Abstract: Background and Objective: The physiological response of corals to ocean warming is an essential component of their overall resiliency to climate change. It is important to understand how increasing sea surface temperature will affect the mutualistic relationship between corals and their endosymbionts dinoflagellates (Symbiodinium spp.). This study focused on the effects of temperature stress on the photophysiology of two Caribbean corals differing in life history strategies: Orbicella annularis (O. annularis) and Porites astreoides (P. astreoides). Materials and Methods: A total of 12 fragments of each species were collected from the Puerto Morelos, Mexico shoreline. Six fragments of each species were placed under ambient light and a water temperature of 28°C and the other six under ambient light and an elevated temperature of 32°C for 10 days. Maximum pressure over photosytem II (Qm) was estimated daily while chlorophyll content and Symbiodinium densities were measured at the end of the experiment. Results: Qm values were considerably higher in O. annularis at 32°C when compared to O. annularis at 28°C. In contrast, Qm values for P. astreoides did not differ significantly between treatments. Interestingly, Qm values of P. astreoides at 32°C was similar to that of O. annularis at 28°C. Orbicella annularis showed higher Symbiodinium densities and significantly higher concentrations of chlorophyll a at 28°C than at 32°C. In P. astreoides, no differences on chlorophyll content and Symbiodinium densities were found between temperature treatments. Porites astreoides showed higher chlorophyll a content and Symbiodinium densities than O. annularis under high temperature stress. Conclusion: Low Qm values but high chlorophyll concentrations and Symbiodinium densities within P. astreoides after induced temperature stress provides a physiological basis of the opportunistic strategy of this species. However, the significant increase in Qm, lower chlorophyll a concentration and reduced Symbiodinium densities observed in O. annularis at 32°C highlights its vulnerability to ocean warming. If O. annularis, which is considered one of the main builders of Caribbean coral reefs, disappears and is replaced by relatively small and weedy P. astreoides, the structural heterogeneity of Caribbean coral reefs will be reduced.
INTRODUCTION
Corals are particularly vulnerable to climate change because they live near their physiological thermal tolerance, making them susceptible to small but rapid changes in sea surface temperature1. Nonetheless, the observed effects of increasing sea surface temperature on different coral species will depend on their phenotypic plasticity, genetic diversity, availability of habitats and ecological interactions within the different ecosystems1. Reef building corals live in mutualism with algal dinoflagellates from the genus Symbiodinium. The photosynthetic activity of Symbiodinium, which is hosted within the coral tissue, provides up to 90% of the amount of energy or quantity of carbon supplied that coral needs for functioning and the coral in turn provides protection, ammonia and phosphate that the algae assimilate2.
Temperature is considered one of the major physical parameters that can alter the relationship between a coral and its endosymbionts. Some mechanisms on how thermal stress alters the photosynthetic performance of Symbiodinium include the damage of the D1 protein within the PS II reaction centers3-5, impairment of the calvin cycle6, damage of the rubisco site7, thylakoid membranes deterioration8 and the inhibition of light harvesting antennae proteins9. Thermal stress can also interrupt the coral/algal symbiosis by heat-induced bleaching10, a phenomenon that refers to the reduction or loss of Symbiodinium. Indeed, the increase in ocean temperature has caused catastrophic mass bleaching events, such as the episode of 1998 which severely damaged 16% of coral reefs worldwide11. The Caribbean has also experienced these events including the bleaching event in 2005 that severely impacted the population dynamics and demography of Orbicella annularis, an important frame boulder coral species12. A reduction in the photosynthetic efficiency of corals may compromise their rates of calcification, survival and reproduction13 increasing their susceptibility to coral diseases and lowering the capability to compete for space with macroalgae. Current climate change scenarios project increases of between 1.2 and 2.6°C on average ocean surface temperature for the year 21001. It thus becomes essential to identify which coral species will have the physiological capacity (i.e., phenotypic plasticity) to cope with the expected increases in temperature.
The objectives of this study included a comparison of the photosynthetic performance of algal symbionts of two of the most common scleractinians in Caribbean coral reefs, Orbicella annularis and Porites astreoides, under elevated temperature stress. Porites astreoides is characterized by high population growth rates14, high coral cover15, weedy life-history traits16,17, high fecundity18-20 and the production of brooded larvae19,20 that normally settle at elevated densities regardless of depth20. Orbicella annularis, on the other hand, is characterized by slow growth, longer generation times, high fecundity (but low recruitment) and large corallites17. A comparison of Symbiodinium densities, chlorophyll content and photochemical efficiency based on the maximum excitation pressure over photosystem II (Qm) was done. Qm represents the interaction of photochemical and non-photochemical processes in the algal symbiont21. The main hypothesis was that under high thermal stress Symbiodinium within the weedy coral P. astreoides would perform better showing higher densities, higher Chl a and c content and lower Qm values. This research was a controlled experiment which explored the effects of elevated temperature on the physiology of corals. Even though the study was completed 2.5 years ago, it contributes to the advancement of knowledge because of the current increase of sea surface temperature that is affecting the mutualistic relationship between corals and their endosymbionts dinoflagellates (Symbiodinium spp.). Furthermore, ocean warming is predicted to continue affecting this symbiosis altering coral reef ecosystem dynamics. Therefore, the study provides essential data that can be used by coral reef managers to deal with the challenge of ocean warming.
MATERIALS AND METHODS
Sample collection: Twenty four O. annularis (n = 12) and P. astreoides (n = 12) fragments (2-4 cm2) were collected using a hammer and a chisel at a coral nursery in Puerto Morelos, Mexico (2-4 m, 20°52' 48.9" N, 86°50' 59.34" W) in October, 2014. Fragments of each species belonged to three different parent colony (genets). The fragments were acclimated for 7 days under ambient temperature and light conditions in a seawater table at the Universidad Nacional Autónoma de Mexico, Unidad de Sistemas Arrecifales in Puerto Morelos.
Experimental design: Six fragments of each species were placed at the center of the control seawater table (temperature range of 28.4-30.6°C), while the other six fragments were placed at the experimental seawater table with increased temperature (temperature range of 31.0-32.2°C). Fragments were placed in two rows of, 2-3 cm apart of each other and alternating species. Temperature within the experimental seawater table was maintained using a NEMA 4X Electronic Temperature Control (Ranco, Plain City, Ohio, USA). Temperature in both seawater tables were monitored twice a day using a thermometer. These measurements were taken at different points around the two rows of fragments in each seawater table to avoid exposing the fragments to different temperature regimes. In this way, it was tried to compensate for the lack of independent temperature replication of the colonies, a limitation of that experiment. The daily irradiance exposure was 481.17±10.59 μmol photons m–2 sec–1 (Mean±SE) (data provided by the Servicio Academico de Monitoreo Meteorología y Oceanografía de la UASA del ICML de la UNAM). The experiment was run for 10 days after acclimation.
Fluorescence determinations: Fluorescence measurements were performed everyday (for 7 days) using an underwater pulse amplitude fluorometer or diving-PAM (Walz, Effeltrich, Germany). It was measured that the light-dependent reductions of the effective quantum yield of photosystem II (F/Fm’) at noon and at dusk (Fv/Fm). These two values were used to calculate the maximum excitation pressure over photosystem II (Qm) or 1-[(F/Fm’ at noon)/(Fv/Fm at dusk)]22.
Coral tissue extraction and processing: Coral tissue was removed using an airbrush and filtered sea water. The resulting slurry containing coral tissue and symbiotic algae was homogenized and centrifuged (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 2000 rpm for10 min. A total of 5 mL of supernatant was removed for protein analysis and stored samples in freezer. Afterwards, discarded the remaining supernatant. The pellet was re-suspended in 5 mL filtered seawater and removed 0.5 mL for cell counts. Samples were preserved in 200 μL iodine. Finally, we removed 3 replicates of 0.3 mL subsamples for chlorophyll analysis.
Pigment analysis and Symbiodinium densities: Three replicates of the 0.3 mL subsamples were placed into 3 Eppendorf tubes (300 μL each), centrifuged at 13,000 rpm for 1 min. After this, samples were processed in the dark at 4°C. Then added 50 μL DMSO and vortex the samples. Then, added 950 μL Acetone and vortex once again. Tubes were wrapped in aluminum foil and stored them at 4°C for 12 h. After the 12 h, we centrifuged at maximum speed for 5 min and read absorption (630, 663 and 750 nm) in 1 mL glass cuvettes. Chlorophyll a and c concentrations were determined by using the equations of Jeffrey and Humphrey23. Symbiodinium densities were determined with triplicate counts in a hemacytometer (LW Scientific, Lawrenceville, Georgia, USA).
Estimated absorbance (De) calculations based on reflectance measurements: Reflectance measurements were done following Enríquez et al.24. The reflectance spectra of corals were measured between 400 and 750 nm using a fiber optic attached to an USB4000 Spectrometer (Ocean Optics, Winter Park, Florida, USA). Coral samples were submerged in seawater in a small glass container with a black bottom. Uniform lighting was provided by an incandescent light source placed 25 cm above the coral surface (see Enriquez et al.24 for further details). The fiber optic was placed underwater, 1 cm away from the sample. Reflectance measurements were expressed as the ratio of the radiance measured from the coral surface relative to the radiance obtained from a reference white coral skeleton24. These data were used to calculate the absorbance (De) which was expressed as De = LOG10 (1/Reflectance). Chl a specific absorption coefficient was calculated by relating:
| ρ | = | Pigment content per surface area (mg cm–2) |
| De | = | Estimated absorbance described above but using only values at 675 nm24-25 |
At this wavelength, the reflectance of the skeleton is closer to one and it reduces the intrusion of accessory algal and animal pigments24-25.
Statistical analyses: Fluorescence data were analyzed using a two-way repeated measures ANOVA, followed by Holm-Sidak multiple comparison tests. Factors included type of species, temperature treatments and number of days. Chlorophyll a and c and Symbiodinium densities were analyzed using a two-way ANOVA followed by Holm-Sidak multiple comparison tests. Factors included type of species and temperature treatments (p<0.05).
RESULTS
Fluorescence measurements: The mean maximum pressure over photosystem II (Qm) in O. annularis was significantly lower (p<0.05) between the 28°C (mean 0.145±0.029 SE) and 32°C (mean 0.277±0.041 SE) water temperature treatments during the 7 days that these measurements were taken (Fig. 1, Table 1). There was also a significant temporal heterogeneity in Qm, with significantly lower values in P. astreoides) and 28°C treatment on days 2-3 and 5-7. No significant differences were found in Qm of P. astreoides colonies at 28°C and those at 32°C (Fig. 2).
Chlorophyll a content: Porites astreoides at 28°C had significantly more chlorophyll a (19.23 mg cm–2±1.18 SE) than at 32°C (11.83 mg cm–2±1.15 SE). Orbicella annularis exhibited the same pattern, with significantly higher concentrations of chlorophyll a at 28°C (10.82 mg cm–2± 0.88 SE) than at 32°C (3.28 mg cm–2±0.27 SE) (Fig. 3, Table 2). Also, chlorophyll a content in P. astreoides at 32°C and O. annularis at 28°C were significantly higher (p<0.05) than O. annularis at 32°C. However, no significant differences in chlorophyll a content were found between P. astreoides at 32°C and O. annularis at 28°C.
Chlorophyll a specific absorption coefficient: Results on the Chl a specific absorption coefficient showed the highest absorption coefficient in O. annularis at 32°C (0.460 cm2 mg–1 Chl a) and the lowest absorption coefficient in P. astreoides 28°C (0.076 cm2 mg–1 Chl a) while P. astreoides at 32°C and O. annularis at 28°C showed similar values (0.140 and 0.130 cm2 mg–1 Chl a, respectively) (Table 3).
Chlorophyll c content: Porites astreoides had significantly more (p<0.05) chlorophyll c at 28°C (9.35 mg cm–2±0.48 SE) than at 32°C (5.72 mg cm–2±0.60 SE). Orbicella annularis followed the same pattern, with more chlorophyll a at 28°C (4.59 mg cm–2±0.52 SE) than at 32°C (2.24 mg cm–2±0.63 SE) (Fig. 4, Table 4). The results of the effect of temperature on chlorophyll c content were similar to those of chlorophyll a, in P. astreoides and O. annularis. However, no significant differences in chlorophyll c content were found between P. astreoides at 32°C and O. annularis at 28°C.
| Fig. 1: | Qm (±SE) values for O. annularis |
| Fig. 2: | Qm (±SE) values for P. astreoides |
| Table 1: | Two way-repeated measures ANOVA results for the fluorescence data (statistical analyses only for 7 days) |
| *Subject: Number of fragments | |
| Table 2: | Results for the two-way ANOVA analysis for chlorophyll a data |
| Table 3: | Chl a specific absorption coefficient values for each treatment |
Symbiodinium densities: Porites astreoides colonies at 28°C had significantly higher (p<0.05) Symbiodinium densities cm–2 (3.07×106±5.64×105 SE) than at 32°C (1.51×106±3.44×105 SE).
| Fig. 3: | Chlorophyll a content (mg cm–2) (±SE) for both species and treatments |
| Different letters denote statistical difference (p<0.05) | |
| Fig. 4: | Chlorophyll c (mg cm–2) (±SE) for both species and treatments |
| Different letters denote statistical difference (p<0.05) | |
Orbicella annularis followed the same pattern, with higher Symbiodinium densities at 28°C (1.49×106±3.18×105 SE) than at 32°C (3.64×105±1.02×105 SE) (Fig. 5, Table 5).
DISCUSSION
Algal symbionts within O. annularis exposed to the elevated temperature (32°C) showed the highest maximum pressure over photosystem II (Qm) in comparison to symbionts in O. annularis exposed to normal temperature conditions, while P. astreoides exposed to both temperature treatments did not differ significantly in Qm. Differences in Qm values have been used to explain the vertical distribution of corals in the Pacific22. As Qm approaches 1.0, a higher fraction of PS II reaction centers are closed, causing photoinhibition22. Warner et al.21 found similar patterns in Belize when comparing symbionts within the same species. In that study pressure over PS II or Qm in P. astreoides was significantly lower than in O. annularis colonies at 8 m without any thermal stress.
| Fig. 5: | Symbiodinium densities (±SE) for both species and treatments |
| Different letters denote statistical difference (p<0.05) | |
| Table 4: | Two-way ANOVA results for chlorophyll c content |
| Table 5: | Two-way ANOVA results for Symbiodinium densities |
Also, when exposed briefly to elevated temperature, a higher loss in PS II activity was found in Orbicella colonies compared to P. astreoides colonies. However, Qm values from P. astreoides and O. annularis at 25 m reported by Warner et al.21 were similar to values from colonies from thermal induced stress study which were collected at much shallower depths (2-4 m). A relationship between thermal stress and the disruption of photosystem II (based on the Qm values) especially in Orbicella colonies was found, which concurs with Warner et al.21. These authors also found that P. astreoides exposed to higher temperatures exhibited a significantly lower loss in activity of photosystem II than colonies of Orbicella faveolata exposed to the same conditions.
Mechanisms of photo-acclimation of Symbiodinium to light variability include changes of pigment concentration and the shifting of the photosynthetic response curve (e.g., P:I curve) and photosynthetic units (e.g., reaction center+ light-harvesting pigments), changes in algal densities, size and clade within the host due to spatial (depth), temporal (seasons) and nutrient concentrations within the coral reef26-30. Iglesias-Prieto and Trench27 found that 3 clades of Symbiodinium increased their photosynthetic unit when exposed to low photon flux density levels. Colonies of Turbinaria mesenterina contained lower chlorophyll concentration within their dinoflagellates when exposed to levels higher than their acclimation irradiance26. Symbiodinium can also acclimatize to increased temperatures29,31-34. Robinson and Warner29 found that diverse cultures of Symbiodinium under high light but normal temperature (26°C) conditions showed lower values of Fv/Fm than cultures under low light conditions. However, when they increased the temperature to 32°C, Fv/Fm values of some Symbiodinium acclimated to high light conditions showed an intense drop and the authors found that D1 protein inactivated the PS II. D1 protein degradation within the reaction centers of PS II has been proposed as the primary cause of photoinhibition35. The results by Robinson and Warner29 concur with the hypothesis that increased temperature causes photoinactivation within the reaction centers of the PS II due to a low capacity of D1 repair when corals suffer from bleaching5. Non-photochemical quenching (e.g., dissipation of excess of light as heat) is another way of photoprotection of the PS II when thermal stress occurs because when the Fv/Fm decreases, an increase in the non-photochemical pathways occur while the net photosynthesis remains high36. Symbionts in O. annularis appear to have less physiological plasticity to warming. This is supported by the non-significant results in Qm, Chl a aborption coefficient and algal densities between P. astreoides under normal conditions (28°C) and P. astreoides under elevated temperature stress (32°C) found in this study and the significant differences between temperature treatments found in O. annularis. Perhaps the efficiency of the D1 repair of the PS II of Symbiodinium within P. astreoides during thermal stress may confer these colonies the phenotypic plasticity to cope with ocean warming.
As anticipated, the estimated absorbance analysis was dominated by absorption bands of Chl a and Chl c. Based on the results of the Chl a specific absorption coefficient Symbiodinium within P. astreoides at 32°C absorbed most of the incident light (0.75) with 11.8 mg cm–2 Chl a whereas O. annularis at 28°C absorbed the least (0.61) with 10.8 mg cm–2 Chl a. P. astreoides at 28°C showed 19 mg cm–2 Chl a with and absorbance of 0.64. Other studies have found values of 19 mg cm–2 Chl a associated to absorbance values closer to 1 in Porites branneri after a natural coral bleaching event24. In this context, P. branneri seems to be more efficient in absorbing light than P. astreoides but further research and comparison among both species should be done. Also, in that study, other P. branneri colonies showed concentration values of 3.3 mg cm–2 Chl a associated to approximately 0.1 of absorbance values. In the current study, 3.3 mg cm–2 Chl a was related to absorbance values of 0.66 in O. annularis at 32°C. This suggests that under thermal stress O. annularis colonies may be more efficient in absorbing a higher fraction of incident light than P. branneri with comparable Chl a concentration in both species. Results on the Chl a absorption coefficient supports this as well with the highest values found on O. annularis at 32°C. Increases in the light harvesting carotenoid associated to chlorophyll and peridinin enhances the light capturing capability of Symbiodinium37. This may explain differences in the absorbance values. However, this was not addressed in this study. Porites astreoides under thermal stress have shown no significant differences in Chl a values when compared to colonies in normal conditions38. It has found contrasting results since colonies at 32°C showed a significant reduction in Chl a and c in relation to control P. astreoides, perhaps due to a reduction in Symbiodinium densities at 32°C. Also, O. annularis at 28°C showed similar pigment values to P. astreoides at 32°C. The same patterns were found when comparing absorption coefficient values of control O. annularis showing similar values to treatment P. astreoides. Venn et al.38 found no significant differences when comparing algal densities from P. astreoides under normal and stressful conditions. Significant differences were found in the current study but we only exposed corals to thermal stress while these authors also increased irradiance, which may explain these differences.
Fitt et al.39 found that typical algal symbiont densities within O. annularis are approximately 2×106 cells cm–2. Similar algal densities within O. annularis at 28°C were found but significant reduction when exposed to elevated temperature. Porites astreoides at 32°C, on the other hand, showed comparable algal densities to O. annularis at 28°C. While Symbiodinium/coral symbiosis is essential for the corals’ adaptations to thermal stress, host distinctiveness such as DNA, mucus production, skeletal structure and tissue biomass also matters40. Thornhill et al.41 suggest that there is a strong and significant positive correlation between tissue biomass (e.g., tissue excluding skeleton) of P. astreoides and Symbiodinium densities. However, this correlation weakens within O. annularis. These authors explain that the nutritional relationship between the coral and its algal symbionts is essential to understand this correlation. That study also found that low coral tissue biomass within O. annularis increases its susceptibility to death following stress which is contrasting to the idea that O. annularis is stress-tolerant17. Corals with thicker tissue have been proposed to resist coral bleaching42 and Porites spp. are known to have high tissue biomass43.
In the current study, P. astreoides under thermal stress (i.e., 32°C) showed similar values in photopigments, Chl a absorption coefficient and algal densities to O. annularis under normal conditions (i.e., 28°C). These results demonstrate the higher likelihood of these colonies to colonize warmer localities within reefs or of coping better to warmer episodes than O. annularis17. This supports the hypothesis that P. astreoides will continue to strive under ocean warming scenarios14,15,44. This resilience to heat stress could be either a trait of the Porites genus or to the relationship this coral has with Symbiodinium clade A38, considered a bleaching resistant clade due to its tolerance to high light45 and temperature46. However, other studies have found thermally resistant and highly derived clade C (C15) within massive Porites from the Pacific47 which is transmitted vertically (e.g., obtained directly from parents) to the host48. This type of coral-Symbiodinium association creates an intrinsic mutualism within Porites causing a high fidelity symbiosis which helps the coral to thrive under stressful conditions47 and this may be happening with Porites astreoides .
Weedy corals, such as P. astreoides may be better survivors and colonizers in disturbed environments than non-weedy corals due to their high reproductive output and because of the set of life history traits that weedy species exhibit in comparison to other species17. Colonies from the genus Porites are known to cope well with thermal stress events49 and are considered "ecological winners" after coral bleaching episodes due to their specificity with their symbiotic partners47.
The "stress tolerant" classification of O. annularis by Darling et al.17 do not include any physiological trait and responses to elevated temperature conditions. In the case of O. annularis, several studies point towards a high susceptibility of this species to thermal stress and bleaching. After the massive coral bleaching event in the Eastern Caribbean in 2005, O. annularis species complex (O. annularis, O. faveolata and O. franksi ) suffered a 51% cover decline in St. John50. In Puerto Rico, more than 90% of O. annularis bleached during 200551 and over the last 15 years bleaching and disease have reduced coral cover of this species up to 80% within two important natural reserves52. Edmunds and Elahi53 found a 57% decline in the cover of O. annularis during 15 years in St. John and predicted a possible local extinction of this species in next 50 years. Demographic analysis and modeling indicates that the vital rates of O. annularis are very sensitive to bleaching and that interval of mayor events of less than 17 years will result in population reduction that will seriously compromise the viability of O. annularis populations12. The results of this study provide a physiological basis for the documented sensitivity of O. annularis to thermal stress. In this context, we propose that O. annularis should not be considered a "stress tolerant" species as categorized by Darling et al.17 but a species "sensitive to thermal stress".
CONCLUSION
One of the most important findings in the current study is that under thermal stress, the photophysiology of P. astreoides is similar to O. annularis exposed to normal conditions. This advances our understanding on coral resilience and vulnerability and enlightens us about the potential community structure of Caribbean coral reefs in the future due to current climate change effects. This shows a novel comparison with results that can be used to improve coral reef management and conservation initiatives. Moreover, the overall findings of the current study support the hypothesis of future increase in P. astreoides and decline of O. annularis population under the expected global warming scenarios. If O. annularis, which is considered one of the main builders of Caribbean coral reefs, disappears and is replaced by relatively small and weedy P. astreoides, the structural heterogeneity of Caribbean coral reefs will be reduced. This will result in less habitat availability for fish and other highly important commercial species altering species composition and dynamics within these reefs. This shift in species composition will affect directly coastal communities due to less food availability, less coastal protection from hurricanes, loss of sources for pharmaceutical products and a reduction in biodiversity. Local and global anthropogenic inputs altering coral reef habitats are and will continue to be one of the most important problems to address when managing these ecosystems.
SIGNIFICANCE STATEMENT
The results of the experiment significantly advance understanding of the photosynthetic response to elevated water temperature of the symbionts associated with this two coral hosts. In this study, results were interpreted within the context of life history strategies of corals and coral reef resilience to ocean warming. The paper should be of interest to readers in the areas of coral physiology and coral reef resilience.
One of the most important findings in the current study is that under thermal stress, the photophysiology of P. astreoides is similar to O. annularis exposed to normal conditions. This advances our understanding on coral resilience and vulnerability and enlightens us about the potential community structure of Caribbean coral reefs in the future due to current climate change effects. It is believed that RJES readers will benefit from this study because it shows a novel comparison with results that can be used to improve coral reef management and conservation initiatives.
ACKNOWLEDGMENTS
The authors would like to give special thanks to Roberto Iglesias-Prieto, Tim Scheufen, Claudia Galindo, Tomás López and Luis González for help in the laboratory and the field. Also, thanks to Susana Enríquez, Miriam Schutter, Román Vásquez, Nadine Schubert for help with logistics and Francisco Gerardo Ruiz Rentería and Jose Edgar Escalante Mancera for the meteorological data. Thanks to the University of Puerto Rico-Puerto Rico Center for Environmental Neuroscience faculty, staff and students. Funding for this study was provided by the NSF PRCEN HRD-1137725 and NSF IGERT #0301577 grants.