ Os (Grottoli et al. 2005). Therefore some of the observed differences between this study and Heikoop et al. (2000a) may have been caused by methodological differences. Further, the type of samples selected could also have played a role. Importantly, corals in this study were exposed to elevated temperature resulting in coral bleaching, whereas Heikoop et al. (2000a) collected corals across natural light and depth gradients. Specifically, coral bleaching may produce stress-related responses that affect coral isotopes in unknown ways, whereas corals collected over natural light and depth gradients would have been acclimated to the environmental conditions and thus not be influenced by stress responses. It is further possible that the significant correlation of 13 18 skeletal δ C and δ O is not the best indicator of kinetic isotope effects, and/or that other growth-related factors such as linear extension rate are involved in kinetic fractionation. 13 13 However, given the small number of significant correlations between δ Ch and δ Csorig 13 and δ Cscorr, it is unlikely that any of these factors had a strong influence on the observed results.

3.5.3 Comparison of measured and isotope-based P/R ratios

Measured and isotope-based calculated P/R ratios were generally in poor agreement independent of how oxygen isotopic equilibrium was calculated or whether 13 18 δ Cs was corrected or not. Further, isotope-based P/R ratios calculated using δ Oeq after Grossman and Ku (1986) tended to underestimate P/R ratios in all coral species, whereas 18 isotope-based P/R ratios calculated using δ Oeq after Maier (2004) were typically higher than measured P/R ratios (Fig. 3.4, 3.5). This was particularly evident in the Mexican

121 coral species (Fig. 3.5, Tables 3.6, 3.7), where all isotope-based P/R ratios differed significantly from P/R ratios measured by respirometry, and were not correlated with them in any significant way. In the Hawaiian corals (Fig. 3.4, Tables 3.4, 3.5), isotope- 18 based P/R ratios of P. compressa calculated using δ Oeq after Maier (2004) were negatively correlated with measured P/R ratios indicating that the calculated P/R values were nowhere close to the measured values. This was further corroborated by the t-tests which indicated that all isotope-based P/R ratios differed significantly from measured P/R ratios. Similarly, all isotope-based P/R ratios of M. capitata differed significantly from measured P/R ratios with the exception of isotope-based P/R ratios calculated using 13 18 δ Cscorr and δ Oeq after Grossman and Ku (1986). Only in P. lobata were isotope-based 18 P/R ratios calculated using δ Oeq after Grossman and Ku (1986) statistically the same as measured P/R (Tables 3.4, 3.5). This was independent of whether original or corrected 13 δ Cs was used. Some factors may have contributed to the observed differences between isotope- based and measured P/R ratios. Paired measurements of P/R ratios and stable isotopes on the exact same coral fragment were not always possible due to logistical reasons, but they were nevertheless performed on fragments of the same coral population for each species that had undergone the exact same experimental treatment. Rather, estimating carbon and/or oxygen isotopic equilibrium values based on literature values may have 13 18 confounded the isotope-based P/R ratios. Both δ CDIC and δ O of seawater from Puerto Morelos, Mexico are unknown and had to be estimated based on literature values from 13 other locations in the Caribbean. However, δ CDIC was measured for Hawaiian corals in 2006/07, and isotope-based P/R ratios were nevertheless in poor agreement with measured P/R ratios. Therefore, it is unlikely that these factors ultimately caused the significant differences observed in this study. Further studies are needed to determine if isotope-based P/R ratios calculated using measured isotopic equilibrium values result in better agreement with P/R ratios determined via respirometry. Although skeletal δ13C has often been viewed as an indicator of P/R ratios (e.g. Swart et al. 1996b; Grottoli, Wellington 1999; Swart et al. 2005), this is the first time that

122 the accuracy of calculated isotope-based P/R ratios was tested by comparing them to measured P/R ratios. While the findings of this study clearly demonstrate that isotope- based P/R ratios do not reliably trace differences between bleached and non-bleached 18 corals, and are significantly affected by the choice of δ Oeq, they may nevertheless be useful to estimate relative changes in P/R over extreme environmental gradients. For example, Lesser et al. (2010) calculated isotope-based P/R ratios for Montastraea cavernosa ranging from 3 to 91 m depth. They found that P/R ratios significantly decreased with depth, and that P/R was greater than 1 up to a depth of 61 m. This relative decrease with depth as well as the transition towards heterotrophy below a specific depth (60 m) is certainly realistic. However, given the findings from this study, it is likely that 18 their reported P/R ratios significantly overestimated P/R as they calculated δ Oeq after Maier (2004) (M. Lesser, pers. comm.). As a consequence, the transition towards heterotrophy likely occurred at shallower depth than 60 m.

3.5.4 Implications for paleo-climate reconstruction

Overall, the findings of this study demonstrate that the data correction proposed by Heikoop et al. (2000a) did not effectively remove kinetic isotope effects in the Caribbean and Pacific coral species studied here, and that the metabolic effect of the bleaching signal did not improve due to the data correction. It seems therefore unlikely that the data correction can improve the accuracy of skeletal δ13C as a paleo-climate proxy or the reconstruction of past bleaching events from coral skeletons, as was suggested by Heikoop et al. (2000a). Since both O. faveolata and P. lobata are mounding coral species that are used for paleo-climate reconstruction, this is disappointing news. While the data correction may nevertheless be useful in improving correlations of skeletal δ13C with environmental variables in some species and/or locations (e.g. Heikoop et al. 2000a), a routine application without prior evaluation of its effectiveness cannot be recommended. Further, isotope-based P/R ratios should be interpreted with great caution, especially when seawater δ13C and δ18O are unknown.

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Acknowledgements. In Mexico, we thank R. Iglesias-Prieto, A. Banaszak, S. Enriquez, R. Smith, and the staff of the Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, in Puerto Morelos for their generous time and logisitical support. We also thank Y. Matsui, J. Baumann, T. Huey, D. Borg, E. Zebrowski, J. Scheuermann, and M. McBride for help in the field and laboratory. In Hawaii, we thank the Hawaii Institute of Marine Biology, Academy of Natural Sciences, Department of Land and Natural Resources Hawaii, P. Jokiel, J. Stimson, L. Bloch, J. Palardy, C. Malachowski, D. Velinsky, O. Gibb, M. Cathey, P. Petraitis, R. Gates, J.A. Leong, J. Flemming, T. Pease, A. Chrystal, D. Gulko, S. Hughes, L. Hurley, F. Lugo, R. Michelli, R. Moyer, C. Paver, and Z. Rosenbloom. . This work was funded by the National Science Foundation (Mexico: grants no. 0825490 and 0825413, Hawaii: grants no. 0426022, 0542415, and 0610487). All work undertaken in this study complied with the current laws of Mexico and the United States of America.

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3.6 REFERENCES

Allison N, Tudhope AW, Fallick AE (1996) Factors influencing the stable carbon and oxygen isotopic composition of Porites lutea coral skeletons from Phuket, South Thailand. Coral Reefs 15:43-57

Allison N, Finch AA, EIMF (2012) A high resolution δ13C record in a modern Porites lobata coral: insights into controls on skeletal δ13C. Geochimica et Cosmochimica Acta 84:534-542.

Brown BE (1997a) Adaptations of reef corals to physical environmental stress. Advances in Marine Biology 31:222-299

Brown BE (1997b) Coral bleaching: causes and consequences. Coral Reefs 16 suppl:s129-s138

Budd AF, Fukami H, Smith ND, Knowlton N (2012) Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia). Zoological Journal of the Linnean Society 166:465-529

Cohen AL, Hart SR (1997) The effect of colony topography on climate signals in coral skeleton. Geochimica et Cosmochimica Acta 61:3905-3912

Druffel ERM (1997) Geochemistry of corals: Recorders of past ocean chemistry, ocean circulation and climate. Proceedings of the National Academy of Sciences of the USA 94:8354-8361

Epstein S, Mayeda T (1953) Variation of O18 content of waters from natural sources. Geochimica and Cosmochimica Acta 4:213-224

Fairbanks RG, Dodge RE (1979) Annual periodicity of the 18O/16O and 13C/12C ratios in the coral Montastea annularis. Geochimica and Cosmochimica Acta 43:1009- 1020

125

Fitt WK, Brown BE, Warner ME, Dunne RP (2001) Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs 20:51-65

Fitt WK, Warner ME (1995) Bleaching patterns of four species of Caribbean reef corals. Biological Bulletin 189:298-307

Gagan MK, Chivas AR, Isdale PJ (1994) High-resolution isotopic records from corals using ocean temperature and mass-spawning chronometers. Earth and Planetary Science Letters 121:549-558

Glynn PW (1993) Coral reef bleaching: ecological perspectives. Coral Reefs 12:1-17

Glynn PW (1996) Coral reef bleaching: facts, hypotheses and implications. Global Change Biology 2:495-509

Goreau TF, Goreau NI (1959) The physiology of skeleton formation in corals. II. Calcium deposition by hermatypic corals under different conditions. Biological Bulletin 117:239-250

Grossman EL, Ku T-L (1986) Oxygen and carbon isotope fractionation in biogenic aragonite: Temperature effects. Chemical Geology: Isotope Geoscience section 59:59-74

Grottoli AG (2002) Effect of light and brine shrimp levels on skeletal 13C values in the Hawaiian coral Porites compressa: A tank experiment. Geochimica and Cosmochimica Acta 66:1955-1967

Grottoli AG (1999) Variability of stable isotopes and maximum linear extension in reef- coral skeletons at Kaneohe Bay, Hawaii. Marine Biology 135:437-449

Grottoli AG, Eakin CM (2007) A review of modern coral 18O and 14C proxy records. Earth-Science Reviews 81:67-91

Grottoli AG, Rodrigues LJ, Matthews KA, Palardy JE, Gibb OT (2005) Pre-treatment effects on coral skeletal 13C and 18O. Chemical Geology 221:225-242

126

Grottoli AG, Rodrigues LJ, Palardy JE (2006) Heterotrophic plasticity and resilience in bleached corals. Nature 440:1186-1189

Grottoli AG, Wellington GM (1999) Effect of light and zooplankton on skeletal C values in the Eastern Pacific corals Pavona clavus and Pavona gigantea. Coral Reefs 18:29-41

Heikoop JM, Dunn JJ, Risk MJ, Schwarcz HP, McConnaughey TA, Sandeman IM (2000) Separation of kinetic and metabolic isotope effects in carbon-13 records preserved in reef coral skeletons. Geochimica et Cosmochimica Acta 64:975-987

Hoegh-Guldberg O (1999) Climate change, coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research 50:839-866

Jokiel PL, Coles SL (1990) Response of Hawaiian and other Indo-Pacific reef corals to elevated temperature. Coral Reefs 8:155-162

Jones RJ (1997) Changes in zooxanthellar densities and chlorophyll concentrations in corals during and after a bleaching event. Marine Ecology Progress Series 158:51- 59

Kaandorp JA, Sloot PMA, Merks RMH, Bak RPM, Vermeij MJA, Maier C (2005) Morphogenesis of the branching reef coral Madracis mirabilis. Proceedings of the Royal Society B: Biological Sciences 272:127-133

Leder JJ, Swart PK, Szmant AM, Dodge RE (1996) The origin of variations in the isotopic record of scleractinian corals: I. Oxygen. Geochimica et Cosmochimica Acta 60:2857-2870

Leder JJ, Szmant AM, Swart P (1991) The effect of prolonged "bleaching" on skeletal banding and stable isotopic composition in Montastraea annularis. Coral Reefs 10:19-27

127

Lesser MP (2004) Experimental coral reef biology. Journal of Experimental Marine Biology and Ecology 300

Lesser MP (1997) Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16:187-192

Lesser MP, Slattery M, Stat M, Ojimi M, Gates RD, Grottoli A (2010) Photoacclimatization by the coral Montastraea cavernosa in the mesophotic zone: light, food, and genetics. Ecology 91:990-1003

Levas SJ (2012) Biogeochemistry and Physiology of Bleached and Recovering Hawaiian and Caribbean Corals. PhD Thesis, The Ohio State University, p 238

Maier C (2004) Exploring stable isotopic variation in scleractinian coral tissue and skeleton: Proxies for climate, ecological plasticity and coral functioning. PhD thesis, University of Amsterdam, p 120

Maier C, Paetzold J, Bak RPM (2003) The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus Madracis. Coral Reefs 22:370-380

Maier C, Weinbauer MG, Paetzold J (2010) Stable isotopes reveal limitations in C and N assimilation in the Caribbean reef corals Madracis auretenra, M. carmabi and M. formosa. Marine Ecology Progress Series 412:103-112

McConnaughey T (1989a) 13C and 18O isotopic disequilibrium in biological carbonates: I. Patterns. Geochimica et Cosmochimica Acta 53:151-162

McConnaughey T (1989b) 13C and 18O isotopic disequilibrium in biological carbonates: II. In vitro simulation of kinetic isotope effects. Geochimica et Cosmochimica Acta 53:163-171

McConnaughey TA, Burdett J, Whelan JF, Paull CK (1997) Carbon isotopes in biological carbonates: Respiration and photosynthesis. Geochimica et Cosmochimica Acta 61:611-622

Moran MD (2003) Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos 100:403-405

128

Moyer RP, Grottoli AG (2011) Coral skeletal carbon isotopes (13C and 14C) record the delivery of terrestrial carbon to the coastal waters of Puerto Rico. Coral Reefs 30:791-802

Ostlund HG, Craig H, Broecker WS, Spenser D (1987) GEOSECS Atlantic, Pacific and Indian Ocean expeditions: Shorebased Data and Graphics, vol. 7. National Science Foundation, Washington, D.C.

Ourbak T, DeLong K, Correge T, Malaize B, Kilbourne H, Caquineau S, Hollander D (2008) The significance of geochemical proxies in corals, does size (age) matter? Proceedings of the 11th International Coral Reef Symposium, Session number 4:82-86

Porter JW, Fitt WK, Spero HJ, Rogers AD, White MW (1989) Bleaching in reef corals: physiological and stable isotopic responses. Proceedings of the National Academy of Sciences of the USA 86:9342-9346

Quinn GP, Keough MJ (2002) Experimental Design and Data Analysis for Biologists. Cambridge University Press, 557 p, New York

Raymond PA, Bauer JE (2001) DOC cycling in a temperate estuary: A mass balance approach using natural 14C and 13C isotopes. Limnology and Oceanography 46:655-667

Risk MJ, Sherwood OA, Heikoop JM, Llewellyn G (2003) Smoke signals from corals: isotopic signature of the 1997 Indonesian 'haze' event. Marine Geology 202:71-78

Rodrigues LJ, Grottoli AG (2007) Energy reserves and metabolism as indicators of coral recovery from bleaching. Limnology and Oceanography 52:1874-1882

Rodrigues LJ, Grottoli AG, Lesser MP (2008) Long-term changes in the chlorophyll fluorescence of bleached and recovering corals from Hawaii. The Journal of Experimental Biology 211:2502-2509

Romanek CS, Grossman EL, Morse JW (1992) Carbon isotopic fractionation in synthetic aragonite and calcite: Effects of temperature and precipitation rate. Geochimica et Cosmochimica Acta 56:419-430

129

Schmidt GA (1999) Forward modeling of carbonate proxy data from planktonic foraminifera using oxygen isotope tracers in a global ocean model. Paleoceanography 14:482-497

Schmidt GA, Bigg GR, Rohling EJ (1999) Global seawater oxygen-18 database - v1.21. http://data.giss.nasa.gov/o18data/

Suzuki A, Gagan MK, Fabricius K, Isdale PJ, Yukino I, Kawahata H (2003) Skeletal isotope microprofiles of growth perturbations in Porites corals during the 1997- 1998 mass bleaching event. Coral Reefs 22:357-369

Suzuki A, Hibino K, Iwase A, Kawahata H (2005) Intercolony variability of skeletal oxygen and carbon isotope signatures of cultured Porites corals: Temperature- controlled experiments. Geochimica et Cosmochimica Acta 69:4453-4462

Suzuki A, Nakamura T, Yamasaki H, Minoshima K, Kawahata H (2008) Influence of water flow on skeletal isotopic composition in the coral Pocillopora damicornis. Coral Reefs 27:209-218

Swart P, Leder JJ, Szmant AM, Dodge RE (1996b) The origin of variations in the isotopic record of scleractinian corals: II. Carbon. Geochimica and Cosmochimica Acta 60:2871-2885

Swart P, Szmant AM, Porter JW, Dodge RE, Tougas JI, Southam JR (2005) The isotopic composition of respired carbon dioxide in scleractinian corals: Implications for cycling of organic carbon in corals. Geochimica and Cosmochimica Acta 69:1495-1509

Swart PK (1983) Carbon and oxygen isotope fractionation in scleractinian corals: A review. Earth-Science Reviews 19:51-80

Watanabe T, Winter A, Oba T, Anzai R, Ishioroshi H (2002) Evaluation of the fidelity of isotope records as an environmental proxy in the coral Montastraea. Coral Reefs 21:169-178

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3.7 TABLES

Table 3.1. Seawater dissolved inorganic carbon (DIC) concentration and isotopic 13 composition (δ CDIC) from Kaneohe Bay, Hawaii, from September 2006 to August 2007. Stdev = standard deviation.

13 Collection CO2 δ CDIC Date (μmol kg-1) (‰, v-PDB)

9/18/2006 2155 -0.628 9/18/2006 2155 -0.643 10/6/2006 2058 0.089 10/9/2006 2069 0.427 10/9/2006 2050 0.469 1/27/2007 2092 0.252 1/30/2007 2023 0.469 8/15/2007 2072 0.258 8/17/2007 2030 0.347 Average 2078 0.116 Stdev. 48.33 0.443

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13 Table 3.2. Correlation analyses of original skeletal carbon isotope (δ Csorig) versus 18 13 13 13 skeletal oxygen isotopes (δ Os), δ Csorig versus δ C of the animal host (δ Ch), and 13 13 13 corrected δ Cs (δ Cscorr) versus δ Ch for the Hawaiian coral species Porites compressa, Montipora capitata, and Porites lobata. Analyses were computed (A) pooled for species and treatments, and (B) individually for each treatment and recovery interval. Significant p-values (p≤0.05) are highlighted in bold. Treat. = treatment, NB = non-bleached, BL = singly bleached, 0, 1, 1.5, 4, 5, 8, 11 = months of recovery, N = sample size, r = Spearman’s correlation coefficient.

Treat. 13 18 13 13 13 13 And δ Csorig vs. δ Os δ Csorig vs. δ Ch δ Cscorr vs. δ Ch Recovery N r p N r p N r p

(A) Pooled Analyses

All species 127 0.68 <0.0001 100 0.44 <0.0001 102 0.40 <0.0001

P. compressa All corals 44 0.20 0.1923 30 0.15 0.4365 31 0.14 0.4680 NB 24 -0.57 0.0040 14 0.65 0.0115 14 0.42 0.1369 BL 20 0.71 0.0005 16 0.02 0.9396 17 0.15 0.5540

M. capitata All corals 41 0.44 0.0040 28 -0.01 0.9570 29 0.06 0.7684 NB 20 0.18 0.4482 13 0.17 0.5780 14 0.22 0.4549 BL 21 0.57 0.0066 15 -0.19 0.4907 15 -0.17 0.5409

P. lobata All corals 42 0.58 <0.0001 42 0.59 <0.0001 42 0.43 0.0048 NB 24 0.61 0.0015 24 0.56 0.0041 24 0.33 0.1136 BL 18 0.54 0.0207 18 0.73 0.0007 18 0.66 0.0029

(B) By Treatment and Recovery Interval

P. compressa NB - 0 6 0.55 0.2574 4 1.00 <0.0001 4 0.40 0.6000 BL - 0 6 0.49 0.3287 4 0.40 0.6000 4 -0.40 0.6000 NB – 1.5 7 0.11 0.8175 N < 4 BL – 1.5 4 -0.32 0.6838 5 -0.63 0.3675 5 -0.30 0.6238 NB - 4 6 0.78 0.0657 N < 4

continued 132

Table 3.2 continued

BL - 4 6 0.83 0.0416 5 -0.50 0.3910 5 0.40 0.5046 NB - 8 5 -0.67 0.2189 4 0.80 0.2000 4 -0.20 0.8000 BL - 8 4 0.80 0.2000 N < 4

M. capitata NB - 0 6 0.83 0.0416 4 -0.40 0.6000 4 -0.80 0.2000 BL - 0 6 0.77 0.0724 4 0.00 1.0000 4 0.40 0.6000 NB – 1.5 6 -0.09 0.8717 N < 4 BL – 1.5 6 0.84 0.0361 4 -1.00 <0.0001 4 -0.80 0.2000 NB - 4 4 0.20 0.8000 4 -0.50 0.6667 4 0.40 0.6000 BL - 4 5 0.70 0.1881 4 0.80 0.2000 4 0.00 1.0000 NB - 8 4 0.40 0.6000 N < 4 BL - 8 4 0.80 0.2000 N < 4

P. lobata NB - 0 6 0.20 0.7040 6 0.60 0.2080 6 0.77 0.0724 BL - 0 6 0.94 0.0048 6 0.83 0.0416 6 -0.03 0.9572 NB – 1 6 0.43 0.3965 6 0.77 0.0724 6 -0.60 0.2080 BL – 1 6 0.66 0.1562 6 0.66 0.1562 6 0.66 0.1562 NB - 5 6 0.54 0.2657 6 -0.03 0.9572 6 -0.09 0.8717 BL - 5 N < 4 NB - 11 6 0.66 0.1562 6 0.03 0.9572 6 0.83 0.0416 BL - 11 N < 4

133

13 Table 3.3. Correlation analyses of original skeletal carbon isotope (δ Csorig) versus 18 13 13 13 skeletal oxygen isotopes (δ Os), δ Csorig versus δ C of the animal host (δ Ch), and 13 13 13 corrected δ Cs (δ Cscorr) versus δ Ch for the Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata. Analyses were computed (A) pooled for species and treatments, and (B) individually for each treatment and recovery interval. Significant p-values (p≤0.05) are highlighted in bold. Treat. = treatment, NB = non- bleached, BL = repeat bleached, 0, 1.5, 11 = months of recovery, N = sample size, r = Spearman’s correlation coefficient.

Treat. 13 18 13 13 13 13 And δ Csorig vs. δ Os δ Csorig vs. δ Ch δ Cscorr vs. δ Ch Recovery N r p N r p N r p

(A) Pooled Analyses

All species 131 0.91 <0.0001 131 0.47 <0.0001 131 0.41 <0.0001

O. faveolata All corals 48 0.90 <0.0001 48 0.34 0.0171 48 0.12 0.3995 NB 24 0.92 <0.0001 24 0.26 0.2214 24 -0.04 0.8527 BL 24 0.82 <0.0001 24 0.48 0.0172 24 0.32 0.1329

P. astreoides All corals 51 0.65 <0.0001 51 0.52 0.0001 51 0.55 <0.0001 NB 27 0.69 <0.0001 27 0.56 0.0024 27 0.52 0.0058 BL 24 0.45 0.0269 24 0.42 0.0388 24 0.58 0.0032

P. divaricata All corals 32 0.67 <0.0001 32 0.56 0.0010 32 0.34 0.0581 NB 14 0.52 0.0558 14 0.48 0.0814 14 0.28 0.3260 BL 18 0.73 0.0006 18 0.63 0.0050 18 0.35 0.1611

(B) By Treatment and Recovery Interval

O. faveolata NB – 0 9 0.97 <0.0001 9 0.58 0.0922 9 -0.32 0.4064 BL - 0 8 0.81 0.0149 8 0.07 0.8665 8 -0.07 0.8665 NB – 1.5 9 1.00 <0.0001 9 -0.30 0.4328 9 0.35 0.3558 BL – 1.5 8 0.99 <0.0001 8 0.28 0.5091 8 -0.24 0.5678 NB - 11 6 0.74 0.1108 6 0.60 0.2080 6 -0.09 0.8717 BL - 11 8 0.41 0.3199 8 0.38 0.3518 8 0.36 0.3851

continued 134

Table 3.3 continued

P. astreoides NB – 0 9 0.68 0.0448 9 -0.22 0.5755 9 0.33 0.3807 BL - 0 8 0.45 0.2604 8 0.77 0.0265 8 0.24 0.5678 NB – 1.5 9 0.70 0.0366 9 0.09 0.8284 9 0.12 0.7631 BL – 1.5 9 0.65 0.0581 9 0.48 0.1875 9 0.62 0.0769 NB - 11 9 0.54 0.1373 9 0.88 0.0016 9 0.90 0.0009 BL - 11 7 0.43 0.3374 7 -0.71 0.0713 7 0.31 0.5040

P. divaricata NB – 0 5 1.00 <0.0001 5 0.70 0.1881 5 -0.70 0.1881 BL - 0 7 1.00 <0.0001 7 0.54 0.2152 7 -0.50 0.2532 NB – 1.5 6 0.66 0.1562 6 0.49 0.3287 6 0.49 0.3287 BL – 1.5 6 -0.09 0.8717 6 0.54 0.2657 6 0.03 0.9572 NB - 11 N < 4 BL - 11 5 0.30 0.6238 5 0.90 0.0374 5 0.90 0.0374

135

Table 3.4. Correlation analyses of measured (meas.) and isotope-based P/R ratios for the Hawaiian coral species Porites compressa, Montipora capitata, and Porites lobata. 13 13 Isotope-based P/R ratios were computed with both δ Csorig (orig. P/R) and δ Cscorr (corr. 18 P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. N = sample size, r = Spearman’s correlation coefficient.

Correlation N r p-value

P. compressa Meas. P/R vs. Orig. P/R (ref. 1) 10 0.10 0.7770 Meas. P/R vs. Corr. P/R (ref. 1) 10 0.07 0.8548 Meas. P/R vs. Orig. P/R (ref. 2) 10 -0.76 0.0111 Meas. P/R vs. Corr. P/R (ref. 2) 10 -0.71 0.0217

M. capitata Meas. P/R vs. Orig. P/R (ref. 1) 18 0.08 0.7537 Meas. P/R vs. Corr. P/R (ref. 1) 18 0.03 0.8932 Meas. P/R vs. Orig. P/R (ref. 2) 18 -0.17 0.5095 Meas. P/R vs. Corr. P/R (ref. 2) 18 -0.11 0.6654

P. lobata Meas. P/R vs. Orig. P/R (ref. 1) 28 0.71 <0.0001 Meas. P/R vs. Corr. P/R (ref. 1) 28 0.70 <0.0001 Meas. P/R vs. Orig. P/R (ref. 2) 28 0.26 0.1848 Meas. P/R vs. Corr. P/R (ref. 2) 28 0.29 0.1413

136

Table 3.5. Results from paired t-tests comparing measured (meas.) and isotope-based P/R ratios from Hawaiian coral species Porites compressa, Montipora capitata, and Porites 13 lobata. Isotope-based P/R ratios were computed with both δ Csorig (orig. P/R) and 13 18 δ Cscorr (corr. P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. df = degrees of freedom.

Comparison df t-statistic p-value

P. compressa Meas. P/R vs. Orig. P/R (ref. 1) 9 3.78 0.0043 Meas. P/R vs. Corr. P/R (ref. 1) 9 3.28 0.0096 Meas. P/R vs. Orig. P/R (ref. 2) 9 -2.77 0.0216 Meas. P/R vs. Corr. P/R (ref. 2) 9 -2.94 0.0166

M. capitata Meas. P/R vs. Orig. P/R (ref. 1) 17 -2.96 0.0087 Meas. P/R vs. Corr. P/R (ref. 1) 17 -2.07 0.0541 Meas. P/R vs. Orig. P/R (ref. 2) 17 -13.42 <0.0001 Meas. P/R vs. Corr. P/R (ref. 2) 17 -9.87 <0.0001

P. lobata Meas. P/R vs. Orig. P/R (ref. 1) 27 -0.05 0.9576 Meas. P/R vs. Corr. P/R (ref. 1) 27 0.00 0.9982 Meas. P/R vs. Orig. P/R (ref. 2) 27 -21.42 <0.0001 Meas. P/R vs. Corr. P/R (ref. 2) 27 -14.21 <0.0001

137

Table 3.6. Correlation analyses of measured (meas.) and isotope-based P/R ratios for the Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata. 13 13 Isotope-based P/R ratios were computed with both δ Csorig (orig. P/R) and δ Cscorr (corr. 18 P/R). Further, they were computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Recovery intervals were pooled for each species. Significant p-values (p≤0.05) are highlighted in bold. N = sample size, r = Spearman’s correlation coefficient.

Correlation N r p-value

O. faveolata Meas. P/R vs. Orig. P/R (ref. 1) 18 0.08 0.7632 Meas. P/R vs. Corr. P/R (ref. 1) 18 0.04 0.8643 Meas. P/R vs. Orig. P/R (ref. 2) 18 -0.10 0.6922 Meas. P/R vs. Corr. P/R (ref. 2) 18 -0.13 0.5952

P. astreoides Meas. P/R vs. Orig. P/R (ref. 1) 17 0.12 0.6594 Meas. P/R vs. Corr. P/R (ref. 1) 17 -0.03 0.9219 Meas. P/R vs. Orig. P/R (ref. 2) 17 -0.25 0.3253 Meas. P/R vs. Corr. P/R (ref. 2) 17 -0.10 0.7082

P. divaricata Meas. P/R vs. Orig. P/R (ref. 1) 18 -0.03 0.9013 Meas. P/R vs. Corr. P/R (ref. 1) 18 -0.22 0.3828 Meas. P/R vs. Orig. P/R (ref. 2) 18 -0.35 0.1532 Meas. P/R vs. Corr. P/R (ref. 2) 18 -0.33 0.1762

138

Table 3.7. Results from paired t-tests comparing measured (meas.) and isotope-based P/R ratios from Mexican coral species Orbicella faveolata, Porites astreoides, and Porites divaricata after 0 month of recovery from repeat bleaching. Isotope-based P/R ratios 13 13 were computed with both δ Csorig (orig. P/R) and δ Cscorr (corr. P/R). Further, they were 18 computed using δ Oeq values either after Grossman and Ku 1986 (ref. 1) or after Maier 2004 (ref. 2). Significant p-values (p≤0.05) are highlighted in bold. df = degrees of freedom.

Comparison df t-statistic p-value

O. faveolata Meas. P/R vs. Orig. P/R (ref. 1) 17 2.97 0.0086 Meas. P/R vs. Corr. P/R (ref. 1) 17 2.17 0.0446 Meas. P/R vs. Orig. P/R (ref. 2) 17 -17.99 <0.0001 Meas. P/R vs. Corr. P/R (ref. 2) 17 -13.68 <0.0001

P. astreoides Meas. P/R vs. Orig. P/R (ref. 1) 16 4.35 0.0005 Meas. P/R vs. Corr. P/R (ref. 1) 16 2.48 0.0249 Meas. P/R vs. Orig. P/R (ref. 2) 16 -18.30 <0.0001 Meas. P/R vs. Corr. P/R (ref. 2) 16 -10.85 <0.0001

P. divaricata Meas. P/R vs. Orig. P/R (ref. 1) 17 6.54 <0.0001 Meas. P/R vs. Corr. P/R (ref. 1) 17 5.55 <0.0001 Meas. P/R vs. Orig. P/R (ref. 2) 17 6.08 <0.0001 Meas. P/R vs. Corr. P/R (ref. 2) 17 -2.41 0.0275

139

Treat. And δ13C vs. δ13C δ13C vs. δ13C Recovery sorig e scorr e N r p N r p

(A) Hawaii: Pooled Analyses

All species 100 0.41 <0.0001 102 0.35 0.0003

P. compressa All corals 30 0.26 0.1742 31 0.33 0.0710 NB 14 0.66 0.0099 14 0.54 0.0470 BL 16 0.14 0.6168 17 0.30 0.2373

M. capitata All corals 28 0.02 0.9229 29 0.15 0.4236 NB 13 0.32 0.2835 14 0.29 0.3138 BL 15 -0.09 0.7418 15 -0.08 0.7757

P. lobata All corals 42 0.68 <0.0001 42 0.56 0.0001 NB 24 0.66 0.0005 24 0.47 0.0204 BL 18 0.75 0.0003 18 0.68 0.0019

(B) Hawaii: By Treatment and Recovery Interval

P. compressa NB - 0 4 0.80 0.2000 4 0.20 0.8000 BL - 0 4 0.80 0.2000 4 -0.20 0.8000 NB – 1.5 N < 4 N < 4 BL – 1.5 4 -0.61 0.3880 5 0.30 0.6238 NB - 4 N < 4 N < 4

continued

140

Table 3.8 continued

BL - 4 5 -0.10 0.8729 5 -0.10 0.8729 NB - 8 4 0.60 0.4000 4 -0.05 0.9531 BL - 8 N < 4 N < 4

M. capitata NB - 0 4 0.40 0.6000 4 -0.80 0.2000 BL - 0 4 0.20 0.8000 4 0.40 0.6000 NB – 1.5 N < 4 N < 4 BL – 1.5 4 -1.00 <0.0001 4 -0.80 0.2000 NB - 4 N < 4 4 0.75 0.2494 BL - 4 4 0.00 1.0000 4 0.20 0.8000 NB - 8 N < 4 N < 4 BL - 8 N < 4 N < 4

P. lobata NB - 0 6 0.77 0.0724 6 0.83 0.0416 BL - 0 6 0.83 0.0416 6 -0.03 0.9572 NB – 1 6 0.77 0.0724 6 -0.60 0.2080 BL – 1 6 0.66 0.1562 6 0.66 0.1562 NB - 5 6 -0.54 0.2657 6 0.09 0.8717 BL - 5 N < 4 N < 4 NB - 11 6 0.41 0.4247 6 0.17 0.7417 BL - 11 N < 4 N < 4

All species 131 0.10 0.2451 131 0.04 0.6713

O. faveolata All corals 48 0.25 0.0889 48 -0.13 0.3887 NB 24 0.23 0.2786 24 -0.11 0.5995 BL 24 0.49 0.0155 24 0.03 0.8893

P. astreoides All corals 51 0.43 0.0015 51 0.47 0.0005 NB 27 0.41 0.0361 27 0.48 0.0119 BL 24 0.37 0.0768 24 0.35 0.0964

P. divaricata All corals 32 0.52 0.0021 32 0.45 0.0095

continued 141

Table 3.8 continued

NB 14 0.52 0.0586 14 0.54 0.0449 BL 18 0.51 0.0319 18 0.38 0.1166

(D) Mexico: By Treatment and Recovery Interval

O. faveolata NB – 0 9 0.52 0.1544 9 -0.23 0.5457 BL - 0 8 0.26 0.5309 8 -0.48 0.2329 NB – 1.5 9 0.00 1.0000 9 0.10 0.7980 BL – 1.5 8 0.31 0.4528 8 -0.30 0.4713 NB - 11 6 -0.09 0.8717 6 -0.09 0.8717 BL - 11 8 0.67 0.0710 8 0.24 0.5702

P. astreoides NB – 0 9 -0.45 0.2242 9 0.13 0.7324 BL - 0 8 0.41 0.3199 8 -0.29 0.4927 NB – 1.5 9 0.32 0.3980 9 0.19 0.6183 BL – 1.5 9 0.03 0.9489 9 0.22 0.5739 NB - 11 9 0.90 0.0009 9 0.93 0.0002 BL - 11 7 0.00 1.0000 7 0.41 0.3553

P. divaricata NB – 0 5 0.10 0.8729 5 -0.10 0.8729 BL - 0 7 0.54 0.2152 7 0.07 0.8790 NB – 1.5 6 0.37 0.4685 6 0.60 0.2080 BL – 1.5 6 0.37 0.4685 6 -0.09 0.8717 NB - 11 N < 4 N < 4 BL - 11 5 0.50 0.3910 5 0.70 0.1881

142

3.8 FIGURES

PC non-bleached A) Hawaii Resp. Eq. 0 PC bleached MC non-bleached Wi -1 Resp. MC bleached KIE PL non-bleached -2 Su PL bleached Eq. KIE Grossman and KIE Ku (1986) -3 BL Slow KIE Maier (2004) -4

-5

-6 P BL NB Fast

(‰, v-PDB) (‰, Resp. s B) Mexico OF non-bleached O 0 Eq. OF repeat bleached

 PA non-bleached Wi PA repeat bleached -1 Resp. KIE PD non-bleached Su PD repeat bleached -2 Eq. KIE Grossman and Ku (1986) -3 BL KIE Slow KIE Maier (2004) -4

-5

-6 P BL NB Fast

-6 -5 -4 -3 -2 -1 0 1 2 3 4 13C (‰, v-PDB) s orig 18 13 Figure 3.1. Plot of skeletal δ Os vs. δ Csorig for (A) non-bleached and singly bleached Hawaiian corals, and (B) non-bleached and repeat bleached Mexican corals. KIE marks the trend along which kinetic isotope effects occur. Eq represents isotopic 18 equilibrium composition based on two different methods to calculate δ Oeq (Grossman and Ku 1986, Maier 2004). Wi and Su represent winter and summer isotopic equilibrium composition, respectively. Resp and P indicate the carbon isotopic offset from the KIE line due to respiration and photosynthesis, respectively. Slow and Fast refer to calcification rates. NB = non-bleached control, BL = bleached, PC = Porites compressa, MC = Montipora capitata, PL = Porites lobata OF = Orbicella faveolata, PA = Porites astreoides, PD = Porites divaricata. Please see text for further explanation.

143

P. compressa M. capitata P. lobata C -3 A NBNB E NB NB -3 -3 -4 -4 -4 -5 -5 -5 -6

-3 B D BL F BL (‰, v-PDB) (‰, BL s -3 -3

18 -4 BL  -4 -4 -5 -5 -5 -6 -7 -6 -5 -4 -3 -2 -5 -4 -3 -2 -1 0 1 -6 -5 -4 -3 -2 -1 13  Csorig (‰, v-PDB)

I K -3 G NB NB NB -3 -3 -4 -4 -4 -5 -5 -5 -6

-3 H J L BL (‰, v-PDB) (‰, BL s BL -3 -3

18 -4  -4 -4 -5 -5 -5 -6 -7 -6 -5 -4 -3 -2 -5 -4 -3 -2 -1 0 1 -6 -5 -4 -3 -2 -1 13C (‰, v-PDB) scorr

18 18 13 13 Figure 3.2. Plots of skeletal δ O (δ Os) vs. (A-F) original skeletal δ C (δ Csorig) and 13 13 (G-L) corrected skeletal δ C (δ Cscorr) for non-bleached and singly bleached Porites compressa, Montipora capitata, and Porites lobata from Hawaii throughout 8-11 months of recovery. ● = 0 month of recovery, ● = 1 or 1.5 months of recovery, ● = 4 or 5 months of recovery, and ● = 8 or 11 months of recovery. NB = non-bleached control, BL = singly bleached. – 18 = KIE line leading to δ Oeq after Grossman and Ku (1986), --- = KIE line leading to 18 δ Oeq after Maier (2004). + = outliers excluded from statistical analyses.

144

O. faveolata P. astreoides P. divaricata A C E -1 NB NB NB -4.0 -4.5 -2 -5.0 -3 -4.5 -4 -5.5 -5.0 -5 -6.0

B D F

(‰, v-PDB) (‰, -1 BL BL s -4.0 -4.5 O -2 18 -5.0 BL  -3 -4.5 -4 -5.5 -5.0 -5 -6.0

-4 -3 -2 -1 0 1 -4 -3 -2 -1 -6 -5 -4 -3

13  Csorig (‰, v-PDB)

G I K -1 NB NB NB -4.0 -4.5 -2 -5.0 -3 -4.5 -4 -5.5 -5.0 -5 -6.0

H J L BL (‰, v-PDB) (‰, -1 BL s BL -4.0 -4.5 O -2 18 -5.0  -3 -4.5 -4 -5.5 -5.0 -5 -6.0

-4 -3 -2 -1 0 1 -4 -3 -2 -1 -6 -5 -4 -3 13C (‰, v-PDB) scorr

18 18 13 13 Figure 3.3. Plots of skeletal δ O (δ Os) vs. (A-F) original skeletal δ C (δ Csorig) and 13 13 (G-L) corrected skeletal δ C (δ Cscorr) for non-bleached and repeat bleached Orbicella faveolata, Porites astreoides, and Porites divaricata from Mexico throughout 11 months of recovery. ● = 0 month of recovery, ● = 1.5 months of recovery, and ● = 11 months of recovery. 18 NB = non-bleached control, BL = repeat bleached. – = KIE line leading to δ Oeq after 18 Grossman and Ku (1986), --- = KIE line leading to δ Oeq after Maier (2004).

145

P. compressa M. capitata P. lobata A B C 8 r = -0.76 8 6 6 4 4

2 P/R 2 0 0

original P/R P/R original r = 0.71 -2 isotope-based -2

(isotope-based) F 8 D r = -0.71 8 E 6 6 4 4

2 P/R 2 0 0

isotope-based r = 0.70 corrected P/R P/R corrected -2 -2

(isotope-based)

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 measured P/R measured P/R measured P/R

Figure 3.4. Correlations of measured and isotope-based P/R ratios for non-bleached and bleached Porites compressa, Montipora capitata, and Porites lobata from Hawaii. 13 Isotope-based P/R ratios were computed with both (A-C) δ Csorig (original P/R) and (D- 13 18 F) δ Cscorr (corrected P/R). Further, they were computed using δ Oeq values either after Grossman and Ku (1986) (filled symbols) or after Maier (2004) (open symbols). Dotted line indicates perfect agreement of isotope-based and measured P/R ratios. Treatments and recovery intervals were pooled for each species. r = Spearman’s correlation coefficient when correlation was statistically significant. + = outliers excluded from statistical analyses.

146

O. faveolata P. astreoides P. divaricata 8 A 8 B C 6 6 4 4 2 2 0 P/R 0 -2 -2

original P/R P/R original

isotope-based

(isotope-based) -4 -4 8 D 8 E F 6 6 4 4 2 2 0 P/R 0 -2 -2

isotope-based

corrected P/R P/R corrected

(isotope-based) -4 -4 -1 0 1 2 3 4 5 -1 0 1 2 3 4 5 -1 0 1 2 3 4 5 measured P/R

Figure 3.5 Correlations of measured and isotope-based P/R ratios for non-bleached and repeat bleached Orbicella faveolata, Porites astreoides, and Porites divaricata from Mexico at 0 month of recovery. 13 Isotope-based P/R ratios were computed with both (A-C) δ Csorig (original P/R) and (D- 13 18 F) δ Cscorr (corrected P/R). Further, they were computed using δ Oeq values either after Grossman and Ku (1986) (filled symbols) or after Maier (2004) (open symbols). Dotted line indicates perfect agreement of isotope-based and measured P/R ratios. Treatments were pooled for each species. r = Spearman’s correlation coefficient when correlation was statistically significant.

147

4. CORAL ENERGY RESERVES AND CALCIFICATION IN A

HIGH-CO2 WORLD AT TWO TEMPERATURES

Verena Schoepf1, Andréa G. Grottoli1, Mark E. Warner2, Wei-Jun Cai3,*, Todd F. Melman4, Kenneth D. Hoadley2, Daniel T. Pettay2, Xinping Hu3,†, Qian Li3,‡, Hui Xu3,∆, Yongchen Wang3, Yohei Matsui1, Justin H. Baumann1

1. School of Earth Sciences, The Ohio State University, Columbus, OH, USA 2. School of Marine Science and Policy, University of Delaware, Lewes, DE, USA. 3. Department of Marine Sciences, University of Georgia, Athens, GA, USA 4. Reef Systems Coral Farm, New Albany, OH, USA

* Present address: School of Marine Science and Policy, University of Delaware, Newark, DE, United States † Present address: Department of Physical and Environmental Sciences, Texas A&M University, Corpus Christi, TX, United States ‡ Present address: State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China ∆ Present address: Department of Ocean Science and Engineering, Zhejiang University, Hangzhou, China

Chapter 4 has been accepted for publication in PLoS ONE

148

4.1 ABSTRACT

Rising atmospheric CO2 concentrations threaten coral reefs globally by causing ocean acidification (OA) and warming. Yet, the combined effects of elevated pCO2 and temperature on coral physiology and resilience remain poorly understood. While coral calcification and energy reserves are important health indicators, no studies to date have measured energy reserve pools (i.e., lipid, protein, and carbohydrate) together with calcification under OA conditions under different temperature scenarios. Four coral species, Acropora millepora, Montipora monasteriata, Pocillopora damicornis, Turbinaria reniformis, were reared under a total of six conditions for 3.5 weeks, representing three pCO2 levels (382, 607, 741 μatm), and two temperature regimes (26.5,

29.0°C) within each pCO2 level. After one month under experimental conditions, only A. millepora decreased calcification (-53%) in response to seawater pCO2 expected by the end of this century, whereas the other three species maintained calcification rates even when both pCO2 and temperature were elevated. Coral energy reserves showed mixed responses to elevated pCO2 and temperature, and were either unaffected or displayed nonlinear responses with both the lowest and highest concentrations often observed at the mid-pCO2 level of 607 μatm. Biweekly feeding may have helped corals maintain calcification rates and energy reserves under these conditions. Temperature often modulated the response of many aspects of coral physiology to OA, and both mitigated and worsened pCO2 effects. This demonstrates for the first time that coral energy reserves are generally not metabolized to sustain calcification under OA, which has important implications for coral health and bleaching resilience in a high-CO2 world. Overall, these findings suggest that some corals could be more resistant to simultaneously warming and acidifying oceans than previously expected.

149

4.2 INTRODUCTION

Anthropogenic climate change threatens many marine ecosystems today, and coral reefs are among the most sensitive to current changes in ocean biogeochemistry (Hoegh-Guldberg et al. 2007; Hoegh-Guldberg, Bruno 2010). Rising atmospheric carbon dioxide (CO2) concentrations have already caused an increase of 0.6°C in the average temperature of the upper layers of the ocean over the past 100 years (IPCC 2007), and about one third of all anthropogenic CO2 has been absorbed by the ocean, causing ocean acidification (OA) (Caldeira, Wickett 2003; Sabine et al. 2004b). Since scleractinian corals are calcifying organisms that already live close to their upper thermal tolerance limits (Fitt et al. 2001), both ocean warming and acidification severely threaten their survival and role as reef ecosystem engineers (Hoegh-Guldberg et al. 2007; Wild et al. 2011).

The uptake of anthropogenic CO2 by the ocean changes the carbonate chemistry + - of seawater by increasing proton (H ) and bicarbonate (HCO3 ) concentrations, while at 2- the same time decreasing the concentration of carbonate (CO3 ). Consequently, seawater + 2+ 2- pH (i.e., -log[H ]) and the saturation state with respect to aragonite (Ωarag = [Ca ][CO3 2+ 2- ]/Ksp with Ksp being the ionic product of [Ca ] and [CO3 ] under solution-mineral equilibrium) decrease. As aragonite is the form of calcium carbonate (CaCO3) precipitated by modern corals, this process compromises marine calcification (Kleypas et al. 1999; Feely et al. 2004; Orr et al. 2005). Over the past century, Ωarag in the tropics has decreased from 4.6 to 4.0 (Kleypas et al. 1999) and is expected to decrease to 2.5 – 3.0 by the year 2100 (Kleypas et al. 1999; Hoegh-Guldberg et al. 2007; Feely et al. 2009). Further, it has been estimated that scleractinian calcification rates may drop by up to 35%-40% by the end of this century (Kleypas et al. 1999; Langdon et al. 2000). Coral calcification typically decreases in response to experimentally reduced seawater pH (Marubini, Atkinson 1999; Leclercq et al. 2000; Langdon, Atkinson 2005; Renegar, Riegl 2005; Schneider, Erez 2006; Anthony et al. 2008; Jokiel et al. 2008; Marubini et al. 2008; Krief et al. 2010; Edmunds et al. 2012; Holcomb et al. 2012;

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Comeau et al. 2013b) but not always (Reynaud et al. 2003; Jury et al. 2010; Rodolfo- Metalpa et al. 2010; Edmunds 2011; Rodolfo-Metalpa et al. 2011; Edmunds et al. 2012; Houlbreque et al. 2012; Comeau et al. 2013a; Comeau et al. 2013b). Seawater temperature also influences calcification (Clausen 1971; Clausen, Roth 1975; Jokiel, Coles 1977a; Marshall, Clode 2004; Cantin et al. 2010), resulting in potentially interactive effects of temperature and OA on coral calcification. For example, negative effects of elevated seawater pCO2 on calcification are often exacerbated when temperature is simultaneously increased (Reynaud et al. 2003; Anthony et al. 2008; Rodolfo-Metalpa et al. 2011), suggesting a synergistic interactive effect. However, this is not always observed (Langdon, Atkinson 2005; Rodolfo-Metalpa et al. 2010; Edmunds 2011) and in one study even the opposite was shown (Muehllehner, Edmunds 2008). Clearly, further studies are required to gain a better understanding of the interactive effects of elevated temperature and pCO2 on coral calcification and its resistance to OA. Much less is known about how combined OA and warming will influence other aspects of coral physiology such as energy reserves and tissue biomass. If calcification becomes energetically more costly under elevated pCO2 due to a decreased aragonite saturation state (Cohen, Holcomb 2009; Erez et al. 2011; Pandolfi et al. 2011), then the extra energy needed to maintain calcification might be drawn from one or more of the following sources: 1) Coral energy reserves (i.e., lipids, protein, carbohydrates), 2)

Enhanced endosymbiotic algal production due to CO2 fertilization (Brading et al. 2011), and 3) Increased heterotrophy (i.e., zooplankton, particulate and/or dissolved organic carbon) (Edmunds 2011; Drenkard et al. 2013). These responses may be even more extreme with the simultaneous increases in seawater temperature because tissue biomass, energy reserves, and endosymbiotic algal density are typically lowest when temperature (and irradiance) is highest on seasonal timescales (e.g. Stimson 1997; Fitt et al. 2000; Thornhill et al. 2011) and under bleaching scenarios (e.g. Grottoli et al. 2006; Rodrigues, Grottoli 2007; Levas et al. 2013). Although tissue biomass and energy reserves are important indicators of coral health (Rodrigues, Grottoli 2007; Levas et al. 2013) and play a significant role in

151 promoting resilience to bleaching (Anthony et al. 2009), no studies to date have measured all three energy reserve pools (i.e., lipid, protein, and carbohydrate) under OA conditions at elevated temperature. While protein concentrations were either unaffected (Reynaud et al. 2003; Rodolfo-Metalpa et al. 2010) or increased in response to elevated pCO2 alone (Fine, Tchernov 2007; Krief et al. 2010), the effects of OA, or OA plus elevated temperature, on coral lipids and carbohydrates are unknown. Studies specifically addressing all three energy reserve pools are needed to get a better understanding of how OA affects coral energetics and their overall resistance to future climate change. Finally, the algal endosymbiont (Symbiodinium sp.) provides healthy corals with up to 100% of their daily metabolic energy demand via photosynthesis (e.g. Muscatine et al. 1981). If algal productivity is enhanced under OA due to CO2 fertilization (Brading et al. 2011), this might help maintain calcification rates and/or energy reserves under OA as energetic costs for calcification increase. Further, Symbiodinium sp. exhibit high sensitivity to elevated seawater temperature (e.g. Hoegh-Guldberg 1999). Thus, it is important to monitor endosymbiont and chlorophyll a concentrations in studies manipulating both pCO2 and temperature.

Here, we studied the single and interactive effects of pCO2 (382, 607, 741 μatm) and temperature (26.5 and 29.0°C) on coral calcification, energy reserves (i.e., lipid, protein, and carbohydrate), chlorophyll a, and endosymbiont concentrations in 4 species of Pacific coral with different growth morphologies. It was hypothesized that 1) calcification and energy reserves decrease in response to elevated pCO2 and elevated temperature, 2) decreases are larger when pCO2 and temperature are elevated simultaneously, and 3) that physiological responses are species-specific. We show that only one of the four coral species studied here decreased calcification in response to average ocean acidification levels expected by the second half of this century (741 μatm), even when combined with elevated temperature (+2.5°C). Further, we show for the first time that energy reserves were largely not metabolized in order to sustain calcification under elevated pCO2 and temperature, suggesting that some coral species will be more resistant to combined ocean acidification and warming than previously expected.

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4.3 MATERIAL AND METHODS

4.3.1 Experiment

Six parent colonies of Acropora millepora, Pocillopora damicornis, Montipora monasteriata, and Turbinaria reniformis were purchased from Reef Systems Coral Farm (New Albany, Ohio, USA) which is a CITES permit holder. The parent colonies were specifically collected for this experiment from 3-10 m in northwest Fiji (17°29′19″S, 177°23′39″E) in April 2011. Colonies of the same species were collected at least 10 m apart to increase the probability that different genotypes of the same species were selected. All colonies were shipped to Reef Systems Coral Farm and maintained in recirculating indoor aquaria with natural light (greenhouse, 700-1000 μmol quanta m-2 s- 1) and commercially available artificial seawater (Instant Ocean Reef Crystals) for 2.5 months until the start of the experiment. From April 22 - May 19, 2011, six fragments were collected from each parent colony and mounted on PVC tiles for a total of 144 fragments (4 species x 6 colonies x 6 fragments; Fig. 4.1). Starting on June 19, 2011, corals were gradually acclimated to a custom-made artificial seawater (ESV Aquarium Products Inc.), which was designed to mimic the chemical composition and alkalinity of natural reef seawater. On July 8 and 9, 2011, all 144 fragments were transferred to the experimental recirculating indoor aquaria with artificial light (Tek Light T5 actinic lights, 275 μmol quanta m-2 s-1, 9:15 hrs light:dark cycle) and allowed to acclimate to the artificial light conditions for 10 days under ambient seawater conditions (i.e., 26.5°C and pCO2 of 382 μatm). Photosynthesis to irradiance (P/E) curves performed on Acropora millepora showed that photosynthesis was fully saturated at these light levels. Due to logistical reasons, P/E curves were not performed on the other species. For each of the 6 treatments, the recirculating tank system consisted of one 905 L sump and six aquaria of 57 L each. One fragment per parent colony per species was put in one of the 6 aquaria in each system such that there were a total of 4 fragments (one of

153 each species) in each aquarium, and each parent colony of each species was represented in each system. By placing the same genotypes in each treatment, genotypic variation between treatments was minimized and our ability to detect treatment effects was optimized. Replication of treatments and independent tanks within treatments was not possible due to the complexity and cost of operating tanks under modified pH conditions. While this is, strictly speaking, a pseudo-replicated design (sensu Hurlbert 1984), the disadvantages of this design are outweighed by the advantages of being able to simultaneously manipulate six combinations of temperature and pH. To optimize the experimental design conditions, coral fragments were rotated daily within tanks and every 3 days among tanks within each system to minimize any tank or positional effects within each system. Further, tanks were cleaned every three days, and great care was taken to ensure similar conditions across treatments except for carbonate chemistry and temperature. Experimental treatments were assigned to each system as follows: 26.5°C and 382 μatm, 26.5°C and 607 μatm, 26.5°C and 741 μatm, 29.0°C and 382 μatm, 29.0°C and

607 μatm, and 29.0°C and 741 μatm (Fig 1). The three pCO2 levels – 382, 607, and 741

μatm – were designed to represent present day pCO2, and two pCO2 levels expected by the second half of the 21st century, respectively. The control temperature (26.5°C) represents the current average annual temperatures in Fiji (http://www.ospo.noaa.gov/Products/ocean/index.html), whereas 29.0°C represents the upper limit of current summer temperatures but is still below the bleaching threshold at that location. Therefore, the 26.5°C and 382 μatm treatment served as control. The experiment lasted for 24 days from July 19 – August 12, 2011. Temperature was controlled by titanium aquarium heaters submerged in each system sump (Aqua Medic) and connected to a digital control system (Neptune Systems Apex AquaController). Temperature loggers (Onset Hobo Pro v2) were placed in each sump and recorded temperature every 5 minutes. Seawater pCO2 was controlled by bubbling in pure CO2, CO2-free air, or ambient air delivered by an outdoor air pump

(Sweetwater, Aquatic Eco-Systems Inc.) into each system sump. CO2-free air was

154 achieved by moving ambient air through CO2-scrubbers consisting of a 1.5 m long tube (10 cm diameter) filled with soda lime (SodaSorb HP). Supply of all gases was controlled via a pH stat system using custom designed software (KSgrowstat, written by K. Oxborough, University of Essex). Seawater pH was measured every 5 seconds by microelectrodes (Thermo Scientific Orion Ross Ultra pH glass electrode), which were calibrated daily. For the elevated temperature (29.0°C) treatments, temperature was gradually increased over several days until the desired temperature was reached. For the medium

(607 μatm) and high (741 μatm) pCO2 treatments, pCO2 was gradually increased over several days starting from 382 μatm until the final pCO2 was achieved. Recirculating seawater flow rate was 210-230 l/hour and little pumps (Accela Powerheads) created additional water circulation within each aquarium. A quarter of the entire water volume of each treatment system was exchanged every 3 days. Non-carbonate ceramic filter media (MarinePure High Performance Biofilter Media, CerMedia) were placed in the sumps to filter the water. Tanks were cleaned every 3 days or as needed. Since healthy corals in situ can acquire up to 46% of their daily metabolic energy demands by feeding on zooplankton (Grottoli et al. 2006; Palardy et al. 2008), corals were fed every three days with 48 h old brine shrimp nauplii (Artemia sp., Carolina Biological Supply). Corals were allowed to acclimate to the dark for 30 min before feeding was conducted. They were fed for one hour in separate, partially submerged plastic containers containing water from their respective treatment, and at a concentration of approximately 1 brine shrimp ml-1 which is representative of zooplankton concentrations on natural Pacific reefs (Grottoli 2002). At the end of the hour, brine shrimp nauplii remained in the feeding chambers indicating that the corals had not captured all brine shrimp nauplii available to them. Following feeding, the corals were placed back in their respective aquaria and the feeding container water discarded so as not to introduce brine shrimp into the recirculating systems.

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4.3.2 Monitoring of Seawater Chemistry

Temperature and salinity were measured daily (YSI 63), and salinity was adjusted daily to 35 ppt. Daily water samples were taken using screw-top high-density polyethylene bottles for pH and alkalinity analyses. After equilibration at 25°C in a ® recirculating water bath (30 min), sample pHNBS was measured with an Orion Ross glass electrode (precision 0.01 pH units) (Wang, Cai 2004), which was calibrated daily at 25°C. Total alkalinity (TA) was titrated with HCl on the same samples using an AS- ALK2 (Apollo SciTech Inc.) alkalinity titrator (Cai et al. 2010) (precision 0.1%). The HCl solution was calibrated with Certified Reference Material (CRM) from A.G. Dickson (Scripps).

Treatment xCO2 (dry air), aragonite saturation state (Ωarag), and pHT were calculated using the program CO2SYS (Lewis, Wallace 1998) based on measured pHNBS and alkalinity at the respective temperature. xCO2 was converted to pCO2 using the equation in Weiss et al. (1985). Carbonate dissociation constants were taken from

Millero et al. (2006). In addition, a custom-made CO2 analyzer based on a LI-COR 820 was used weekly to crosscheck with calculated sump xCO2 values according to methods by Wang & Cai (2004), and indicated good agreement of measured and calculated values (r2 = 0.97, n=66).

4.3.3 Laboratory analyses

Calcification Net calcification was determined using the buoyant weight technique (Jokiel et al. 1978). Each coral fragment was buoyantly weighed at the beginning, middle (after 11 experimental days), and end of the experiment (after 23 experimental days). As such, it was possible to assess if calcification rates varied during the experiment. Daily calcification rates were calculated as the difference between initial, middle, and final

156 weights, divided by the respective number of days elapsed, and standardized to surface area (see below). For tissue analyses, corals were frozen at -80°C and a total of three branch tips or growing edge pieces were saved from each fragment for lipid, protein/carbohydrate, and tissue biomass analyses, respectively (Fig. 4.1). The remaining tissue was airbrushed for chlorophyll a and endosymbiont density measurements.

Chlorophyll a and endosymbiont density Coral tissue was stripped off the coral skeleton with a waterpik (Johannes, Wiebe 1970) containing 40 ml of synthetic seawater (Instant Ocean). The endosymbionts were isolated from the host tissue via centrifugation and then resuspended in 10 ml of synthetic seawater. For chlorophyll a concentrations, 1 ml of this algal suspension was pelleted and the cells lysed in 1 ml of 4°C methanol using a bead-beater for 60 seconds. Samples were then immediately placed on ice and allowed to extract for one hour in the dark. Samples were centrifuged to remove cellular debris and measured spectrophotometrically (λ = 652, 665 & 750) on a 96-well plate reader. The equations for chlorophyll a in methanol described by Porra et al. (1989), along with path length correction (Warren 2008), were used to calculate chlorophyll a concentrations (pg/cell), and were then standardized to surface area (see below). Another 1 ml subsample of the algal suspension was preserved with 10 μl of 1% glutaraldehyde solution for endosymbiont quantification, which was calculated using 6 independent replicate counts on a hemocytometer, using a Nikon microphot-FXA epifluorescent microscope at 100x magnification. Photographs were analyzed through Image J using the analyze particles function.

Energy reserves and tissue biomass For all energy reserve and tissue biomass measurements, only branch tips or samples with a growing edge were used. While tissue composition may vary across the surface of a coral (Oku et al. 2002), this approach was used to allow for comparison with previously published studies (Grottoli et al. 2004; Rodrigues, Grottoli 2007; Levas et al.

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2013). Soluble lipids (referred to hereafter simply as lipids) were extracted from a whole, ground coral sample (skeleton + animal tissue + algal endosymbiont) in a 2:1 chloroform:methanol solution for 1 hour (Grottoli et al. 2004; Rodrigues, Grottoli 2007) washed in 0.88% KCl followed by 100% chloroform and another wash with 0.88% KCl. The extract was dried to constant weight under a stream of pure nitrogen (UPH grade 5.0) and standardized to the ash-free dry weight. Animal soluble protein and carbohydrate (referred to hereafter simply as protein and carbohydrate, respectively) were extracted from grinding a whole second branch tip of the same fragment (Rodrigues, Grottoli 2007). Briefly, Milli-Q water was added to the ground coral sample and the resulting slurry was sonicated (5 min) and then centrifuged twice (5000 rpm, 10 min) to separate the animal tissue from the skeleton and endosymbiotic algae. Protein and carbohydrate were extracted from the animal tissue only. One subsample of this animal tissue slurry was used for protein extraction using the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as a standard (Pierce BCA Protein Assay Kit). A second subsample was used for carbohydrate quantification using the phenol-sulfuric acid method (Dubois et al. 1956) with glucose as a standard. Soluble animal protein and carbohydrate concentrations were standardized to the ash-free dry weight. Tissue biomass was measured by drying a third branch tip of whole coral sample (skeleton + animal tissue + algal endosymbiont) to constant dry weight (24 hours, 60°C) and burning it (6 hours, 450°C). The difference between dry and burned weight was the ash free dry weight which was standardized to the surface area of this branch tip.

Surface area Surface area of plating M. monasteriata and T. reniformis fragments was determined using the aluminum foil technique (Marsh 1970), whereas surface area of branching A. millepora and P. damicornis fragments was determined using the single wax dipping technique (Stimson, Kinzie 1991; Veal et al. 2010) after the tissue had been removed. Natural wooden blocks of varying sizes and shapes were used as calibration

158 standards (Veal et al. 2010). Wax dipping was conducted using household paraffin wax (Gulf Wax, Royal Oak Enterprises) heated to 65°C. Dried coral skeletons and wooden calibration standards were maintained at room temperature prior to weighing.

4.3.4 Statistical analyses

Three-way mixed-model analyses of variance (ANOVA) tested the effects of pCO2, temperature, and parent colony on calcification rates in the first and second half of the experiment, chlorophyll a, algal endosymbiont density, lipid, protein, carbohydrate, and tissue biomass. Temperature and pCO2 were fixed and fully crossed, whereas parent colony was a random factor. The ANOVAs were run for each species separately. All data were normally distributed according to plots of residuals versus predicted values for each variable, or transformed to meet the condition of normality. Outlier values greater than 3 times the interquartile range were excluded. Post hoc Tukey tests were performed when main effects were significant (p≤0.05). A posteriori slice tests (e.g., tests of simple effects, Winer 1971) determined if the ambient (26.5°C) and elevated (29.0°C) temperature treatment averages significantly differed within each pCO2 level. Bonferroni corrections were not applied (Quinn, Keough 2002; Moran 2003), therefore significant model p-values >0.0016 (0.05/32 tests) should be interpreted with caution. Statistical analyses were performed using SAS software, Version 9.2 of the SAS System for Windows.

4.4 RESULTS

All corals appeared healthy throughout the experiment. No visible paling and no mortality occurred. The average seawater temperature, pHT, pCO2, saturation state, and total alkalinity for all six treatments are summarized in Table 4.1.

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4.4.1 Calcification

In Acropora millepora, calcification rates during the first half of the experiment were overall unaffected by both temperature (p=0.36) and pCO2 (p=0.79) (Fig. 4.2A;

Table 4.2). However, at the highest pCO2 level (741 μatm) calcification was 43% lower at 29.0°C than at 26.5°C (Fig. 4.2A). During the second half of the experiment, calcification rates were significantly affected by pCO2 (p=0.001) but not temperature (p=0.42), and were lower by 53% at the highest compared to the lowest pCO2 level (Fig. 4.2B; Table 4.2).

In Pocillopora damicornis, a significant interaction of pCO2 and temperature (p<0.001) was observed for calcification rates during the first half of the experiment (Fig. 4.2C, Table 4.2). During the second half of the experiment, calcification rates were generally unaffected by temperature (p=0.06) and pCO2 (p=0.07). However, at ambient seawater pCO2 (382 μatm) corals kept at elevated temperature (29.0°C) calcified 91% more compared to those kept at 26.5°C (Fig. 4.2D, Table 4.2). Calcification rates of Montipora monasteriata were affected by temperature

(p=0.04) but not pCO2 (p=0.42) during the first half of the experiment (Fig. 4.2E-F, Table 4.2), with corals calcifying 18% more at elevated compared to ambient temperature. This was largely driven by significant temperature differences at both 382 and 607 μatm but not 741 μatm. During the second half of the experiment, calcification rates were unaffected by both temperature (p=0.82) and pCO2 (p=0.14). In contrast, calcification rates of Turbinaria reniformis during both the first and the second half of the experiment (Fig. 4.2G-H, Table 4.2) did not respond to changes in seawater temperature (p=0.45 and 0.17) or pCO2 (p=0.36 and 0.09). Notably, the two plating species (M. monasteriata and T. reniformis) calcified more than twice as fast as the two branching species (A. millepora and P. damicornis).

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4.4.2 Chlorophyll a and endosymbiont density

The chlorophyll a concentrations of A. millepora were significantly affected by pCO2 (p<0.001) but not temperature (p=0.054), with concentrations being 51% lower at 607 μatm than at either 382 or 741 μatm (Fig. 4.3A, Table 4.4). Endosymbiont densities were not affected by either seawater temperature (p=0.07) or pCO2 (p=0.03 but overall model p=0.24) (Fig. 4.3B, Table 4.3).

In P. damicornis, a significant interaction of temperature and pCO2 was observed for both chlorophyll a concentrations and endosymbiont densities (p<0.001 and p=0.02, respectively) (Fig. 4.3C-D, Table 4.3). When temperature was elevated, chlorophyll a concentrations were higher by 19% and 67%, respectively, at both 382 and 741 μatm. At 607 μatm, endosymbiont densities decreased by 36% at 29°C compared to concentrations at ambient temperature.

In M. monasteriata, a significant interaction of seawater temperature and pCO2 was observed for chlorophyll a concentrations (p=0.01) (Fig. 4.3E, Table 4.3), with concentrations being 45% and 30% lower at elevated compared to ambient temperature, respectively, under both 382 and 741 μatm conditions. Endosymbiont densities were significantly affected by both temperature (p<0.001) and pCO2 (p=0.01) but the interaction term was not significant (p=0.38) (Fig. 4.3F, Table 4.3). Densities were 32% lower at elevated compared to ambient temperature, and were lowest overall at 607 μatm

(-25%) compared to the other two pCO2 levels.

Chlorophyll a concentrations of T. reniformis were affected by pCO2 (p=0.03) but not temperature (p=0.11), with concentrations being 38% lower at 607 compared to 382 μatm (Fig. 4.3G, Table 4.3). Endosymbiont densities were not affected by either temperature (p=0.90) or pCO2 (p=0.21) but were lower by 45% at elevated compared to ambient temperature under 741 μatm conditions (Fig. 4.3H, Table 4.3).

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4.4.3 Energy reserves and tissue biomass

Lipid concentrations of A. millepora were affected by seawater pCO2 (p=0.01) but not temperature (p=0.053), with concentrations being 28% and 21% higher at 607 and

741 μatm, respectively, compared to concentrations at ambient pCO2 (Fig. 4.4A, Table

4.4). A significant interaction of seawater pCO2 and temperature was observed for protein concentrations (p=0.01) (Fig. 4.4B, Table 4.4). Carbohydrate concentrations were affected by both temperature (p=0.001) and pCO2 (p<0.001) but the interaction term was not significant (p=0.99) (Fig. 4.4C, Table 4.4). Across all pCO2 treatments, carbohydrate concentrations were 15% lower at 29.0°C compared to 26.5°C, and 18% lower at 607 μatm than at either 382 or 741 μatm. Tissue biomass was unaffected by changes in seawater temperature (p=0.99) and pCO2 (p=0.07) (Fig. 4.4D, Table 4.4).

In P. damicornis, lipid concentrations were affected by seawater pCO2 (p=0.01) but not temperature (p=0.53) (Fig. 4.4E, Table 4.4), with concentrations being 41% and 18% higher at 607 and 741 μatm, respectively, compared to concentrations at ambient pCO2 (Fig. 4.4E, Table 4.4). Neither protein, nor carbohydrate concentrations or tissue biomass were affected by seawater temperature (p=0.55, 0.97, 0.33, respectively) and pCO2 (p=0.54, 0.48, 0.41, respectively) (Fig. 4.4F-H, Table 4.4). The lipid concentrations of M. monasteriata were unaffected by both seawater temperature (p=0.38) and pCO2 (p=0.23) (Fig. 4.4I, Table 4.4). A significant interaction of temperature and pCO2 was observed for protein concentrations (p<0.001): at 382 μatm, they decreased (-19%) at elevated compared to ambient temperature, whereas at 607 and741 μatm, they increased (+24% and +33%, respectively) (Fig. 4.4J, Table 4.4). Carbohydrate concentrations were overall unaffected by seawater temperature (p=0.08) and pCO2 (p=0.62) (Fig. 4.4K, Table 4.4), but the concentrations were 29% higher at elevated than at ambient temperature under 382 μatm conditions. Tissue biomass was also unaffected by both seawater temperature (p=0.78) and pCO2 (p=0.12) (Fig. 4.4L, Table 4.4).

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In T. reniformis, none of the measured energy reserve pools responded to changes in seawater temperature and pCO2 (Fig. 4.4M-O, Table 4.4). Tissue biomass was also unaffected by both temperature (p=0.62) and pCO2 (p=0.58), but was 21% lower at 29.0°C compared to 26.5°C at 382 μatm (Fig. 4.4P, Table 4.4).

4.4.4 Effects of parent colony

Parent colony was a significant effect in many of the measured variables, but no single parent colony or group of specific parent colonies was consistently different from all other parent colonies in any of the species studied (Tables 4.2-4.4).

4.5 DISCUSSION

Coral calcification has been predicted to decrease dramatically by the end of this century, thus threatening the existence of coral reefs in the future. Although the response of coral calcification is not uniform across species, most studies have found that calcification decreases with increasing seawater pCO2 (Kroeker et al. 2010; Chan, Connolly 2012; Harvey et al. 2013). Here, we show that only one of the four Pacific coral species studied here decreased calcification in response to average ocean acidification levels expected by the second half of this century (741 μatm), even when combined with elevated temperature (+2.5°C). Further, we investigated for the first time the effects of OA on coral energy reserves and show that they were largely not metabolized in order to sustain calcification under elevated pCO2 and temperature. Acropora millepora was the only coral out of the four species studied here that decreased calcification rates in response to OA (Fig. 4.2B). While calcification rates were not affected by elevated pCO2 and/or temperature during the first half of the experiment, they declined by 53% during the second half of the experiment due to acidification alone. As the second half of the experiment is more likely to reflect the long term response of corals to ocean acidification, this negative response to OA is consistent with other studies 163 on Acropora sp. (Marubini et al. 2003; Schneider, Erez 2006; Anthony et al. 2008; Comeau et al. 2013b), although the amount of decline differs between species. The absence of any change in calcification of Pocillopora damicornis is consistent with another study (Comeau et al. 2013b), whereas declines in calcification of 50% in P. meandrina were reported (Muehllehner, Edmunds 2008). The lack of any change in calcification rates of Montipora monasteriata and Turbinaria reniformis due to acidification (Fig. 4.2D, F, H) is in contrast to other studies which reported a 15-20% decline in Montipora capitata (Jokiel et al. 2008), and a 13% decline in T. reniformis

(albeit at pCO2 levels that were considerably higher than those in the present study)

(Marubini et al. 2003). Although a significant interaction of pCO2 and temperature was observed in P. damicornis during the first half of the experiment (Fig. 4.2C), this was not observed during the second half. Similarly, M. monasteriata calcified more at elevated compared to ambient temperature during the first half (Fig. 4.2E), but not during the second half of the experiment. Thus, it appears that with the exception of A. millepora, these species may be resistant to changes in pCO2 and temperature within the parameter ranges investigated in this study. In the current study, elevated temperature did not exacerbate or counteract the negative effects of OA on calcification in A. millepora, and did not have an overall negative affect on calcification in the other three species. This is in contrast to other studies where elevated temperature was found to mitigate negative OA effects. For example, Anthony et al. (2008) found that elevated temperature (28-29°C vs. 25-26°C) prevented a decline of calcification in A. intermedia at elevated pCO2 (520-705 μatm).

Muehllehner and Edmunds (2008) showed that the negative effects of elevated pCO2 (720 μatm) were fully mediated in P. meandrina when OA was combined with elevated temperature (29°C vs. 27°C). Overall, these findings add to the growing body of evidence that the response of coral calcification to OA is highly species specific, and that some coral species may maintain calcification under combined ocean acidification and warming in the future.

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Although the current study was conducted using artificial seawater, it is unlikely that this influenced the observed responses of calcification to ocean acidification. The carbonate chemistry of the custom-made seawater mimicked natural conditions very well (Table 1), and calcification rates – as well as chlorophyll a concentrations, endosymbiont densities, energy reserves, and tissue biomass – were within the range observed in the field and/or other experimental studies using natural seawater (Stambler et al. 1991; Muller-Parker et al. 1994; Takabayashi, Hoegh-Guldberg 1995; Fitt et al. 2000; Reynaud- Vaganay et al. 2001; Marubini et al. 2003; D'Croz, Mate 2004; Grottoli et al. 2004; Dove et al. 2006; Rodrigues, Grottoli 2007; Treignier et al. 2008; Ferrier-Pages et al. 2010; Krief et al. 2010; Edmunds 2011; Thornhill et al. 2011; Tolosa et al. 2011; Edmunds 2012; Levas 2012).

While many studies note a decline in coral calcification with increasing pCO2 (Kroeker et al. 2010; Chan, Connolly 2012; Harvey et al. 2013), there is considerable among-study variation (Kroeker et al. 2010; Chan, Connolly 2012), and some species are more resistant than others (e.g. this study, Fabricius et al. 2011; Edmunds et al. 2012). Such differences may be due to experimental duration, how seawater carbonate chemistry is altered (i.e., bubbling CO2 vs. acid addition), and how calcification is measured (i.e., buoyant weight vs. total alkalinity anomaly technique). Meta-analyses have shown that experimental duration or the method of carbonate chemistry manipulation did not explain the large variability of responses observed among studies (Kroeker et al. 2010; Chan, Connolly 2012). While this study suggests that experimental duration can influence the response of calcification to OA in some species (i.e., calcification of A. millepora decreased only during the second half of the second half), it is likely that biological aspects have a stronger influence on the sensitivity of coral calcification to OA than differences in methodology. Important biological aspects include energetic status and feeding (Cohen, Holcomb 2009; Edmunds 2011), enhanced algal production (Herfort et al. 2008; Brading et al. 2011), and cellular pH control (Ries 2011; Venn et al. 2011; McCulloch et al. 2012; Venn et al. 2013).

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Despite the assumption that calcification becomes energetically more costly under OA (Cohen, Holcomb 2009; Erez et al. 2011; Pandolfi et al. 2011), energy reserves did not decline with increasing pCO2 (Fig. 4.4). Lipid concentrations increased under OA conditions in both A. millepora and P. damicornis, and were fully maintained in M. monasteriata and T. reniformis. Protein, carbohydrate, and tissue biomass were overall maintained under OA conditions in all species. Further, temperature did not negatively affect energy reserves and tissue biomass except for carbohydrate concentrations in A. millepora, which were lower at elevated compared to ambient temperature. Importantly, energy reserves and tissue biomass were fully maintained or even increased at the highest pCO2 level in A. millepora despite dramatic decreases in calcification rates. These findings suggest that (1) energy reserves are generally not metabolized under OA conditions or OA at elevated temperature, and (2) that either energy reserves do not play a role in sustaining calcification under OA conditions, or that the increased energetic costs of maintaining calcification under OA are relatively insignificant. This is consistent with other work showing that calcification likely does not become energetically more costly under OA conditions (Edmunds 2012), and that the extra energy required to up- regulate pH at the site of calcification under OA conditions is <1% of that produced by photosynthesis (McCulloch et al. 2012). Further, from an energetic standpoint of view, the total amount of energy reserves present in a coral species did not seem to be related to their calcification response to OA. The energetic content of lipid, protein, and carbohydrates is better assessed from an energetic point of view (Gnaiger, Bitterlich 1984; Lesser 2013), as specific enthalpies of combustion differ significantly among energy reserve pools: -39.5 kJ g-1 for lipid, -23.9 kJ g-1 for protein, and -17.5 kJ g-1 for carbohydrate (Gnaiger, Bitterlich 1984). When the total amount of energy available to each species was calculated (i.e., the sum of lipid, protein, and carbohydrate expressed in kJ g-1 tissue biomass), A. millepora had the lowest amount of all species in the control treatment (14.3 vs. up to 19.3 kJ g-1 in T. reniformis), -1 but the second-highest amount in the high-CO2 treatment (19.1 vs. 19.5 kJ g in P. damicornis), and a similar amount as M. monasteriata in the high-CO2 – high

166 temperature treatment (16.8 kJ g-1 versus 16.6 kJ g-1). It is therefore unlikely that high levels of energy reserves per se help corals maintain calcification rates under OA conditions. However, maintaining energy reserves and tissue biomass under ocean acidification does have crucial implications for other aspects of coral health and resistance to stressors such as coral bleaching. For example, maintenance of lipid concentrations may enable corals to maintain their reproductive output (e.g. Ward 1995), even under future OA and warming. This may be critical considering that many other processes involved in coral reproduction such as fertilization, settlement success, and metamorphosis are compromised under OA (e.g. Albright et al. 2010; Nakamura et al. 2011). Furthermore, maintenance of energy reserves has been shown to be associated with higher resistance to coral bleaching and to promote recovery from bleaching (Rodrigues, Grottoli 2007; Anthony et al. 2009), which could prove critical as bleaching events will increase in frequency over the coming decades (Donner 2009). Heterotrophy is known to promote energy storage, tissue synthesis, and skeletal growth in healthy and bleached corals (e.g. Grottoli et al. 2006; Houlbreque, Ferrier- Pages 2009; Levas et al. 2013) as well as corals subjected to OA (Edmunds 2011; Drenkard et al. 2013). Therefore, biweekly feeding in this study (intended to mimic zooplankton contribution to the coral diet on the reef) may have helped corals to sustain energy reserves and tissue biomass under these conditions. It has further been suggested that coral tissue reacts to availability of such resources faster than skeletal growth (Anthony et al. 2002; Houlbreque et al. 2004), which could explain why tissue biomass - but not necessarily calcification – was maintained or even increased in all four species irrespective of pCO2 or temperature conditions. As feeding rates and heterotrophic plasticity are highly species-specific (Palardy et al. 2005; Grottoli et al. 2006; Palardy et al. 2008), it is likely that heterotrophic carbon intake differed significantly among the species studied here, potentially contributing to their differential responses to OA.

Enhanced algal productivity due to CO2-fertilization (Herfort et al. 2008; Brading et al. 2011) may help corals to maintain calcification under OA conditions. Although

167 chlorophyll a concentrations and endosymbiont density were unaffected at the highest pCO2 level (except for chlorophyll in T. reniformis), CO2-fertilization may nevertheless have played a role in helping corals to maintain energy reserves and/or calcification.

Increased availability of CO2(aq) under OA conditions may enhance algal productivity, especially in Symbiodinium phylotypes with less efficient carbon-concentrating mechanisms, which rely to a greater extent on the passive, diffusive uptake of CO2(aq) (Brading et al. 2011). Thus, a potentially increased translocation of autotrophic carbon to the animal host may have contributed to the maintenance of energy reserves and tissue biomass observed here. Interestingly, both chlorophyll a concentrations and endosymbiont density were often lowest at 607 μatm, showing a non-linear relationship with increasing pCO2. Nevertheless, the lack of any significant difference in chlorophyll a and/or symbiont density at 741 μatm versus ambient pCO2 concentrations (except for chlorophyll in T. reniformis) is consistent with other studies (Marubini et al. 2008; Edmunds 2011; Godinot et al. 2011; Houlbreque et al. 2012). The reason for the observed minima at ~600 μatm is unknown. Similar non-linear responses were not observed for calcification rates, tissue biomass, and most energy reserve pools, suggesting that this did not translate into a decreased performance of the animal host. Edmunds (2012) also observed a non- linear pCO2 threshold between 756 and 861 μatm affecting photochemistry and respiration in massive Porites corals, thus highlighting the importance of studying multiple pCO2 levels in OA experiments in order to assess non-linear physiological responses and to better forecast physiological responses over the coming century as the oceans continue to warm and acidify.

In addition to energetic status and enhanced algal productivity due to CO2 fertilization, other factors such as the amount of control over the carbonate chemistry at the site of calcification may explain the observed differences in susceptibility of calcification to OA. Corals have the ability to significantly up-regulate the pH at the site of calcification compared to ambient seawater, even under OA conditions (Al-Horani et al. 2003; Cohen et al. 2009; Ries 2011; Venn et al. 2011; McCulloch et al. 2012; Venn et

168 al. 2013). Yet, the degree to which corals are able to control the pH at the site of calcification likely varies among species (Ries et al. 2009; Ries 2011; McCulloch et al. 2012). Acropora spp. may have the lowest capacity to up-regulate pH at the site of calcification based on boron isotopic measurements (Trotter et al. 2011; McCulloch et al. 2012). Further, crystallization under OA was most compromised in Acropora verweyi and least compromised in T. reniformis (Marubini et al. 2003). Thus, we hypothesize that A. millepora has a weaker proton pump than the other coral species studied here, making its calcification rate more sensitive to future OA. Although pH up-regulation has not been studied in P. damicornis, M. monasteriata, or T. reniformis, it can be hypothesized that they have stronger control over the pH at the site of calcification and were therefore able to maintain calcification under the pCO2 levels studied here. As physiological responses of both the animal host and algal endosymbiont to combined OA and warming were strongly species-specific, a wide range of susceptibility patterns can be expected resulting in ecological “winners and losers” (Loya et al. 2001; Fabricius et al. 2011). Branching Acropora corals, which are important reef builders, are likely to be “losers” on future coral reefs because they are highly susceptible to both bleaching (e.g. Loya et al. 2001) and OA (this study, Schneider, Erez 2006; Anthony et al. 2008; Albright et al. 2010; Fabricius et al. 2011). This can be expected to have severe impacts on reef diversity, structural complexity, and overall reef functioning. Nevertheless, some corals could be more resistant to combined ocean acidification and warming expected by the end of this century than previously thought, as three of the four species fully maintained calcification under elevated pCO2 and temperature without compromising overall energy reserves or biomass. Further, the immediate effects of rising seawater temperature and ocean acidification may be tolerable for some species.

Acknowledgements. We thank E. Zebrowski, M. Berzelis, M. Ringwald, S. Levas, and the staff at Reef Systems Coral Farm for their field and laboratory support. A. G. G., M. E. W., and W.-J. C. thank the National Science Foundation Program in Ocean

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Acidification (Grant No. NSF-EF-1041124, 1040940, and 1041070, respectively) for funding support.

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4.6 REFERENCES

Al-Horani FA, Al-Moghrabi SM, de Beer D (2003) The mechanism of calcification and its relation to photosynthesis and respiration in the scleractinian coral Galaxea fascicularis. Marine Biology 142:419-426

Albright R, Mason B, Miller M, Langdon C (2010) Ocean acidification compromises recruitment success of the threatened Caribbean coral Acropora palmata. Proceedings of the National Academy of Sciences of the USA 107:20400-20404

Anthony KRN, Connolly SR, Willis BL (2002) Comparative analysis of energy allocation to tissue and skeletal growth in corals. Limnology and Oceanography 47:1417-1429

Brading P, Warner ME, Davey P, Smith DJ, Achterberg EP, Suggett DJ (2011) Differential effects of ocean acidification on growth and photosynthesis among phylotypes of Symbiodinium (Dinophyceae). Limnology and Oceanography 56:927-938

Cai W-J, Hu X, Huang W-J, Jian L-Q, Peng T-H, Zhang X (2010) Alkalinity distribution in the western North Atlantic Ocean margins. Journal of Geophysical Research 115:C08014

Caldeira K, Wickett ME (2003) Anthropogenic carbon and ocean pH. Nature 425:365

Cantin NE, Cohen AL, Karnauskas KB, Tarrant AM, McCorkle DC (2010) Ocean warming slows coral growth in the Central Red Sea. Science 329:322-325

Chan NCS, Connolly SR (2012) Sensitivity of coral calcification to ocean acidification: a meta-analysis. Global Change Biology:doi: 10.1111/gcb.12011

Clausen C (1971) Effects of temperature on the rate of 45Ca uptake by Pocillopora damicornis. In: Lenhoff HM, Muscatine L, Davis LV (eds) Experimental Coelenterate Biology. University of Hawaii Press, Honolulu, pp 246-260

171

Clausen C, Roth AA (1975) Effect of temperature and temperature adaptation on calcification rataes in the hermatypic coral Pocillopora damicornis. Marine Biology 33:93-100

Cohen AL, Holcomb M (2009) Why corals care about ocean acidification - uncovering the mechanism. Oceanography 22:118-127

Cohen AL, McCorkle DC, de Putron S, Gaetani GA, Rose KA (2009) Morphological and compositional changes in the skeletons of new coral recruits reared in acidified seawater: Insights into the biomineralization response to ocean acidification. Geochemistry, Geophysics, Geosystems 10:Q07005, doi:07010.01029/02009GC002411

Comeau S, Carpenter R, Edmunds P (2013a) Effects of feeding and light intensity on the response of the coral Porites rus to ocean acidification. Marine Biology 160:1127-1134

Comeau S, Edmunds PJ, Spindel NB, Carpenter RC (2013b) The responses of eight coral reef calcifiers to increasing partial pressure of CO2 do not exhibit a tipping point. Limnology and Oceanography 58:388-398

D'Croz L, Mate JL (2004) Experimental responses to elevated water temperature in genotypes of the reef coral Pocillopora damicornis from upwelling and non- upwelling environments in Panama. Coral Reefs 23:473-483

Donner SD (2009) Coping with commitment: Projected thermal stress on coral reefs under different future scenarios. PLoS ONE 4:e5712

Dove S, Ortiz J-C, Enriquez S, Fine M, Fisher P, Iglesias-Prieto R, Thornhill DJ, Hoegh- Guldberg O (2006) Response of holosymbiont pigments from the scleractinian coral Montipora monasteriata to short-term heat stress. Limnology and Oceanography 51:1149-1158

Drenkard EJ, Cohen AL, McCorkle DC, Putron SJ, Starczak VR, Zicht AE (2013) Calcification by juvenile corals under heterotrophy and elevated CO2. Coral Reefs:DOI 10.1007/s00338-00013-01021-00335

Dubois M, Giles KA, Hamilton JK, Pebers PA, Smith F (1956) Colorimetric method for determination of sugar and related substances. Analytical Chemistry 28:350-356

Edmunds PJ (2012) Effect of pCO2 on the growth, respiration, and photophysiology of massive Porites spp. in Moorea, French Polynesia. Marine Biology 159:2149- 2160

172

Edmunds PJ (2011) Zooplanktivory ameliorates the effects of ocean acidification on the reef coral Porites spp. Limnology and Oceanography 56:2402-2410

Edmunds PJ, Brown D, Moriarty V (2012) Interactive effects of ocean acidification and temperature on two scleractinian corals from Moorea, French Polynesia. Global Change Biology 18:2173-2183

Erez J, Reynaud S, Silverman J, Schneider K, Allemand D (2011) Coral calcification under ocean acidification and global change. In: Stambler N, Dubinsky Z (eds) Coral Reefs: An Ecosystem in Transition. Springer, 552 p, pp 552

Fabricius KE, Langdon C, Uthicke S, Humphrey C, Noonan S, De'ath G, Okazaki R, Muehllehner N, Glas MS, Lough JM (2011) Losers and winners on coral reefs acclimatized to elevated carbon dioxide concentrations. Nature Climate Change 1:165-169

Feely RA, Doney SC, Cooley SR (2009) Ocean acidification: present condition and future changes in a high-CO2 world. Oceanography 22:36-47

Feely RA, Sabine CL, Lee K, Berelson W, Kleypas J, Fabry VJ, Millero FJ (2004) Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 305:362-366

Ferrier-Pages C, Rottier C, Beraud E, Levy O (2010) Experimental assessment of the feeding effort of three scleractinian coral species during a thermal stress: Effect on the rates of photosynthesis. Journal of Experimental Marine Biology and Ecology 390:118-124

Fine M, Tchernov D (2007) Scleractinian coral species survive and recover from decalcification. Science 315:1811

Fitt WK, Brown BE, Warner ME, Dunne RP (2001) Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs 20:51-65

Gnaiger E, Bitterlich G (1984) Proximate biochemical composition and caloric content calculated from elemental CHN analysis: a stoichiometric concept. Oecologia 62:289-298

173

Godinot C, Houlbreque F, Grover R, Ferrier-Pages C (2011) Coral uptake of inorganic phosphorus and nitrogen negatively affected by simultaneous changes in temperature and pH. PLoS ONE 6:e25024

Grottoli AG (2002) Effect of light and brine shrimp levels on skeletal d13C values in the Hawaiian coral Porites compressa: A tank experiment. Geochimica and Cosmochimica Acta 66:1955-1967

Grottoli AG, Rodrigues LJ, Palardy JE (2006) Heterotrophic plasticity and resilience in bleached corals. Nature 440:1186-1189

Herfort L, Thake B, Taubner I (2008) Bicarbonate stimulation of calcification and photosynthesis in two hermatypic corals. Journal of Phycology 44:91-98

Hoegh-Guldberg O (1999) Climate change, coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research 50:839-866

Hoegh-Guldberg O, Bruno JF (2010) The impact of climate change on the world's marine ecosystems. Science 328:1523-1528

Houlbreque F, Ferrier-Pages C (2009) Heterotrophy in tropical scleractinian corals. Biological Reviews 84:1-17

174

Houlbreque F, Tambutte E, Allemand D, Ferrier-Pages C (2004) Interactions between zooplankton feeding, photosynthesis and skeletal growth in the Scleractinian coral Stylophora pistillata. Journal of Experimental Biology 207:1461-1469

Hurlbert SH (1984) Pseudoreplication and the Design of Ecological Field Experiments. Ecological Monographs 54:187-211

Johannes RE, Wiebe WJ (1970) A method for determination of coral tissue biomass and composition. Limnology and Oceanography 21:540-547

Jokiel PL, Coles SL (1977a) Effects of temperature on the mortality and growth of Hawaiian reef corals. Marine Biology 43:201-208

Jokiel PL, Maragos JE, Franzisket L (1978) Coral growth: buoyant weight technique. In: Stoddart DR, Johannes RE (eds) Coral Reefs: Resesarch Methods. UNESCO, Paris, pp 529-541

Jokiel PL, Rodgers KS, Kuffner IB, Andersson AJ, Cox EF, T. Mackenzie F (2008) Ocean acidification and calcifying reef organisms: a mesocosm investigation. Coral Reefs 27:473-483

Kleypas J, Buddemeier RW, Archer D, Gattuso J-P, Langdon C, Opdyke BN (1999) Geochemical consequences of increased atmospheric carbon dioxide on coral reefs. Science 284:118-120

Kroeker KJ, Kordas RL, Crim RN, Singh GG (2010) Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms. Ecology Letters 13:1419-1434

Langdon C, Atkinson MJ (2005) Effect of elevated pCO2 on photosynthesis and calcification on corals and interactions with seasonal change in 175

temperature/irradance and nutrient enrichment. Journal of Geophysical Research 110:C09S07, doi:10.1029/2004JC002576

Langdon C, Takahashi T, Sweeney C, Chipman D, Goddard J, Marubini F, Aceves H, Barnett H (2000) Effect of calcium carbonate saturation state on the calcification rate of an experimental coral reef. Global Biogeochemical Cycles 14:639-654

Leclercq N, Gattuso J-P, Jaubert J (2000) CO2 partial pressure controls the calcification rate of a coral community. Global Change Biology 6:329-334

Lesser MP (2013) Using energetic budgets to assess the effects of environmental stress on corals: are we measuring the right things? Coral Reefs 32:25-33

Levas SJ (2012) Biogeochemistry and Physiology of Bleached and Recovering Hawaiian and Caribbean Corals. PhD Thesis, The Ohio State University, p 238

Lewis E, Wallace D (1998) Program Developed for CO2 System Calculations. ORNL/CDIAC-105. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee.

Loya Y, Sakai K, Yamazato K, Nakan Y, Sambali H, van Woesik R (2001) Coral bleaching: the winners and the losers. Ecology Letters 4:122-131

Marsh JA (1970) Primary productivity of reef-building calcareous red algae. Ecology 51:255-263

Marshall AT, Clode P (2004) Calcification rate and the effect of temperature in a zooxanthellate and an azooxanthellate scleractinian reef coral. Coral Reefs 23:218-224 Marubini F, Atkinson MJ (1999) Effects of lowered pH and elevated nitrate on coral calcification. Marine Ecology Progress Series 188:117-121

Marubini F, Ferrier-Pages C, Cuif J-P (2003) Suppression of skeletal growth in scleractinian corals by decreasing ambient carbonate-ion concentration: a cross- family comparison. Proceedings of the Royal Society B 270:179-184

Marubini F, Ferrier-Pages C, Furla P, Allemand D (2008) Coral calcification responds to seawater acidification: a working hypothesis towards a physiological mechanism. Coral Reefs 27:491-499 176

McCulloch MT, Falter J, Trotter J, Montagna P (2012) Coral resilience to ocean acidification and global warming through pH up-regulation. Nature Climate Change:doi:10.1038/nclimate1473

Millero FJ, Graham TB, Huang F, Bustos-Serrano H, Pierrot D (2006) Dissociation constants of carbonic acid in seawater as a function of salinity and temperature. Marine Chemistry 100:80-94

Moran MD (2003) Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos 100:403-405

Muller-Parker G, McCloskey LR, Hoegh-Guldberg O, McAuley PJ (1994) Effect of ammonium enrichment on animal and algal biomass of the coral Pocillopora damicornis. Pacific Science 48:273-283

Muscatine L, McCloskey LR, Marian RE (1981) Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration. Limnology and Oceanography 26:601-611

Nakamura M, Ohki S, Suzuki A, Sakai K (2011) Coral larvae under ocean acidification: survival, metabolism, and metamorphosis. Public Library of Science One 6:e14521. doi:14510.11371/journal.pone.0014521

Oku H, Yamashiro H, Onaga K, Iwasaki H, Takara K (2002) Lipid distribution in branching coral Montipora digitata. Fisheries Science 68:517-522

Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, Feely RA, Gnanadesikan A, Gruber N, Ishida A, Joos F, Key RM, Lindsay K, Maier-Reimer E, Matear R, Monfray P, Mouchet A, Najjar RG, Plattner G-K, Rodgers KB, Sabine CL, Sarmiento JL, Schlitzer R, Slater RD, Totterdell IJ, Weirig M-F, Yamanaka Y, Yool A (2005) Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437:681-686

Palardy JE, Grottoli AG, Matthews KA (2005) Effects of upwelling, depth, morphology and polyp size on feeding of three species of Panamanian corals. Marine Ecology Progress Series 300:79-89

177

Pandolfi JM, Connolly SR, Marshall DJ, Cohen AL (2011) Projecting coral reef futures under global warming and ocean acidification. Science 333:418-422

Porra RJ, Tompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta 975:384-394

Quinn GP, Keough MJ (2002) Experimental Design and Data Analysis for Biologists. Cambridge University Press, 557 p, New York

Reynaud-Vaganay S, Juillet-Leclerc A, Jaubert J, Gattuso J-P (2001) Efect of light on skeletal d13C and d18O, and interaction with photosynthesis, respiration and calcification in two zooxanthellate scleractinian corals. Palaeogeography, Palaeoclimatology, Palaeoecology 175:393-403

Ries JB (2011) A physicochemical framework for interpreting the biological calcification response to CO2-induced ocean acidification. Geochimica and Cosmochimica Acta 75:4053-4064

Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology 37:1131-1134

Rodrigues LJ, Grottoli AG (2007) Energy reserves and metabolism as indicators o f coral recovery from bleaching. Limnology and Oceanography 52:1874-1882

Sabine CL, Feely RF, Gruber N, Key RM, Lee K, Bullister JL, Wanninkhof R, Wong CS, Wallace DWR, Tilbrook B, Millero FJ, Peng T-H, Kozyr A, Ono T, Rios AF (2004) The oceanic sink for anthropogenic CO2. Science 305:367-371

Schneider K, Erez J (2006) The effect of carbonate chemistry on calcification and photosynthesis in the hermatypic coral Acropora eurystoma. Limnology and Oceanography 51:1284-1293

Stambler N, Popper N, Dubinsky Z, Stimson J (1991) Effects of nutrient enrichment and water motion on the coral Pocillopora damicornis. Pacific Science 45:299-307

Stimson J (1997) The annual cycle of density of zooxanthellae in the tissue of field and laboratory-held Pocillopora damicornis (Linnaeus). Journal of Experimental Marine Biology and Ecology 214:35-48

Stimson J, Kinzie RA (1991) The temporal pattern and rate of release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus) under nitrogen-enrichment and control conditions. Journal of Experimental Marine Biology and Ecology 153:63-74

Takabayashi M, Hoegh-Guldberg O (1995) Ecological and physiological differences between two colour morphs of the coral Pocillopora damicornis. Marine Biology 123:705-714

Treignier C, Grover R, Ferrier-Pages C, Tolosa I (2008) Effect of light and feeding on the fatty acid and sterol composition of zooxanthellae and host tissue isolated from

179

the scleractinian coral Turbinaria reniformis. Limnology and Oceanography 53:2702-2710

Trotter J, Montagna P, McCulloch MT, Silenzi S, Reynaud S, Mortimer G, Martin S, Ferrier-Pages C, Gattuso J-P, Rodolfo-Metalpa R (2011) Quantifying the pH 'vital effect' in the temperate zooxanthellate coral Cladocora caespitosa: Validation of the boron seawater pH proxy. Earth and Planetary Science Letters 303:163-173

Veal CJ, Carmi M, Fine M, Hoegh-Guldberg O (2010) Increasing the accuracy of surface area estimation using single wax dipping of coral fragments. Coral Reefs 29:893- 897

Venn A, Tambutte E, Holcomb M, Allemand D, Tambutte S (2011) Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater. PLoS ONE 6:e20013 Venn AA, Tambutte E, Holcomb M, Laurent J, Allemand D, Tambutte S (2013) Impact of seawater acidification on pH at the tissue-skeleton interface and calcification in reef corals. Proceedings of the National Academy of Sciences 110:1634-1639

Wang ZA, Cai W-J (2004) Carbon dioxide degassing and inorganic carbon export from a marsh-dominated estuary (the Duplin River): a Marsh CO2 pump. Limnology and Oceanography 49:341-354

Ward S (1995) Two patterns of energy allocation for growth, reproduction and lipid storage in the scleractinian coral Pocillopora damicornis Coral Reefs 14:87-90

Warren CR (2008) Rapid measurement of chlorophylls with a microplate reader. Journal of Plant Nutrition 31:1321-1332

Weiss RF, Bullister JL, Gammon RH, Warner MJ (1985) Atmospheric chlorofluoromethanes in the deep equatorial Atlantic. Nature 314:608-610

Winer BJ (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York

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4.7 TABLES

400 ppm target 600 ppm target 800 ppm target ambient elevated ambient elevated ambient elevated temp. temp. temp. temp. temp. temp. Temp. 26.45 ± 29.31 ± 26.37 ± 28.53 ± 26.61 ± 28.93 ± (°C) 0.01 0.02 0.01 0.02 0.01 0.02

8.07 ± 8.04 ± 7.90 ± 7.89 ± 7.83 ± 7.81 ± pH T 0.01 0.01 0.01 0.01 0.01 0.01

pCO2 364.31± 400.62 ± 598.37 ± 616.08 ± 732.04 ± 749.63 ± (μatm) 9.69 16.83 18.50 24.24 22.37 26.21

TA 2269.4 ± 2270.1 ± 2303.8 ± 2288.3 ± 2306.3 ± 2304.5 ± (µmol 10.84 11.15 9.34 10.43 10.64 9.08 kg-1)

3.69 ± 3.79 ± 2.75 ± 2.91 ± 2.40 ± 2.52 ± Ω arag 0.07 0.09 0.05 0.05 0.06 0.06

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Table 4.2. Results of 8 two-way ANOVAs for average calcification rate during the first and second half of the experiment. Four species (Acropora millepora, Pocillopora damicornis, Montipora monasteriata, Turbinaria reniformis) were compared at three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold).

F- Variable Effect df SS p-value Tukey statistic

Acropora millepora

1st Half Model 10, 35 0.1282 1.28 0.2955 Error 25 0.2512 Temp 1 0.0086 0.86 0.3625 pCO2 2 0.0047 0.23 0.7938 Colony 5 0.0752 1.50 0.2265 Temp x pCO2 2 0.0397 1.98 0.1597

2nd Half Model 10, 33 0.0187 4.78 0.0009 Error 23 0.0090 Temp 1 0.0003 0.67 0.4203 pCO2 2 0.0037 9.45 0.0010 382=60 > 607=741 5 0.0021 5.25 0.0023 3=2=4=5=1 > Colony 2=4=5=1=6 Temp x pCO2 2 0.0003 0.76 0.4769

Pocillopora damicornis

1st Half Model 10, 35 73.7080 20.04 <0.0001 Error 25 9.1967 Temp 1 0.0687 0.19 0.6694 pCO2 2 5.7149 7.77 0.0024 Colony 5 54.9847 29.89 <0.0001 4=6 > 6=5 > 2=3=1 Temp x pCO2 2 12.9417 17.59 <0.0001

2nd Half Model 10, 34 0.0684 3.82 0.0035 Error 24 0.0430

continued

182

Table 4.2 continued

Temp 1 0.0073 4.06 0.0551 pCO2 2 0.0106 2.96 0.0709 5 0.0388 4.33 0.0060 4=2=6=5 > Colony 2=6=5=1=3 Temp x pCO2 2 0.0106 2.95 0.0716

Montipora monasteriata

1st Half Model 10, 35 0.2514 3.55 0.0049 Error 25 0.1768 Temp 1 0.0348 4.91 0.0360 29.0 > 26.5 pCO2 2 0.0128 0.91 0.4166 Colony 5 0.1687 4.77 0.0034 6=4=2=1 > 4=2=1=5 > 2=1=5=3 Temp x pCO2 2 0.0351 2.48 0.1042

2nd Half Model 10, 35 0.5361 5.71 0.0002 Error 25 0.2348 Temp 1 0.0005 0.05 0.8208 pCO2 2 0.0396 2.11 0.1427 6=4 > 4=2 > Colony 5 0.4800 10.22 <0.0001 2=5=1=3 Temp x pCO2 2 0.0160 0.85 0.4379

Turbinaria reniformis

1st Half Model 10, 35 0.1838 4.51 0.0011 Error 25 0.1020 Temp 1 0.0024 0.59 0.4492 pCO2 2 0.0086 1.06 0.3618 Colony 5 0.1619 7.94 0.0001 5=3=4=1=2 > 6 Temp x pCO2 2 0.0109 1.34 0.2812

continued

183

Table 4.2 continued

2nd Half Model 10, 34 0.1828 2.89 0.0160 Error 24 0.1516 Temp 1 0.0125 1.97 0.1729 pCO2 2 0.0339 2.68 0.0887 5 0.1308 4.14 0.0075 5=3=2=1=4 > Colony 2=1=4=6

Temp x pCO2 2 0.0044 0.35 0.7094 df = degrees of freedom, SS = sum of squares of the effects

184

Table 4.3. Results of 8 two-way ANOVAs for average chlorophyll a concentrations and symbiont density. Four species (Acropora millepora, Pocillopora damicornis, Montipora monasteriata, Turbinaria reniformis) were compared at three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold).

F- Variable Effect df SS p-value Tukey statistic

Acropora millepora

Chlorophyll a Model 10, 34 0.4018 3.34 0.0075 Error 24 0.2889 Temp 1 0.0495 4.11 0.0539 382=741 > pCO2 2 0.3121 12.96 0.0002 607 Colony 5 0.0277 0.46 0.8022 Temp x pCO2 2 0.0048 0.20 0.8193

Symbiont Dens. Model 10, 34 0.3188 1.41 0.2354 Error 24 0.5431 Temp 1 0.0847 3.74 0.0650 pCO2 2 0.1844 4.07 0.0300 Colony 5 0.0319 0.28 0.9183 Temp x pCO2 2 0.0132 0.29 0.7496

Pocillopora damicornis

Chlorophyll a Model 10, 35 17.0200 10.45 <0.0001 Error 25 4.0703 Temp 1 2.7279 16.75 0.0004 pCO2 2 8.2183 25.24 <0.0001 Colony 5 2.4594 3.02 0.0288 5=4=6=2=3 > 4=6=2=3=1 Temp x pCO2 2 3.6145 11.10 0.0004

continued

185

Table 4.3 continued

Symbiont Dens. Model 10, 35 0.0911 2.71 0.0210 Error 25 0.0840 Temp 1 0.0011 0.33 0.5681 pCO2 2 0.0435 6.48 0.0054 Colony 5 0.0171 1.02 0.4275 Temp x pCO2 2 0.0293 4.36 0.0238

Montipora monasteriata

Chlorophyll a Model 10, 35 69.6441 5.47 0.0003 Error 25 31.8195 Temp 1 31.7254 24.93 <0.0001 pCO2 2 17.4752 6.86 0.0042 Colony 5 7.5874 1.19 0.3413 Temp x pCO2 2 12.8561 5.05 0.0144

Symbiont Dens. Model 10, 35 1.7103 5.15 0.0004 Error 25 0.8302 Temp 1 0.9684 29.16 <0.0001 26.5 > 29.0 382=741 > pCO2 2 0.4408 6.64 0.0049 607 Colony 5 0.2345 1.41 0.2541 Temp x pCO2 2 0.0667 1.00 0.3809

Turbinaria reniformis

Chlorophyll a Model 10, 35 42.6673 2.72 0.0207 Error 25 39.2306 Temp 1 4.3642 2.78 0.1079 pCO2 2 13.5374 4.31 0.0246 382=741 >741=607 Colony 5 16.7740 2.14 0.0939 Temp x pCO2 2 7.9917 2.55 0.0985

continued

186

Table 4.3 continued

Symbiont Dens. Model 10, 35 1.0202 2.11 0.0638 Error 25 1.2110 Temp 1 0.0008 0.02 0.8976 pCO2 2 0.1621 1.67 0.2079 Colony 5 0.3812 1.57 0.2038 Temp x pCO2 2 0.4760 4.91 0.0159 df = degrees of freedom, SS = sum of squares of the effects

187

Table 4.4. Results of 16 two-way ANOVAs for average soluble lipid, animal soluble protein, animal soluble carbohydrate concentrations, and tissue biomass. Four species (Acropora millepora, Pocillopora damicornis, Montipora monasteriata, Turbinaria reniformis) were compared at three pCO2 concentrations (382, 607, 741 μatm) and two temperature levels (26.5, 29.0°C) with colony as a random factor. Post hoc Tukey tests were used when main effects were significant. Effects were considered significant when p≤0.05 (highlighted in bold).

F- p- Variable Effect df SS Tukey statistic value

Acropora millepora

Lipid Model 10, 35 0.0229 3.54 0.0050 Error 25 0.0162 Temp 1 0.0027 4.13 0.0529 pCO2 2 0.0068 5.28 0.0123 607=741 > 741=382 Colony 5 0.0120 3.69 0.0122 6=2=5=4=3 > 2=5=4=3=1 Temp x pCO2 2 0.0015 1.12 0.3423

Protein Model 10, 35 0.1065 3.07 0.0112 Error 25 0.0869 Temp 1 0.0000 0.00 0.9471 pCO2 2 0.0499 7.18 0.0034 Colony 5 0.0110 0.63 0.6752 Temp x pCO2 2 0.0456 6.56 0.0051

Carbohydrate Model 10, 34 0.0001 4.78 0.0008 Error 24 0.0000 Temp 1 0.0000 13.49 0.0012 26.5 > 29.0 pCO2 2 0.0000 9.76 0.0008 382=741 > 607 Colony 5 0.0000 3.15 0.0251 4=2=1=5=6 > 1=5=6=3 Temp x pCO2 2 0.0000 0.00 0.9991

continued

188

Table 4.4 continued

Tissue Biomass Model 10, 35 9.0170 0.98 0.4864 48.631 Error 25 7 Temp 1 0.0319 0.02 0.8991 11.465 pCO2 2 9 2.95 0.0709 Colony 5 3.9026 0.40 0.8433 Temp x pCO2 2 3.6165 0.93 0.4079

Pocillopora damicornis

Lipid Model 10, 35 0.0591 4.21 0.0017 Error 25 0.0351 Temp 1 0.0006 0.41 0.5261 pCO2 2 0.0146 5.22 0.0128 607=741 > 741=382 Colony 5 0.0411 5.86 0.0010 1=2=3=4=5 > 3=4=5=6 Temp x pCO2 2 0.0028 0.98 0.3887

Protein Model 10, 35 0.0101 0.43 0.9203 Error 25 0.0595 Temp 1 0.0009 0.37 0.5469 pCO2 2 0.0030 0.63 0.5415 Colony 5 0.0050 0.42 0.8331 Temp x pCO2 2 0.0013 0.27 0.7633

Carbohydrate Model 10, 35 0.0000 0.49 0.8811 Error 25 0.0000 Temp 1 0.0000 0.00 0.9672 pCO2 2 0.0000 0.75 0.4824 Colony 5 0.0000 0.63 0.6817 Temp x pCO2 2 0.0000 0.13 0.8780

continued

189

Table 4.4 continued

Tissue Biomass Model 10, 35 0.0487 1.11 0.3959 Error 25 0.1102 Temp 1 0.0044 1.00 0.3257 pCO2 2 0.0081 0.92 0.4101 Colony 5 0.0281 1.27 0.3066 Temp x pCO2 2 0.0081 0.92 0.4124

Montipora monasteriata

Lipid Model 10, 35 0.0031 0.89 0.5544 Error 25 0.0087 Temp 1 0.0003 0.79 0.3816 pCO2 2 0.0011 1.57 0.2280 Colony 5 0.0017 0.96 0.4582 Temp x pCO2 2 0.0001 0.08 0.9254

Protein <0.00 Model 10, 35 0.2040 6.67 01 Error 25 0.0765 Temp 1 0.0135 4.42 0.0457 pCO2 2 0.0070 1.15 0.3322 Colony 5 0.1047 6.85 0.0004 6=4=3 > 4=3=2=1=5 Temp x pCO2 2 0.0787 12.86 0.0001

Carbohydrate Model 10, 35 0.0001 2.45 0.0337 Error 25 0.0001 Temp 1 0.0000 3.25 0.0834 pCO2 2 0.0000 0.49 0.6201 Colony 5 0.0001 2.86 0.0354 6=4=5=3=2 > 5=3=2=1 Temp x pCO2 2 0.0000 2.98 0.0690

continued

190

Table 4.4 continued

Tissue 48.089 Biomass Model 10, 31 5 2.24 0.0572 45.039 Error 21 4 Temp 1 0.1734 0.08 0.7789 pCO2 2 9.9874 2.33 0.1221 Colony 5 37.684 3.51 0.0183 Temp x pCO2 2 8.5097 1.98 0.1625

Turbinaria reniformis

Lipid Model 10, 35 0.0034 0.88 0.5612 Error 25 0.0096 Temp 1 0.0003 0.75 0.3950 pCO2 2 0.0004 0.48 0.6220 Colony 5 0.0026 1.36 0.2715 Temp x pCO2 2 0.0001 0.15 0.8642

Protein Model 10, 34 0.0520 2.22 0.0537 Error 24 0.0564 Temp 1 0.0036 1.54 0.2262 pCO2 2 0.0167 3.56 0.0443 Colony 5 0.0190 1.61 0.1943 Temp x pCO2 2 0.0100 2.10 0.1444

Carbohydrate Model 10, 35 0.0001 0.86 0.5830 Error 25 0.0002 Temp 1 0.0000 0.66 0.4233 pCO2 2 0.0000 0.21 0.8137 Colony 5 0.0000 0.93 0.4806 Temp x pCO2 2 0.0000 1.43 0.2592

continued

191

Table 4.4 continued

Tissue Biomass Model 10, 35 101.41 2.41 0.0362 Error 25 105.14 Temp 1 1.0452 0.25 0.6225 pCO2 2 4.7408 0.56 0.5762 Colony 5 72.994 3.47 0.0161 3=4=2=1 > 4=2=1=5=6 Temp x pCO2 2 22.628 2.69 0.0875 df = degrees of freedom, SS = sum of squares of the effects

192

4.8 FIGURES

193

A. millepora P. damicornis M. monasteriata T. reniformis (a) (c) (e) (g) 0.10 0.25 26.5°C 0.5 * * 0.5 0.08 * 0.20 29.0°C 0.4 0.4 0.06 0.15 0.3 0.3 0.04 ± 1 SE) ± 1 0.10 0.2 0.2

First half First -2 0.02 0.05 0.1 0.1

-1 (d) (h) 0.10 (b) a ab b 0.25 0.5 (f) 0.5 0.08 0.20 * 0.4 0.4 Calcification rate Calcification 0.06 0.15 0.3 0.3

(mg day (mg 0.04 0.10 0.2 0.2

Second half Second 0.02 0.05 0.1 0.1

382 607 741 382 607 741 382 607 741 382 607 741 Seawater pCO (atm) 2

Figure 4.2. Average daily calcification rate during the first and the second half of the experiment for (a, b) Acropora millepora, (c, d) Pocillopora damicornis, (e, f) Montipora monasteriata, and (g, h) Turbinaria reniformis. Averages ± 1 SE are shown for three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate significant differences between 26.5 and 29.0°C within a given pCO2 level (determined by a posteriori slice tests). The letters a and b indicate results of the post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 5 and 6. Statistical details can be found in Table 4.2.

194

A. millepora P. damicornis M. monasteriata T. reniformis 12 (c) (e) (g)

a (a) 10 * * 8 ± 1 SE) ± a b ab -2 6 a b a * * 4

g cm

Chlorophyll Chlorophyll

 2

(

6 (d) (f) (h) 1.5 (b) 26.5°C a b a 1.2 29.0°C * * *

± 1 SE) ±

-2 0.9 * 0.6 0.3 *

(cells cm (cells

Symb. density x10 density Symb. 382 607 741 382 607 741 382 607 741 382 607 741 Seawater pCO (atm) 2

Figure 4.3. Average chlorophyll a concentrations and symbiont density for (a, b) Acropora millepora, (c, d) Pocillopora damicornis, (e, f) Montipora monasteriata, and (g, h) Turbinaria reniformis. Averages ± 1 SE are shown for three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate significant differences between 26.5 and 29.0°C within a specific pCO2 level (determined by a posteriori slice tests). The letters a and b indicate results of the post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 5 and 6. Statistical details can be found in Table 4.3.

195

A. millepora P. damicornis M. monasteriata T. reniformis (e) 0.4 (a) (i) (m) 26.5°C b a ab b a ab 29.0°C ± 1 SE) 0.3

-1 Lipid 0.2 * 0.1

(g gdw (f) (n) 0.8 (b) (j) 0.6 * * * *

± 1 SE) -1 0.4

Protein 0.2

(g gdw (c) (g) (k) (o) 0.020 * 0.015 a b a

± 1 SE)

-1 0.010 0.005

Carbohydrate

(g gdw 15 (d) (h) (l) (p) 12 * 9

± 1 SE)

-2 6 3

Tissue biomass Tissue (mg cm (mg 382 607 741 382 607 741 382 607 741 382 607 741

Seawater pCO2 (atm)

Figure 4.4. Average lipid, protein, carbohydrate concentrations, and tissue biomass of (a- d) Acropora millepora, (e-h) Pocillopora damicornis, (i-l) Montipora monasteriata, and (m-p) Turbinaria reniformis. Averages ± 1 SE are shown for three pCO2 levels and two temperature regimes (26.5, 29.0°C). Asterisks indicate significant differences between 26.5 and 29.0°C within a specific pCO2 level (determined by a posteriori slice tests). The letters a and b indicate results of the post hoc Tukey tests when there was a significant pCO2 effect. Sample sizes ranged between 4 and 6. Statistical details can be found in Table 4.4.

196

5. SUMMARY AND FUTURE RESEARCH

5.1 SUMMARY

Coral reefs are in serious decline worldwide due to a combination of natural and anthropogenic stressors. Climate change in particular threatens their existence beyond the 21st century by causing ocean acidification, ocean warming, and mass bleaching events. Ocean acidification can compromise coral calcification and reef accretion, whereas bleaching due to high seawater temperatures results in the breakdown of the symbiosis between corals and their algal endosymbionts. This significantly threatens their survival in a future of climate change. As atmospheric CO2 concentrations keep rising, mass bleaching events are expected to increase in frequency and intensity, while at the same time oceans continue to acidify. However, the impact of repeated coral bleaching as well as the effects of combined ocean acidification and warming on coral physiology and biogeochemistry remain largely unknown. Therefore, the goals of this dissertation research were as follows:

1. Assess the impact of annually repeated coral bleaching on the physiology and biogeochemistry of three Caribbean coral species. 2. Re-evaluate metabolic and kinetic isotope effects in coral paleo-climate proxies using coral bleaching as a case study. 3. Assess the effects of combined ocean acidification and warming on the physiology of four Pacific coral species.

197

We conducted two controlled, replicated tank experiments to simulate annually recurring coral bleaching on ecologically relevant timescales as well as combined ocean acidification and warming as projected for the middle and end of this century. The three main findings of this dissertation research are summarized below.

Annually repeated coral bleaching has the potential to dramatically alter thermal tolerance in corals, and can both increase and lower bleaching resistance to future temperature stress. For example, branching Porites divaricata was moderately resistant to single bleaching, but largely unaffected by repeat bleaching the following year. Its rapid acclimation was likely due to phenotypic plasticity in both animal host and algal endosymbiont, and involved high levels of energy reserves combined with increases in thermally tolerant endosymbiont type A4. In contrast, mounding Porites astreoides was highly resistant to single bleaching, yet suffered dramatic declines in many aspects of its physiology after repeat bleaching. This increased bleaching susceptibility was likely caused by a loss of heterotrophic plasticity combined with a lack of flexibility to associate with different endosymbiont types and low total energy reserve concentrations. Finally, key reef-builder Orbicella faveolata was affected similarly by single and repeat bleaching despite significant changes in its endosymbiont community. Increases in endosymbiont type D1a resulted in improved performance of the algal endosymbiont, but appears to incur significant short term costs to the animal host. While both O. faveolata and P. divaricata were able to fully recover from repeat bleaching within one year, energy reserves and calcification of P. astreoides were still impacted after 11 months of recovery. This suggests that while some coral species will be able to acclimate to annual bleaching events, others may face significant decline.

Kinetic isotope effects can mask bleaching signals (i.e., metabolic isotope effects) in coral skeletons, but the alteration of the isotopic signature is not sufficiently systematic to allow for a simple data correction. Correlations between 13 13 13 skeletal stable carbon isotopes (δ Cs) and the δ C the animal host (δ Ch) did not

198 improve when the kinetic isotope effect correction (Heikoop et al. 2000) was applied. Further, by comparing the corrected data to measured changes in physiology, we were able to determine that the data correction did not reveal masked metabolic isotope effects. As a consequence, photosynthesis to respiration (P/R) ratios calculated from skeletal and tissue isotopes are in poor agreement with P/R ratios measured by respirometry, and typically over- or underestimated P/R. For these reasons, widespread application of this data correction in paleo-climate reconstruction cannot be recommended, and it is unlikely that coral skeletons can be used as recorders of past bleaching events.

Some coral species will be more resistant to combined ocean acidification and warming than previously expected. Three out of four Pacific coral species (Pocillopora damicornis, Montipora monasteriata, and Turbinaria reniformis) were able to maintain calcification rates under ocean acidification levels expected by the end of this century (741 μatm), even when combined with elevated seawater temperature (+2.5°C). Only Acropora millepora calcified 53% less in acidified seawater, and elevated temperature neither improved nor worsened this. Further, energy reserves (i.e., lipid, protein, and carbohydrate) were generally maintained in all four species, suggesting that they are not catabolized to provide the additional energy required to calcify under high CO2- conditions. Overall, these findings suggest that some coral species will be able to persist and potentially thrive on future coral reefs for the coming decades. However, significant shifts in coral community composition and diversity are likely as the major reef building coral in the Pacific (Acropora sp.) appears to be highly susceptible to both ocean acidification and coral bleaching conditions expected towards the end of the century and beyond.

Overall, the findings from this dissertation research highlight that the majority of coral species will be able to survive near-future climate change. Moderate levels of ocean acidification as well as bleaching events that allow the majority of corals to recover before the next bleaching event will enable many coral species to survive over the

199 coming decades. Nevertheless, the combination of these stressors will likely erode their resistance to other disturbances such as diseases, overfishing, storms, and eutrophication, and prolong recovery from stress events. However, as ocean acidity increases and coral bleaching becomes more and more frequent, temperature- and CO2-sensitive species will likely face significant demise and potentially local extinction. While some corals seem to be able to acclimate to simultaneously warming and acidifying oceans, they may not be able to maintain the high calcification rates required to build reefs in excess of erosion and dissolution. Further, they may not be able to provide the high structural complexity seen on pristine reefs today. Since this is expected to have dramatic consequences for coral reef diversity and overall reef functioning, the future of coral reefs beyond the 21st century may be uncertain.

5.2 FUTURE RESEARCH

The findings presented in this dissertation lead to the following questions and lines of future research:

1. We showed for the first time that Caribbean coral species have the potential to acclimate to increasing seawater temperature, which is encouraging in the light of

rising atmospheric CO2 concentrations. However, it has to be considered that the repeat bleaching experiment simulated mild bleaching events (i.e., +1°C for 2.5 weeks) that cannot be compared to severe mass bleaching events affecting coral reefs worldwide. As a consequence, both the observed acclimation potential as well as the increased bleaching susceptibility are conservative estimates. As mass bleaching events are expected to occur annually sometime later this century (Frieler et al. 2012), further studies are needed to determine how recurring severe bleaching events (e.g., +3°C for 4 weeks) will affect thermal tolerance, acclimation, and long term recovery.

200

2. The repeat bleaching experiment simulated mild bleaching events occurring in two consecutive years, and we showed that two out of the three species studied were able to fully recover within one year after repeat bleaching. However, later this century coral bleaching is likely to occur annually over much longer timescales than two years, i.e. corals will experience bleaching every year for many years to come. This significantly challenges their capacity to recover between single bleaching events and their overall resilience, and needs to be addressed in modeling studies and/or long term annually repeating bleaching experiments spanning multiple years.

3. As atmospheric CO2 concentrations keep increasing, corals will not only experience more frequent and intense bleaching events, but also live in increasingly more acidic seawater. While my dissertation research has shown that some corals will be able to persist despite repeat bleaching and ocean acidification, the question is how corals will cope with more frequent bleaching events when simultaneously living in acidified seawater. Further studies are needed to determine how different seawater

pCO2 concentrations affect bleaching tolerance and recovery capacity, and to assess

thresholds and tipping points for both seawater temperature and pCO2 levels.

4. Four Pacific coral species were able to maintain coral energy reserves under combined ocean acidification and warming, independent of whether they maintained calcification rates or not. This indicates that coral energy reserves are not catabolized to maintain calcification under ocean acidification, either because this type of stored energy cannot be allocated towards skeletal growth and/or because no or only little additional energy is required to calcify under ocean acidification conditions. Since the actual energetic costs of coral calcification remain largely unknown even under current environmental conditions, this represents a major knowledge gap especially in the context of ocean acidification research.

201

5. Due to logistical constraints, most ocean acidification experiments - including the one performed for this dissertation - are conducted over relatively short time scales that typically range from several days to weeks. This significantly limits our

understanding of how corals will be able to acclimate to elevated seawater pCO2 levels over long time scales. For example, we showed that after 11 days of exposure to acidified seawater, Acropora millepora was able to maintain calcification rates unless temperature was elevated as well. However, after 23 days of exposure, calcification rates decreased by 53% independent of temperature. This indicates that the duration of exposure to ocean acidification influences the observed physiological response. Further studies are needed to understand if corals will be able to acclimate to ocean acidification when given enough time, and on what time scales and rates of ocean acidification acclimation can or cannot occur.

202

LIST OF REFERENCES

Albright R, Langdon C (2011) Ocean acidification impacts multiple early life history processes of the Caribbean coral Porites astreoides. Global Change Biology 17:2478-2487

Albright R, Mason B, Langdon C (2008) Effect of aragonite saturation state on settlement and post-settlement growth of Porites astreoides larvae. Coral Reefs 27:485-490

Allemand D, Tambutte E, Zoccola D, Tambutte S (2011) Coral calcification, cells to reefs. In: Dubinsky Z, Stambler N (eds) Coral Reefs: An Ecosystem in Transition. Springer, pp 119-150

Allison N, Finch AA, Facility EIM (2012) A high resolution 13C record in a modern Porites lobata coral: Insights into controls on skeletal 13C. Geochimica and Cosmochimica Acta 2012:534-542

Allison N, Tudhope AW, Fallick AE (1996) Factors influencing the stable carbon and oxygen isotopic composition of Porites lutea coral skeletons from Phuket, South Thailand. Coral Reefs 15:43-57

Andersson AJ, Gledhill DK (2013) Ocean acidification and coral reefs: Effects on breakdown, dissolution, and net calcification. Annual Review of Marine Science 5:1.1-1.28

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