Resilience and acclimation to bleaching stressors in the scleractinian coral Porites cylindrica

Journal of Experimental Marine Biology and Ecology 349 (2007) 35 – 44 www.elsevier.com/locate/jembe

Resilience and acclimation to bleaching stressors in the scleractinian coral Porites cylindrica Shakil Visram 1 , Angela E. Douglas ⁎ Department of Biology (Area 2), University of York, PO Box 373, York, YO10 5YW, UK Received 21 November 2006; received in revised form 28 March 2007; accepted 17 April 2007

Abstract ‘Resilience’, the capacity of the coral symbiosis with dinoflagellate algal symbionts (‘zooxanthellae’) to recover after bleaching, is a little-studied but crucial aspect of coral responses to bleaching stressors. This study investigated the response of the zooxanthella population in the coral Porites cylindrica after bleaching either naturally on a shallow subtidal reef or experimentally in response to elevated temperature and darkness. Coral resilience was influenced by the nature and duration of the stressor. Corals strongly bleached by natural stressors were less resilient than those that had been partially bleached; and a similar recovery profile was obtained for corals experimentally bleached by exposure to elevated temperature, in which recovery was slower for corals thermally-stressed 96 h than for 72 h. The opposite trend was evident for corals exposed to darkness, indicating that the bleaching trigger had a strong impact on coral resilience. When P. cylindrica recently recovered from bleaching was subjected to a repetition of bleaching stressors, it did not display acclimation, i.e. experience-mediated acquisition of resistance to bleaching stressors. The zooxanthella populations in all corals tested throughout the experiments were typed by PCR-RFLP as clade C, indicating that coral responses were not accompanied by any substantial change in zooxanthella composition at the cladal level. © 2007 Elsevier B.V. All rights reserved. Keywords: Acclimatisation; Coral bleaching; Porites cylindrica; Resilience; Symbiodinium; Zooxanthellae

1. Introduction Coral bleaching, the paling of tissues resulting from the drastic decline in zooxanthella densities and/or the loss of photosynthetic pigments, is a classic stress response of corals and allied symbiotic marine animals to perturbations in environmental conditions (Glynn, 1993;

⁎ Corresponding author. Tel.: +44 1904 328610; fax: +44 1904 328505. E-mail address: [email protected] (A.E. Douglas). 1 Current Address: CORDIO East Africa, PO Box 10135 Mombasa 80101, Kenya. 0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2007.04.014

Brown, 1997). Among the various triggers of bleaching, elevated seawater temperature often in combination with high irradiance is of global significance (Glynn, 1993; Rowan et al., 1997; Brown et al., 2000), and has been predicted to result in global collapse of coral reef ecosystems by 2050 (Hoegh-Guldberg, 1999). These predictions are based on the simplifying assumption that the bleaching response of corals to elevated temperature is fixed. In principle, however, corals vary in their response to bleaching stressors in three ways: their degree of resistance, resilience and acclimatisation. A detailed understanding of these three sources of variation is vital to the development of more accurate predictions of impacts of global climate change on coral reefs.

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Resistance refers to the ability of corals to withstand bleaching stressors without bleaching (West and Salm, 2003). It is convenient to consider resistance as the alternative response to susceptibility, where the coral does bleach; but in reality, corals display a continuum of responses of varying degrees of resistance/susceptibility. Factors contributing to resistance include antioxidant enzymes and fluorescent pigments in the coral (Lesser, 1996, 1997; Salih et al., 2000; Brown et al., 2002a) and physiological characteristics of the zooxanthellae (Rowan et al., 1997; Perez et al., 2001), possibly including efficient xanthophyll cycling (Brown et al., 1999). Resilience relates to bleaching-susceptible corals and refers to the capacity of a coral to recover from bleaching. Corals in which the zooxanthella population increases rapidly to the density occurring before bleaching are described as having high resilience. In principle, both the proliferation of the resident zooxanthella population and invasion of the coral tissue by free-living zooxanthellae either from the water column or living on or near the reef bottom (Berkelmans and van Oppen, 2006) can contribute to this process. Corals with low resilience (i.e. with a zooxanthella population that increases very slowly or not at all) generally die in the field. Acclimatisation refers to experience-mediated increase in resistance to bleaching, i.e. on first exposure to a bleaching stressor, a coral colony is susceptible and resilient, but is more resistant to a second exposure. Acclimatisation may be mediated by changes in the physiological/biochemical traits of the coral or its zooxanthellae (Brown et al., 2000, 2002a,b) or by either the replacement of bleaching-susceptible zooxanthellae by genetically distinct, resistant zooxanthellae (Rowan, 2004; Baker et al., 2004), or by shifts in the dominant members of zooxanthella populations (Berkelmans and van Oppen, 2006). Experience-mediated change in response to a bleaching stressor under experimental conditions is known as acclimation. To date, most study of the variability in coral response to bleaching stressors has focused on resistance, and the little research on resilience and acclimatisation has addressed the underlying processes. The patterns of coral recovery and acclimatisation are very poorly documented, even though they are crucial to the development of more accurate and realistic models of projected coral responses to global climate change. This study was conducted on the scleractinian coral Porites cylindrica, which is known to be susceptible to bleaching in Kenya (Obura, 2001a), can be maintained readily under experimental conditions, and has a regular cylindrical morphology and shallow calices of corallites suitable for accurate quantification of zooxan-

thella density. Zooxanthella density defines the bleaching status of a coral, and was adopted as the index for all the experiments. Two bleaching stressors were used: elevated temperature and darkness. Elevated temperature is the primary environmental trigger for the extensive bleaching and mortality of corals in recent decades (Hughes et al., 2003; Sheppard, 2003) and is widely used in experimental studies of bleaching. The primary lesion leading to temperature-induced bleaching has been identified as damage to the photosynthetic apparatus of the zooxanthellae (Warner et al., 1999), but damage to host tissues, particularly in the endoderm, has also been reported (Gates et al., 1992; Dunn et al., 2002). The second bleaching stressor, incubation in darkness, is not generally ecologically important, but it is experimentally valuable because it allows one to explore recovery of zooxanthella populations without the confounding factor of damage to host tissues or the photosynthetic machinery of zooxanthellae. We investigated the relationship between the density of the residual zooxanthella population in bleached corals and the subsequent resilience of the corals. We also studied acclimation to bleaching stressors in corals by reexposing corals that had recently recovered from bleaching to the experimental bleaching stressors. The specific purpose of these investigations was to test two hypotheses: (1) that bleached corals with a large residual population of zooxanthellae are more resilient than corals with a small residual population, independent of the bleaching stressor, and (2) that bleached corals that recover are subsequently resistant to the same bleaching stressor through acclimation. In addition, the zooxanthellae in the corals were subjected to molecular typing by PCR-RFLP to establish whether resilience or acclimation was accompanied by a shift in zooxanthella clade. 2. Materials and methods 2.1. Collection and maintenance of corals Experiments were conducted between April and August 2003. Coral fragments (4–5 cm long) were collected from adult colonies of P. cylindrica Dana 1846, each separated by at least 5 m on the shallow subtidal reef at Kanamai near Mombasa, Kenya (3.97°S, 39.58°E). Fragments were glued (Superglue, Alpha Techno Co., Japan) at their base onto individual dead coral stones. They were maintained under natural light (12 h dark/light cycle) and shaded from direct solar irradiance in plastic tanks (10–12 l) containing unfiltered seawater that was aerated continuously and exchanged daily. Positions of tanks were changed once a

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week. All corals were maintained in these conditions for at least one week before experiments to allow corals to acclimate. 2.2. Zooxanthella density measurements Coral tissue was blasted off the skeleton using a water-pik with seawater filtered through a GF/C filter (Whatman), and homogenised with a hand-held homogeniser. Zooxanthella density was counted in duplicate samples by microscopy at x160 magnification in a Sedgwick–Rafter counting chamber (PhycoTech Inc., USA), and normalised to coral skeletal area by the aluminium foil technique of Marsh (1970). 2.3. Resilience of naturally-bleached corals In April 2003, mild-to-moderate bleaching was observed along the Kenyan coastline and in northern Tanzania. At the collection site, bleaching may have been triggered by the combination of anomalously high temperature, calm weather conditions and spring low tides immediately prior to bleaching. Corals were visually inspected and assigned to one of three bleaching states according to coloration; bleached (pale yellow), partially bleached (tan) and unbleached (chocolate brown). Twenty fragments from each of three coral colonies for each of the three bleaching categories were collected. Of the 20 fragments per colony collected from the reef, 10 were used in this experiment and 4 in subsequent (acclimation) experiments, with the extra 6 fragments used to insure against possible mortality. For each of the three bleaching categories, 10 randomly selected fragments from each colony were held in each of two replicate tanks, making a total of 30 fragments in each of 6 tanks. Completely-bleached, partiallybleached and unbleached corals were housed in independent tanks. Zooxanthella density was measured for one randomly selected fragment for each colony for each tank, to give 6 readings per bleaching category immediately on collection from the reef (day 0), and thereafter on days 7, 21, 42 and 63.

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the coral fragments from each of the three bleaching states described above were then exposed to elevated temperature of 32.5 °C for 72 h, which is slightly above the maximum seawater temperatures of 31–32 °C reported on Kenyan reefs annually at the end of the warm north-east monsoon season (Obura, 2001b). The remaining fragments were maintained at ambient temperature as controls, making a total of 6 experimental groups. Two fragments from each of three colonies for each of the experimental groups (i.e. 36 fragments) were assayed immediately on termination of treatment. 2.5. Temperature-treatment of corals Sixty fragments from each of two coral colonies were collected from the reef, and 10 randomly selected fragments from each colony were divided into each of 6 tanks. After acclimation for 24 days, two tanks were randomly selected as controls, and the temperature in two treatment tanks was raised over 6 h from ambient (approximately 28 °C) to 32.5 ± 0.2 °C, and thereafter kept constant for 96 h. Two days later, the coral fragments in the remaining pair of treatment tanks were temperature-treated at 32.5 °C for 48 h. Treatment was terminated simultaneously for both treatments and the zooxanthella density in one fragment from each colony for each tank was assayed on days 0, 7, 21, 42 and 63. At the start of the experiment, and at four-week intervals thereafter, a fragment from each coral colony in each control tank was also assayed. 2.6. Acclimation of corals to temperature-induced bleaching At 63 days after exposure to 32.5 °C for 96 h, coral fragments were subjected to elevated seawater temperature (32.5 °C) for 24 h with control corals held at ambient temperature (28 °C) conditions. Two fragments from each of two coral colonies for each of four experimental groups were assayed for zooxanthella density immediately on termination of treatment. 2.7. Dark-treatment of corals

2.4. Acclimation of corals to natural bleaching stressors To investigate the impact of recovery of zooxanthella populations from natural bleaching on the bleaching response to elevated temperature, coral fragments that had been collected from the reef in bleached, partiallybleached and unbleached states were first maintained for 63 days under the ambient temperature regime to promote recovery of zooxanthella populations. Half

Dark-treatment comprised enclosure in lightproof heavy-duty black polythene, with no alteration to seawater exchange and aeration. Treatments were commenced on different days, but terminated on the same day. This allowed for simultaneous measurements of zooxanthella densities on designated days post-exposure to light on fragments from all treatment tanks. Ten fragments of each of two coral colonies were transferred to each of

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8 tanks. After acclimation for 7 days, two tanks were randomly selected as ‘controls’, and two tanks were darktreated for 21 days. Seven and 14 days later, two tanks were dark-treated for each of 14 days and 7 days respectively. Immediately on removal of the polythene sheet, one fragment from each colony in each treatment tank was assayed for zooxanthella density (day 0) and again on days 7, 21 and 42. The control for dark-treatment was the same as the control for temperature-treatment of corals, and one fragment per colony in each control tank was assayed at the start of the experiment and at fourweek intervals thereafter. 2.8. Acclimation of corals to dark-induced bleaching At 42 days after exposure to 21 days of darkness, coral fragments were exposed to elevated seawater temperature (32.5 °C) for 72 h, or incubated in darkness for 21 days, with control corals held at ambient temperature (28 °C) throughout. Two fragments from each of two coral colonies for each of the four experimental groups were assayed for zooxanthella density immediately on termination of treatment. 2.9. Assessment of zooxanthella clade For each experiment, 2–4 coral samples were taken from each treatment group at the start and end of each experiment and stored in DMSO-fixative (Seutin et al., 1991) prior to analysis. DNA was extracted with the DNeasy® Plant Mini Kit (Qiagen) according to the manufacturer's instructions, and ca. 650 bp zooxanthella LSU rRNA gene fragment was amplified using the universal primers 24D1F51 (5′-TTA AGC ATA TAA GTA AGC GGA GGA-3′) and 24D23R1 (5′-CTC CTT GGT CCG TGT TTC AAG ACG-3′) (Baker et al., 1997). Zooxanthella clade was identified from the restriction fragments obtained by digestion with the enzyme HpyCh4IV (New England Biolabs) with the predicted diagnostic bands of sizes: 350, 180 and 120 base pairs (bp) for clade A, 650 bp for clade B (undigested PCR product), 400 and 250 bp for clade C, and 520 bp for clade D. 2.10. Statistical analysis Data were tested for assumptions of normal distribution using the Anderson–Darling test and homogeneity of variances with Bartlett and Levene tests before testing for significant differences by t-test or ANOVA. As coral fragments from bleached, partially bleached and unbleached corals in the field were derived from

separate colonies, nested ANOVA, with colony nested within bleaching state, was used to test for acclimation in corals recovered from natural bleaching. Fisher's LSD test was used as the post hoc test. 3. Results 3.1. Resilience to bleaching stressors The ‘unbleached’ corals collected from the reef bore a mean of 3.02 × 106 zooxanthellae cm− 2 coral surface area, and the zooxanthella density in corals designated ‘partially-bleached’ and ‘bleached’ was reduced by 45% and 68%, respectively; the differences were statistically significant (one-way ANOVA, F2,15 = 15.12, p b 0.001). At the end of the recovery period of 63 days, there were no significant differences in zooxanthella density between the three groups (one-way ANOVA: F2, 15 = 2.76, p N 0.05) (Fig. 1a). Analysis by two-way ANOVA of zooxanthella density in the partially-bleached and bleached groups over days 0–21 during recovery revealed highly significant effects of coral group (F1,30 = 112.23, p b 0.001) and time (F2,30 = 36.52, p b 0.001), but not for the interaction term (F2,30 = 1.95, p N 0.05). The significant terms could be attributed to greater resilience of corals in the partially-bleached corals than in the bleached group, as confirmed by the post hoc test (Fig. 1a). These results are consistent with hypothesis-1 (see Introduction) that bleached corals with a large residual population of zooxanthellae are more resilient than corals with a small residual population. At the end of the experiment, the colonies on the reef from which the coral fragments had been collected were examined. The partially-bleached and unbleached colonies had survived but two of the three bleached colonies at the field site had died. In the experiments on coral fragments exposed to the elevated temperature of 32.5 °C, the corals paled with a significant reduction in zooxanthella density by 53% in the 48 h treatment and 68% in the 96 h treatment (Fig. 1b). Over the first 7 days after return to ambient temperature of 28 °C, zooxanthella density in the 48 hour treatment corals did not change significantly and it declined by a further 63% in the 96 hour treatment corals; this effect was statistically significant (one-way ANOVA: F3, 12 = 30.43, p b 0.001). The zooxanthellae in both groups subsequently increased. By day-42, the mean density in corals of 96 hour treatment was not significantly different from the control corals and the mean density of 48 hour treatment corals was significantly higher than the controls, as determined by one-way ANOVA (F2, 9 = 15.86, p b 0.01) and post hoc analysis. The control colonies in this

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Fig. 1. Resilience of bleaching-susceptible zooxanthellae, as quantified by zooxanthella densities (mean values ± 1 SE) in (a) corals recovering from natural bleaching; sample size n = 6, and experimental bleaching induced by (b) exposure to elevated seawater temperature; n = 4 or (c) incubation in darkness; n = 4. Letters indicate homogenous subsets from post-hoc analysis with Fisher's LSD test. Statistical analysis was performed for the first 7 days (b) or 21 days (a) and (c) of recovery.

experiment displayed a progressive reduction in zooxanthella density over the 63-day experiment, from 2.9 × 106 to 1.9 × 106 cells cm− 2 coral surface area. Regular observation of the coral fragments throughout the experiment revealed expanded and feeding polyps and no sign of tissue regression. This suggested that the decline in zooxanthella density was not a consequence of poor coral health, but the causes were not investigated further. Corals incubated in continuous darkness displayed a progressive paling and loss of zooxanthellae with increased duration of treatment with darkness for up to 21 days, and the greatest reduction in zooxanthellae density, to 39% of the control corals, was in the 21-day treatment group (Fig. 1c). For logistical reasons, coral resilience was investigated over 42 days, and not

63 days, in this experiment. Changes in zooxanthella densities over the first 21 days of recovery varied with the duration of dark treatment (two-way ANOVA, interaction term F4, 27 = 6.60, p b 0.001). Corals held in darkness for 21 days displayed significant increases in zooxanthella population densities between day-7 (1.47 × 106 cells cm− 2) and day-21 (2.05 × 106 cells cm− 2) of recovery, showing the highest zooxanthella resilience of all treatments. The least resilient corals were those incubated in darkness for the shortest time, 7 days; this material displayed a decline in zooxanthella density over the 42-day experiment. These results are the reverse of the prediction from hypothesis-1. The zooxanthellae in all coral samples from all treatments taken at the start and end of each experiment were identified as clade C by PCR-RFLP.

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Fig. 2. Acclimation of zooxanthellae to bleaching stressors, quantified by zooxanthella densities (mean values ± 1 SE). Grey bars: treatment (elevated temperature or darkness); black bars: controls under ambient conditions (12 L:12 D at 28 °C) regime. (a) Temperature treatment of corals collected from the reef in unbleached, partially bleached and bleached states; sample size n = 6. (b) Temperature treatment of corals pretreated with elevated temperature (32.5 °C: pretreatment corals); n = 4 (c) Temperature treatment of corals pretreated with darkness; n = 4. (d) Darkness treatment of corals pretreated with darkness; n = 4. Arrows show mean zooxanthellae densities at the start of treatment.

3.2. Acclimation to bleaching stressors To assess coral acclimation to bleaching stressors, three experiments were conducted to quantify the response to elevated temperature of corals that had recovered from a natural bleaching event and from experimental bleaching triggered by elevated temperature and darkness (Fig. 2a–c). Linked to the third experiment, the impact of darkness on corals recovered from darkinduced bleaching was also investigated (Fig. 2d).

The starting material for the experiment using naturallybleached corals was samples of the ‘bleached’, ‘partiallybleached’ and ‘unbleached’ groups that had recovered over 63 days under experimental conditions, as in Fig. 1a. Their mean zooxanthella densities did not differ significantly [unbleached corals: 3.05± 0.20, partially bleached corals: 3.17 ± 0.18, bleached corals: 2.50 ± 0.26, mean density (×106) ± 1 SE, n = 6; one-way ANOVA: F2, 15 = 2.76, p N 0.05]. The response of the corals to elevated temperature varied significantly among the three groups (the

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interaction term in the nested two-way ANOVA was significant: F2,24 = 3.94, p b 0.05), with the ‘bleached’ and ‘partially bleached’ groups, but not the ‘unbleached’ group, displaying reduced zooxanthella density (Fig. 2a). The zooxanthella density in coral samples that had recovered from prior exposure to elevated temperature (‘pretreatment’ corals) did not differ significantly from the ‘pretreatment-control’ corals that had not previously been exposed to elevated temperature [pretreatment corals: 1.93 ± 0.40; pretreatment-control corals: 1.91 ± 0.17, mean density (× 106 ) ± 1 SE, n = 4; two-sample t-test: t4 = 0.05, p N 0.05]. Exposure to elevated temperature resulted in a mottled appearance to the coral fragments from the pretreatment-control group within the first 12–24 h. This was indicative of mortality of some polyps, and the experiment was therefore terminated at 24 h. Over this timescale, elevated temperature had no statistically significant impact on the density of zooxanthellae (two-way ANOVA: treatment: F1, 12 = 1.11, p N 0.05; pretreatment: F1, 12 = 1.45, p N 0.05; interaction: F1, 12 = 2.81, p N 0.05; Fig. 2b). The density of zooxanthellae in corals used to explore the impact of prior exposure to darkness on bleaching resistance to elevated temperature or darkness did not differ significantly between corals that had previously been incubated in the dark (pretreatment corals; recovered over 42 days from 21 days of darkness) and pretreatment-control corals: [pretreatment corals: 1.89 ± 0.14; pretreatment-control corals: 2.20 ± 0.08, mean density (× 106) ± 1 SE, n = 4; t4 = 2.18, p N 0.05]. The corals treated with elevated temperature paled and had significantly depressed zooxanthella density (Fig. 2c; two-way ANOVA, F1,12 = 12.77, p b 0.01). Prior dark exposure had no significant effect on zooxanthella density (F1,12 = 0.24, p N 0.05). The interaction term in the two-way ANOVA was not significant (F1,12 = 0.34, p N 0.05). Treatment with darkness resulted in a visible paling of coral tissues and a reduction of zooxanthellae density by more than 50% to levels significantly below treatment control corals (Fig. 2d; two-way ANOVA; treatment: F1, 12 = 54.77, p b 0.001). Previous exposure to darkness had no significant effect on zooxanthellae density (two-way ANOVA; pretreatment: F1, 12 = 2.15, p N 0.05). Coral responses to darkness were independent of levels of pretreatment, as indicated by the non-significant interaction term (two-way ANOVA; interaction: F1, 12 = 0.03, p N 0.05). The zooxanthellae in the corals were typed at the start and end of each acclimation experiments and were all clade C. These experiments provide no evidence for acclimation of P. cylindrica to bleaching stressors under

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controlled conditions, and are contrary to the expectation of hypothesis-2 (see Introduction). 4. Discussion A first objective of this study was to establish the relationship between the capacity for corals to recover from bleaching, i.e. resilience, and the density of residual symbionts in corals immediately after bleaching. Our results indicate that this relationship varies with the nature of the bleaching stressor. Where the causative agent for bleaching was elevated temperature, the relationship was direct; low densities of residual zooxanthellae corresponded with diminished resilience. However, for corals exposed to darkness, there was an inverse relationship between resilience and density of residual zooxanthellae. The differences in resilience properties of corals exposed to elevated temperature and corals incubated in the dark suggest that the mode of coral bleaching may be an important factor governing resilience. Whereas darkness is not known to directly damage coral tissues or the photosynthetic machinery of their symbionts, elevated temperature is associated with damage to the photosynthetic apparatus of zooxanthellae (IglesiasPrieto et al., 1992; Warner et al., 1996; Warner et al., 1999; Jones et al., 2000) and has also been linked to structural damage and disruption of coral endoderm tissue (Lasker et al., 1984; Glynn et al., 1985; Hayes and Bush, 1990; Gates et al., 1992; Dunn et al., 2002), where the zooxanthellae reside. Resilience is therefore predicted to decline with increased duration or magnitude of exposure to elevated temperature, with impaired photosynthetic function combined with the deterioration in condition of endoderm tissues. Other environmental factors may also affect resilience. For example, Grottoli et al. (2006) have demonstrated that access to food enhances the resilience of three coral species after temperature-induced bleaching. The resilience of the P. cylindrica in this study may also have been promoted by planktonic food items in the unfiltered sea water used for their maintenance but, beyond assuring that the corals fed holozoically, this issue was not addressed systematically. The mechanistic basis for the different impacts of temperature and darkness on resilience can be explored further by considering the zooxanthella populations in corals at the cellular level. In principle, corals may regain their zooxanthellae by two routes: proliferation of endodermal cells bearing zooxanthellae coupled to the division and distribution of their zooxanthellae to daughter cells; and infection of uninfected endodermal cells by zooxanthellae from the gastric cavity which had

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either been expelled from infected cells or derived from the exterior (Berner et al., 1993; Fitt et al., 1993; Jones and Yellowlees, 1997). (The uninfected endodermal cells could be vacant cells that had lost zooxanthellae during bleaching but that remain competent, and/or newly differentiating from stem cells replacing host cells lost during the bleaching event.) Prolonged darkness treatment would be expected to increase the availability of uninfected but infectible endodermal cells available for colonization by extracellular zooxanthellae, so promoting recovery. By contrast, prolonged temperature treatment is predicted to reduce the availability of such host cells, through damage to the endodermal layer. Clearly, analysis of these cellular processes, including an assessment of the relative importance of proliferation of infected cells and zooxanthella colonization of uninfected cells for recovery after bleaching, is an important priority for future studies on recovery from bleaching. The corals used in our experiments did not acclimate to bleaching stressors, and on recovery were susceptible to bleaching stressors when exposed to elevated temperature or darkness. These results are different from the evidence for acclimatisation in other studies. In particular, coral-mediated acclimatisation has been reported from shallow-water reef flats in Thailand, where high levels of photosynthetically active radiation (PAR: 400–700 nm wavelength) were found to enhance the resistance of corals to thermal bleaching in a range of intertidal corals (Dunne and Brown, 2001). This type of cross-protection between solar and thermal stressors was first observed in the scleractinian Goniastrea aspera (Brown et al., 2000). Resistance to thermal bleaching following solar bleaching and recovery in this coral was attributed to enhanced expression of antioxidant enzymes and heat stress proteins by the host (Brown et al., 2002a). In light of these studies demonstrating a link between high-light and tolerance to high temperature, the opposite response might be expected in corals with a recent history of low-light conditions. This in fact is what we report here, where darkness-induced bleached coral colonies had low resistance on exposure to high temperature in ambient light. In general, variation in acclimatory response can be predicted, varying with zooxanthella genotype, coral species and environmental factors; and it is important for this variability to be included in any assessment of the impact of climate change on coral community composition. Several studies indicate that the genetic identity of zooxanthellae may be a key factor in influencing susceptibility to bleaching stressors, and that this is

linked to observed changes in the genetic composition of zooxanthellae on reefs after major cycles of bleaching (Rowan et al., 1997; Baker, 2001; Toller et al., 2001; Baker, 2003; Baker et al., 2004). These changes in the genetic composition of zooxanthella populations in corals have been proposed to promote coral resistance to thermal stress in a future environment of elevated ocean temperatures. It must be noted, however, that the majority of corals are thought not to change their symbionts (Goulet, 2006). In this study, no change in zooxanthella composition in P. cylindrica was observed. However, post-bleaching genotype switches of zooxanthellae within a clade have been reported in corals (Thornhill et al., 2006), and the PCR-RFLP method adopted in our study would not have detected withinclade changes of the zooxanthella population. A limitation of our study is that zooxanthella density was the sole index by which coral bleaching and recovery were assessed. Changes in zooxanthella pigment composition is commonly associated with bleaching (Brown, 1997; Venn et al., 2006), and the pigment profile of zooxanthellae in symbioses recovering from bleaching is an important topic for future research. The experimental approach adopted here raises a general issue: are the acute and well-defined laboratory stressors used in experimental studies equivalent to the chronic and multiple stressors that corals are exposed to in their natural habitat? The fate of the naturally bleached corals used in our experiments suggests not. These survived and recovered their zooxanthellae in the laboratory, but two of the three bleached colonies died in the field. As well as providing assurance of the suitability of the culture methods for maintaining healthy corals, these results suggest that coral resilience and their long-term response to bleaching stressors in the field are shaped by complex interactions between the corals and their environment that are difficult to replicate in the laboratory. As a result, interpretation and extrapolation of results from laboratory investigations of bleaching responses of corals requires extreme caution. Nevertheless, it is only by combining laboratory analyses, with their application of tightly controlled sets of conditions on a select group of study organisms, with broader field studies that we will emerge with a more realistic picture of the likely impacts of climate change on coral reef systems. Acknowledgements We thank the R.E. Train Education for Nature (EFN) Program of WWF (USA), the ORS Scholarship (UK Universities) and a scholarship from the University of York for financial support. The research was conducted

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with permission from the Government of Kenya through a research permit. [SS] References Baker, A., 2001. Reef corals bleach to survive change. Nature 411, 765–766. Baker, A., 2003. Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of Symbiodinium. Annu. Rev. Ecol. Evol. Syst. 34, 661–689. Baker, A.C., Rowan, R., Knowlton, N., 1997. Diversity of symbiotic dinoflagellates (zooxanthellae) in scleractinian corals of the Caribbean and Eastern Pacific. Proc. 8th Internat. Coral Reef Symp. Panama 2, 1301–1306. Baker, A.C., Starger, C.J., McClanahan, T.R., Glynn, P.W., 2004. Corals' adaptive response to climate change. Nature 430, 741. Berkelmans, R., van Oppen, M.J.H., 2006. The role of zooxanthellae in the thermal tolerance of corals: a ‘nugget of hope’ for coral reefs in an era of climate change. Proc. Roy. Soc. Lond. B 273, 2305. Berner, T., Baghdasarian, G., Muscatine, L., 1993. Repopulation of a sea anemone with symbiotic dinoflagellates: analysis by in vivo fluorescence. J. Exp. Mar. Biol. Ecol. 170, 145–158. Brown, B.E., 1997. Coral bleaching: causes and consequences. Coral Reefs 16, S129–S138. Brown, B.E., Ambarsari, I., Warner, M.E., Fitt, W.K., Dunne, R.P., Gibb, S.W., Cummings, D.G., 1999. Diurnal changes in the photochemical efficiency and xanthophyll concentrations in shallow water reef corals: evidence for photoinhibition and photoprotection. Coral Reefs 18, 99–105. Brown, B.E., Dunne, R.P., Goodson, M.S., Douglas, A.E., 2000. Bleaching patterns in reef corals. Nature 404, 142–143. Brown, B.E., Downs, C.A., Dunne, R.P., Gibb, S.W., 2002a. Exploring the basis of thermotolerance in the reef coral Goniastrea aspera. Mar. Ecol. Prog. Ser. 242, 119–129. Brown, B.E., Goodson, M.S., Dunne, R.P., Douglas, A.E., 2002b. Experience shapes the susceptibility of a reef coral to bleaching. Coral Reefs 21, 119–126. 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. Dunne, R.P., Brown, B.E., 2001. The influence of solar radiation on bleaching of shallow water reef corals in the Andaman Sea, 1993– 1998. Coral Reefs 20, 201–210. Fitt, W.K., Spero, H.J., Halas, J., White, M.W., Porter, J.W., 1993. Recovery of the coral Montastraea annularis in the Florida Keys after the 1987 Caribbean ‘bleaching event’. Coral Reefs 12, 57–64. Gates, R.D., Baghdasarian, G., Muscatine, L., 1992. Temperature stress causes host cell detachment in symbiotic cnidarians: implications for coral bleaching. Biol. Bull. 182, 324–332. Glynn, P.W., 1993. Coral reef bleaching: ecological perspectives. Coral Reefs 12, 1–17. Glynn, P.W., Peters, E.C., Muscatine, L., 1985. Coral tissue microstructure and necrosis: relation to catastrophic coral mortality in Panamá. Dis. Aquat. Org. 1, 29–37. Goulet, T.L., 2006. Most corals may not change their symbionts. Mar. Ecol. Prog. Ser. 321, 1–7. Grottoli, A.G., Rodrigues, L.J., Palardy, J.E., 2006. Heterotrophic plasticity and resilience in bleached corals. Nature 440, 1186–1189.

43

Hayes, R.L., Bush, P.G., 1990. Microscopic observations of recovery in the reef building scleractinian coral, Montastraea annularis, after bleaching on a Cayman reef. Coral Reefs 8, 203–209. Hoegh-Guldberg, O., 1999. Climate change, coral bleaching and the future of the world's coral reefs. Mar. Freshw. Res. 50, 839–866. Hughes, T.P., Baird, A.H., Bellwood, D.R., Card, M., Connolly Folke, C., Grosberg, R., Hoegh-Guldberg, O., Jackson, J.B.C., Kleypas, J., Lough, J.M., Marshall, P., Nyström, M., Palumbi, S.R., Pandolfi, J.M., Rosen, B., Roughgarden, J., 2003. Climate change, human impacts, and the resilience of coral reefs. Science 301, 929–933. Iglesias-Prieto, R., Matta, J.L., Robins, W.A., Trench, R.K., 1992. Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc. Natl. Acad. Sci. U. S. A. 89, 10302–10305. Jones, R.J., Yellowlees, D., 1997. Regulation and control of intracellular algae(=zooxanthellae) in hard corals. Phil. Trans. Roy. Soc. Lond. 352, 457–468. Jones, R.J., Ward, S., Amri, A.Y., Hoegh-Guldberg, O., 2000. Changes in quantum efficiency of photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event. Mar. Freshw. Res. 51, 63–71. Lasker, H.R., Peters, E.C., Coffroth, M.A., 1984. Bleaching of reef coelenterates in the San Blas Islands, Panama. Coral Reefs 3, 183–190. Lesser, M.P., 1996. Elevated temperatures and ultraviolet radiation causes oxidative stress and inhibit photosynthesis in symbiotic dinoflagellates. Limnol. Oceanogr. 41, 271–283. Lesser, M.P., 1997. Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16, 187–192. Marsh Jr., J.A., 1970. Primary productivity of reef-building calcareous red algae. Ecology 51, 255–263. Obura, D.O., 2001a. Can differential bleaching and mortality among coral species offer useful indicators for assessment and management of reefs under stress? Bull. Mar. Sci. 69, 777–791. Obura, D.O., 2001b. Kenya. Mar. Pollut. Bull. 42, 1264–1278. Perez, S.F., Cook, C.B., Brooks, W.R., 2001. The role of symbiotic dinoflagellates in the temperature induced bleaching response of the subtropical sea anemone Aiptasia pallida. J. Exp. Mar. Biol. Ecol. 256, 1–14. Rowan, R., 2004. Thermal adaptation in reef coral symbionts. Nature 430, 742. Rowan, R., Knowlton, N., Baker, A., Jara, J., 1997. Landscape ecology of algal symbionts creates variation in episodes of coral bleaching. Nature 388, 265–269. Salih, A., Larkum, A., Cox, G., Kuhl, M., Hoegh-Guldberg, O., 2000. Fluorescent pigments in corals are photoprotective. Nature 408, 850–853. Seutin, G., White, B.N., Boag, P.T., 1991. Preservation of avian blood and tissue samples for DNA analyses. Can. J. Zool. 69, 82–90. Sheppard, C.R.C., 2003. Predicted recurrences of mass coral mortality in the Indian Ocean. Nature 425, 294–297. Thornhill, D.J., LaJeunesse, T.C., Schmidt, G.W., Fitt, W.K., 2006. Multi-year seasonal geotypic surveys of coral-dinoflagellate symbioses reveal prevalent stability of postbleaching reversion. Mar. Biol. 148, 711–722. Toller, W.W., Rowan, R., Knowlton, N., 2001. Repopulation of zooxanthellae in the Caribbean corals Montastraea annularis and M. faveolata following experimental and disease-associated bleaching. Biol. Bull. 201, 360–373. Venn, A.A., Wilson, M.A., Trapido-Rosenthal, H.G., Keely, B.J., Douglas, A.E., 2006. The impact of coral bleaching on the pigment

44

S. Visram, A.E. Douglas / Journal of Experimental Marine Biology and Ecology 349 (2007) 35–44

profile of the symbiotic alga, Symbiodinium. Plant Cell Environ. 29, 2133–2142. Warner, M.E., Fitt, W.K., Schmidt, G.W., 1996. The effects of elevated temperature on the photosynthetic efficiency of zooxanthallae in hospite from four different species of reef coral: a novel approach. Plant Cell Environ. 19, 291–299.

Warner, M.E., Fitt, W.K., Schmidt, G.W., 1999. Damage to photosystem II in symbiotic dinoflagellates: a determinant of coral bleaching. Proc. Natl. Acad. Sci. U. S. A. 96, 8007–8012. West, J.M., Salm, R.V., 2003. Resistance and resilience to coral bleaching: implications for coral reef conservation and management. Cons. Biol. 17, 956.