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墾丁地區密集迷紋珊瑚(Leptoria phrygia)和鐘形微孔珊瑚(Porites lutea)對不同溫度環境的生理差異及馴化過程

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(1)國立臺灣師範大學理學院生命科學系 中央研究院生物多樣性國際研究生博士學位學程 博士論文 Department of Life Science, College of Science Taiwan International Graduate Program on Biodiversity, Academia Sinica. National Taiwan Normal University Doctoral Dissertation. 墾丁地區密集迷紋珊瑚(Leptoria phrygia)和鐘形微孔珊瑚(Porites lutea) 對不同溫度環境的生理差異及馴化過程 Physiological differences and acclimatisation processes in two scleractinian corals: Leptoria phrygia and Porites lutea, from two sites with contrasting temperature regimes in Kenting National Park, southern Taiwan. 高洛德 Rodrigo Enrique Carballo Bolaños 指導教授:陳昭倫 博士 Advisor: Chen, Chaolun Allen, Ph.D.. 中華民國 109 年 02 月 February, 2020.

(2) Acknowledgements I would like to thank my supervisor Dr. Allen Chen for accepting me in his lab and for all his support during my thesis. I am also grateful to my advisory committee and my defense committee for all their comments that help to improve this thesis. Many thanks to all the members of the Coral Reef Evolutionary Ecology and Genetics Laboratory for all their help, specially during my field work (Weber, Sophia, Steve, Lauriane, Christine, Sophie, MingJay, Stephane, YaYi) and for their advice and support (Derek, Juliet, Garmin, ChaiHsia, YenWen, Shanky, WenHua, Aichi, SungYin, CY). Special thanks to Dr. Vianney Denis from NTU for lending me equipment to analyse my samples, for all the productive talks we had and his statistical expertise. Also to the Stable Isotope lab in NTU for helping me with my samples (Dr. Nina Zhang and LingWen). I also would like to thank Dr. Meng in the National Museum of Marine Biology and Aquarium for providing space in his wet laboratory to keep the corals and the Kenting National Park for issuing collection permits. I am grateful to TIGP-Biodiversity program (Academia Sinica and National Taiwan Normal University) for the funding of my PhD, all professors within the program and the program assistants (Jane, Sage and Vanessa). I want to thank all my friends in Taiwan, my fellow classmates from the TIGP-Biodiv program, my friends from the NTNU gymnastics team, and all my friends in El Salvador and around the world for their support and advice during all this years. Last but not least this PhD would not have been possible without the constant support of my family, my parents, my sister and cousins, who kept encouraging me even through the distance. 謝謝!. i.

(3) Abstract: The frequency and severity of global bleaching events are increasing, due to the rise of anthropogenic CO2 in the atmosphere. As our oceans keep warming up, understanding the mechanisms driving thermal tolerance in reefbuilding corals is of outstanding importance. In Kenting National Park, southern Taiwan, there is a ‘Variable Site’ (VS) that is influenced by the constant hot-water effluent from a nuclear power plant outlet and temperatures during the summer can be 3 °C higher that at any other site within Kenting. It is also influenced by a monthly upwelling that makes seawater temperature to fluctuate up to 8 °C in one day. In this dissertation, I collected samples of Leptoria phrygia from the VS and from a ‘Stable Site’ (SS) that is not affected by high temperature or high temperature fluctuations within Kenting and compared physiological parameters to elucidate the mechanisms this species has to survive in the VS (Chapters II and III). Results suggest that L. phrygia is a species that presents multi-symbiont association and inter-colony variation in SS: most colonies associated with Cladocopium spp. (stress-sensitive), some colonies had co-dominance between Durusdinium glynnii and Cladocopium spp., and very few associated only with D. glynnii (stress-resistant). Meanwhile in the VS, L. phrygia associated all year long with D. glynnii (>90% dominance). I found out that only those colonies with a co-dominance exhibited temporal variation, and I hypothesize that those co-dominant colonies might be able to survive future scenarios of climate change by modifying the relative abundance of both symbionts. If the environment becomes stressful, it becomes Durusdiniumdominant similar to the current situation in the VS. Furthermore, I performed a reciprocal transplantation experiment with L. phrygia and Porites lutea between both sites, to understand the acclimatisation processes to thermal stress (in the summer) and to high temperature variation throughout the year (Chapter IV). Results indicate that both species have different mechanisms to resist stress and to acclimatise to their new environment. Leptoria phrygia is dependent on the different Symbiodiniaceae association and is able to acclimatise faster than P. lutea,. ii.

(4) but only if it presents co-dominance of Cladocopium spp. and Durusdinium spp. within the colony. If they are >90% Cladocopium-dominant, then they cannot survive high temperatures in the summer in VS. In contrast, P. lutea acclimatise to the new environment slower and modified both partners physiology to confront changes in the environment. The results of this dissertation increase our knowledge on coral physiology and specifically on the differences between species. Even though both species are able to acclimatise to rapid changes in climate using different mechanisms, it is imperative to change completely our societal dependence on fossil fuels, in order to address the root causes of climate change.. Keywords: Leptoria phrygia, Acclimatisation, High temperature variability, Kenting, Porites lutea, Durusdinium, Cladocopium.. iii.

(5) Table of Contents List of Tables……………………………………………………….…..ix List of Figures…………………………………………………………...x CHAPTER 1: General introduction………….………………….….1 Background………………………………………………………….….1 Mechanisms to survive thermal stress………………………….……3 1. Thermally tolerant endosymbionts…………………….…………3 2. Acclimatisation (phenotypic plasticity)……………………..…….6 2.1 Thermal stress acclimatisation………………….…….7 2.2 Acclimatisation to high temperature variability..............9 3. Adaptation……………………...………………………………12 4. Heterotrophy (Mixotrophy)………………………………………15. Research overview……………………………………………………16 CHAPTER 2: Temporal variation and photochemical efficiency of species in Symbiodiniaceae associated with coral Leptoria phrygia (Scleractinia; Merulinidae) exposed to contrasting temperature regimes……..…………………………………………20 Abstract……………………………………………………...…………20 Introduction…………………………………………………………….22 Materials and methods……………………………………………….27 Study sites and temperature data………………………………….27. iv.

(6) Sample collection, photochemical efficiency and preservation……..29 Laboratory analyses………………………………...…………….30 Surface area……….……………………………………………..30 Symbiont density……………….…………………………………31 Chlorophyll a concentration………………………………………..31 Molecular analysis……….………………………………………..32 DNA extraction……………………………………………..32 Quantitative PCR (qPCR)…………..………………………32 Symbiont identification – (DGGE)…………………………..33 Statistical analysis…………….…………………………………..34. Results…………………………………………………………………35 Seawater temperature differences between sites………………….35 Symbiodiniaceae association……………..………………………36 Correlation between Durusdinium spp. and temperature…………..39 Physiological parameters……………..…………………………..40. Discussion……………………………………………………………..41 Supplemental Information……………………………………………49 CHAPTER 3: Differences in δ13C and δ15N isotopic niches of Leptoria phrygia associated with Cladocopium spp. or Durusdinium spp. symbionts……………………………………..51 Abstract………………………………………………………………..51 Introduction……………………………………………………………52. v.

(7) Materials and methods…………………………………………….…56 Study sites and coral sample collection………..…………….…….56 Coral sample preparation……………..…………………….…….57 Stable isotope analysis…………………………….………….…..59 Molecular analysis: DNA extraction and qPCR…………………….60 Statistical analysis………………………………….……………..60. Results…………………………………………………………………62 Temperature differences between sites………………...………….62 Strength of the symbiotic relationship within C-dominant and Ddominant corals…………..…………………………..…………..62 Differences in isotopic niche, δ13C and δ15N between C- dominant and D-dominant corals……………………………………..………….63 Temporal variation in isotopic niche………………..…..…….……68 Correlation between δ13C/ δ15N and the percentage of Durusdinium spp………………………………..………………………..…….70. Discussion……………………………………………………………..71 Supplemental information……………………………………………75 CHAPTER 4: Acclimatisation processes in Leptoria phrygia and Porites lutea when transplanted to a habitat with different seawater temperature variability in southern Taiwan…...……78 Abstract…………………………………………………………...……78 Introduction…………………………………………………………….79. vi.

(8) Materials and methods…………………………………….…………84 Study sites and temperature data……..………..…………………84 Experimental design……………………………………………....85 Photochemical efficiency………………………………………….87 Laboratory analyses……………………………………………....87 Surface area……….………………………………………87 Symbiont density……………….…………………………..88 Chlorophyll a concentration…………………………………88 Lipid concentration………………………………...……….89 Protein concentration……………………………………….89 Molecular analysis……….………………………………………..90 DNA extraction and qPCR………………………………….90 Statistical analysis………………………….……………………..90. Results…………………………………………………………………92 Seawater temperature differences between sites………………….92 QPCR……………………….……………………………………94 Mortality………………………………………………….……….94 Photochemical efficiency………………………………………….95 Symbiont density………………………..………………………..97 Chlorophyll a concentration……………….………………………99 Lipid concentration (algae and coral host fractions)………………100 Protein concentration (coral host fraction)……………………..…103. vii.

(9) Discussion……………………………………………………………105 Supplemental information…………………………………………..110 CHAPTER 5: Conclusions………………………………….…….111 References………………………………………………..………….117. viii.

(10) List of Tables: CHAPTER 1…………………………………….………………………1 Table 1. Summary of the different mechanisms of resistance to thermal stress…………………………………………………………..6 Table 2. Studies performed to test acclimatisation to thermal stress and high temperature variability at different locations around the world……………………………………………………………………10 CHAPTER 2…………………………………………………………...20 Table 1. Studies reporting co-dominance of different symbiont genera within a single colony………………………………………..26 Table 2. Dominant symbiont identified with DGGE and subsequent sequencing at both sites……………………………………………..37 S1 Table. Seawater temperature information at both sites from June 2016 to June 2017…………………….…...…………………..49 S2 Table. Percentage of Durusdinium spp. at both sites…………50 CHAPTER 3………………………………………………………...…51 Table 1. δ13C and δ15N mean values ± s.d. (‰) of algae and coral fraction by symbiont association: either C-dominant or D-dominant at each sampling time………………………………………………..68 CHAPTER 4…………………………………………………………..78 Table 1. Seawater temperature information at both sites from March 2015 to March 2017………………………………………….92 Table 2. Relative fitness calculated for all physiological parameters measured at both sites in both species after 12 and 18 months, including the significance of the site-origin interaction from the linear mixed model……………………………………………………96. ix.

(11) List of Figures: CHAPTER 1…………………………………………………………….1 Figure 1. A. Onset of a bleaching process in a colony of Acropora spp., in Kenting, Taiwan 2015; B. Bleaching event showing colonies with fluorescent pigments as a protective mechanism (a) and already bleached colonies (b), in Okinawa, Japan 2016; C. Intra‐specific: between Montipora spp. colonies (a) and between Isopora palifera colonies (b), inter‐specific: between Montipora spp. and I. palifera colonies (c) and intra‐colony: within Leptoria phrygia colony (d) responses to thermal stress in Kenting, Taiwan 2016…..………………………………………………………………….5 CHAPTER 2…………………………………………………………...20 Figure 1. Study sites in Nanway Bay, southern Taiwan………….29 Figure 2. Seawater daily temperatures recorded for June 2016 to July 2017 at both sites………………………………………………..36 Figure 3. Pie charts showing symbiont associations between Cladocopium spp. and/or Durusdinium spp……….……………….38 Figure 4. Denaturing gradient gel electrophoresis (DGGE) from selected colonies at each site……………………………………….39 Figure 5. Correlation between mean maximum temperature and the percentage of Durusdinium spp. (A) and between delta temperature (mean monthly maximum – mean monthly minimum) and the percentage of Durusdinium spp. (B)..……………………..40 Figure 6. Physiological parameters measured photochemical efficiency (Fv/Fm) (A), symbiont density (million cells cm-2) (B) and chlorophyll a concentration (pg per symbiont cells) (C)………….43 CHAPTER 3………………………………………………………...…51 Figure 1. Study sites in Kenting National Park, southern Taiwan (A). Temperature variability at both sites during each sampling time (B)……..…………………………………………………………58. x.

(12) Figure 2. δ15N vs. δ13C (‰) bi-plots displaying the isotopic niche for C-dominant corals at each site during August 2016 (A), December 2016 (B) and March 2017 (C)………………………….64 Figure 3. δ15N vs. δ13C (‰) bi-plots displaying the isotopic niche for D-dominant corals at each site during August 2016 (A), December 2016 (B) and March 2017 (C)………………………….65 Figure 4. Density plots showing the confidence intervals of the Bayesian Standard Ellipse Areas in August 2016 (A), December 2016 (B) and March 2017 (C)……………………………………….66 Figure 5. δ15N vs. δ13C (‰) bi-plots of standardized isotopic values (Δδ13Chost-symbiont and Δδ15Nhost-symbiont) displaying the isotopic niche according to the symbiont association at each site during August 2016 (A), December 2016 (B) and March 2017 (C)………………69 SI Figure 1. δ13C (‰; top) and δ15N (‰; bottom) for algae fraction (A and C) and coral fraction (B and D) during each sampling time grouped by symbiont association…………………………………..75 SI Figure 2. Correlation between δ13C and the percentage of Durusdinium sp. in algae fraction (A) and coral fraction (B)……..76 SI Figure 3. Correlation between δ15N and the percentage of Durusdinium sp. in algae fraction (A) and coral fraction (B)……..77 CHAPTER 4…………………………………………………………..78 Figure 1. Study sites in Nanway, southern Taiwan….…………...85 Figure 2. Daily seawater temperatures recorded for March 2015 to March 2017 at both sites……………………………………………..93 Figure 3. Pie charts showing symbiont associations between Cladocopium spp. and/or Durusdinium spp………………………..95 Figure 4. Mean photochemical efficiency (Fv/Fm) per group measured in L. phrygia (A) and P. lutea (B) at each sampling time……………………………………………………………………..98 Figure 5. Symbiont density (million cells cm-2) per group measured in L. phrygia (A) and P. lutea (B) at each sampling time………..100. xi.

(13) Figure 6. Chlorophyll a concentration (pg/symbiont cells) per group measured in L. phrygia (A) and P. lutea (B) at each sampling time……………………………………………………………………101 Figure 7. Lipid concentration (mg cm-2) in algae fraction (A) and coral fraction (B) per group measured in L. phrygia at each sampling time………………………………………………………..103 Figure 8. Lipid concentration (mg cm-2) in algae fraction (A), coral fraction (B) and both fractions (C) per group measured in P. lutea at each sampling time………………………………………………104 Figure 9. Protein concentration (mg cm-2) per group measured in L. phrygia (A) and P. lutea (B) at each sampling time……………..105 SI Fig 1. Experimental design of the reciprocal transplantation experiment (RTE), showing two racks as control at each site (VS control and SS control) and two reciprocally transplanted racks (from VS to SS: SS transplant; from SS to VS: VS transplant). Each rack has 5 columns and each column represents one colony…………………………………………………………………110. xii.

(14) Chapter I: General Introduction. CHAPTER I: General Introduction Background Since the last century, scleractinian coral reef ecosystems have undergone a decrease in biodiversity and ecological functioning (Wilkinson 2000, Gardner et al. 2003, Pandolfi et al. 2003, Bruno and Selig 2007, De’ath et al. 2012), formerly attributed to the direct and indirect effects of overfishing (Jackson et al. 2001, Valentine and Heck 2005), pollution from agriculture, sewage runoff, and land development (Dubinsky and Stambler 1996, McCulloch et al. 2003, Fabricius 2005). Currently, along with the exponential increase of the human population (Cohen 1995) and our societal dependence on carbon fossil fuels, these local threats have been compounded by the impacts of global climate change in the oceans (Hughes et al. 2003, HoeghGuldberg et al. 2007, D’Angelo and Wiedenmann 2014). The impact of increasing greenhouse gases in the atmosphere is leading to a global increase in seawater temperatures that has caused mass bleaching events (Hoegh-Guldberg 1999, Fitt et al. 2001, Coles and Brown 2003, Hughes et al. 2003, Hoegh-Guldberg et al. 2007). These global bleaching events are becoming more frequent (1998, 2010 and 2014–17) and severe (HoeghGuldberg 1999, Hoegh-Guldberg et al. 2007, Heron et al. 2016, Hughes et al. 2017a, Hughes et al. 2017b, Eakin et al. 2019, Skirving et al. 2019), leaving coral reefs vulnerable and unable to recover. The 2014–2017 mass bleaching event, which lasted 36 months and spanned four calendar years, was the longest-lasting, most widespread, and probably most damaging event on record (Ku‘ulei et al. 2017, Frade et al. 2018, Burt et al. 2019, Eakin et al.. 1.

(15) Chapter I: General Introduction 2019, Harrison et al. 2019, Head et al. 2019, Raymundo et al. 2019, Skirving et al. 2019, Vargas-Ángel et al. 2019), and stands out as unique by spanning all phases of the El Niño-Southern Oscillation cycle of 2017, being the warmest non-El Niño year ever recorded (Hartfield et al. 2018, Eakin et al. 2019). Therefore, it is of great importance to understand the mechanisms corals have to survive thermal stress and to study which species will be able to survive these bleaching events. Coral bleaching is defined as the loss of colour, due to the partial or total loss of Symbiodiniaceae dinoflagellates and/or the reduction of their photosynthetic pigments, that exposes the white calcium carbonate of the coral skeleton (Figure 1A) (Glynn 1993, Douglas 2003). Bleaching is a generalized stress response to environmental perturbations such as aerial exposure, sedimentation, eutrophication, exposure to heavy metals, high UV radiation, and extreme changes in salinity and temperature (Glynn 1993, Brown 1997b, Coles and Brown 2003, Obura 2009), however, at large scales is triggered by high seawater temperatures (exceeding normal summer maxima) in combination with high solar radiation (Glynn 1993, Brown 1997b, Fitt et al. 2001, Coles and Brown 2003, Douglas 2003, Hughes et al. 2003, Jokiel 2004). Scleractinian corals possess molecular protective mechanisms, such as heat shock proteins and antioxidant enzymes to resist thermal stress (Brown 1997b, Coles and Brown 2003, Lesser 2006), or mycosporine amino acids (MAA) and fluorescent pigments to resist light stress (Figure 1B) (Brown 1997b, Salih et al. 2000, Shick and Dunlap 2002, Coles and Brown 2003). The cellular mechanism of bleaching starts with the photoinhibition process within the photosynthetic apparatus of the endosymbionts, which results in the. 2.

(16) Chapter I: General Introduction build-up of free electrons that react to form reactive oxygen species (ROS) (Tchernov et al. 2004, Weis 2008). The proliferation of harmful ROS leads to the degradation, exocytosis, or apoptosis of symbiont cells by the coral host (Weis 2008), in order to avoid cellular damage (Lesser 2006). If the duration of the thermal stress extends beyond their physiological ability to recover, corals cannot survive without their main symbiotic partners (Glynn 1993, Fitt et al. 2001, Jokiel 2004). Even though the molecular process of bleaching is similar across coral species, variations in the mechanism to resist and survive thermal stress exist among different coral species (Figure 1B,C) (Coles and Brown 2003, Douglas 2003, Jokiel 2004).. Mechanisms to survive thermal stress 1. Thermally tolerant endosymbionts By associating with stress-resistant symbionts some coral species are able to acquire increased thermal tolerance (Table 1). Within the Symbiodiniaceae, species like Durusdinium spp. (previously clade D) (Rowan 2004, Berkelmans and van Oppen 2006, Stat and Gates 2011), Cladocopium C15 (Levas et al. 2013) and C. thermophilum C3 (Hume et al. 2015, Hume et al. 2016) are resistant to thermal stress. Dinoflagellates in the genus Durusdinium are extremophiles inhabiting stressful environments of thermal stress, high temperature fluctuations, sedimentation and high-latitudinal marginal reefs (Chen et al. 2003, Chen et al. 2005, Lien et al. 2007, LaJeunesse et al. 2010a, Hsu et al. 2012, Keshavmurthy et al. 2012 , Keshavmurthy et al. 2014, Wham et al. 2017, Kao et al. 2018, LaJeunesse et. 3.

(17) Chapter I: General Introduction al. 2018, Carballo-Bolaños et al. 2019). In recent decades, Durusdinium spp. have generated a lot of interest because it proliferates in bleached. 4.

(18) Chapter I: General Introduction Figure 1. A. Onset of a bleaching process in a colony of Acropora spp., in Kenting, Taiwan 2015 (Photo: J. Wei); B. Bleaching event showing colonies with fluorescent pigments as a protective mechanism (a) and already bleached colonies (b), in Okinawa, Japan 2016 (Photo: S.-Y. Yang); C. Intra‐specific: between Montipora spp. colonies (a) and between Isopora palifera colonies (b), inter‐specific: between Montipora spp. and I. palifera colonies (c) and intra‐colony: within Leptoria phrygia colony (d) responses to thermal stress in Kenting, Taiwan 2016 (Photo: R. Carballo-Bolaños). corals (Jones et al. 2008, Lajeunesse et al. 2009, Hsu et al. 2012, Silverstein et al. 2015, Kao et al. 2018), protecting against thermal stress by providing 1 to 1.5 °C of thermal tolerance (Berkelmans and van Oppen 2006). Durusdinium spp. maintain high photochemical efficiency when exposed to high temperatures compared to symbionts from other genera (Breviolum or Cladocopium) (Rowan 2004, Oliver and Palumbi 2011a, Cunning et al. 2017) and are able to fix more carbon and assimilate more nitrogen (Baker et al. 2013b). Furthermore, D. trenchii has been found to provide tolerance to cold stress too (LaJeunesse et al. 2010b, Silverstein et al. 2017). Some species of corals are capable of shifting the relative abundance of their dominant symbionts when exposed to thermal stress; background symbionts, which can represent <10% of the overall Symbiodiniaceae community (Mieog et al. 2007, Kao et al. 2018), become dominant, conferring thermal tolerance to the holobiont. Even though many coral species are able to associate with a heterogeneous community of Symbiodiniaceae (Baker 2003, Silverstein et al. 2012), others do not change their dominant symbiont even when bleaching (Goulet 2006), showing a long-term symbiotic adaptation between coral host and its dominant symbiont (Thornhill et al. 2006a, Thornhill et al. 2006b, Stat et al. 2009, Thornhill et al. 2009).. 5.

(19) Chapter I: General Introduction Table 1. Summary of the different mechanisms of resistance to thermal stress.. Mechanism. Acclimatisation. Association with thermally tolerant endosymbionts Thermal stress High temperature variability Adaptation Heterotrophy (Mixotrophy). Description. References. Increased thermal tolerance by association with stress resistant symbionts, e.g.: Durusdinium spp. After experiencing thermal stress, corals show a ‘thermal memory’ and react better to subsequent thermal stress Corals living in places with high temperature variability show better respond to thermal stress Changes in the genetic composition transfer to the next generation through natural selection. (Chen et al. 2003, Rowan 2004, Chen et al. 2005, Berkelmans and van Oppen 2006, Hsu et al. 2012, Keshavmurthy et al. 2014, Cunning et al. 2015, Silverstein et al. 2015, Wham et al. 2017, Kao et al. 2018, Carballo-Bolaños et al. 2019). Corals ingest all nutrients that cannot receive from the symbionts. (Maynard et al. 2008, Middlebrook et al. 2008, Bellantuono et al. 2012, Guest et al. 2012, Brown et al. 2015, Putnam and Gates 2015, Fisch et al. 2019) (Thompson and van Woesik 2009, Oliver and Palumbi 2011a, Mayfield et al. 2013, Palumbi et al. 2014, Safaie et al. 2018). (Van Oppen et al. 2011, Howells et al. 2012, Fine et al. 2013, Dixon et al. 2015, Chakravarti et al. 2017, Krueger et al. 2017, Torda et al. 2017, Eirin-Lopez and Putnam 2019) (Grottoli et al. 2006, Rodrigues and Grottoli 2007, Borell and Bischof 2008, Borell et al. 2008, Anthony et al. 2009, Hughes et al. 2010, Hughes and Grottoli 2013, Baumann et al. 2014, Grottoli et al. 2014, Levas et al. 2016). 2. Acclimatisation (phenotypic plasticity) Phenotypic plasticity refers to different phenotypes that can be generated from a single genotype in response to different environmental conditions (Pigliucci et al. 2006). These phenotypic changes are reversible and dependent on the boundaries of each organism’s genotype (Coles and Brown 2003). In this context, acclimatisation refers to the phenotypic changes of corals in their natural environment, while acclimation refers to short-term phenotypic changes under manipulative experimental conditions in the laboratory. Previously, in coral reef ecology studies the term ‘phenotypic adaptation’ has been used as substitute for ‘acclimatisation’ due to the life cycle of most coral species that span many decades (Buddemeier and Fautin 1993, Brown 1997a, b, Brown and Cossins 2011), but recently with technological advances most studies are recognizing that what used to be called adaptive processes are in fact acclimatisation processes (Edmunds and Gates 2008).. 6.

(20) Chapter I: General Introduction Reciprocal transplantation experiments (RTE) are a well-known method to quantify acclimatisation mechanisms by measuring differences in physiological parameters across environmentally different sites, locations or regions. For example, a RTE of Porites lobata between a fore reef (site impacted by high wave action, oceanic swells and storms) and back reef (sheltered site) at two neighbouring islands in American Samoa, showed phenotypic plasticity in mean annual skeletal extension rates, mean bulk densities and mean annual calcification rates after only six months, with all three variables measured in the transplanted corals approximating values to the corals originally from the site (Smith et al. 2007). In another study in the Red Sea, Sawall et al. (2015) found optimal calcification rates at 28–29 °C throughout all populations of Pocillopora verrucosa with evident differences in temperature fluctuations between the northern (21–27 °C) and southern (28– 33 °C) parts of the Red Sea, supporting high phenotypic plasticity due to low genetic divergence between north and south coral host populations.. 2.1 Thermal stress acclimatisation Multiple studies have identified a direct link between thermal preconditioning and bleaching susceptibility (Table 1) (Maynard et al. 2008, Middlebrook et al. 2008, Bellantuono et al. 2012, Guest et al. 2012, Brown et al. 2015, Putnam and Gates 2015, Hawkins and Warner 2017, Coles et al. 2018, Fisch et al. 2019, Hughes et al. 2019). After exposing corals to shortterm thermal preconditioning experiments, only preconditioned corals did not bleach during a heat-stress experiment (Table 2) (Middlebrook et al. 2008, Bellantuono et al. 2012), despite maintaining their Symbiodiniaceae and the. 7.

(21) Chapter I: General Introduction bacterial community (Bellantuono et al. 2012). Moreover, other studies have compared coral responses of the first major mass bleaching event in 1998 with subsequent stronger bleaching events (Maynard et al. 2008, Guest et al. 2012). Maynard et al. (2008) surveyed the same sites in 1998 and after a more severe bleaching event in 2002, which featured exposure to twice as many degree heating weeks (DHW) and 15% higher solar irradiance, corals acclimatised, and exhibited less bleaching than in 1998. In a similar study, Guest et al. (2012) demonstrated how coral bleaching was less severe after the 2010 large-scale bleaching event in Southeast Asia in locations that previously showed high bleaching in 1998 (Singapore and Malaysia), and had greater historical temperature variability and lower rates of warming. Meanwhile, corals in Indonesia were unaffected by bleaching in 1998, but showed high mortality in 2010. Consequently, corals acclimatised to previous thermal stress events, but also those living in sites with highly variable temperatures presented higher tolerance (Guest et al. 2012). Brown et al. (2015) demonstrated ‘long-term environmental memory’ during the bleaching event in 2010. In 2000, coral colonies were rotated 180° in a manipulative experiment (Brown et al. 2000). During the bleaching event of 2010, the sides of colonies exposed to high solar radiation before rotation in the 2000 experiment, retained four times as many symbionts than the sides exposed to low solar radiation, despite experiencing higher radiation for 10 years (Brown et al. 2015). These experiments provide evidence that long-term acclimatisation to local conditions enhances thermal tolerance during bleaching events (Table 2). Coles et al. (2018) showed evidence of acclimatisation to increasing seawater temperatures by replicating a bleaching. 8.

(22) Chapter I: General Introduction experiment from 1970 at the same location in 2010. Because sea-surface temperature (SST) has steadily increased 1.13 °C over the last four decades, the authors experimentally increased 2.2 °C of ambient temperatures. Corals in 2017 showed higher calcification rates, delayed bleaching, and mortality compared to corals in 1970 (Table 2) (Coles et al. 2018). Unfortunately, despite increased temperature tolerance in local corals, Hawaii suffered high coral mortality (34%) during the 2014–2017 global bleaching event, showing that high-temperature acclimatisation processes may not be occurring quickly enough to mitigate the projected length and intensity of future bleaching events (Coles et al. 2018).. 2.2 Acclimatisation to high temperature variability A series of backreef pools exhibiting tidal temperature variability on the island of Ofu, American Samoa, present a unique environment to study physiological differences between conspecific corals at small-spatial scales (Thomas et al. 2018). Using genetically identical coral fragments in a heatstress experiment from both pools, Oliver and Palumbi (2011a) provided evidence of increased thermal tolerance when corals have acclimatised to high temperature variability (Table 2). Corals from the highly variable (HV) pool showed lower mortality and higher photochemical efficiency, while those from the moderately variable (MV) pool suffered increased mortality and lower photochemical efficiency related to symbiont species. Corals associated with Durusdinium spp. exhibited an intermediate decline in photochemical efficiency, while those associated with Cladocopium spp. showed the highest decline (Oliver and Palumbi 2011a).. 9.

(23) Chapter I: General Introduction Table 2. Studies performed to test acclimatisation to thermal stress and high temperature variability at different locations around the world. Temp/ DHW. Duration. 28 °C (precond.). 10 d. 31 °C (HSE). 8d. 31 °C (precond.) 34 °C (HSE). 2d. Comparison 1998/2002 BE. DHW = 2002 > 1998. Bleaching survey. GBR, Australia. Comparison 1998/2010 BE. DHW = Malaysia + Singapore > Indonesia. Bleaching survey. Indonesia, Malaysia, Singapore. -. Bleaching survey. Study Precondition + HSE. Precondition + HSE. Survey 2010 BE. Comparison 1970/2017 HSE. 31.4 °C. Location. Species. Main results. Ref.. GBR, Australia. Acropora millepora. No bleaching in preconditioned corals. (Bellantuo no et al. 2012). GBR, Australia. Acropora aspera. No bleaching in preconditioned corals. (Middlebr ook et al. 2008). Less mortality in 2002. (Maynard et al. 2008). Low bleaching in Malaysia and Singapore. (Guest et al. 2012). Thailand. Coelastrea aspera. Less bleaching in high irradiance colony sides (decadal environmental ‘memory’). (Brown et al. 2015). Hawaii, USA. Montipora capitata, Pocillopora damicornis, Lobactis scutaria. Higher calcification, delayed bleaching and mortality in 2017. (Coles et al. 2018). 6d. 31 d. Acropora spp., Pocillopora spp., Porites spp. Acropora spp., Pocillopora spp.,. (Oliver and Palumbi 2011a). HSE (HV and MV). 31.5 °C. 5d. American Samoa. HSE from RT (HV and MV). 34 °C. 3h. American Samoa. Acropora hyacinthus. HSE (HV and LV). 30 °C. 270 d. Taiwan. Pocillopora damicornis,. Acclimatised: HV = control in all parameters. (Mayfield et al. 2013). Multiple species. Less bleaching in HV sites. (Thompso n and van Woesik 2009). Multiple species. Less bleaching in HV sites. (Safaie et al. 2018). Comparison 1998/2005-06 BE (HV - LV). -. Bleaching survey. Comparison multiple BEs (HV LV). -. Bleaching survey. Egypt, Madagascar, Seychelles, Australia, Guam, Kiribati, Cook Islands Western Indian Ocean, Pacific Ocean, Caribbean Sea, GBR, Red Sea. Acropora hyacinthus. Mortality and photochemical efficiency decline: HV+Durusdinium< MV+Durusdinium< MV+Cladocopium Acclimatised: MV to HV increased heat resistance; HV to MV reduced chl a retention; Different expression of 74 genes. HSE = Heat Stress Experiment, HV = High Variable, MV = Moderate Variable, LV = Low Variable, RT = Reciprocal Transplantation, BE = Bleaching Event, DHW = Degree Heating Weeks, d = days, h = hours, GBR = Great Barrier Reef.. 10. (Palumbi et al. 2014).

(24) Chapter I: General Introduction Palumbi et al. (2014) performed reciprocal transplantations of corals between HV and MV pools and subjected those corals to a heat stress experiment to test for acclimatisation responses to thermal stress (Table 2). Corals acquired heat sensitivity based on the pool they were transplanted to: MV pool corals acquired heat resistance when moved to HV pool, but not to the same extent of HV conspecifics, while HV to MV transplantees experienced reduced chlorophyll a retention, similar to the levels of native corals (Palumbi et al. 2014). Mayfield et al. (2013) performed a thermal stress experiment with corals from a site in Taiwan exhibiting high daily temperature fluctuations and found that, under HV conditions, physiological parameters behaved similarly to those in control corals, suggesting that individuals living under HV temperatures can acclimate to high temperatures that would cause bleaching and mortality in unacclimated corals from other regions (Table 2) (Mayfield et al. 2013). Some studies have compiled data of past bleaching events, in an effort to link patterns of bleaching susceptibility within sites under high temperature variability, in a worldwide context (Thompson and van Woesik 2009, Safaie et al. 2018). Sites characterized by a high-frequency pattern of temperature variability experienced higher thermal stress during both bleaching events, with extensive bleaching reported during 1998. However, in 2005–2006, these sites experienced reduced bleaching compared to sites under low frequency patterns, due to the acclimatisation of corals to thermal stress after the 1998 bleaching event and selective adaptation of resilient corals that survived the bleaching event (Thompson and van Woesik 2009). Safaie et al. (2018) explored this concept further by collecting in situ data with remotely sensed. 11.

(25) Chapter I: General Introduction datasets from different reef locations around the globe, along with spatiotemporally coincident quantitative coral bleaching observations. Corals regularly exposed to temperature fluctuations on daily or tidal timescales became acclimatised to thermal stress and resistant to bleaching events. More importantly, these patterns of high-frequency temperature variability to bleaching occur in many reefs worldwide (Safaie et al. 2018).. 3. Adaptation Adaptation, strictly defined, refers to changes in the genetic composition of a population that are passed onto the next generation through natural selection (Table 1) (Brown 1997a, Coles and Brown 2003, Edmunds and Gates 2008). The major concern regarding global climate change is that the current rate of environmental changes will outpaced the evolutionary capabilities of corals to adapt (Hoegh-Guldberg 1999, Hughes et al. 2003, Hoegh-Guldberg et al. 2007, Hoegh-Guldberg 2012, Hughes et al. 2017a). Recent evidence has shown that, in addition to phenotypic plasticity and acclimatisation, other adaptive responses in corals, such as transgenerational plasticity (Torda et al. 2017), epigenetics (Durante et al. 2019, Eirin-Lopez and Putnam 2019), and somatic mutations (Van Oppen et al. 2011) might contribute to resilience under thermal stress. Moreover, the fast rate of asexual reproduction within the Symbiodinaceae (days to weeks in hospite) (Wilkerson et al. 1988) in combination with large population sizes within corals (~ 1-5 x 106 cells cm-2) (Fitt et al. 2000) provides the potential for random mutations to develop that might enable corals to resist thermal stress (Van Oppen et al. 2011).. 12.

(26) Chapter I: General Introduction Few studies have examined adaptation to local thermal history in Symbiodiniaceae dinoflagellates (Howells et al. 2012, Chakravarti et al. 2017) Howells et al. (2012) demonstrated adaptive capacity with C. goreaui (formerly type C1) in corals from two sites in the GBR with dissimilar thermal histories. Corals hosting C. goreaui from the cooler site presented photodamage and bleaching, while those from the hotter site exhibited no signs of stress and greater growth (Howells et al. 2012). Chakravarti et al. (2017) tested adaptation to thermal tolerance of C. goreaui through experimental evolution. Dinoflagellates were cultured in vitro at elevated temperature of 31 °C for ~80 generations (2.5 y), while wild-types were reared at 27 °C ambient temperature, then both cultures were tested at both temperatures. To measure physiological responses in hospite, both types (thermally selected and wild types) were inoculated into aposymbiotic recruits of three coral species and were exposed to both temperatures similar to in vitro experiments (Chakravarti et al. 2017). Symbionts reared in vitro performed better in photophysiology and growth at both temperatures, and showed lower levels of extracellular ROS. In contrast, wild-type symbionts were unable to photosynthesise or grow at high temperatures, and produced 17 times more extracellular ROS (Chakravarti et al. 2017). The differences were less obvious in hospite than in vitro. Cultures of corals inoculated with the thermally tolerant symbionts showed no difference in growth between 27 and 31 °C, while those inoculated with wild-types showed a negative growth trend at 31 °C, confirming an adaptation to thermal stress in C. goreaui after many generations living under high temperature (Chakravarti et al. 2017).. 13.

(27) Chapter I: General Introduction Dixon et al. (2015) revealed genetic data from the coral host that forms the heritable basis of temperature tolerance by performing a cross-fertilization experiment with coral colonies from two thermally divergent locations in GBR. The authors measured heat tolerance using the survivorship rate of larvae exposed to high temperatures and found that parents from the warmer location conferred significantly higher thermo-tolerance to their offspring, up to 10 fold increase in odds of survival, in comparison to parents from the cooler location. Dixon et al. (2015) also identified “tolerance-associated genes” (TAG’s), whose expression before stress predicted high survivorship rates in larvae under thermal stress, dissimilar from frontloaded genes (Barshis et al. 2013). When TAG expression was compared with parental colonies after three days of heat stress, they were negatively correlated with long-term heat stress response similar to the larval response, indicating that the larval heat tolerance results from the absence of pre-existing stress and not from prior up-regulation of heat stress genes through frontloading (Dixon et al. 2015). Krueger et al. (2017) presented evidence that Stylophora pistillata underwent selection for heat tolerance in the Red Sea, after spending 47 days at 1–2 °C above their long-term summer maximum and showed an increase in primary productivity. Fine et al. (2013) demonstrated how different corals species showed no signs of stress after exposure to 33 °C for four weeks and proposed that corals that colonised the Gulf of Aqaba after the last ice age had to cross exceedingly warm waters (> 32 °C in the summer) at the entrance of the Red Sea, maintaining this adaptation to heat tolerance until the present day.. 14.

(28) Chapter I: General Introduction. 4. Heterotrophy (Mixotrophy) Heterotrophic carbon can become a significant energy source for some coral species when phototrophic carbon is unavailable, such as during a bleaching event (Table 1) (Houlbrèque and Ferrier‐Pagès 2009). Some studies have shown how heterotrophy replenished energy reserves in corals exposed to high temperatures (Grottoli et al. 2006) and during the recovery phase (Rodrigues and Grottoli 2007). Similarly, Borell and Bischof (2008) showed higher photochemical efficiency in fed corals compared to unfed corals after a mild thermal stress experiment. Also, Borell et al. (2008) demonstrated how heterotrophy sustained photosynthetic activity and energy reserves in thermally stressed corals. In a study which developed an energy-budget model linking coral bleaching and mortality risk, authors concluded that the time between the start of severe bleaching and the beginning of mortality is influenced by the amount of lipid stores corals have before the bleaching event and their capacity to acquire energy through heterotrophy (Anthony et al. 2009). With a stable isotope. 13. C pulse-chase labelling experiment, Hughes et al. (2010). demonstrated that, after exposure to high temperatures, coral hosts incorporated heterotrophic labelled carbon for storage and to stimulate endosymbiont recovery. Even after recovery from bleaching, 75% of carbon in newly acquired lipids was sourced heterotrophically (Baumann et al. 2014), and corals continued assimilating heterotrophic carbon for up to 11 months after the bleaching experiment (Hughes and Grottoli 2013). Nonetheless, the capacity for heterotrophic plasticity is compromised after two consecutive bleaching events (Levas et al. 2016). Researchers experimentally bleached. 15.

(29) Chapter I: General Introduction corals for 2.5 weeks, transferred corals to the field for recovery, and then repeated the bleaching experiment after one year. After the first thermal stress experiment, zooplankton and dissolved organic carbon (DOC) allowed the metabolic demand of bleached corals to be met; however, neither form of heterotrophic carbon was able to contribute to the energy budget of both species after the second bleaching experiment, suggesting that the capacity for heterotrophic plasticity is compromised under annual bleaching events (Levas et al. 2016), and corals need to depend on their energy reserves and/or symbiont association to survive repeated bleaching (Grottoli et al. 2014).. Research overview The frequency of global bleaching events are increasing, due to the rise of anthropogenic CO2 in the atmosphere (Hughes et al. 2017b). As our oceans keep warming up, understanding the mechanisms driving thermal tolerance in reef-building corals is of outstanding importance. Furthermore, those coral species thriving in present day extreme thermal environments, including sites influenced by high thermal stress or sites with high levels of temperature variation at fine special-scales (Thomas et al. 2018), offer important insight into the mechanisms of thermal tolerance and the future capacity for coral species to cope with a rapidly warming planet (Palumbi et al. 2014, Hughes et al. 2017a, Hughes et al. 2017b). In Kenting National Park (KNP), southern Taiwan, there is a site that is influenced by the constant hot-water effluent from a nuclear power plant outlet. At this ‘Variable Site’ (VS), temperatures during the summer can be 2 –. 16.

(30) Chapter I: General Introduction 3 °C higher that at any other site within KNP, and it is also influenced by a monthly upwelling that makes seawater temperature to fluctuate up to 8 °C in one day (Lee et al. 1997, Peir 2011, Keshavmurthy et al. 2019). By comparing physiological parameters of conspecific corals living in this climate-changed simulator with a ‘Stable Site’ (SS) within KNP that is not affected by high temperatures or high temperature fluctuations, I raised the questions: ! How do conspecific corals live in two environments with different temperature regimes? ! What are the physiological differences amongst corals from different sites? ! What mechanisms allow them to survive in variable environmental conditions? ! How does symbiont identity influenced the physiology of corals living in different environmental conditions?. In this dissertation I focused on two species of corals: Leptoria phrygia and Porites lutea. Both species have been reported to associate each with different stress-resistant symbionts in KNP (Keshavmurthy et al. 2014). Thus, the ‘association with thermally tolerant symbionts’ is one of the mechanisms that I am interested to investigate, at the same time complement it with ‘acclimatisation’ mechanisms to a thermally stressful environment that these two species have and their nutritional changes (autotrophy/heterotrophy). To answer the research questions of this dissertation, I set up two general aims:. 17.

(31) Chapter I: General Introduction 1. To determine physiological differences in Leptoria phrygia as a function of symbiont identity when living at two sites with contrasting seawater temperature regime; and 2. To determine the acclimatisation processes with the use of a reciprocal transplantation experiment by comparing physiological parameters between Leptoria phrygia and Porites lutea for 18 months when living at two sites with contrasting seawater temperature regime.. I introduce my research topic in Chapter I; in Chapter II, I report the results on the physiological differences of L. phrygia from both sites and how the seawater temperature influenced the symbiont association and its responses in photochemical efficiency, symbiont density and chlorophyll a concentration, so as to achieve the general objective 1. In Chapter III, I present some insights in the differences in δ13C and δ15N isotopic niche when L. phrygia associates Cladocopium spp. or Durusdinium spp, as a continuation from Chapter II. In Chapter IV, I report the results of 18 months reciprocal transplantation. experiment. design. to. understand. the. acclimatisation. processes to thermal stress (in the summer) and to high temperature variation that these two species associated with stress resistant symbionts have (L. phrygia with D. glynnii and P. lutea with Cladocopium C15), in order to achieve the general objective 2. Different physiological parameters were used from the symbiont: photochemical efficiency, symbiont density and chlorophyll a concentration; from the coral host: total soluble proteins; and lipid concentration from both fractions. In Chapter V, I present the main. 18.

(32) Chapter I: General Introduction conclusions from my research and I discuss how corals might respond to future climate change scenarios, according to my main findings.. * A substantial part of this chapter was published in December 2019 in the special issue ‘Coral Reef Resilience’ as: Carballo-Bolaños C, Soto D, Chen CA (2020) Thermal stress and resilience of corals in a climate-changing world. J. Mar. Sci. Eng. 8(1): 15; https://doi.org/10.3390/jmse8010015. 19.

(33) Chapter II: Symbiodiniaceae community of Leptoria phrygia. CHAPTER II: Temporal variation and photochemical efficiency of species in Symbiodiniaceae associated with coral Leptoria phrygia (Scleractinia; Merulinidae) exposed to contrasting temperature regimes 1. Abstract: The Symbiodinaceae are paradoxical in that they play a fundamental role in the success of scleractinian corals, but also in their dismissal when under stress. In the past decades, the discovery of the endosymbiont’s genetic and functional diversity has led people to hope that some coral species can survive bleaching events by associating with a stress-resistant symbiont that can become dominant when seawater temperatures increase. The variety of individual responses encouraged us to scrutinize each species individually to gauge its resilience to future changes. Here, we analyse the temporal variation in the Symbiodinaceae community associated with Leptoria phrygia, a common scleractinian coral from the Indo-Pacific. Coral colonies were sampled from two distant reef sites located in southern Taiwan that differ in temperature regimes, exemplifying a ‘variable site’ (VS) and a ‘steady site’ (SS). We investigated changes in the relative abundance of the dominant symbiont and its physiology every 3-4 months from 2016-2017. At VS, 11 of the 12 colonies were dominated by the stress-resistant Durusdinium spp. (>90% dominance) and only one colony exhibited co-dominance between Durusdinium spp. and Cladocopium spp. Every colony displayed high photochemical efficiency across all sampling periods, while showing temporal differences in symbiont density and chlorophyll a concentration. At SS, seven. 20.

(34) Chapter II: Symbiodiniaceae community of Leptoria phrygia colonies out of 13 were dominated by Cladocopium spp., five presented codominance between Durusdinium spp./Cladocopium spp. and only one was dominated by Durusdinium spp. Colonies showed temporal differences in photochemical efficiency and chlorophyll a concentration during the study period. Our results suggest that VS colonies responded physiologically better to high temperature variability by associating with Durusdinium spp., while in SS there is still inter-colonial variability, a feature that might be advantageous for coping with different environmental changes.. 1. This chapter was published in June 2019 as: Carballo-Bolaños R, Denis V, Huang Y-Y,. Keshavmurthy S, Chen CA (2019) Temporal variation and photochemical efficiency of species in Symbiodinaceae associated with coral Leptoria phrygia (Scleractinia; Merulinidae) exposed. to. contrasting. temperature. regimes.. PLoS. ONE. 14(6):. e0218801.. https://doi.org/10.1371/journal. pone.0218801. 21.

(35) Chapter II: Symbiodiniaceae community of Leptoria phrygia. Introduction The success of coral reefs in tropical oligotrophic waters is often attributed to the symbiotic relationship between scleractinian corals and dinoflagellates algae. The Symbiodiniaceae are intracellular photosynthetic organisms that supply up to 95% of the coral host’s energy requirements in optimum conditions (Muscatine 1990). However, this subtle relationship is commonly disrupted under stressful environmental conditions, such as abnormally high seawater temperatures, in what is known as coral bleaching. Due to climate change and anthropogenic increases in seawater surface temperatures, bleaching events are becoming more frequent and severe (Hughes et al. 2017b). Many coral reefs worldwide are now recurrently affected by mass bleaching events and mortality and, consequently, may become scarce within the next 20-30 years (Hoegh-Guldberg 1999). One well-known asset that some coral species have to survive bleaching events is their ability to associate with a functionally diverse community of symbionts, and to adjust their relative abundances to favour those better fitted to endure temperature variations (Buddemeier and Fautin 1993, Berkelmans and van Oppen 2006, Silverstein et al. 2015). The family Symbiodiniaceae is highly diverse and corals typically associate with members from the genera Symbiodinium (formerly Clade A), Breviolum (formerly Clade B), Cladocopium (formerly Clade C), and Durusdinium (formerly Clade D) (LaJeunesse et al. 2018). Durusdinium spp. are extremophile endosymbionts that have been found in stressed habitats, such as those with high temperature (LaJeunesse et al. 2010a, Oliver and Palumbi 2011b, Hsu et al. 2012, Keshavmurthy et al. 2014), high turbidity. 22.

(36) Chapter II: Symbiodiniaceae community of Leptoria phrygia (LaJeunesse et al. 2010a) or located in high latitudinal marginal reefs (Chen et al. 2003, Lien et al. 2007, Lien et al. 2013). In order to survive, some corals are able to increase the relative abundance of Durusdinium trenchii during and after bleaching events (Baker et al. 2013a). Some examples of this mechanism are found in species such as Acropora millepora in Australia (Jones et al. 2008); Orbicella annularis, Siderastrea siderea, Agaricia spp., and Montrastraea cavernosa in Barbados (Lajeunesse et al. 2009); and Isopora palifera in Taiwan (Hsu et al. 2012). It is a species- and locationspecific mechanism that changes with environmental conditions (Stat et al. 2009, Thornhill et al. 2009, Baker et al. 2013a). Some examples include Pocillopora damicornis, Seriatopora hystrix, Stylophora pistillata, Favites abdita, Goniastrea favulus, A. millepora, I. palifera in Australia (Stat et al. 2009) and O. faveolata and O. annularis in the Florida Keys and the Bahamas (Thornhill et al. 2009), which did not change their symbiotic composition during. and. after. thermal. stress.. Because. the. systematics. of. the. Symbiodinaceae family is still a work in progress, there are many dinoflagellates that have not yet been identified to the species level. Therefore, the former sub-clade or type is used after the genera. Within the Cladocopium genus there are some species that have been recognized to be thermally resistant, such as Cladocopium C15. In Australia, Porites lutea colonies. revealed. that,. when. experimentally. heated,. corals. hosting. Cladocopium C15 maintained higher maximum photochemical efficiency (Fv/Fm) than those hosting Cladocopium C3 (Fisher et al. 2012). Similar results were found in Porites lobata from Hawaii; colonies that were experimentally bleached could maintain gross photosynthetic rates similar to. 23.

(37) Chapter II: Symbiodiniaceae community of Leptoria phrygia control colonies when hosting Cladocopium C15. Moreover, this thermally tolerant symbiont helped the coral recover faster from bleaching by contributing 96% of the host’s daily metabolic demand, even when chlorophyll a levels were significantly lower than in the control (Levas et al. 2013). The capacity to associate with multiple Symbiodinaceae genera is considered a widespread phenomenon (Baker 2003, Silverstein et al. 2012). Some coral species can maintain a stable association with their dominant symbiont across their lifetime (Goulet 2006), including during and after stress events (Stat et al. 2009, Thornhill et al. 2009). Alternatively, some species are capable of shifting the relative abundance of their dominant partner to background symbionts when exposed to stress (Berkelmans and van Oppen 2006, Silverstein et al. 2015, Cunning et al. 2017). Those background symbionts are usually considered to initially represent <10% of the overall Symbiodinaceae community within one colony (Mieog et al. 2007, Mieog et al. 2009). A third scenario, documented for a very few number of species is to have a different symbiont dominating distinct areas within the coral colony (Table 1). In this study, I refer to multi-symbiont dominance on the colony scale, but each microhabitat within the colony can have its own dominant symbiont. For example, the dominant symbiont may be different between the top part of the colony and the lowest part. In the Caribbean, this has been documented in the Orbicella spp. complex (including O. annularis, O. faveolata and O. franski) since the 90’s (Table 1) (Rowan and Knowlton 1995, Rowan et al. 1997, Toller et al. 2001, Kemp et al. 2015). These massive corals associate with multiple dominant symbionts in response to different light gradients, creating different microhabitats within the same coral colony.. 24.

(38) Chapter II: Symbiodiniaceae community of Leptoria phrygia In O. faveolata—the top part of the colony, with high-irradiance—was dominated by Symbiodinium sp. and Breviolum sp., while the side or shaded parts of the colony were dominated by Cladocopium sp. (Rowan et al. 1997, Kemp et al. 2015). The same pattern was observed between colonies living at different depths: those living in shallow waters with high light intensity were dominated by Symbiodinium sp. and Breviolum sp., whereas Cladocopium sp. dominated those colonies living in deep waters (Rowan et al. 1997). In the Pacific, Isopora palifera colonies from southern Taiwan presented multisymbiont dominance between Cladocopium C3 and D. trenchii (Table 1) (Chen et al. 2005, Hsu et al. 2012). The relative abundance of both symbionts varied across the study period and D. trenchii became highly abundant in some colonies after the 1998 bleaching event (Chen et al. 2005). The relative abundance of D. trenchii decreased afterwards and Cladocopium C3 became dominant almost 10 years after the bleaching event (Hsu et al. 2012). These differences in the dominant symbiont between Symbiodiniaceae genera explain why certain colonies, or parts of the colony, bleach and some do not during natural bleaching events (Rowan et al. 1997); they also explain how Symbiodiniaceae can cope with and recover from a bleaching event by shuffling their relative abundance (Hsu et al. 2012). These species are able to cope with environmental fluctuations, presenting a long-term ecological and evolutionary coral-Symbiodinaceae specialization strategy (Rowan et al. 1997, Kemp et al. 2015).. 25.

(39) Chapter II: Symbiodiniaceae community of Leptoria phrygia Table 1. Studies reporting co-dominance of different symbiont genera within a single colony. Host species. Genetic method for ID. Symbiont genera/species. Study site. Orbicella annularis O. faveolata. Symbiodinium, Breviolum, Cladocopium Symbiodinium, Breviolum, Cladocopium. San Blas Archipelago, Panama. srRNA -RFLP . O. annularis. O. faveolata. O. franski. San Blas Archipelago, Panama. srRNA -RFLP . Acropora tenuis A. valida. Symbiodinium, Breviolum, Cladocopium, Durusdinium Symbiodinium, Breviolum, Cladocopium, Durusdinium Symbiodinium, Breviolum, Cladocopium, Durusdinium Cladocopium, Durusdinium Cladocopium, Durusdinium. Great Barrier Reef, Australia. rDNA-ITS1, SSCP. A. valida. Cladocopium, Durusdinium. Great Barrier Reef, Australia. rDNA-ITS1, SSCP. Isopora palifera. Cladocopium, Durusdinium. Kenting National Park, Taiwan. lsrRNA -RFLP . O. faveolata O. annularis. O. franski. Siderastrea siderea O. annularis O. faveolata. Stephanocoenia intersepta . A. valida. B. minutum, Cladocopium C12 B. minutum, Brevolium B10, Cladocopium C3, D. trenchii B. minutum, Cladocopium C12 B. minutum, Brevolium B10, Cladocopium C3, D. trenchii Brevolium B5a, Cladocopium C3 B. minutum, C7, Cladocopium C3 Symbiodinium A3, B. minutum, Brevolium B17, Cladocopium C7 and D. trenchii Symbiodinium A3b, Symbiodinium A3, Cladocopium C3, Cladocopium C16, Cladocopium C54 C. goreaui, Cladocopium C2 and Symbiodinium. Lee Stoking Islands, Bahamas Upper Florida Keys, USA. Lee Stoking Islands, Bahamas Upper Florida Keys, USA. Upper Florida Keys, USA Carrie Bow Cay, Belize. ITS2-DGGE. ITS2-DGGE. (Warne r et al. 2006). Great Barrier Reef, Australia. rDNA-ITS1, SSCP. I. palifera. Cladocopium C3, D. trenchii. Kenting National Park, Taiwan. ITS2-DGGE. O. faveolata. Symbiodinium A3, Brevolium B17, Cladocopium C7, D. trenchii. Puerto Morelos, Mexico. ITS2-DGGE. O. faveolata. B. minutum, Cladocopium C7a, D. trenchii Symbiodinium A3, B. minutum, Brevolium B17, Cladocopium C7 Symbiodinium A3, B. minutum, Brevolium B17, Cladocopium C7, D. trenchii B. minutum, Cladocopium C3. Exuma Cay, Bahamas. Carrie Bow Cay, Belize. Puerto Morelos, Mexico. Upper Florida Keys, USA. ITS2-DGGE. (Ulstru p et al. 2008) (Hsu et al. 2012) (Kemp et al. 2014) (Kemp et al. 2015). Studies in light grey are from the Indo-West Pacific Ocean.. 26. Ref. (Rowan and Knowlt on 1995, Rowan et al. 1997) (Toller et al. 2001). (van Oppen et al. 2001) (Ulstru p and van Oppen 2003) (Chen et al. 2005) (Thorn hill et al. 2006b).

(40) Chapter II: Symbiodiniaceae community of Leptoria phrygia In the present chapter, I describe the temporal dynamics of Cladocopium spp. and Durusdinium spp. associated with the sub-massive brain coral Leptoria phrygia (Ellis and Solander, 1786) in Kenting National Park, southern Taiwan. I monitored symbiont community changes and performance in coral colonies from two shallow reef flats exposed to contrasting seawater temperature regimes. The average shallow water temperatures at the ‘Outlet’ reef flat can be up to 2 - 3 °C higher than other coral reef sites during the summer (Fan 1991, Peir 2011). Furthermore, a tidal upwelling (Lee et al. 1997, Jan and Chen 2009) causes daily temperature fluctuation that can reach 6 - 8 °C; we considered this our “variable site” (VS). Temperatures at the ‘Wanlitong’ reef flat are steadier (daily seawater temperature fluctuations <3 °C); I considered this my “stable site” (SS). My specific objectives were to: (1) investigate whether the relative abundance of endosymbionts changes are in line with temporal changes in local environments from both sites, and (2) characterise the dynamics of the endosymbionts’ physiology (photochemical efficiency, symbiont density, and chlorophyll a concentration) caused by these temporal changes.. Materials and methods Study sites and temperature data Both Variable Site (VS) and Stable Site (SS) are located within Kenting National Park (KNP) in southern Taiwan (Fig. 1). They are less than 20 km apart but have very different seawater temperature regimes. VS is located next to a nuclear power plant outlet in Nanwan Bay (21° 55' 53.7" N - 120° 44'. 27.

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