Exaptation in corals to high seawater temperatures: Low concentrations of apoptotic and necrotic cells in host coral tissue under bleaching conditions

Journal of Experimental Marine Biology and Ecology 369 (2009) 31–42

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Exaptation in corals to high seawater temperatures: Low concentrations of apoptotic and necrotic cells in host coral tissue under bleaching conditions Kevin B. Strychar a,⁎, Paul W. Sammarco b a b

Dept. Life Sciences, Texas A&M University – Corpus Christi, 6300 Ocean Drive – Unit 5869, Corpus Christi, Texas 78412 USA Louisiana Universities Marine Consortium (LUMCON), 8124 Hwy. 56, Chauvin, LA 70344 USA

a r t i c l e

i n f o

Article history: Received 31 January 2008 Received in revised form 7 August 2008 Accepted 16 October 2008 Keywords: Apoptosis Bleaching Climate change Coral tissue Exaptation Necrosis Temperature Zooxanthellae

a b s t r a c t Scleractinian corals are known to suffer bleaching or loss of their symbiotic zooxanthellae under conditions of elevated seawater temperatures often associated with climate change (i.e. global warming). This can occur on a massive scale and has caused the decimation of reefs on a global basis. During the bleaching process, the expelled zooxanthellae suffer cell damage from heat stress, characterized by irreversible ultrastructural and physiological changes which are symptomatic of cell degeneration and death (called apoptosis) or necrosis. A question that remains unanswered, however, is whether the coral hosts themselves are sensitive to seawater temperatures, and, if so, to what degree? In a controlled experiment, we exposed corals Acropora hyacinthus (Dana, 1846) and Porites solida (Forskål, 1775) with their symbiotic zooxanthellae (Symbiodinium sp.) to temperatures of 28 °C (control), 30 °C, 32 °C, and 34 °C for 48 h and also to 36 °C for 12 h. We assessed coral and zooxanthellar cells in-situ for symptoms of apoptosis and necrosis using transmission electron microscopy (TEM), fluorescent microscopy (FM), and flow cytometry (FC). We found that the coral host cells in-situ exhibited, for the most part, little or no mortality from increased seawater temperatures. Damage to the coral hosts only occurred under conditions of prolonged exposure (≥12 h) at high temperatures (34 °C), or at exceptionally high temperatures (e.g. 36 °C). On the other hand, we found high levels of apoptosis and necrosis in the zooxanthellae in-situ under all treatment conditions of elevated seawater temperatures. We found that during bleaching, the host cells are not experiencing much mortality – but the zooxanthellae, even while still within the host, are. The host corals exhibit exaptation to accommodate temperatures as high as ≥34 °C. Temperature stress within these highly specific and coevolved symbiotic systems is derived not from host sensitivity to temperature, but from the symbiont's sensitivity and the loss of the coral's endosymbiotic partners. Published by Elsevier B.V.

1. Introduction 1.1. Coral Bleaching Coral bleaching due to increased sea surface temperatures (SSTs) has caused the decimation of reefs world-wide (Hoegh-Guldberg, 1999; Done et al., 2003; Donner et al., 2005). Although increased SST is not the sole contributor to this decline (e.g.- mass mortality of grazers Hughes, 1994; coral disease - Rosenberg and Loya, 2004; Weil et al., 2006), it is certainly a primary force (Glynn, 1991; Brown, 1997; Goreau et al., 2000). Bleaching is the discoloration of some reef organisms, particularly corals, due to a loss of endosymbiotic algae and/or pigments; it is a stress response by symbiotic corals to a variety of environmental stressors that cause internal physiological imbalance (Brown and Suharsono, 1990; Obura, 2005). It has been suggested that the maximum temperature that tropical Great Barrier Reef corals can endure is 2.0−2.5 °C above their maximum mean summer temperatures (Berkelmans and Willis,1999; Berkelmans, 2002). Coral mortality ⁎ Corresponding author. Tel.: +1 361 825 5883; fax: +1 361 825 2025. E-mail address: [email protected] (K.B. Strychar). 0022-0981/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jembe.2008.10.021

is correlated with both the intensity and duration of the temperature anomaly, and various metrics have been used to describe this relationship (Podesta and Glynn, 1997; Winter et al., 1998; VargasAngel et al., 2001; Sammarco et al., 2006). To date, most investigations of bleaching have focused on the algal symbiont, not the coral host. One of the specific causes of bleaching has been hypothesized to be differential species-specific sensitivity of zooxanthellae to temperature in different coral species. Many investigators have examined this sensitivity in various corals. For example, it is now known that Acropora spp. are often the first corals to succumb to small increases in SSTs as opposed to Porites spp. which are less sensitive (Williams and Bunkley-Williams, 1988; HoeghGuldberg, 1994). Another suspected cause of variation in bleaching is resistance to temperature increases by different strains of zooxanthellae (Kinzie et al., 2001). Hermatypic corals may be recolonized by zooxanthellae after their loss and thus survive the temperature perturbation via the re-establishment of the symbiont-host relationship (Baker, 2001; Hoegh-Guldberg et al., 2002; Estes et al., 2003). This implies that the coral hosts are more resistant to temperature increases than their symbiotic zooxanthellae, although few data exist on this question.

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Corals can survive the loss of their zooxanthellae after bleaching. Recolonization by the zooxanthellae must occur, however, within some critical time period, since Symbiodinium (zooxanthellae) provide as much as 95% of the host's nutritional requirements. Feeding by polyps alone does not provide sufficient nutrition (Schlichter et al., 1983; McCloskey et al., 1994). Thus, the temperature tolerance of the host is critical to the survival of the coral colony and the species as a whole. Some investigators suggest that the loss of Symbiodinium cells helps corals adapt to changing environmental conditions by allowing them to “shuffle” their complement of zooxanthellae, thereby benefiting the corals (termed the “adaptive bleaching hypothesis”; Buddemeier and Fautin, 1993; Baker, 2001). This can only occur if the coral host is robust enough to withstand the temperatures that the zooxanthellae could not. Dennis (2002) and Hoegh-Guldberg et al. (2002) claim that Symbiodinium shuffling does not occur quickly enough to be of benefit, and that expulsion of the symbiont instead usually causes death of the coral host. We believe that this will only occur if the coral host does not have sufficient tolerance to accommodate the high temperatures or a means of acquiring sufficient nutrients to survive the interim period. A debate has also existed for some time regarding whether loss of Symbiodinium from a coral is due to the zooxanthellae actively disengaging from the coral host and returning to open water, or whether the zooxanthellae, due to temperature-stress, compromise their symbiotic relationship with the coral and are actively expelled. Recent experimentation has demonstrated that under high temperature stress, the expelled zooxanthellae exhibit symptoms of cell death (Strychar et al., 2004a,b). This indicates that bleaching is a manifestation of zooxanthellar pathology. The question of this same pathology occurring in the coral host in-situ, however, has remained open. With respect to the zooxanthellae, Bhagooli and Hidaka (2003, 2004a,b) examined photosynthetic activity in in-situ and released cells immediately after a bleaching episode, using the PS-II system as an indicator. They found that this system was not affected, and the zooxanthellae exhibited no change in morphology at 30 °C and 32 °C. They proposed that the host was the stressed partner. In this case, however, no data were collected from the host cells. Secondly, the zooxanthellar samples were taken immediately after the stress treatment. Hill and Ralph (2007) demonstrated that, although sampled zooxanthellae show normal morphology immediately after a bleaching event, significant photosynthetic damage is not evident via the loss of structural integrity of the cells until after 6-12 hrs – a period not experienced by the zooxanthellae in Bhagooli and Hidaka's (2003, 2004a,b) experimental corals. Additional evidence exists suggesting that the zooxanthellae are more sensitive to temperature stress than the coral hosts, albeit with some variance. Sandeman (2006) presents evidence that, under conditions of thermal stress, coral gastrodermal tissue actively expels zooxanthellar cells in association with some of its own enveloping tissue, implying that the zooxanthellae are compromised in this symbiotic relationship – not the host. This was consistent with the results of Dunn et al. (2007), who worked on the symbiotic anemone Aiptasia pallida. They also demonstrate that the nuclei of the coral cells exhibit no condensation of chromatin normally associated with apoptosis. Lesser and Farrell (2004), on the other hand, found damage to both the zooxanthellae and the coral host cells under solar radiation and thermal stress. Such discrepancies may be due to a number of factors, including types of experimental treatments, type of host and/ or symbiont clade present, thermal acclimation, methodology, etc.

et al., 2002; terrestrial plants van Cruchten and van den Broeck 2002). Programmed cell death (PCD; e.g. Dunn et al., 2004; Ainsworth et al., 2007) is mortality of cells that has been triggered by either external or internal factors (Vaux and Strasser, 1996; Lim et al., 2007). It encompasses several forms of cell death including, but not limited to, apoptosis and paraptosis (Franklin et al., 2006; Reape et al., 2008). Apoptosis is a term that has sometimes been used synonymously with PCD, which to a certain degree is incorrect (Bursch et al., 2000). We use the term apoptosis as it was first described by Kerr et al. (1972) and further expanded by Darzynkiewicz et al. (1997) and Kanduc et al. (2002). We define apoptosis as the process by which a cell dies, characterized by pre-programmed biochemical and pathological events that form an integral part of a cell's physiology, helping to regulate abundant cell populations. Apoptosis implies that cell death will always occur in a controlled, regulated fashion, with the cells playing an active role in their own death (Ameisen, 2002; Strychar 2002; Dash 2008). Franklin et al. (2006) described cell death in phytoplankton and stated that the term PCD should only be used where molecular and morphological markers have demonstrated the specific type of cell death involved (apoptosis or paraptosis). Increased SST may trigger bleaching and apoptosis in corals but does not necessarily trigger PCD. The symptoms of these processes will always be identical. For example, the loss of webbing between mammalian fingers in-utero is PCD, but the death of a virally infected cell within the same organism is apoptosis, because a stimulus (virus) is required to induce cell death (Strychar et al., 2004a). Necrosis, on the other hand, is a passive, accidental, and unordered occurrence of cellular death (Strychar et al., 2004a). This concept has been applied to degenerative cell processes in corals by Gates and Edmunds (1999) and clarified by Strychar et al. (2001, 2004a,b). These types of cell death can be inferred from the high mortality rates (95− 99%) of expelled zooxanthellae observed in cnidarians such as alcyonacean and scleractinian corals under experimental temperatures of 32−34 °C for 9−48 h (ibid.), or during conditions of prolonged exposure to moderately increased temperatures of 30 °C (Coles et al., 1976; Warner et al., 1996). Thus, whether the zooxanthellae remain within the coral host or are expelled into open-water, after they have been exposed to unusually high temperature stress, they generally die. If expelled while still alive, most of these cells will probably not survive. Are the host corals also temperature sensitive, or do they exhibit exaptation to temperatures well beyond those that their symbiotic zooxanthellae cannot tolerate? Exaptation is defined as a character that has evolved for another function, or for no function at all, but which has been co-opted for a new use (Gould and Vrba, 1982, c.f. Futuyma, 1998). Ravindran and Raghukumar (2006) examined the responses of host and zooxanthellar cell to a coral disease. They found that the host did not exhibit symptoms of apoptosis – or irreversible ultrastructural or physiological changes characteristic of cell degeneration and death. The symbiont, on the other hand, did. The question we raise here is whether host coral tissues also experience cell damage and death upon exposure to high seawater temperatures, at what temperatures, and to what degree. Under temperature stress, are the host corals also suffering physiological damage, e.g. cell necrosis - moreso than the zooxanthellae? Is one of the two symbiotic partners suffering disproportionately? And do the levels of sensitivity vary between species of corals? Here we will demonstrate that corals do exhibit exaptation to high seawater temperatures and that this varies species-specifically with respect to the coral host.

1.2. Apoptosis, Necrosis, and Programmed Cell Death (PCD)

2. Materials and methods

In marine and terrestrial organisms, apoptosis and necrosis are two well-studied forms of cell death. They result from various physiological stimuli within an organism, e.g. tissue homeostasis and immune system development (e.g. sea anemones - Dunn et al., 2002; mammals - Kanduc

2.1. Target species The scleractinian corals Acropora hyacinthus (Dana, 1846) and Porites solida (Forskål, 1775) and their endosymbionts (Symbiodinium sp.) were

K.B. Strychar, P.W. Sammarco / Journal of Experimental Marine Biology and Ecology 369 (2009) 31–42

collected at ∼8.5 m depths from Barren Reef (23° 10' S, 151° 55'E) and Outer Rock Reef (23° 4.3'S, 151° 57.2'E), Great Barrier Reef, located off central Queensland, Australia. Acropora was chosen because its species members are often the first to succumb to even small increases in sea temperature (Marcus and Thorhaug, 1981; Edmunds and Davies, 1989; Goreau et al., 2000), as opposed to species of Porites, which are less sensitive (Williams and Bunkley-Williams, 1988; Hoegh-Guldberg, 1994; Marshall and Baird, 2000). The responses of expelled cells of Symbiodinium sp., but not the host, to these experimental conditions of increased seawater temperature have been reported in detail elsewhere (Strychar et al., 2004b). For orientation of the reader, however, we will summarize these methods and results below, where necessary, for comparative purposes. 2.2. Experimental design A detailed account of the techniques used to isolate and examine expelled Symbiodinium cells from corals using a flow through design is presented in Strychar et al. (2004b). Data presented here are derived from the same set of experiments but, we have shifted the focus for analyses to in-situ host and Symbiodinium cells, as opposed to expelled cells, which have been covered in earlier publications. We will present and discuss the present results in the context of those earlier results. We exposed host coral cells to various experimental seawater temperatures. The experiment followed a random blocks design (Zar, 1998). Filtered seawater (FSW) was first pumped through a series of 10 and 1 μm filters and deposited into four aerated 250 l header tanks (HT); water in each tank was mixed using submersible pumps. A peristaltic pump was then used to force the FSW at a constant flow rate of 20 ml min− 1 through 1 μm Millipore© depth filters to remove potential algal growth. It was then distributed into a series of eight two-liter plastic holding chambers. The FSW was mixed with an octagonally shaped magnetic stir bar within each chamber, to provide water movement for the corals. Corals were subjected to elevated heat stress for 48 h using 30, 32, or 34 °C, and 12 h at 36 °C and with a 28 °C control. In these studies, acclimation and control temperatures were set at 28 °C. This temperature was chosen because monthly SSTs collected from satellite, ship, and buoy instruments from Integrated Global Ocean Services System (IGOSS; Reynolds and Smith, 1994) yielded this temperature as the yearly peak average for the region. This control temperature plus the higher experimental temperatures were chosen based upon blended data from IGOSS (Reynolds and Smith, 1994). Strychar (2002) statistically analyzed these data to predict SSTs near these reefs from 1981 to 2100. An auto-regressive, integrated, movingaverage model (ARIMA) was employed, using Systat V 9.0; most severe bleaching events have occurred since 1981 (Reaser et al., 2000; Grimsditch and Salm, 2005). The model predicted that mean temperatures between 1981 and 2100 would be a yearly average of 26.97 °C and a yearly peak average of 31.85 °C. We also tested the extreme temperatures of 34 °C and 36 °C because Hoegh-Guldberg (1999) and Goreau et al. (2000) predict that global warming will produce temperatures similar to these between 2001 and 2100. Furthermore, Podesta and Glynn (1997) and Sandeman (2006) observed more noticeable symptoms of stress within corals at temperatures higher than 30 °C, allowing for better characterization of bleaching events. Although temperatures of 36 °C have not been observed on the Great Barrier Reef, such high temperatures are common in the Persian Gulf (N 33 °C; Sotka and Thacker, 2005), and are known to fluctuate between 17 and 36 °C in the Arabian Gulf of South Africa (Riegl, 2003). 2.3. Sample preparation for Transmission Electron Microscopy (TEM) Approximately 5 mm2 samples of excised coral tissue were taken from the coral colony with the assistance of a scalpel and a pair of fine

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dissecting forceps. In addition, zooxanthellae were also collected from the natural overflow from each plastic holding container. At each sampling interval, separate aliquots of coral host tissue and Symbiodinium cells were fixed in 0.2 M phosphate buffered saline (PBS, pH 7.2; ICN Biomedicals, Irvine, CA, USA) containing 3% glutaraldehyde for 24 hrs. Following fixation, both host tissue and Symbiodinium cell aliquots were centrifuged (2000× g) and washed three-times in 0.1 M Sorensen's phosphate buffer (pH 7.2; Electron Microscopy Sciences EMS, WA, USA) for 10 min. Samples were centrifuged to concentrate the Symbiodinium cells, estimated at a concentration of 1–2 × 106 cells ml− 1. In order to examine host cells by transmission electron microscopy (TEM), decalcification was performed using a solution of sodium citrate (10%) and formic acid (20%) following the methods of Rinkevich and Loya (1979) and Banin et al. (2000). After ∼24 hrs of decalcification, any remaining host tissue was gently crushed, and the solution centrifuged and similarly washed thrice. After the third wash and removal of the supernatant, the individual host samples including their zooxanthellae were embedded in 0.2 ml agar. This allowed us to analyze both symbiotic partners in the same samples. Expelled symbiont cells were embedded in a similar manner (Strychar et al., 2004b). When the agar solidified, the agar-embedded samples were post-fixed in 1% osmium tetroxide (EMS) in 0.1 M PBS for 14–24 h at 48 °C, after which the samples were centrifuged (500× g) and the supernatant removed. Samples were dehydrated in a graded series of acetone washes (30, 50, 70, 90 and 3× at 100%) at 30 min intervals. Once dehydrated, the embedded samples were suspended in a series of graded acetone–Spurr's resin (EMS) to ensure complete infiltration of the resin into all tissue. The samples were then polymerized into a mold by incubation at 60 °C for 3 d. Thin sections of each sample were obtained using an Ultracut T ultra-microtome (Leica Microsystems, Austria). Thin sections were placed on 3 mm copper grids (size 5, 300 mesh) coated with 1.2% (w/v) formvar in trichloromethane. They were then stained for 5 min with 2% (w/v) aqueous uranyl acetate followed by 1.5% (w/v) lead citrate for 5 min. After this, the samples were observed with a JEOL-1010 or Hitachi 7000 TEM. One hundred host tissue cells and 100 zooxanthellar cells were examined for each time interval per temperature per host - to analyze their ultrastructural characteristics. This included logging cell characteristics symptomatic of apoptosis, necrosis, and viability. Quantitative measurements of organelle structures were also taken for symbiont cells but not for host cells, as the responses of the latter were negligible in almost all cases. All data presented here are new. Some comparisons will be made with data published earlier and, in those cases, will be cited accordingly. 2.4. Sample preparation for Fluorescent Microscopy (FM) Fluorescent microscopy (FM) was used initially to confirm appropriate binding and fluorescent properties of dyes used to characterize apoptosis and necrosis, respectively, prior to the use of flow cytometry (FC). The stain used was a mixture of approximately 4 μl of propidium iodide (PI; 100 mg ml− 1; Sigma–Aldrich, Castle Hill, NSW, Australia) and 5 μl of Annexin V-fluor (AV-fluor; Bioscientific, Kirawee, NSW, Australia). AV-fluor was used to identify cells undergoing apoptosis (Strychar et al., 2004b). The conjugates bind to phosphatidylserine in cells undergoing apoptosis and fluoresce green when excited with a blue light (Martin et al., 1995). This mixture was added to aliquots containing 100 μl of Symbiodinium cell suspension (1.8 × 105 cells) and separate aliquots containing host cells. Aliquots containing stain were placed in the dark at room temperature for 2030 min, after which 10 ml of cell suspension were examined under a FM to ensure dye-binding specificity and distinct auto-fluorescence of each conjugate with respect to the host cells and the zooxanthellae. A control with no additives was used to compare and contrast results obtained from cultures containing stained CS-156 (Symbiodinium) cells.

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2.5. Sample preparation for Flow Cytometry (FC) Aliquots of AV-fluor and PI-stained in-situ coral host cells and Symbiodinium cells were examined with an EPICS Profile-II cytometer to discriminate between apoptotic and necrotic cells, as expelled zooxanthellar cells were analyzed in an earlier study (Strychar et al., 2004b). Visually, host and Symbiodinium cells processed by FC fluoresce red when stained with PI, green when stained with AVfluor, and will not stain with any of the dyes if viable (Strychar, 2002; Strychar et al., 2004b). Coral host cells were filtered to remove any remnants of crushed coral skeleton. In each study, a minimum of 100,000 events were examined by FC, comparing each group of replicates from each time period (e.g. 3, 9, 12 h) under each treatment (28, 30, 32, 34, and 36 °C), and assessing their physiological state (viable, apoptotic, necrotic). This was repeated for each coral species nine colonies per treatment. FC controls for both positive (viable) and negative (necrotic) cells were prepared fresh before each experimental period. Analysis of filtered host cells collected from crushed coral samples (see TEM preparation above) was problematic because the flow cell assembly (intake nozzle and micro-tubing) of the FC instrument frequently became clogged with CaCO3 sediment from the coral. Thus, data regarding apoptosis and necrosis in host cells was collected via TEM. 2.6. Statistical analyses Data were analyzed by multivariate ANOVA using SPSS V15.0, comparing each host, group of replicates, temperature, and time period against their associated physiological state (e.g., viable, apoptotic, and necrotic). Only statistically significant data and higher-order interactions will be discussed below. Lack of response to heat stress was also documented and will be discussed; those data, however, will not be presented in the figures. Details of statistical analytical results may be found in figure legends. 3. Results 3.1. Coral host and Symbiodinium cells – main effects There was no significant difference between the 28 °C control and the 30 °C treatment in either the host coral cell fraction or Symbiodinium fraction with respect to main effects (p N 0.05, MANOVA). Significantly higher numbers of host coral cells and Symbiodinium cells exhibiting apoptosis and necrosis characteristics were observed, however, from each of the corals tested at 32 and 34 °C, in comparison to the control temperature. 3.2. Transmission Electron Microscopy (TEM) 3.2.1. Response of Acropora hyacinthus host cells in-situ at 28-36 °C through time The percentage of viable host and symbiont cells in-situ was generally high at 28 °C (Fig. 1). As experimental temperatures were increased from 28 to 34 °C, however, the percentage of viable host cells remained constant while the number of viable symbiont cells decreased significantly (Fig. 1). Only negligible levels of apoptosis occurred at 2832 °C in in-situ host cells of A. hyacinthus (∼1% at 28 °C to ∼7% at 34 °C; Fig. 2). Necrosis levels were also very low in host cells (∼1% at 28 °C to ∼5% at 34 °C; Figs. 3 and 4) and did not increase significantly until the highest experimental temperature of 36 °C. On the other hand, in-situ Symbiodinium cells exhibited significantly increased frequencies of apoptosis (∼7% at 28 °C to ∼75% at 34 °C; Fig. 2), once a temperature of 30 °C was surpassed. This could also be seen within the host's symbiosome (Fig. 4E). Symptoms of necrosis were negligible in Symbiodinium cells (∼2% at 28 °C to ∼7% at 34 °C) until 36 °C (Figs. 5A-D, 3

Fig. 1. Effect of temperature on the percentage of viable Acropora hyacinthus host cells and Symbiodinium cells in-situ, collected over 12 h. Corals exposed to 28 °C (control), 30 °C, 32 °C, 34 °C, and 36 °C. (No significant difference found between control and 30 °C treatment; therefore, latter is not shown here.) (○) = observed viable Acropora host cells. (■) = observed viable Symbiodinium cells. Data presented as mean with 95% confidence intervals from nine separate experiments. Some confidence limits are too small to be seen. Each point represents the percentage (%) of different cell types analyzed via transmission electron microscopy (TEM). Time intervals correspond to peak periods where corals bleached at elevated temperatures, and where A. hyacinthus tissues were sampled and prepared for TEM analysis. 100 zooxanthellar cells were randomly examined per experimental temperature at each time interval, to help characterize normal, apoptotic, and necrotic processes. With respect to the coral host: Highly significant difference between temperatures (p b 0.001, MANOVA) and between time intervals (p b 0.001, MANOVA) exists; a highly significant interaction between temperature and time (p b 0.001) is also present. A posteriori tests (Tukey HSD and Bonferroni) indicate that all time intervals were significantly different from each other (p b 0.05-0.001). With respect to temperature, 28 °C, 30 °C, and 32 °C were not significantly different from each other; 34 °C and 36 °C, however, were significantly different from each other and all other temperatures (p b 0.001). With respect to Symbiodinium: Highly significant differences between temperatures (p b 0.001, MANOVA) and between time intervals (p b 0.001, MANOVA) exist; a highly significant interaction between temperature and time (p b 0.001) also occurs. A posteriori tests (Tukey HSD and Bonferroni) indicate that all time intervals are significantly different from each other (p b 0.05-0.001). Results from all experimental temperatures are significantly different (p b 0.001), except between 28 °C and 30 °C (p N 0.05), indicating a differential sensitivity between the host and its symbiont.

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∼65% (Figs. 2 and 4). Cell viability of the symbiont was also significantly lower at these higher temperatures (∼10%; Figs. 3 and 4). Both the host and symbiont cells exhibited similar symptoms of rapid cell death over 12 h, including the formation of apoptotic-like bodies (Fig. 5E).

Fig. 2. Effect of temperature on the percentage of apoptotic Acropora hyacinthus host cells and Symbiodinium cells in-situ, collected over 12 h. Corals exposed to 28 °C (control), 30 °C, 32 °C, 34 °C, and 36 °C. (No significant difference found between control and 30 °C treatment; therefore, latter is not shown here.) (○) = observed apoptotic host cell subpopulations. (■) = observed apoptotic Symbiodinium cell sub-populations. Data presented as mean plus 95% confidence intervals from nine separate experiments. Some confidence limits are too small to be seen. (See legend of Fig.1 for additional details). With respect to the coral host: Highly significant difference between temperatures (p b 0.001, MANOVA) and between time intervals (p b 0.001, MANOVA), and a highly significant interaction between temperature and time (p b 0.001). A posteriori tests (Tukey HSD and Bonferroni) indicate that all time intervals are significantly different from each other (p b 0.01−0.001). With respect to temperature, tests indicated no significant differences between 28 °C, 30 °C, and 32 °C, exist; significant differences, however, between 34 °C, and 36 °C, and between those and all other temperatures. With respect to Symbiodinium: Highly significant differences between temperatures (p b 0.001, MANOVA) and between time intervals (p b 0.001, MANOVA); highly significant interaction between temperature and time (p b 0.001). A posteriori tests (Tukey HSD and Bonferroni) indicate that all time intervals are significantly different from each other (p b 0.01−0.001), and that results from all experimental temperatures are significantly different (p b 0.001) with respect to apoptosis, except between 28 °C, and 30 °C, and between 34 °C, and 36 °C, (p N 0.05).

and 4). At 36 °C, the host still exhibited high frequencies of viable cells (∼75%; Fig. 1) and low frequencies of apoptotic cells (∼20%; Fig. 2). The in-situ symbiont cells, however, exhibited significantly higher levels of apoptosis even at 30 °C, and plateaued at 34 °C, to a maximum level of

Fig. 3. Effect of temperature on the percentage of necrotic Acropora hyacinthus host cells and Symbiodinium cells in-situ, collected over 12 h. Corals exposed to 28 (control), 30 °C, 32 °C, 34 °C, and 36 °C. (No significant difference found between control and 30 °C, treatment; therefore, latter is not shown here.) (○) = observed necrotic host cell subpopulations. (■) = observed necrotic Symbiodinium cell sub-populations. Data presented as mean plus 95% confidence intervals from nine separate experiments. Some confidence limits are too small to be seen. (See legend of Fig.1 for additional details). With respect to the coral host: Highly significant differences between temperatures (p b 0.001, MANOVA) and time intervals (p b 0.001, MANOVA); highly significant interaction between temperature and time (p b 0.001). A posteriori tests (Tukey HSD and Bonferroni) indicate that the 12 hr time interval is highly significantly different from all others (p b 0.001), indicating that necrosis is not detectable in host cells until 12 hrs after exposure to high temperatures. A posteriori tests did not detect significant differences between temperatures until 36 °C, which was significantly different from all others (p b 0.001). With respect to Symbiodinium: Highly significant differences between temperatures (p b 0.001, MANOVA) but not between time intervals (p b 0.001, MANOVA) occurs. A highly significant interaction, however, between temperature and time (p b 0.001). A posteriori tests (Tukey HSD and Bonferroni) indicated that significant differences in time were not strong enough to be picked up by these tests (p N 0.05) due to highly significant interactions with temperature. A posteriori tests detected significant differences between 36 °C, and all other temperatures (p b 0.001).

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Fig. 4. Transmission electron micrographs showing Acropora hyacinthus host cells and symbiont (Symbiodinium) cells in-situ at 28-36 °C. Host tissue appears normal at 28-34 °C (A−D); symptoms of apoptosis or necrosis not observed in host cells. After 12 h at 32 °C (C), many Symbiodinium cells exhibited such symptoms. After 12 h at 34 °C, early signs of apoptosis and necrosis began to appear (D) in the host tissue and within the symbiosome associated with Symbiodinium (E). At 36 °C, host cells exhibit symptoms of cell death (F). Symbols: cl = chloroplast, m = mitochondria, n = nucleus, sg = starch grain. 100 zooxanthellar cells were randomly examined per experimental temperature at each time interval, to help characterize normal, apoptotic, and necrotic processes. Scale bar provided for each micrograph.

3.2.2. Ultrastructural changes in the expelled Symbiodinium cells of Acropora hyacinthus and Porites solida in response to temperature (28-34 °C) At the lower experimental temperatures of 28-30 °C (Figs. 5A,B and 6A,B), Symbiodinium cells exhibited ‘normal’ characteristics after expulsion from A. hyacinthus and P. solida (see Kevin et al., 1969; Strychar et al., 2004a for greater detail). The cells were primarily viable and coccoid in shape (12 ± 1.3 μm; mean ± 16 95% CI) and

possessed multi-lobed and elongated chloroplasts with distinct lamellae. At 32 °C and 34 °C, cytoplasmic organelles normally found as ‘loose’ aggregates in a viable Symbiodinium cell (e.g. chloroplasts, nuclei, and mitochondria), began to fragment and become less distinct (Figs. 5C,D and 6C,D). Those cells that became apoptotic also became smaller in size (6.5 ± 2.4 μm, e.g. Figs. 5E and 6E), and the cytoplasmic organelles within them fused together, forming large organelle bodies. At later stages of apoptosis, separate chloroplast organelles could no

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Fig. 5. Transmission electron micrographs of Symbiodinium cells expelled from Acropora hyacinthus at temperatures 28-34 °C. Most Symbiodinium cells appeared normal at 28-30 °C (A,B). Symptoms of reversible early- to mid-stage apoptosis (C) or necrosis (D) occurred at 32 °C within 12 h. After 9 h at 34 °C, many Symbiodinium cells exhibited symptoms of late non-reversible apoptosis (E⁎) and necrosis (F). Symbols: ab = apoptotic body, chr = chromosome, cl = chloroplast, cw = cell wall, gx = glyoxysome (see Lesser and Shick, 1990; Clode and Marshall, 2002 for discussion of these organelles in anemones and corals) m = mitochondria, n = nucleus, py = pyrenoid, sg = starch grain. * = Panel derived from Strychar et al. (2004a) for comparative purposes; reprinted by permission of Allen Press Publishing Services from Phycologia, by Kevin B. Strychar et al. (2004a), copyright © 2004 International Phycological Society. 100 zooxanthellar cells were randomly examined every time interval per temperature to help characterize normal, apoptotic, and necrotic processes. Scale bar= 500 nm.

longer be discerned (Figs. 5E and 6E). Lamellae within the chloroplasts, however, were occasionally observed as ‘free-floating’ throughout the cell cytoplasm (Fig. 5D) – a symptom of late apoptosis. The final apoptotic stage occurs when the cell membrane ruptured. Elevated temperatures (≥32 °C) also caused necrosis in some cells (Figs. 5F and 6F). This appearance was short in duration since the cell membrane of Symbiodinium disintegrated and cytosolic contents were

released into the symbiosome, coral host tissue, or environment in both A. hyacinthus and P. solida. 3.2.3. Temporal Response of Symbiodinium cells expelled from Acropora hyacinthus and Porites solida at 32 °C Changes in ultrastucture of Symbiodinium cells in Acropora hyacinthus were first clearly observable in the 32 °C treatment. At

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Fig. 6. Transmission electron micrographs of Symbiodinium cells expelled from Porites solida at temperatures 28−34 °C. Symbiodinium cells appeared normal at 28−32 °C (A,B,C). Within 12−18 h of exposure, symptoms of early apoptosis occurred (D⁎). After prolonged exposure (N24 h) at 34 °C, many Symbiodinium cells exhibited symptoms of late nonreversible apoptosis (E⁎) and necrosis (F⁎) symptoms. Symbols: ab = apoptotic body, chr = chromosome, cl = chloroplast, cw = cell wall, dp = disaggregated polysomes, gx = glyoxysome, m = mitochondria, n = nucleus, nl = nucleolus, py = pyrenoid, sg = starch grain. 100 zooxanthellar cells were randomly examined per experimental temperature at each time interval, to help characterize normal, apoptotic, and necrotic processes. Scale bar = 500 nm. * = Panels derived from Strychar et al. (2004a) for comparative purposes; reprinted by permission of Allen Press Publishing Services from Phycologia, by Kevin B. Strychar et al. (2004a), copyright © 2004 International Phycological Society.

higher temperatures, the progression of changes was very similar but accelerated. Here, we will provide a detailed account of the responses within the 32 °C treatment. There were no ultrastructural changes in the expelled Symbiodinium cells in the 32 °C treatment at ≤6 h (Fig. 7A,B). After 9 h, however,

evidence of apoptosis and necrosis in the Symbiodinium cells emerged (Fig. 7C,D, respectively). Exposures between 9 and 12 h (Fig. 7E) resulted in less distinct organelles and their fusion to form one large organelle body, particularly in apoptotic cells. After 12 h, free-floating chloroplast lamellae were observed within the cell - a diagnostic

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Fig. 7. Transmission electron micrographs of Symbiodinium cells expelled from Acropora hyacinthus through a 12 h period at 32 °C, in sequence. Images represent samples taken every 3 h. Onset of specific stages of apoptosis and necrosis within the symbiont cells occurred within 9 h (C and D). Within 12 h, mid- to late- apoptotic (E) and necrotic (F) stages entered non-reversible phases. Symbols: chr = chromosome, cl = chloroplast, gx = glyoxysome, m = mitochondria, n = nucleus, py = pyrenoid, sg = starch grain. 100 zooxanthellar cells were randomly examined per experimental temperature at each time interval, to help characterize normal, apoptotic, and necrotic processes. Scale bar = 500 nm.

characteristic of cell death (Fig. 7F; see Strychar et al., 2004a). At this stage, the cell membrane of Symbiodinium disintegrated, and the cytosolic contents were released. The above changes did not occur in the expelled symbionts derived from Porites solida until temperatures reached 34 °C. Other than this, the observed changes tracked those in A. hyacinthus. Therefore, the detailed responses of Symbiodinium in this coral species will not be considered further.

3.3. Fluorescent Microscopy (FM) Viable cells of either the host or Symbiodinium stained with HO342 fluoresced blue. Those stained with the conjugate AV-fluor fluoresced green. We did not observe any apoptotic or necrotic cells in any of the coral host cells in either the 28 °C control or in any of the higher treatment temperatures, including 34 °C. Host cells did not exhibit symptoms of apoptotic and necrotic cell death until the highest

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Table 1 Percent (%) of viable, apoptotic, and necrotic Symbiodinium cells expelled from the scleractinian corals Acropora hyacinthus and Porites solida at experimental temperatures of 28 °C, 32 °C, and 34 °C, respectively Temperature = 28 °C 3h

Temperature = 32 °C

Temperature = 34 °C

9h

12 h

3h

9h

12 h

3h

9h

12 h

Acropora hyacinthus Cell Viability Viable 78.1 ± 0.32 Apoptotic 7.8 ± 0.28 Necrotic 5.0 ± 0.07

78.9 ± 0.17 7.6 ± 0.29 4.3 ± 0.09

82.9 ± 0.46 5.4 ± 0.26 3.3 ± 0.09

70.0 ± 0.5 1.4 ± 0.1 8.6 ± 0.2

54.7 ± 0.2 9.9 ± 0.1 17 ± 0.1

27 ± 0.4 28.9 ± 0.2 29.7 ± 0.4

18.9 ± 1.0 6.0 ± 0.1 52.5 ± 0.9

19.5 ± 0.3 2.9 ± 0.2 41.2 ± 0.4

27.8 ± 0.4 5.2 ± 0.5 31.4 ± 0.7

Porites solida Cell Viability Viable Apoptotic Necrotic

54.8 ± 0.14 0.03 ± 0.0 4.1 ± 0.19

53.2 ± 0.18 0.06 ± 0.03 4.2 ± 0.03

65.4 ± 0.26 2.1 ± 0.05 5.6 ± 0.01

67.2 ± 0.17 2.1 ± 0.07 6.2 ± 0.22

60.4 ± 0.47 3.1 ± 0.2 9.3 ± 0.26

41.7 ± 1.0 1.1 ± 0.32 16.2 ± 0.21

44.1 ± 0.26 9.2 ± 0.42 11.8 ± 0.56

32.8 ± 1.4 2.4 ± 0.12 19.8 ± 1.1

54.2 ± 0.32 0.1 ± 0.0 4 ± 0.05

Data collected via flow cytometry 3, 9, and 12 hrs after initiation of experiment. Presented as mean ± standard error of the mean, based on a cell population size of n(i) = 100,000. Derived from Strychar et al. (2004b; see that paper for additional details).

experimental temperature of 36 °C. At temperatures as low as 30 °C and above, however, we observed increased concentrations of expelled apoptotic and necrotic Symbiodinium cells in both A. hyacinthus and P. solida corals. This was confirmed using FC. 3.4. Analysis of Symbiodinium via Flow Cytometry (FC) Data are presented here for viability, apoptosis, and necrosis in the cells of Symbiodinium sp. that have been expelled from the host. Data may be found in Table 1, providing mean, standard error of the mean, and sample size under the various treatments used here. Data are summarized from Strychar et al. (2004b) and are provided here to facilitate comparison with the above in-situ cell data. The reader is referred to Strychar et al. (2004b) for further details. It may be seen that the frequency of symbiont cell loss in both Acropora hyacinthus and Porites solida increased with both elevated temperature and prolonged exposure. After 12 hrs, when temperature was increased from 28 °C to 34 °C, the viability of the expelled symbiont cells fell from 80% to 20% in A. hyacinthus and from 55% to 35% in P. solida (Table 1). The frequency of necrotic Symbiodinium cells expelled from A. hyacinthus increased from ∼5% at 28 °C to 60% at 34 °C. This was much higher than that for P. solida (∼4% to ∼15% over these temperatures (Table 1). At 12 hrs, the number of apoptotic symbiont cells expelled from both coral host species increased with higher temperatures and as time progressed. 4. Discussion For the host coral, variability in temperature is clearly not as important a factor for survival as it is for its algal symbionts. An obvious difference between the coral host and its symbiont is the lack of limitations that are associated with being photosynthetic – i.e., sensitivity to changes in light levels, light spectra, and temperature. The reduction in the frequency of viable symbiont cells we observed is consistent with the findings of Rowan (2004). He tested the quantum yield of photosystem II (PSII) in Clade C zooxanthellae and found that when water temperatures were raised from 28.5 °C to 32 °C, the PSII pigments decreased significantly. In Clade D symbionts, however, he noted that the quantum yield of these pigments increased with temperature. Sotka and Thacker (2005) interpreted this to be an adaptive response. Baker et al. (2004) and Muller-Parker et al. (2007) have also shown that different clades of Symbiodinium spp. have broad thermal tolerances. Baker et al. (2004) and Ulstrup et al. (2006) found that Clade D symbionts are more thermally tolerant and dominate the population following heat-induced bleaching episodes. Our results indicate that bleaching in corals is not linked to the coral part of this symbiotic relationship – but to the plant. The coral animals appear to exhibit exaptation to increases in SSTs. The plants, on the other hand, are more restricted in their temperature tolerances and are unable

to cope with these high temperatures. Warner, Schmidt, and Fitt (unpubl., cited in Fitt et al., 2000; and pers. comm. to KBS) monitored the effects of temperature on optimum photosynthetic and growth rates in different species of Symbiodinium. They reported that the zooxanthellae they investigated require different temperature regimes for sustained growth. Some zooxanthellae grew well between 26 and 32 °C; others were suppressed at ≥26 °C. Kinzie et al. (2001) also examined growth rates in different Symbiodinium isolates at 27, 29, and 31 °C. They found that some displayed poor growth rates at higher temperatures, some exhibited no response at all to temperature, and others exhibited higher growth rates at 31 °C - a temperature almost always associated with bleaching (Drollet et al.,1995; Davies et al., 1997; Sammarco et al., 2006). The lower rates of loss of viable, apoptotic, or necrotic cells in the coral host vs. Symbiodinium indicates that host cells are clearly better adapted to elevated temperatures. Apoptosis and necrosis were not observed in the coral host tissue but were commonly observed in Symbiodinium. These results agree with those of Mise and Hidaka (2003) who studied effects of increased temperature on Acropora nasuta. They described rapid degradation of zooxanthellae cells under these conditions, detailing symptoms characteristic of apoptosis and necrosis (see Strychar et al., 2004a). They report that the damaged symbiont cells were removed by the host. We interpret this to mean that the host was in better health. This is similar to observations made by Marlow and Martindale (2007) in the gastrulation of certain coral larvae during the early stages of development preceding settlement. In this case, membrane-bound cellular fragments are passed to the blastocoel where they are eventually phagocytosed and later form future sites of endodermal tissue, encouraging Symbiodinium to invade. This implies that the host controls zooxanthellar movements in-situ. Dunn et al. (2002, 2004) also report apoptosis-like cell death in anemones. They report, however, that cell death occurs simultaneously in the host and symbionts within minutes of heat stress. This disagrees with our results and those of Hill and Ralph (2007), where the loss of cell integrity did not manifest itself until at least 6-12 hrs. after the stress. Dunn et al. (2004) also reported that anemone ectoderm tissue exhibited little damage, but that the endoderm which contains the zooxanthellae exhibited significant apoptosis-like symptoms. These authors suggest that the amount of damage that occurs to host tissues rivals that of zooxanthellate tissues and that the host underwent significant stress. Thus, the host would appear to be more affected by temperature stress than the endosymbionts. Our results suggest that damage occurs only under conditions of prolonged exposure to high temperatures (32 °C for A. hyacinthus and 34 °C for P. solida) or at exceedingly high temperatures (36 °C), and not during short periods of increased SSTs. It is possible that the experimental animals that Dunn et al. (2004, 2006) used vs. ours yielded different responses due to their different respective physiologies. Another interpretation of these data is that the endodermal breakdown described by Dunn et al. (2002, 2004) may be a hypersensitive

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response, like those known to occur in some terrestrial plants infected by a pathogen (Lam et al., 2001). Heat stress may trigger a mechanism by which the host coral rids itself of unwanted symbionts, ensuring the survival of only the most robust symbiont cells within its tissue. The results reported here have important implications for the coralalgal symbiosis in both physiological and evolutionary time. In terms of adaptation, it is clear that the algae are “doing most of the dying” in this symbiotic relationship, allowing for adaptation to occur through natural selection of more robust Symbiodinium individuals. This concurs with the results of Grottoli et al. (2006) who observed resilience of the host coral in bleaching colonies of Montipora capitata. In the absence of its symbionts, over six weeks, the coral fully replenished its energy reserves and biomass by increasing its food intake five-fold. This also indicated that the host is less susceptible to heat stress than the symbiont. By contrast, Day et al. (2008) suggested that coral colonies more susceptible to heat stress are removed from the population through natural selection, increasing representation of more fit colonies in the population and allowing disfavored holobiont alleles (less resistant heat-stress alleles) to be removed from the ecosystem. It is possible for both the coral host and the symbiont to become adapted to extreme environments. Consider the environment of corals in the Red Sea, where temperatures routinely reach 34 °C (Ateweberhan et al., 2005) with salinities up to 40.5 ppt (Okbah et al., 1999; KarakoLampert et al., 2004). It is critical that some variability in physiological tolerance to temperature be present, and that this variability have a genetic basis (Palumbi 1994; Smith-Keune and van Oppen 2006; Ulstrup et al., 2006). This would be particularly important if temperatures exceed the average tolerance range of the host. The coral host is also dependent upon rapid adaptation of its endosymbionts. The zooxanthellae appear to be continually adapting to temperature increases that are tolerable to the host (Figs. 5–7 and Table 1 vs. 1−4). In this set of experiments, the host clearly survived the temperature shifts (Figs. 1–4). 5. Conclusion: Thermal tolerance of coral hosts vs. zooxanthellae Our major conclusion is that the coral hosts appear well adapted to temperature stress and that the endosymbiotic zooxanthellae will either die or experience irreversible cell damage prior to expulsion from the coral. In addition, zooxanthellae are being actively expelled by the coral; they are not leaving of their own volition (Kinzie et al., 2001; Lewis and Coffroth, 2004; Little et al., 2004; Strychar et al., 2005). Based on the data presented here, we hypothesize that these coral species are experiencing little or no mortality from increased SSTs – but a great deal of mortality from the loss of their zooxanthellae. It would appear that the coral hosts exhibit exaptation to temperatures as high as 34 °C. Were it not for the loss of zooxanthellae, some coral hosts might be able to survive indefinitely at these temperatures. In our study, it was not until they reached extraordinarily high temperatures (i.e., 36 °C) that they began to exhibit symptoms of apoptosis, in a species-specific manner. Acknowledgments We thank the Great Barrier Reef Marine Park Authority for grants awarded to KBS. We also thank T. Piva and M. Coates for their contributions to earlier parts of this research and Dr. T. Pesecreta and the Electron Microscopy Center, Dept. Biology, University of Louisiana at Lafayette for usage of their SEM. We also thank Dr. Jeff Levinton for critiquing an earlier version of this manuscript. [SS] References Ainsworth, T.D., Kvennefors, E.C., Blackall, L.L., Fine, M., Hoegh-Guldberg, O., 2007. Disease and cell death in white syndrome of acroporid corals on the Great Barrier Reef. Mar. Biol. 151, 19–29. Ameisen, J.C., 2002. On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ. 9, 367–393.

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Ateweberhan, M., Bruggemann, J.H., Breeman, A.M., 2005. Seasonal patterns of biomass, growth and reproduction in Dictyota cervicornis and Stoechospermum polypodioides (Dictyotales, Phaeophyta) on a shallow reef flat in the southern Red Sea (Eritrea). Bot. Mar. 48, 8–17. Baker, A.C., 2001. Reef corals bleach to survive change. Nature 411, 765–766. Baker, A., Starger, C., McClanahan, T., Glynn, P., 2004. Corals' adaptive response to climate change. Nature 430, 741. Banin, E., Israely, T., Kushmaro, A., Loya, Y., Orr, E., Rosenberg, E., 2000. Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica. Appl. Environ. Microbiol. 66, 3031–3036. Berkelmans, R., Willis, B.L., 1999. Seasonal and local spatial patterns in the upper thermal limits of corals on the inshore Central Great Barrier Reef. Coral Reefs 18, 219–228. Berkelmans, R., 2002. Time-integrated thermal bleaching thresholds of reefs and their variation on the Great Barrier Reef. Mar. Ecol. Prog. Ser. 229, 73–82. Bhagooli, R., Hidaka, M., 2003. Comparison of stress susceptibility of in-hospite and isolated zooxanthellae among five coral species. J. Exp. Mar. Biol. Ecol. 291, 181–197. Bhagooli, R., Hidaka, M., 2004a. Photoinhibition, bleaching susceptibility and mortality in two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata, in response to thermal and light stress. Comp. Biochem. Physiol. 137, 547–555. Bhagooli, R., Hidaka, M., 2004b. Release of zooxanthellae with intact photosynthetic activity by the coral Galaxea fascicularis in response to high temperature stress. Mar. Biol. 145, 329–337. Brown, B.E., 1997. Coral bleaching: causes and consequences. Proc. 8th Coral Reef Symp. 1, 65–74. Brown, B.E., Suharsono, K.W., 1990. Damage and recovery of coral reefs affected by El Niño related seawater warming in the Thousand Islands, Indonesia. Coral Reefs 8, 163–170. Buddemeier, R.W., Fautin, D.G., 1993. Coral bleaching as an adaptive mechanism: a testable hypothesis. BioScience 43, 320–325. Bursch, W., Ellinger, A., Gerner, C., Fröhwein, U., Schulte-Hermann, R., 2000. Programmed cell death (PCD): Apoptosis, autophagic PCD, or others? Ann. NY Acad. Sci. 926, 1–12. Clode, P.L., Marshall, A.T., 2002. Kalisomes in corals: a novel KCl concentrating organelle? Tissue Cell 34, 199–209. Coles, S.L., Jokiel, P.L., Lewis, C.R., 1976. Thermal tolerance in tropical versus subtropical Pacific reef corals. Pac. Sci. 30, 159–166. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1–20. Dash, P., 2008. Apoptosis. http://www.sgul.ac.uk/dept/immunology/~-dash. Davies, J.M., Dunne, R.P., Brown, B.E., 1997. Coral bleaching and elevated sea-water temperature in Milne Bay Province, Papua New Guinea, 1996. Mar. Freshw. Res. 48, 513–516. Day, T., Nagel, L., van Oppen, J.H., Caley, M.J., 2008. Factors affecting the evolution of bleaching resistance in corals. Am. Nat. 171, E72–E88. Dennis, C., 2002. Reef under threat from bleaching outbreak. Nature 415, 947. Done, T., Turak, E., Wakeford, M., De'ath, G., Kininmonth, S., Wooldridge, S., Berkelmans, R., van Oppen, M., 2003. Testing bleaching resistance hypotheses for the 2002 Great Barrier Reef bleaching event. Unpublished report to The Nature Conservancy. Australian Institute of Marine Science, 106 pp. Donner, S.D., Skirving, W.J., Little, C.M., Oppenheimer, M., Hoegh-Guldberg, O., 2005. Global assessment of coral bleaching and required rates of adaptation under climate change. Global Change Biol. 11, 2251–2265. Drollet, J., Faucou, H.M., Martin, P.M.V., 1995. Elevated sea-water temperature and solar UV-B flux associated with two successive coral mass bleaching events in Tahiti. Mar. Freshw. Res. 46, 1153–1157. Dunn, S.R., Bythell, J.C., Le Tissier, M.D.A., Burnett, W.J., Thomason, J.C., 2002. Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp. J. Exp. Mar. Biol. Ecol. 272, 29–53. Dunn, S.R., Thomason, J.C., Le Tissier, M.D.A., Bythell, J.C., 2004. Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration. Cell Death Differ. 11, 1213–1222. Dunn, S.R., Phillips, W.S., Spatafora, J.W., Green, D.R., Weis, V.M., 2006. Highly conserved caspase and Bcl-2 homologues from the sea anemone Aiptasia pallida: lower metazoans as models for the study of apoptosis evolution. J. Mol. Evol. 63, 95–107. Dunn, S.R., Schnitzler, C., Weis, V.M., 2007. Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose. Proc. R. Soc. Lond. B. 274, 3079–3085. Edmunds, P.J., Davies, P.S., 1989. An energy budget for Porites porites (Scleractinia), growing in a stressed environment. Coral Reefs 8, 37–43. Estes, A.M., Kempf, S.C., Henry, R.P., 2003. Localization and quantification of carbonic anhydrase activity in the symbiotic scyphozoan Cassiopea xamachana. Biol. Bull. 204, 278–289. Fitt, W.K., McFarland, F.K., Warner, M.E., Chilcoat, G.C., 2000. Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching. Limnol. Oceanogr. 45, 677–685. Franklin, D.J., Brussaard, C.P.D., Berges, J.A., 2006. What is the role and nature of programmed cell death in phytoplankton ecology? Eur. J. Phycol. 41, 1–14. Futuyma, D.J., 1998. Evolutionary biology, 3rd ed. Sinauer Associates, Sunderland, MA. 763+ pp. Gates, R.D., Edmunds, P.J., 1999. The physiological mechanisms of acclimatization in tropical reef corals. Am. Zool. 39, 30–43. Glynn, P.W., 1991. Coral reef bleaching in the 1980s and possible connections with global warming. TREE 6, 175–179. Goreau, T., McClanahan, T., Hayes, R., Strong, A., 2000. Conservation of coral reefs after the 1998 global bleaching event. Conserv. Biol. 14, 5–16.

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Gould, S.J., Vrba, E.S., 1982. Exaptation - a missing term in the science of form. Paleobiology 8, 4–15. Grimsditch, G.D., Salm, R.V., 2005. Coral reef resilience and resistance to bleaching. IUCN, Gland, Switzerland. Grottoli, A.G., Rodrigues, L.J., Palardy, J.E., 2006. Heterotrophic plasticity and resilience in bleached corals. Nature 440, 186–189. Hill, R., Ralph, P.J., 2007. Post-bleaching viability of expelled zooxanthellae from the scleractinian coral Pocillopora damicornis. Mar. Ecol. Prog. Ser. 352, 137–144. Hoegh-Guldberg, O., 1994. Mass-bleaching of coral reefs in French Polynesia, April 1994. Climate Impacts Series, vol. 1. Greenpeace International. 36 pp. Hoegh-Guldberg, O., 1999. Climate change, coral bleaching, and the future of the world's coral reefs. Green Peace International, Washington, DC, USA. 27 pp. Hoegh-Guldberg, O., Jones, R.J., Ward, S., Loh, W.K., 2002. Is coral bleaching really adaptive? Nature 415, 601–602. Hughes, T.P., 1994. Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef. Science 265, 1547–1551. Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A.A., Natale, C., Santacroce, R., Di Corcia, M.G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simone, S., Bucci, R., Farber, E., 2002. Cell death: apoptosis versus necrosis. Int. J. Oncol. 21, 165–170. Karako-Lampert, S., Katcoff, D.J., Achituv, Y., Dubinsky, Z., Stambler, N., 2004. Do clades of symbiotic dinoflagellates in scleractinian corals of the Gulf of Eilat (Red Sea) differ from those of other coral reefs? J. Exp. Mar. Biol. Ecol. 311, 301–314. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. Kevin, M.K., Hall, W.T., Mclaughlin, J.J.A., Zahl, P.A., 1969. Symbiodinium microadriaticum Freudenthal. A revised taxonomic description ultrastructure. J. Phycol. 5, 341–350. Kinzie, R.A., Takayama, M., Santos, S.R., Coffroth, M.A., 2001. The adaptive bleaching hypothesis: experimental tests of critical assumptions. Biol. Bull. 200, 51–58. Lam, E., Kato, N., Lawton, M., 2001. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848–853. Lesser, M.P., Farrell, J.H., 2004. Exposure to solar radiation increases damage to both host tissues and algal symbionts of corals during thermal stress. Coral Reefs 23, 367–377. Lesser, M.P., Shick, J.M., 1990. Effects of visible and ultraviolet radiation on the ultrastructure of zooxanthellae (Symbiodinium sp.) in culture and in-situ. Cell Tissue Res. 261, 501–508. Lewis, C.L., Coffroth, M.A., 2004. The acquisition of exogenous algal symbionts by an Octocoral after bleaching. Science 304, 1490–1492. Lim, P.O., Kim, H.J., Nam, H.G., 2007. Leaf senescence. Annu. Rev. Plant Biol. 58, 115–136. Little, A.F., van Oppen, M.J., Willis, B.L., 2004. Flexibility in algal endosymbioses shapes growth in reef corals. Science 304, 1492–1494. Marlow, H.Q., Martindale, M.Q., 2007. Embryonic development in two species of scleractinian coral embryos: zooxanthellae localization and gastrulation mechanism. Evol. Dev. 9, 355–367. Marcus, J., Thorhaug, A., 1981. Pacific versus Atlantic responses of the subtropical hermatypic coral Porites spp. to temperature and salinity effects. Proc. 4th Intl. Coral Reef Symp. 2, 15–20. Marshall, P.A., Baird, A.H., 2000. Bleaching of corals on the Great Barrier Reef: Differential susceptibilities among taxa. Coral Reefs 19, 155–163. Martin, S.J., Reutenlingsperger, C.P., McGahon, A.J., Rader, J.A., van Schie, R.C., Laface, D.M., Green, D.R., 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by over expression of Bcl-2 and Ab1. J. Exp. Med. 182, 1545–1556. McCloskey, L.R., Muscatine, L., Wilkerson, F.P., 1994. Daily photosynthesis, respiration and carbon budgets in a tropical marine jellyfish (Mastigias sp.). Mar. Biol. 116, 13–22. Mise, T., Hidaka, M., 2003. Degradation of zooxanthellae in the coral Acropora nasuta during bleaching. Galaxea 5, 33–39. Muller-Parker, G., Pierce-Cravens, J., Bingham, B.L., 2007. Broad thermal tolerance of the symbiotic dinoflagellate Symbiodinium muscatinei (Dinophyta) in the sea anemone Anthopleura elegantissima (Cnidaria) from northern latitudes. J. Phycol. 43, 25–31. Obura, D.O., 2005. Resilience and climate change: lessons from coral reefs and bleaching in the Western Indian Ocean. Estuar. Coast. Shelf Sci. 63, 353–372. Okbah, M.A., Mahmoud, T.H., El-Deek, M.S., 1999. Nutrient salts concentrations in the Gulf of Awa and northern Red Sea. Bull. Nat. Inst. Ocean. Fish. (Egypt). 25, 103–116. Palumbi, S.R., 1994. Genetic divergence, reproductive isolation, and marine speciation. Ann. Rev. Ecol. Syst. 25, 547–572.

Podesta, G.P., Glynn, P.W., 1997. Sea surface temperature variability in Panamá and Galápagos: extreme temperatures causing coral bleaching. J. Geophys. Res. 102, 15749–15759. Ravindran, J., Raghukumar, C., 2006. Histological observations on the scleractinian coral Porites lutea affected by pink-line syndrome. Curr. Sci. 90, 720–723. Reape, T.J., Molony, E.M., McCabe, P.F., 2008. Programmed cell death in plants: distinguishing between different modes. J. Exp. Bot. 59, 435–444. Reaser, J.K., Pomerance, R., Thomas, P.O., 2000. Coral bleaching and global climate change: Scientific findings and policy recommendations. Conserv. Biol. 14, 1500–1511. Reynolds, R.W., Smith, T.M., 1994. Improved global sea surface temperature analyses. J. Climate. 7, 929–948. Riegl, B., 2003. Climate change and coral reefs: different effects in two high-latitude areas (Arabian Gulf, South Africa). Coral Reefs 22, 433–446. Rosenberg, E., Loya, Y., 2004. Coral health and disease. Springer-Verlag, New York. Rinkevich, B., Loya, Y., 1979. The reproduction of the Red Sea coral Stylophora pistillata. I. Gonads and planulae. Mar. Ecol. Prog. Ser. 1, 133–144. Rowan, R., 2004. Coral bleaching: thermal adaptation in reef coral symbionts. Nature 430, 742. Sandeman, I.M., 2006. Fragmentation of the gastrodermis and detachment of zooxanthellae in symbiotic cnidarians: a role for hydrogen peroxide and Ca2+ in coral bleaching and algal density control. Rev. Biol. Trop. 54, 79–96. Sammarco, P.W., Winter, A., Stewart, J., 2006. Coefficient of variation of sea surface temperature (SST) as an indicator of coral bleaching. Mar. Biol. 149, 1337–1344. Schlichter, D., Svoboda, A., Keremer, B.P., 1983. Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host. Mar. Biol. 78, 29–38. Smith-Keune, C., van Oppen, M.J.H., 2006. Genetic structure of a reef-building coral from thermally distinct environments on the Great Barrier Reef. Coral reefs 25, 493–502. Sotka, E.E., Thacker, R.W., 2005. Do some corals like it hot? TRENDS Ecol. Evol. 20, 59–62. Strychar, K.B., 2002. Bleaching in soft and scleractinian corals: comparison of the physiology, genetics, and biochemistry of Symbiodinium. PhD. Thesis, Central Queensland University, Rockhampton, Australia. 294 pp. Strychar, K.B., Coates, M., Piva, T.J., 2001. Effect of water temperature on the viability of Symbiodinium lost from the scleractinian coral Acropora hyacinthus. Austr. Soc. Biochem. Mol. Biol. 61–64. Strychar, K.B., Coates, M.C., Sammarco, P.W., Piva, T.J., 2004a. Apoptotic and necrotic stages of cell death activity: bleaching of soft and scleractinian corals. Phycologia 43, 768–777. Strychar, K.B., Coates, M.C., Sammarco, P.W., Piva, T.J., 2004b. Bleaching as a pathogenic response in scleractinian corals, evidenced by high concentrations of apoptotic and necrotic zooxanthellae. J. Exp. Mar. Biol. Ecol. 304, 99–121. Strychar, K.B., Coates, M.C., Sammarco, P.W., 2005. Loss of Symbiodinium from bleached soft corals Sarcophyton ehrenbergi, Sinularia sp, and Xenia sp. J. Exp. Mar. Biol. Ecol. 320, 159–177. Ulstrup, K.E., Berkelmans, R., Ralph, P.J., van Oppen, M.J.H., 2006. Variation in bleaching sensitivity of two coral species across a latitudinal gradient on the Great Barrier Reef: the role of zooxanthellae. Mar. Ecol. Prog. Ser. 314, 135–148. van Cruchten, S., van den Broeck, W., 2002. Morphological and biochemical aspects of apoptosis, oncosis, and necrosis. Anat. Hist. Embryol. 31, 214–223. Vargas-Angel, B., Zapata, F., Hernandez, H., Jimenez, J., 2001. Coral and coral reef responses to the 1997-1998 El Niño event on the Pacific coast of Colombia. Bull. Mar. Sci. 69, 111–132. Vaux, D.L., Strasser, A., 1996. The molecular biology of apoptosis. Proc. Natl. Acad. Sci. U. S.A. 93, 2239–2244. Warner, M., Fitt, W.K., Schmidt, G.W., 1996. The effects of elevated temperature on the photosynthetic efficiency of zooxanthellae in hospite from four different species of reef coral: a novel approach. Plant Cell. Environ. 19, 291–299. Weil, E., Smith, G., Gil-Agudelo, D.L., 2006. Status and progress in coral reef disease research. Dis. Aquat. Org. 69, 1–7. Williams, E.H., Bunkley-Williams, L., 1988. Bleaching of Caribbean coral reef symbionts in 1987-1988. Proc. 6th Intl. Coral Reef Symp. 3, 313–318. Winter, A., Appeldoorn, R.S., Bruckner, A., Williams Jr., E.H., Goenaga, C., 1998. Sea surface temperatures and coral reef bleaching off La Parguera, Puerto Rico (northeast Caribbean Sea). Coral Reefs 17, 377–382. Zar, J.H., 1998. Biostatistical analysis, 4th ed. Prentice Hall, New Jersey.