Diversity in skeletal architecture influences biological heterogeneity and Symbiodinium habitat in corals

Zoology 116 (2013) 262–269

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Diversity in skeletal architecture influences biological heterogeneity and Symbiodinium habitat in corals Denise M. Yost a , Li-Hsueh Wang b , Tung-Yung Fan b,c , Chii-Shiarng Chen b,d,e , Raymond W. Lee f , Emilia Sogin a , Ruth D. Gates a,∗ a Hawaii Institute of Marine Biology, School of Ocean and Earth Science and Technology, University of Hawaii, 46-007 Lilipuna Road, Kaneohe, HI 96744, USA b National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, Taiwan, ROC c Institute of Marine Biodiversity and Evolution, National Dong Hwa University, Pingtung, Taiwan, ROC d Institute of Marine Biotechnology, National Dong Hwa University, Pingtung, Taiwan, ROC e Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC f School of Biological Sciences, Washington State University, PO Box 644236, Pullman, WA 99164-4236, USA

a r t i c l e

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Article history: Received 10 December 2012 Received in revised form 24 June 2013 Accepted 28 June 2013 Available online 3 August 2013 Keywords: Scleractinian corals Coral architecture Perforate corals Symbiodinium habitat Host–symbiont dynamics

a b s t r a c t Scleractinian corals vary in response to rapid shifts in the marine environment and changes in reef community structure post-disturbance reveal a clear relationship between coral performance and morphology. With exceptions, massive corals are thought to be more tolerant and branching corals more vulnerable to changing environmental conditions, notably thermal stress. The typical responses of massive and branching coral taxa, respectively, are well documented; however, the biological and functional characteristics that underpin this variation are not well understood. We address this gap by comparing multiple biological attributes that are correlated with skeletal architecture in two perforate (having porous skeletal matrices with intercalating tissues) and two imperforate coral species (Montipora aequituberculata, Porites lobata, Pocillopora damicornis, and Seriatopora hystrix) representing three morphotypes. Our results reveal inherent biological heterogeneity among corals and the potential for perforate skeletons to create complex, three-dimensional internal habitats that impact the dynamics of the symbiosis. Patterns of tissue thickness are correlated with the concentration of symbionts within narrow regions of tissue in imperforate corals versus broad distribution throughout the larger tissue area in perforate corals. Attributes of the perforate and environmentally tolerant P. lobata were notable, with tissues ∼5 times thicker than in the sensitive, imperforate species P. damicornis and S. hystrix. Additionally, P. lobata had the lowest baseline levels of superoxide and Symbiodinium that provisioned high levels of energy. Given our observations, we hypothesize that the complexity of the visually obscured internal environment has an impact on host–symbiont dynamics and ultimately on survival, warranting further scientific investigation. © 2013 Elsevier GmbH. All rights reserved.

1. Introduction Scleractinian corals create reef habitats that provide critical ecosystem services worldwide. Corals have persisted over 500 million years, but have become increasingly threatened by the rapid changes in the marine environment linked to climate change and local human activities (Glynn, 1996). Corals have responded dramatically to environmental disturbances within recent decades (Berkelmans et al., 2004) resulting in large-scale global changes in the community structure of reefs. These changes have prompted many to question whether corals have the capacity to buffer,

∗ Corresponding author. Tel.: +1 808 236 7420; fax: +1 808 236 7443. E-mail address: [email protected] (R.D. Gates). 0944-2006/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.zool.2013.06.001

acclimatize and/or adapt to the dynamic environmental conditions predicted to occur as a result of climate change and to survive into the next century. Observed ecological variation in the responses of corals and reef communities provides insight into which corals are likely to persist under challenging environmental conditions (Baker et al., 2004; van Woesik et al., 2011). Corals with massive morphologies are among the most stress-tolerant corals, exhibiting much lower mortality rates following environmental disturbances (e.g., thermal stress) compared to branching and plating corals (Gates and Edmunds, 1999; McClanahan, 2004; Schloder and D’Croz, 2004). Even within the same genus (e.g., Porites), massive coral morphotypes appear to be less vulnerable to bleaching than their branching counterparts (McClanahan et al., 2001; but see also Guest et al. (2012)). Distinctive qualities of branching corals such as

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the combination of shallow tissue depths and limited resources (Loya et al., 2001), along with high metabolic rates (Gates and Edmunds, 1999), have been implicated as key factors that increase the thermal sensitivity of these corals. Branching corals also display stronger responses to ocean acidification compared to massive corals (Anthony et al., 2008). In addition to tissue depth, other strategies for buffering environmental factors may include shuffling of endosymbiotic dinoflagellate (Symbiodinium) communities (sensu Baker, 2003) to optimize performance in response to environmental change. Indeed, the functional integrity and persistence of the intimate associations between corals and Symbiodinium ultimately dictates whether corals survive in the face of changing environmental conditions or not. That said, the biological characteristics that contribute to response variability among corals and how they compare among coral species is not well characterized, but such analyses serve as important context for predicting how corals will respond to rising sea temperatures and ocean acidification. Beneath the commonly known gross morphology of corals lies an interior calcium carbonate skeleton. Carbonate skeletal density (micro-density) and porosity are key features of coral architecture and vary significantly between species, colonies, and even within a single colony (Bucher et al., 1998). Longitudinal sections through perforate (porous) corals reveal skeletal matrices with intercalating tissues, whereas imperforate species typically have a ‘veneer’ or surface covering of tissue that does not penetrate into the skeleton as is the case with perforate corals. Perforate skeletal matrices are characteristic of many species in the dominant reef-building genera such as Acropora, Porites, Montipora and Astreopora, but how skeletal porosity influences the biology and environmental range of corals is not well understood. There are several observations that suggest perforate architectural arrangements may have a positive impact on the survival and physiological dynamics of the symbiosis. Deep tissues that penetrate perforate skeletons are thought to enable the survival (Jokiel et al., 1993) and rapid recovery (Krupp et al., 1993) of corals following stressful events (low salinity, tissue damage), as well as facilitate the calcification process (Buchsbaum-Pearse and Muscatine, 1971; Gladfelter, 1983), by promoting within-colony transport of Symbiodinium cells and potentially maximizing photosynthesis (Santos et al., 2009). Additionally, corals with perforate skeletons and deep tissues appear more physiologically robust to thermal stress through reduced sunlight exposure in tissues and reduced photodamage to Symbiodinium (Santos et al., 2009). Deep tissues are known to co-occur with high levels of tissue-soluble proteins and high Symbiodinium densities in Porites lobata, which may give these corals a competitive advantage over branching species such as Pocillopora damicornis that have lower protein levels and fewer Symbiodinium (Schloder and D’Croz, 2004). In the present study, we address a fundamental gap in our understanding of coral biology by examining biological attributes that are correlated with coral skeletal architecture. Four coral species (Montipora aequituberculata, Porites lobata, Pocillopora damicornis, and Seriatopora hystrix) representing three morphotypes (foliose, massive, and branching) were selected to provide information on a wide range of the structural and biological complexity found in the scleractinians, as well as encompass corals known to have very different environmental thresholds (Loya et al., 2001). To compare the baseline biology of these four coral species, we evaluated their skeletal and tissue architecture and multiple physiological traits, using a variety of analytical approaches. This comparison reveals very high levels of heterogeneity among corals and suggests that perforate skeletons may play an important role in structuring internal architectures that create biologically complex Symbiodinium habitat. Our findings demonstrate that although massive macroarchitectures might suggest that the internal

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architecture of P. lobata is simple, in fact it is not. As a result, intercolating, deep tissues create a habitat within P. lobata that is unique among the coral morphotypes investigated, a key biological feature that in combination with other correlated attributes may explain some of the ecological variation among corals. 2. Materials and methods 2.1. Experimental organisms Qualitative and quantitative comparisons of the structural and biological attributes of M. aequituberculata, P. lobata, P. damicornis, and S. hystrix (n = 32; 8 per species) were conducted using light and confocal microscopy. Additionally, we measured total soluble protein, total chlorophyll, Symbiodinium cell density, superoxide levels and isotopic signatures of intact corals to investigate base state physiology. Corals were selected (March 2011) from aquarium collections maintained at the National Museum of Marine Biology and Aquarium, Taiwan, allowing for high sample numbers (8 per species). The corals originated from the coastal reefs near Hobihou in southern Taiwan. Species-specific differences (e.g., between P. lobata and other species of Porites) were not explored in our study due to the limited number of coral species maintained at the aquarium. All corals were kept in the same large flow-through aquarium and experienced equivalent light and temperature regimes (nutrient levels were not measured). Prior to sampling, M. aequituberculata, P. lobata, P. damicornis and S. hystrix corals were fragmented (7.6 ± 0.8 cm2 ; average ± SEM) using a hammer and chisel, strung on monofilament line and hung in common gardens to acclimate for 1 week. All corals were kept (before and after selection and sampling) under ambient conditions. Temperature data were logged using HOBO temperature loggers (Onset Computer Corp., Cape Cod, MA, USA) and averaged 23.4 ± 0.05 ◦ C. Light conditions were recorded three times daily in the common garden using a handheld probe and averaged 82.4 ± 9.9 ␮mol m−2 s−1 . 2.2. Microscopy Sub-fragments (approximately 1–2 cm2 ) of each coral were fixed in 4% paraformaldehyde for 1 h and then de-calcified in 10% HCl until the tissue tunics (intact biological tissues) were skeletonfree. The tissue tunics were stored in 1× PBS at 4 ◦ C in the dark. Decalcified tissue tunics were bisected with a scalpel and visualized using confocal microscopy. Samples were scanned with excitations of violet (405 nm) and green (498 nm and 543 nm) light, and emissions were collected at 450 nm to visualize host tissues and at 600 nm to visualize autofluorescence of Symbiodinium. Measurements of tissue thickness were taken in triplicate at random locations on each tissue sample to determine a colony average. Coral tissue thickness was characterized by either (i) high biomass, thick tissues anastomosing through highly perforate skeletons of perforate corals or (ii) low biomass, thin tissue ‘veneers’ of imperforate corals. Thus, tissue thickness did not appear to be altered by water absorption mechanisms (e.g., in the gastrovascular cavity) that may alter tissue thickness. 2.3. Physiological metrics Following removal from tanks, corals were immediately airbrushed with filtered natural seawater (0.2 ␮m), and the total volume of the homogenate recorded. After airbrushing, all fragments were dipped briefly in dilute bleach, left to dry, and their surface areas then measured using the paraffin wax technique (Stimson and Kinzie, 1991). Aliquots of the fresh homogenates were immediately frozen in liquid nitrogen and stored at −80 ◦ C for later

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analysis of protein and chlorophyll. Total protein was quantified in thawed homogenates using the BCA assay and bovine serum albumin as a standard (Pierce, Rockford, IL, USA). Chlorophyll was measured by passing 1 ml of thawed tissue homogenate across a Whatman GF/F (glass fiber filter) and the filter was subsequently extracted in 90% acetone for 24 h at 4 ◦ C (Parsons et al., 1984). Chlorophyll in these extracts was measured spectrophotometrically and chlorophyll (a and c2) concentrations were calculated using the equation from Jeffrey and Humphrey (1975). Additional fresh homogenate aliquots were preserved in 3.6% paraformaldehyde to assess Symbiodinium cell density. Before counting, cells in these aliquots were washed three times with filtered natural seawater (0.2 ␮m) and resuspended in 1× PBS. Symbiodinium cell densities were determined using a Scepter handheld automated cell counter (EMD Millipore Corp., Billerica, MA, USA). 2.4. Assay for superoxide ions in intact host tissues by NBT reduction Nitroblue tetrazolium (NBT) is reduced by superoxide ions to form its diformazan derivative (formazan). To determine baseline oxidative loads in each coral, the reduction of NBT to formazan was measured in intact corals exposed to 1.2 × 10−4 M NBT (Nii and Muscatine, 1997) for 20 min. The NBT solution was made using filtered natural seawater (0.2 ␮m) and each coral was submerged in a 100 ml volume of the solution in individual, aerated 200 ml glass beakers. The beakers were floated in ambient temperature tanks in the light and aerated throughout the incubation period. After the incubation, the NBT solution was poured off and each of the corals rinsed by filling the beaker with fresh, filtered natural seawater (0.2 ␮m). Each coral was then immediately airbrushed with filtered seawater (0.2 ␮m) to obtain a homogenate and the total volume recorded. Host and symbiont are difficult to separate; airbrushing, though not a perfect technique, is commonly used to provide representative samples of these fractions and permits the use of different normalization indices (e.g., Szmant and Gassman, 1990; Yost and Mitchelmore, 2010). Each sample homogenate was then centrifuged at 1000 × g for 5 min. The supernatant containing primarily host tissue was removed carefully so as not to disturb the pellet that contained primarily the algae and formazan. The supernatants were frozen in liquid nitrogen and stored at −80 ◦ C for later protein analysis. Symbiodinium pigments that can interfere with the spectrophotometric measurement of the ethanol-insoluble NBT formazan (Nii and Muscatine, 1997) were extracted from the Symbiodinium/formazan pellets using 1 ml of 95% EtOH for 1 h at 4 ◦ C. Samples were centrifuged at 5000 × g for 5 min and the EtOH supernatant removed. The pelleted formazan was dissolved in 1 ml dimethylformamide (DMF) by sonication (3 min). The absorbance of the resulting solution was measured spectrophotometrically at 550 nm. Spectrophotometric examination of the EtOH extract and DMF solution verified the complete removal of algal pigments following EtOH extraction. The concentration of formazan was calculated from the measured absorbance and the molar extinction coefficient of the NBT formazan, with results expressed as nmol NBT formazan produced per milligram of protein (Nii and Muscatine, 1997). 2.5. Inorganic carbon and nitrogen assimilation experiments Intact corals (n = 5 per species) were exposed to 500 ␮M 13 Cbicarbonate and 100 ␮M 15 NH3 for 30 min in individual, aerated 200 ml glass beakers. Beakers were floated (two-thirds submerged) in tanks under ambient conditions. At the end of the incubation period each coral was briefly placed in fresh, filtered natural seawater (0.2 ␮m) before being immediately airbrushed to obtain a homogenate (as detailed above) with deionized water (to minimize

the interference of chloride ions in the isotopic analysis). Diluted homogenates were subsampled and centrifuged at 12,000 × g for 5 min at 4 ◦ C to separate the coral host from Symbiodinium. The colorless supernatant (coral host fraction) was pipetted into a fresh microfuge tube and placed on ice. The remaining pellet (Symbiodinium fraction) was resuspended in ice-cold artificial seawater. This washing procedure was repeated three times, and the pellets containing primarily Symbiodinium were resuspended in 10–20 ml artificial seawater. All samples were immediately frozen or dried for later isotopic analysis. 2.6. Stable isotope analysis Samples for the analysis of 15 N/14 N and 13 C/12 C ratios were prepared by removing unincorporated 15 NH3 and 13 CO2 , with 1 N sodium hydroxide for 24 h, followed by 1 N phosphoric acid. Treated samples were dried and 0.5–1 mg subsamples analyzed by continuous flow isotope ratio mass spectrometry. Samples were combusted and the resulting N2 and CO2 gases separated in an elemental analyzer (EuroVector S.p.A., Milan, Italy); these gases were admitted into a Micromass Isoprime mass spectrometer (GV Instruments, Manchester, UK) for determination of 15 N/14 N and 13 C/12 C ratios. Isotopic analyses are presented in atom % 13 C or 15 N calculated as 15 N/(14 N + 15 N) × 100 or 13 C/(12 C + 13 C) × 100. Raw atom % values were corrected for background (control) values and used to calculate atom % enrichment in the samples. For each of the experiments, unenriched control corals (n = 5 per experiment; no 13 C-bicarbonate or 15 NH3 addition) were sampled and analyzed. Atom % enrichment was calculated by subtracting average control atom % values from experimental coral host and Symbiodinium atom % values. Control atom % values ranged from 1.08974 to 1.09145 atom % 13 C and from 0.36928 to 0.36938 atom % 15 N. 2.7. Statistical analysis All data were checked for normality and homogeneity of variances before statistical analysis. Non-normal data were logtransformed prior to statistical analysis. Data were analyzed by analysis of variance (ANOVA) or Student’s t-tests. Following significant results from ANOVA, pairwise differences were analyzed by the Tukey method. Pearson’s correlation coefficients were calculated to determine correlations between indices (pairwise for the following endpoints: formazan, chlorophyll, Symbiodinium density). All analyses were conducted using Minitab statistical software (Minitab Inc., State College, PA, USA), with ˛ = 0.05 for all tests. 3. Results Distinct skeletal and tissue characteristics were apparent at multiple spatial scales, underlining profound biological differences among morphotypes (Fig. 1). Branching corals have high levels of external structural diversity due to their intricate skeletons that allow for considerable habitat structure that scales with growth. Conversely, the foliose, and massive corals, have condensed threedimensional architectures and comparatively reduced degrees of macro-morphological complexity (Fig. 1, column a). Under a light microscope skeletal micro-architecture reveals a spectrum of polyp arrangements: cup-like calices (branching P. damicornis and S. hystrix), sub-surface polyps (massive P. lobata) and comparatively dispersed calices and elaborate coenosteum (foliose M. aequituberculata) (Fig. 1, column b). In column c of Fig. 1, simple schematic diagrams of longitudinal sections through lobate, massive and branching corals show different patterns of polyp, skeletal and tissue (with Symbiodinium) arrangements. Cross-sectional confocal

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Fig. 1. Visual comparison of M. aequituberculata, P. lobata, P. damicornis and S. hystrix showing colony morphology, skeletal architecture and tissue tunics with Symbiodinium. Column (a): macroscopic photographs of external colony morphology appearing foliose or plate-like with papillae (bumps on skeleton surface) in M. aequituberculata, massive and simple in P. lobata, and branching and more externally complex (multiple branches) in P. damicornis and S. hystrix. Column (b): microscopic photographs of tissue-free skeletal micro-architectures showing the arrangement of corallites (skeleton produced by a single polyp) within the greater skeletal matrix. Column (c): simple schematic diagrams of longitudinal sections through perforate (porous) and imperforate corals in the present study paralleling the confocal micrographs in column (d). Perforate morphology in the plate-like and massive corals reveals porous skeletal matrices with intercalating tissues and Symbiodinium distributed throughout, whereas the imperforate branching species have a ‘veneer’ of tissue that does not penetrate into the skeleton and Symbiodinium populations that are near the surface and more concentrated in the polyps. Column (d): macroscopic confocal micrographs (longitudinal views) of decalcified tissue tunics showing perforate tissues with ‘holes’ where skeleton was removed, or, alternatively, thin coenosarcs (tissue between corallites) that covered imperforate skeletons in branching species. Note different scale bars. Tissue depth measurements are described in Section 2.2; P indicates an individual polyp; S. hystrix tissue tunic is circular due to horizontal sectioning through a branch. Column (e): confocal micrographs of tissue tunic surfaces showing polyp arrangements with the distribution of Symbiodinium that appear concentrated in polyps and near the surface in branching corals (host tissues appear as blue and green hues or gray in black and white micrographs (P. lobata, S. hystrix); Symbiodinium appear red (color micrographs) or black (black and white micrographs). Black and white micrographs are shown with overlays of chlorophyll autofluorescence (red spheres) to emphasize Symbiodinium distribution patterns.

micrographs of decalcified tissues show differences in both the tissue depths and the spatial distribution of Symbiodinium in tissues among coral species (Fig. 1, column d, similar to the schematics in column c). M. aequituberculata and P. lobata corals have perforate skeletons (porous with a three-dimensional mesh-like structure; Santos et al., 2009), whereas the skeletons of the branching P. damicornis and S. hystrix corals are imperforate. The perforate corals have the greatest tissue depths, approximately three (M. aequituberculata) and five (P. lobata) times greater than branching corals, and Symbiodinium are distributed throughout the intricate maze of

coral tissue (Fig. 1, column d, and Table 1). Conversely, the branching corals have shallow tissue layers and Symbiodinium are in close proximity to one another, concentrated in the thin ‘veneer’ of coral polyp and inter-calyx tissues atop their imperforate skeletons. In addition, the distribution of Symbiodinium is more concentrated in the polyps of the imperforate corals (Fig. 1, columns d and e). The massive Porites corals exhibited comparatively high levels of protein, high Symbiodinium densities and low Symbiodinium chlorophyll concentrations (Fig. 2) when compared to the

Table 1 Biological attributes (mean ± SEM) of M. aequituberculata, P. lobata, P. damicornis and S. hystrix corals. Imperforate, branching corals (P. damicornis and S. hystrix) have shallower tissue depths compared to perforate corals. Atom % 13 C ratios are also higher in the branching corals, a notable difference with implications for carbon recycling dynamics in the symbiosis. Other attributes show biological heterogeneity within and between imperforate and perforate corals. Attribute

Montipora

Porites

Pocillopora

Seriatopora

Tissue depth (␮m) Symbiodinium spatial pattern in tissue Total chlorophyll (pg cell−1 ) Symbiodinium cell diameter (␮m) Atom % 13 C ratio pellet:supernatant (primarily Symbiodinium:host) Atom % 15 N ratio pellet:supernatant (primarily Symbiodinium:host)

608 ± 55 Dispersed 3.44 ± 0.50 9.00 ± 0.08 0.30 ± 0.10 0.20 ± 0.01

958 ± 22 Dispersed 0.98 ± 0.12 8.15 ± 0.12 0.36 ± 0.07 0.43 ± 0.01

183 ± 36 Concentrated 3.18 ± 0.38 8.44 ± 0.06 1.65 ± 0.41 0.43 ± 0.01

154 ± 27 Concentrated 1.56 ± 0.11 7.66 ± 0.04 1.84 ± 0.32 0.31 ± 0.04

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Fig. 2. Comparison of physiological metrics in M. aequituberculata (Ma), P. lobata (Pl), P. damicornis (Pd), and S. hystrix (Sh). (a) Soluble protein (mg protein/cm2 ), (b) Symbiodinium density (Symbiodinium/mg protein), (c) Symbiodinium chlorophyll concentrations (␮g chlorophyll/mg protein), and (d) formazan concentration (indicates superoxide radical levels; nmol formazan/mg protein). Letters indicate non-significant (same letter) or significant (different letters) statistical differences between species.

branching and foliose corals, which shared similar (i.e., not significantly different) physiological patterns among coral hosts and Symbiodinium parameters. Total soluble protein levels ranged from 0.25 to 1.10 mg protein−1 cm−2 among all species investigated (Fig. 2). M. aequituberculata and S. hystrix corals contained similar amounts of protein (0.39 ± 0.07; 0.50 ± 0.03 mg protein−1 cm−2 ) and averaged 45% of the 1.10 ± 0.12 mg protein−1 cm−2 for P. lobata (ANOVA P < 0.05); the average protein content of the P. lobata

homogenates was also significantly higher than that of P. damicornis by a margin of 88% (Fig. 2a). Though P. lobata hosted a relatively high number of Symbiodinium (Fig. 2b), the chlorophyll content of those algal cells was significantly lower than that of M. aequituberculata, P. damicornis, and S. hystrix corals (Fig. 2c and Table 1), and was inversely correlated with Symbiodinium densities (Symbiodinium/mg protein; Fig. 2b) in a moderately negative relationship (−0.316, P > 0.05). Among corals, the average diameter of

Fig. 3. 13 C and 15 N atom % enrichment values (atom %/mg protein) in supernatant (primarily coral tissue; black bars) and pellet (primarily Symbiodinium; gray bars) for M. aequituberculata (Ma), P. lobata (Pl), P. damicornis (Pd), and S. hystrix (Sh) corals. Values indicate dynamics of carbon and nitrogen assimilation and cycling in the intact symbiosis (n = 20; 5 corals per species). Letters indicate non-significant (same letter) or significant (different letters) statistical differences for Symbiodinium:host ratios between species.

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Symbiodinium (in ␮m ± SEM) was variable and significantly different between M. aequituberculata and S. hystrix (Table 1). Baseline levels of superoxide ions in intact coral fragments, as measured by the reduction of NBT to formazan (nmol formazan/mg protein), were significantly higher in M. aequituberculata than in P. lobata, P. damicornis or S. hystrix, indicating that M. aequituberculata corals had the highest levels of superoxide ions during the 20 min NBT exposure period (Fig. 2d). Additionally, these results show varying degrees of correlation (Pearson’s correlation coefficients) between superoxide ion production and Symbiodinium densities (Sym) or chlorophyll concentrations (chl) within a species (for all correlations P > 0.05): M. aequituberculata: 0.377 (Sym), −0.066 (chl); P. lobata: 0.598 (Sym), 0.422 (chl); P. damicornis: −0.177 (Sym), −0.465 (chl); S. hystrix: 0.614 (Sym), 0.626 (chl) (Fig. 2b–d). Following the 30 min isotopic exposure (100 ␮M 15 N and 500 ␮M 13 C), both supernatant (primarily coral tissues) and pelleted fractions (primarily Symbiodinium) were enriched with carbon and nitrogen (Fig. 3). 13 C enrichment in the supernatants averaged 0.011 ± 0.001 atom % 13 C/mg protein and there were no significant differences in the levels of enrichment among coral species. In contrast, 13 C enrichment patterns in the pellet fractions isolated from the different coral species were different. The pellets from S. hystrix and P. damicornis corals had the highest levels of 13 C enrichment; significant differences were evident in pellets from S. hystrix (0.023 ± 0.006) and M. aequituberculata (0.005 ± 0.002) or P. lobata (0.003 ± 0.001) (ANOVA P < 0.05). Additionally, pellets isolated from M. aequituberculata and P. lobata were significantly less 13 C enriched than their coral hosts (t-test P < 0.05; Table 1). These data show that the pellets from imperforate, branching species were significantly more enriched with 13 C sodium bicarbonate (13 C pellet to supernatant ratios were more than five times greater) than those in perforate, foliose or massive corals at the end of the incubation period. 15 N enrichment patterns in pellets averaged 0.118 ± 0.010 atom % 15 N/mg protein and were not significantly different among coral hosts (Fig. 3). The supernatant fractions were, however, more enriched with 15 N ammonium chloride compared to their pellets (ANOVA P < 0.05; Table 1) and were significantly different among hosts. M. aequituberculata supernatants averaged approximately 50% more atom % 15 N/mg protein than those of the other coral species and were significantly more enriched (0.695 ± 0.105) than both P. lobata (0.381 ± 0.069) and P. damicornis (0.274 ± 0.069) supernatants.

4. Discussion The present study reveals inherent differences in the basic biology of corals representing four important genera and emphasizes the high levels of structural and biological heterogeneity that exist in corals. The patterns that emerge show different combinations of external and internal physical complexity, characteristics that are correlated with fundamental biological attributes of the coral symbiosis and the quality, quantity and complexity of Symbiodinium habitat. Furthermore, our findings exemplify that skeletal porosity is a trait related to known environmental thresholds in corals. Our investigation of two perforate and two imperforate coral species revealed high variance in the mean values for most of the characteristics investigated. Within-group variation was pronounced in perforate species (e.g., protein and chlorophyll contents and superoxide radical levels) and variance was also high between perforate and non-perforate groups. These results suggest that sampling more species per group would allow further exploration of the observed high levels of variation and how high- and lowvariability traits may pair with different skeletal features across species to influence response variability. In the present study, the

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tradeoff of using a low number of species per group was balanced by a higher sampling number within individual species (n = 8), which led to several possible conclusions notwithstanding high levels of variability. Confocal micrographs of tissue cross-sections show the fundamental differences in tissue architecture between the perforate and imperforate corals. The highly perforated skeletons of the massive P. lobata and foliose M. aequituberculata corals allow the coral tissues to penetrate to much greater depths compared to the imperforate, branching P. damicornis and S. hystrix corals. This internal environment shapes the effective three-dimensional habitat for Symbiodinium, creating more dispersed, or comparatively restricted, spatial arrangements of Symbiodinium within the host coral in perforate versus imperforate corals, respectively. Thus, the relative partitioning of biological complexity is very different among the morphotypes investigated – branching imperforate corals exhibit high levels of external structural complexity and spatial diversity, whereas massive and foliose perforate corals exhibit much simpler external structure and much higher levels of internal complexity. The internal environment that shapes the Symbiodinium habitat in perforate corals adds dimensionality to the interaction between host and symbiont by providing a broader scope for refuge from the external environment, a feature that is absent from imperforate associations. Massive Porites corals exhibit several unique features that distinguish them from the other corals investigated. The comparison of P. lobata and M. aequituberculata suggests that perforate skeletons may be predictive of deeper tissues, but are not necessarily predictive of other fundamental biological traits. Notably, P. lobata had the highest levels of tissue soluble protein, and the lowest levels of total chlorophyll compared to all other corals. Furthermore, only P. lobata showed Symbiodinium densities that were inversely correlated with chlorophyll concentrations in a moderately negative relationship, and levels of superoxide radicals (measured as formazan concentration) in P. lobata were lowest overall. These results suggest that oxygen radical levels may be linked to Symbiodinium densities and/or the chlorophyll content of Symbiodinium cells through positive or negative relationships. Both of these parameters merit concurrent consideration given that a coral’s complement of Symbiodinium is typically regarded as a primary source of cellular reactive oxygen species for the coral host (Warner et al., 1999; Fitt et al., 2001; Weis, 2008). Many of the unique attributes described above appear to be correlated with P. lobata’s highly modified internal environment, an observation that warrants further investigation and suggests a broader relationship between microenvironment and response variability among coral taxa. Specific features related to perforate skeletons might explain some of the links between the physical and biological aspects of these corals. For example, Symbiodinium in perforate corals may encounter lower light levels due to the light absorption properties of thicker coral tissues and higher Symbiodinium densities (Teran et al., 2010). Additionally, intercalating tissues create the opportunity for dispersal and migration of Symbiodinium within the tissues to exploit the different microenvironments afforded by the complexity of the space (Santos et al., 2009). In combination with other traits such as low permeability and high compression strength (Wu et al., 2009), massive Porites’ highly modified internal environment provides a greater scope for uniqueness as a result of its inherently high levels of biological variability. In addition to our observations there are other unique features of P. lobata corals that are likely to contribute to this coral’s ability to survive changing environmental conditions. P. lobata is the only species in the present study with a relatively fixed morphology, whereas the others are known to be phenotypically plastic, changing their external morphological composition in response

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to environmental factors (Lesser et al., 1994; Kaandorp, 1999). P. lobata’s complex internal environment may serve to diversify Symbiodinium habitats despite its simple external architecture. P. lobata is also known to have a primarily autotrophic mode of nutrition, a low growth rate and high fidelity to Symbiodinium type C15 symbionts (Stat et al., 2009 and references therein). Each of the above-mentioned features may in part determine this coral’s notable low bleaching susceptibility or long life span (Loya et al., 2001; van Woesik et al., 2011), but it is likely complex and dynamic groupings of key features that ultimately inform such robustness. The imperforate branching corals show similar patterns of structural and biological organization that were not present among the perforate corals in our study. In addition to their imperforate skeletons and highly complex external architectures, branching corals have thin tissues, and similar chlorophyll levels (though Symbiodinium densities were significantly different) and baseline levels of superoxide radicals. Furthermore, the imperforate corals exhibit similar 13 C enrichment patterns that differ significantly from those of the perforate corals as a group (Symbiodinium more enriched than coral hosts). Corals can rely mainly on autotrophy, heterotrophy or a combination of both (polytrophy) as energy acquisition strategies in nutrition (Holbreque and Ferrier-Pages, 2009). The different atom % 13 C ratios (pellet:supernatant (primarily Symbiodinium:host) fractions) between perforate and imperforate corals are a key difference in this important aspect of symbiosis and may indicate fast or slow recycling (respectively) of carbon between Symbiodinium and the coral host. Additionally, the atom % 15 N ratios (pellet:supernatant) were similar for imperforate corals, but not for perforate corals, where M. aequituberculata supernatants were significantly more 15 N enriched than their Symbiodinium fraction and those of the other corals investigated. These results may indicate a greater capacity for nitrogen assimilation and/or recycling in the M. aequituberculata corals or a potential mechanism to control Symbiodinium proliferation, which may be suppressed by nitrogen limitation (Falkowski et al., 1993). In future studies, mass balance techniques may be used to explicitly determine which partner (symbiont or host) specifically contributes to isotope enrichment patterns over time. Although this factor is beyond the scope of this study, nitrogen limitation (and/or carbon limitation (Franklin et al., 2004)) may influence the oxidative state of Symbiodinium and could be a factor contributing to the high levels of superoxide radicals present in M. aequituberculata. For example, a coral’s reliance on autotrophic (Symbiodinium) and/or heterotrophic sources of nutrition can be assessed using stable isotopes, although the interpretation of our isotopic results is complicated by biological processes innate to the system, including respiration (McConnaughey et al., 1997) and endosymbiotic photosynthesis (Swart et al., 2006). Nonetheless, the contrasting enrichment patterns between perforate and imperforate groups suggest different capacities for Symbiodinium to provision carbon to their host (Stat et al., 2008) and indicate that Symbiodinium performance may be driven in part by subsurface habitat interactions that are not readily discernable from an exterior morphological perspective. It is also likely that the assemblages of Symbiodinium genotypes differed among the coral taxa investigated (Chen et al., 2005). The explicit determination of Symbiodinium genotypes was not a focal point of this study, but known differences among types may explain some differences in the coral attributes investigated, warranting the inclusion of genotyping in future comparisons. Our comparative assessment of perforate and imperforate coral morphotypes demonstrates that perforate skeletons allow for and create fundamentally different arrangements of biological complexity in corals. The fixed external appearance of P. lobata corals veils an inwardly elaborate mosaic of skeleton and tissue that supports a dense population of Symbiodinium that feeds this highly

autotrophic coral. In addition to the unique attributes we describe, the massive Porites are also reported to be slow growing and to exhibit high metabolic rates when compared to branching species (reviewed in Buddemeier and Kinzie, 1976). Collectively, such biological attributes combine to promote enduring symbiotic relationships that show increased resistance to environmental challenges. As corals do not respond uniformly to their environment, analyses that consider the perforate/imperforate nature of coral skeletons in addition to other key aspects of coral biology such as morphology, feeding strategy and Symbiodinium specificity are likely to improve the predictability of coral performance in response to dynamic environmental conditions. Additionally, species-specific comparisons will likely generate further insights into the ecological significance of morphological and physiological differences within genera. Predictions of coral response based on taxonomy will lack the biological resolution of groups assembled according to functional or structural attributes. The use of such groups and the assessment of their relative abundance on reefs will enhance community modeling frameworks and broaden the conceptual context from which predictions of coral community response to environmental change are made.

Acknowledgments We thank all of those at the National Museum of Marine Biology and Aquarium, Taiwan, who assisted in this project. The study was funded by the NSF OISE award #1042509 and represents HIMB contribution number 1560 and SOEST contribution number 8975.

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