Visualization of sub-daily skeletal growth patterns in massive Porites corals grown in Sr-enriched seawater

Journal of Structural Biology 180 (2012) 47–56

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Visualization of sub-daily skeletal growth patterns in massive Porites corals grown in Sr-enriched seawater Kotaro Shirai a,b,⇑, Kohki Sowa c, Tsuyoshi Watanabe c, Yuji Sano d, Takashi Nakamura e, Peta Clode f,g a

Department of Earth and Planetary Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan Increments Research Group, Department of Paleontology, Institute of Geosciences, University of Mainz, Johann-Joachim-Becher-Weg 21, 55099 Mainz, Germany c Department of Natural History Sciences, Graduate School of Science, Hokkaido University, N10W8, Kita-ku, Sapporo 060-0810, Japan d Department of Chemical Oceanography, Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8561, Japan e Department of Mechanical and Environmental Informatics, Graduate School of Information Science and Engineering, Tokyo Institute of Technology, O-okayama 2-12-1-W8-13, Meguro-ku, Tokyo 152-8552, Japan f Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley WA 6009, Australia g Oceans Institute, The University of Western Australia, Crawley WA 6009, Australia b

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Article history: Received 18 January 2012 Received in revised form 22 May 2012 Accepted 23 May 2012 Available online 7 June 2012 Keywords: Scleractinian coral Strontium Calcification Electron probe microanalysis Skeletogenesis

a b s t r a c t We performed high resolution marking experiments using seawater with elevated Sr concentration to investigate the timing and ultrastructure of skeletal deposition by massive Porites australiensis corals. Corals were cultured in seawater enriched with Sr during day-time only, night-time only or for one full-day. Cross sections of skeletal material were prepared and the Sr incorporated into the newly deposited skeleton analyzed by electron probe microanalysis. These regions of Sr incorporation were then correlated with skeletal ultrastructure. Massive Porites coral skeletons are composed of two types of microstructural elements – the ‘‘centers of calcification’’ and the surrounding fibrous structural region. Within the fibrous structural region, alternative patterns of etch-sensitive growth lines and an etch-resistant fibrous layer were observed. In the full-day samples, high-Sr bands extended across both growth lines and fibrous layers. In day-time samples, high-Sr regions corresponded to the fibrous layer, while in the night-time samples high-Sr regions were associated with an outermost growth line. These distinct growth patterns suggest a daily growth pattern associated with the fibrous region of massive P. australiensis corals, where a pair of narrow growth lines and a larger fibrous layer is seen as a daily growth region. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Hermatypic scleractinian corals are widely distributed from tropical to temperate areas, forming calcium carbonate exoskeletons that act as the foundation of coral reef environments. The complex framework formed by coral reefs has resulted in these systems being one of the most diverse and socio-economically important habitats on Earth. One of the key questions that has remained unanswered for more than a century, has been how do these corals grow and deposit their skeleton, in often very fast time frames, to form this complex reef framework? In the face of climate change and ocean acidification, understanding the mechanism of coral calcification has become an important topic in coral reef science (Hallock, 2005; Watanabe et al., 2007; Anthony et al., 2008; Wei et al., 2009; Cohen and Holcomb, 2009). ⇑ Corresponding author at: International Coastal Research Center, Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8564, Japan. E-mail address: [email protected] (K. Shirai). 1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2012.05.017

Considerable debate exists as to the mechanisms underlying the role of symbiotic algae, known as zooxanthellae, in the calcification process. Traditionally, calcification in zooxanthellate corals has been considered ‘light enhanced’, with calcification rates observed to be much higher during day time than night time, with this increase attributed to the photosynthetic activities of the zooxanthellae. However, data from azooxanthellate corals indicates that these corals can calcify at comparable rates to zooxanthellate corals (Marshall, 1996; Marshall and Clode, 2004; Tambutté et al., 2007; Maier et al., 2009), fuelling the ongoing debate as to whether calcification in zooxanthellate corals is truly light enhanced. Further, calcium uptake is known to be light sensitive, although the mechanisms behind this are unclear (de Beer et al., 2000; Al-Horani et al., 2003; Marshall and Clode, 2003). Diurnal patterns of coral biomineralization, such as the cycling of calcification rate and depositional timing of specific ultrastructures, may provide unique opportunities to study the process of coral biomineralization and the potential effects of light upon this. It is well established that coral skeletons are composed of well arranged microstructural elements (e.g. Cuif and Dauphin, 1998; Sto-

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larski, 2003; Nothdurft and Webb, 2007; Janiszewska et al., 2011). It has been accepted for many years that the fundamental ultrastructural units are so called ‘‘centers of calcification’’ surrounded by fibrous skeleton (e.g. Bryan and Hill, 1941; Cohen and McConnaughey, 2003). However, recent advances in microstructural studies suggest that this definition of ultrastructural units is not adequate (Cuif and Dauphin, 1998, 2005a, 2005b; Cuif et al., 2003; Stolarski, 2003; Nothdurft and Webb, 2007; Janiszewska et al., 2011). As our understanding of skeletal ultrastructure and formation further develops, various new terminologies have been introduced, dependent upon the discipline (geo or bio) and on the possible mechanisms of formation. Here we use the traditional terminology of ‘‘center of calcification’’ and fiber because this terminology is, to our knowledge, the most widely recognized and used to date. However it should be noted that recent studies (Cuif and Dauphin, 1998, 2005a,b; Cuif et al., 2003; Stolarski, 2003; Nothdurft and Webb, 2007; Janiszewska et al., 2011) and the current authors do not support idea that the ‘‘center of calcification’’ functions as the name implies. Microstructural observations of the skeletal surface determined by time-series sampling has revealed that deposition of different microstructural elements may occur at day time and night time in a variety of corals, including Manicina aereolota (Barnes, 1972), Acropora cervicornis (Gladfelter, 1982, 1983), and Pocillopora damicornis (Le Tissier, 1988). Using repeated staining with alizarin red Sandeman (2008) also found that the superimposed lamination of optically denser and lighter bands in fibrous region of Agaricia agaricites were formed during the day and night respectively. In contrast, no clear diurnal patterns were observed in Galaxea fascicularis (Hidaka, 1991; Clode and Marshall, 2003a). Raz-Bahat et al. (2006) also reported no diurnal pattern of growth in Stylophora pistillata using a lateral skeleton preparative assay. Such inconsistent results suggest that calcification patterns and diurnal processes may be highly species specific, although this also implies that daylight and zooxanthellae are not central to driving this process. There are several methods that have been used to investigate the skeletal growth patterns of corals, each with advantages and limitations. These include the use of skeletal dyes (Isa, 1986; Böhm et al., 2006) and radioactive tracers (Marshall and Wright, 1998; Tambutté et al., 1996; Furla et al., 2000; Ferrier-Pages et al., 2002), time-lapse photography (Barnes and Crossland, 1980), and ion micro-sensors (Al-Horani et al., 2003; Marshall and Clode, 2003). Skeletal dyes are probably the most straightforward method to visualize growth in a given time. Barnes (1970) and Sandeman (2008) successfully identified skeletal deposition at an hourly scale using alizarin staining. However, the major drawback of this chemical staining method is the potential toxicity of the dye, which may cause decreases in calcification rate (e.g. Dodge et al., 1984; Gaetani et al., 2011). Recently, Houlbreque et al. (2009) used the stable non-toxic isotope tracer 86Sr to visualize skeletal deposition in Porites porites over 3 days by imaging 86Sr distribution within the skeleton using high resolution ion microprobe analyses (NanoSIMS). However, our ICP data show that 86 Sr does not dissolve into seawater when simply added as solid 86 SrCO3 as reported by Houlbreque (unpublished data), questioning the method of Sr uptake into the tissues and skeleton in that study. Understanding the mechanisms of coral skeletal deposition and growth, and the factors affecting these, is also fundamental to the field of coral geochemistry, where Sr/Ca, Mg/Ca and O isotopic ratios are intensively used as past seawater temperature proxies (see reviews by Gagan et al., 2000; Lough, 2004; Correge, 2006; Watanabe et al., 2007). Microanalytical studies have indeed revealed that there are significant compositional heterogeneities that cannot be explained by temperature alone (Cohen et al., 2001; Meibom et al.,

2004, 2006, 2008; Allison and Finch, 2004, 2007; Shirai et al., 2005, 2008; Gaetani and Cohen, 2006; Holcomb et al., 2009; Allison et al., 2010; Gaetani et al., 2011). Since this heterogeneity is strongly associated with skeletal ultrastructure, the source of the heterogeneity is considered to be of, as yet unknown biological origin. In some studies, elemental fractionation is thought to be derived from ultrastructural variation, with compositional differences between ‘‘centers of calcification’’ and fibrous regions of massive Porites corals attributed to diurnal differences, based upon the assumption that these microstructural elements are formed during night and day respectively (Cohen et al., 2001; Cohen and McConnaughey, 2003; Allison and Finch, 2004, 2007). However, diurnal patterns of skeletal growth regions have not been reliably investigated, thus this assumption is largely unfounded. Massive Porites corals are the most commonly used genus for paleoclimate reconstructions, therefore to accurately interpret the mechanisms of microscale elemental fractionation, a detailed understanding of ultrastructural formation is essential. With these considerations in mind, it is clear that more information is needed in regard to the patterns of skeletal deposition and growth in corals and the biotic and abiotic factors that affect and control these. In this study we present a versatile method to visualize sub-daily growth patterns of massive Porites australiensis corals by high temporal resolution marking experiments performed using seawater enriched in Sr concentration. We have correlated this pattern of Sr deposition with skeletal ultrastructure and obtained skeletal deposition patterns over day time and night time.

2. Materials and methods 2.1. Culture experiments Culture experiments were performed at Shiraho Reef, Ishigaki Island, Japan (Fig. 1). Detailed environmental settings of Shiraho Reef are described in Appendix A. Since this study attempted to investigate coral growth over short time scales, experiments were designed to minimize any environmental or mechanical stress on the corals during the sampling process and experimentation. Cuts were made in massive P. australiensis colonies from 1 m depth at several cm intervals and to a depth of 5 cm with a saw. These pieces of coral were allowed to recover for two full days, after which the basal part of the coral colony where no living polyps persist, was mechanically broken. The resulting blocks, of which the top surface was covered with living polyps, were subsequently used for the culture experiments. All replicate experimental blocks were sampled from the same colony. Only corals that extended their tentacles out from the calyx were used for experimentation. The experiment was conducted at the border area between the moat and the inner reef flat of Shiraho Reef. Seawater temperature was measured every hour using a data logger placed within 500 m of the experimental site (Fig. 2). Salinity was measured 2 km north of the experimental site at approximately 16:30 and was found to be 34.5 psu. Solar radiation and tidal data were obtained from the meteorological station located 15 km south west of Shiraho Reef. Relevant environmental data are summarized in Fig. 2. Photon flux density was roughly estimated by the relationship between the values reported by Nakamura and Nakamori (2009) and the solar radiation data (Photon flux density (lmol m 2 s 1) = 446 solar radiation (mJ m 2), n = 14, R2 = 0.93). The low tides were observed at 7:30 and 18:30, and the high tides were observed at 13:40 and 1:00. For incubation, 10 L tightly sealed plastic containers with 84% sunlight transparency were used. Coral samples (n = 4–5) were transferred into these containers without air exposure. Solid

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Fig.2. Observed seawater temperatures, tides (sea surface height from measurement point), and insolation. Estimated photon flux is also shown on the right axis with insolation data (see text). The duration of day-time and night-time experiments are hatched with gray colors, while the full-day experiment extends over both of these periods.

ratory where all samples were removed from the containers and air dried. 2.2. Electron probe microanalysis

Fig.1. Meteorological station and study sites for the incubation experiment at Shiraho fringing reef, Ishigaki Island, Japan.

SrCl2.6H2O (analytical grade) was first dissolved in seawater, then this stock solution was added to the containers, resulting in a Sr concentration double that of normal seawater (16 ppm). It is known that corals demonstrate significant tolerance for high Sr concentrations (Ferrier-Pages et al., 2002). These procedures were performed on a micro-atoll near the experimental site. After addition of the Sr-enriched seawater, the containers were tightly sealed and kept on the seafloor at 2 m depth until all culture experiments were completed. All the experiments were performed within 20 m of the original colony that the samples were collected from, and seawater used for experiments was also collected from that location, thereby closely mimicking local environmental conditions (temperature, pressure, seawater composition, and illumination cycle) naturally experienced by the corals, with strontium concentration, light intensity and water flow being the only changing parameters. To study diurnal growth patterns, three experimental parameters were investigated – day-time growth, night-time growth, and full-day (24 h) growth. The day-time and full-day experiments were started at 10:30. The cover of the day-time container was subsequently opened so that the samples were rinsed in natural seawater at 20:00. The night-time experiment was started at 21:00. At 6:00 the following day, the lids of the full-day and night-time containers were opened and the corals rinsed with natural seawater. Following this, all of the containers were tightly sealed, and the corals were transferred to the marine station labo-

Sub-samples were prepared from the top surfaces of the coral blocks using a diamond saw. The middle portion of the biggest block in each tank was chosen for analysis to minimize any effect of cutting. Coral pieces were cleaned several times with tap water followed by ethanol, and dried again. Based on the etching rate of the Milli-Q water (see below), the use of water and ethanol do not significantly dissolve the skeleton at detectable levels. Sub-samples were then embedded in epoxy resin and coarsely ground until a cross section along the growth direction was exposed. These samples were then polished with a graded series of polishing sheets and finally polished with 1 lm diamond paste. The polished surface was coated with Pt-Pd to avoid charging and sample damage during analysis. Strontium distributions within the skeletons were analyzed using electron probe microanalysis (JXA-8900, JEOL) at the Atmosphere and Ocean Research Institute, University of Tokyo. Measurements were performed with an accelerating voltage of 15 kV, probe current of 50 nA, and 1 lm beam diameter. Composition maps for Sr (La), Mg (Ka), S (Ka), and Ca (Ka) were obtained with a WDS detector and 100 ms acquisition for each 1 lm pixel. Back-scattered electron (BSE) images were also obtained simultaneously. All of the analyses were performed within 500 lm from the growth tip of the coral. Analyzed areas were carefully chosen to avoid any regions with secondary deposits or damage to the coral tip. The spatial resolution of the element analyses were estimated from the minimum width of zoning and found to be 2 lm, which is a typical value for the analytical conditions used. Epoxy resin adjacent to the coral skeleton may exhibit some apparent Sr signal, due to scattering of the electron beam and X-rays, which penetrate readily through the resin, thus the Sr signal can appear blurred at the resin-skeleton interface. Since the BSE images reflect structural information and have higher spatial reso-

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lution (1 lm) than X-rays, areas with low BSE signal are indicative of regions where the X-ray data are not correlated directly with the skeleton and thus should not be considered. 2.3. Skeletal ultrastructural observations To observe the coral skeletal structure with SEM, the polished skeletal surface must be etched with an acid or chelating agent to generate surface structural relief. However, if too much of the surface layer is etched, the skeletal structures observed will no longer correspond to that of the Sr-analyzed plane. Therefore, to compare Sr marking as close as possible to the ultrastructure, etching was performed gently. After the EPMA analyses, the Pt-Pd surface coating was removed by polishing with 1 lm diamond paste. Duration of the polishing was carefully checked by watching the surface color change from gray (Pt–Pd) to white (CaCO3). The sample was then immersed in Milli-Q water at room temperature for 2 days. Since Milli-Q water is undersaturated with calcium carbonate, Milli-Q water dissolves the skeletal surface very slowly. While this results in what appears to be worse presentation of the ultrastructure compared to conventionally used etching solutions, the method is easily controllable to obtain very shallow etching depths from analyzed surfaces, ensuring that the ultrastructure revealed matches that of the analyzed region. After minimal etching, the samples were checked with an optical microscope and re-coated with Pt–Pd. The skeletal ultrastructure corresponding to the Sr marking was subsequently observed and documented using SEM (S-4500, HITACHI) at the Atmosphere and Ocean Research Institute, University of Tokyo. Since some structural features were not always clearly evident in the SEM images, pictures by reflected light microscopy with co-axial light are also shown when possible. Precision of the alignment can be estimated based on the distance between the outline of BSE and that of SEM (or reflected microscopic) images. This distance is less than 1 lm in most cases. There are some regions where the distance appears larger, however this is due changes in shape caused by etching. Thus, the error of the alignment is estimated at less than two microns, which is below the analytical resolution. 3. Results The SEM images reveal typical ultrastructural features expected for Porites coral skeletons (Nothdurft and Webb, 2007), including etch-sensitive ‘‘centers of calcification’’ and fibrous skeletal regions radiating from these (Fig. 3). The fibrous skeletal region is composed of sequential layers of narrow growth lines (negative etching relief) and fibrous layers (positive etching relief wherein the skeletal fibers are coordinated parallel to crystal growth direction), which were alternatively deposited perpendicular to the crystal growth direction. Following incubation in Sr-enriched seawater, high-Sr bands were clearly detectable toward the outer edge of the skeleton across all experimental time periods (Figs. 4–6, Figs. S1–S4). Not surprisingly, high-Sr bands were more evident in the longer full-day experiments compared to the shorter time periods for the day-time and night-time experiments (Figs. 4–6, Figs. S1–S4). The majority of Sr intensities from non-labeled regions were 30–40 counts per pixel, reflecting natural Sr abundance, while those from labeled areas were 60–90 counts per pixel. Thus, the intensity ratio between labeled and non-labeled areas was almost two, which is closely proportional to the difference in the natural and experimental seawater Sr concentrations. Since the precision of each pixel measurement were estimated to be 18% based on counting statistics, analytical conditions employed in the present study can be reliably expected to detect >18% variation in Sr enrichment.

Fig.3. Typical skeletal ultrastructural features of the massive Porites coral. Etchsensitive growth lines are indicated by white dotted lines. Etch-resistant fibrous layers are indicated by white solid lines. Scale bar = 10 lm.

In the full-day experiments, the high-Sr bands were typically co-located with the outermost fibrous layer seen in the SEM images (Fig. 4, Figs. S1 and S2). The width of the high-Sr band ranged from 2–8 lm, which is consistent with the thickness of typical fibrous layers. The inner boundary of the high-Sr bands (i.e. beginning of Sr-enriched deposition) was typically located along the growth lines, while the outer boundary (i.e. end of Sr-enriched deposition) usually ended at the outer edge of the skeleton. In some cases, the high-Sr bands did not always start exactly from a growth line (Fig. S1) and this may reflect that skeletal growth had begun in this region prior to enriched Sr incubation. Additionally, normal skeletal growth subsequent to culture periods was also observed adjacent to the high-Sr bands (Fig. 4), but this degree of growth was not uniform along the skeletal edge. Post-culture skeletal growth generally started from a growth line, which corresponds to the outer boundary of the high-Sr marking. In night-time experiments, high-Sr lines were only observed within or close to a growth line (Fig. 5 and Fig. S3). The width of these high-Sr bands was usually less than 3 lm. In day-time experiments, high-Sr bands were located toward the middle to outer region of the fibrous layer (Figs. 6, Fig. S4). Again, this may reflect that skeletal growth within the incremental region had begun prior to enriched Sr incubation. The high-Sr bands during day-time experiments are up to 3–4 lm in width. Similar to full-day experiments, continued normal skeletal growth following culture periods could be seen deposited outside the high-Sr bands in distinct, isolated regions (Fig. S4). The observed patterns of Sr deposition in the massive P. australiensis coral, over full-day, day-time and night-time incubations, are largely consistent with a diurnal cycle of skeletal growth, where narrow growth lines are formed during night time calcification, while fibrous layers form during day time. However, some heterogeneous skeletal deposition inconsistent with this diurnal pattern was also observed, suggesting a more complex deposition process. 4. Discussion 4.1. Diurnal patterns and skeletal ultrastructure The observed ultrastructural patterns are largely consistent with diurnal cycles where growth lines and fibrous layers are

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Fig.4. Skeletal ultrastructure and Sr distribution in the full-day experiment. (a) Skeletal ultrastructure. (b) Skeletal ultrastructure and Sr distribution with 30% transparency overlay (c) Back-scattered electron image and Sr distribution with 40% transparency overlay (d) Sr distribution (e) Corresponding reflected light microscopy image (f) Enlargement of (b). White dotted lines indicate growth lines. PstCG: post-culture growth. Color scale of the total count of Sr analyses per pixel is shown at the bottom of the figures. Note good alignment indicated by yellow arrows showing small curving at same position. Scale bar = 10 lm.

Fig.5. Skeletal ultrastructure and Sr distribution in the night-time experiment. (a) Skeletal ultrastructure. (b) Skeletal ultrastructure and Sr distribution with 30% transparency overlay (c) Sr distribution. (d) Enlargement of (b). White dotted lines indicate growth lines. COC: ‘‘center of calcification’’. Color scale of the total count of Sr analyses per pixel is shown at the bottom of the figures. Note good alignment indicated by yellow arrows showing small curving at same position. Scale bar = 10 lm.

deposited during night and day time respectively. Such diurnal growth patterns are also consistent with the diurnal pattern observed in Agaricia agaricites using alizarin red staining (Sandeman, 2008). At the colony scale, calcification rate in the light is significantly higher than in the dark (e.g. Gattuso et al., 1999). At the ultrastructure scale, the skeleton is dominated by fibrous layers compared to growth lines, thus there appears to be a discreet correlation between calcification rate, light intensity and ultrastructural components.

However heterogeneous deposition was also found, which is inconsistent with this diurnal cycle. Importantly, there are some factors potentially affecting the co-location of Sr-labeling with the precise diurnal cycle of ultrastructural deposition. Since the experimental time schedule was not perfectly correlated with daylight/darkness, the full-day, day-time and night-time experiment do not precisely reflect calcification during full day, daylight and night time, respectively. This is difficult with the daylight hours observed in Japan at the time of experimentation. For example,

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Fig.6. Skeletal ultrastructure and Sr distribution in the day-time experiment. (a) Skeletal ultrastructure. (b) Skeletal ultrastructure and Sr distribution with 30% transparency overlay (c) Back-scattered electron image and Sr distribution with 40% transparency overlay (d) Sr distribution (e) Corresponding reflected light microscopy image (f) Enlargement of (6b). White dotted lines indicate growth lines. Color scale of the total count of Sr analyses per pixel is shown at the bottom of the figures. Note good alignment indicated by yellow arrows showing small curving at same position. Scale bar = 10 lm.

some of the high Sr marking in the full-day and day-time experiments did not match exactly with the front of a growth line, but began in the central region of a fibrous layer (Figs. S1 and S4). As our experiments began 4–6 h after dawn, day time skeletal growth would have already begun before Sr was incorporated into the skeleton. Consistent with this, Moya et al. (2006) reported that changes of calcification mode from night to day occur in 25 min in Stylophora pistillata. Post-culture growth was, on occasion, observed to be heterogeneous in small areas (not observed parallel to the previous growth line) with crystal growth appearing faster (almost comparable thickness with a typical fibrous layer even though they were formed in 4 h). Therefore, some inconsistencies regarding the diurnal growth-line/fibrous-layer pattern can perhaps be attributed to the difficulties of matching the experimental schedule with the light/dark cycle. Additionally, corals experienced

detectable light intensities at the very start of the night-time experiment and at the very end of the night-time experiment (due to the limited hours of darkness in Okinawa) when the corals were returned to natural conditions. When we consider that it is probable that enriched Sr was still present within the coelenteric cavities, then some Sr deposited during these times may not necessarily truly reflect night time calcification. This effect may cause a slight over-estimation in the width of skeletal deposition, and/or cause a slight shift in the position of the high Sr band. However, thickness of the high Sr band in the night-time experiment (<3 lm) suggests that any contribution of such effects are negligible. Another possibility is that ultrastructural deposition does not correspond directly to the light/dark cycle, and that the heterogeneous deposition reflects a short time lag between the skeletal deposition and the light/dark changes. Further experiments with

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finer marking intervals and better control over the light/dark regime will help to reveal whether a short time lag between night/ day cycle and growth-line/fibrous-layer deposition exists.

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mediation of element incorporation into the coral skeleton, but the mechanisms for this remain elusive. 4.4. Species variability

4.2. Ultrastructure formation mechanisms We have demonstrated that daily growth patterns are observed as alternative patterns of etch-sensitive growth lines and fibrous layers in the massive Porites coral. A similar pattern of growth has also been observed in other biogenic carbonate such as bivalve shells (Lutz and Rhoads, 1980) and fish otoliths (e.g. Morales-Nin, 2000). In bivalve shells, etch-resistant growth lines are associated with an insoluble-organic-rich slow-growing portion, while etchsensitive growth increments are associated with an insoluble-organic-depleted fast-growing portion. Growth patterns associated with organic material may be common in massive Porites corals. Cuif et al. (2003) revealed using X-ray absorption near-edge spectroscopy that organic sulfate concentration is high in the ‘‘early mineralized zone’’ (termed ‘‘center of calcification’’ in this study), and that the sulfate is seen as a banding pattern in the fibrous skeletal region of scleractinian coral skeletons. Cuif and Dauphin (2005b) later suggested a model of coral skeletogenesis where the coral deposits organic material at the growth front, which is then followed by carbonate mineralization, and that this continues in a sequential process. Growth of crystals upon an organic matrix at the skeletal growth front has been visualized by Clode and Marshall (2002, 2003c). Stolarski (2003) also found, using acridine orange, that ‘‘rapid accretion deposits’’ (termed ‘‘centers of calcification’’ in this study) and etch-sensitive growth lines are organic-rich. Since the ‘‘centers of calcification’’ and growth lines are thought to be rich in organic material (perhaps sulfate polysaccharide), which is soluble in the etching solution we used they are not well preserved or recognized in the SEM after etching. This difference in solubility would result in the micro-growth patterns visualized by the etching process. The present study suggests that such cyclical biomineralization process observed in fibrous skeletal regions is strongly associated with the daily light cycle. Unfortunately, we could not verify whether ‘‘centers of calcification’’ are only deposited at night. Reliably identifying the ‘‘centers of calcification’’ at the very growth tip proved difficult, especially following etching, and we were unable to culture the corals post-experimentation for longer periods. Further investigation to reveal the timing of ‘‘center of calcification’’ deposition is still needed. 4.3. Microscale geochemical heterogeneity Several studies have hypothesized that the chemical and isotopic composition of ‘‘centers of calcification’’ in massive Porites corals reflect a more precise environmental record than surrounding fibrous layers, because the centers of calcification are formed slowly during night under conditions close to chemical/isotopic equilibrium, while surrounding fibrous layers are precipitated quickly during day-time when kinetic effects dominate skeletal composition (Cohen et al., 2001; Cohen and McConnaughey, 2003). On the contrary, the results obtained in this study indicate that the diurnal cycle of coral growth is likely recorded as alternative patterns of etch-sensitive growth lines and etch resistant fibrous layers within the fibrous skeletal region, rather than alternating deposition of centers of calcification and fibrous regions between night and day. From our data, the impact of factors such as day and night time upon incorporation of elements and the resulting skeletal fibrous-layer/center of calcification structures (Cohen et al., 2001; Cohen and McConnaughey, 2003) requires further investigation. The compositional differences observed between the fibrous layers and growth lines are indicative of biological

In P. porites, skeletal growth resulting from 3 days in 86Sr-enriched seawater showed strong spatial heterogeneity, with discontinuous mineral deposition along the growth front (Houlbreque et al., 2009). This is consistent with structural heterogeneity identified at the calcifying interface of the coral Galaxea fascicularis (Clode and Marshall, 2002). The massive Porites coral examined in this study shows a more continuous pattern of skeletal growth, with marking strongly associated with skeletal ultrastructure. Since the coral skeletal ultrastructure may show variation at the species level, it is not surprising to find that diurnal patterns of skeletal growth may also show variation. It is also possible that this difference in growth is due to the different environmental conditions used for experimentation between the studies. While aquaria have been routinely used for coral experimentation, reductions in growth rates have been observed in aquariums when compared to natural conditions (Houlbreque et al., 2009). Clode and Marshall (2003b) also demonstrated that skeletons of G. fascicularis polyps maintained in aquaria have distinctly different ultrastructure than those growing on the reef. 4.5. Evaluation of the Sr marking method When looking at skeletal growth over short time frames (hours) the success of marking uninhibited skeletal growth is dependent upon the degree of stress experienced by the coral. Our ability to readily visualize rapid skeletal deposition, even at low rates during darkness, confirm that enriched Sr is a suitable, low stress tracer for high temporal studies of skeletal formation. The marking results indicate that Sr is rapidly assimilated into the coral skeleton, which is consistent with previous Sr uptake experiments (FerrierPages et al., 2002). From our experiments high Sr bands can be observed with approximately 2 lm resolution, and this is largely limited by the resolution of EPMA analysis and X-ray scattering. Assuming a skeletal growth rate of approximately 5–8 lm/20 h, estimated from the full-day experiments, the 2 lm resolution reflects skeletal deposition across a few-hour scale in day time corals. The present study attempted to culture corals as close to natural conditions as possible and is likely to closely reflect natural skeletal growth patterns. Such semi-controlled culturing technique have been employed (e.g. Grottoli and Wellington, 1999; Grottoli, 2002), while the present study performed shorter (sub-daily) time scale. Advantages of the current experiments compared to aquarium culture techniques include (1) seawater temperature and composition, and light cycle accurately and naturally maintained, (2) easy to perform, with no major facilities required, (3) significant analogy to natural conditions. Since the aim of this study was to understand diurnal patterns of coral growth in relation to light, a natural illumination cycle with a gradual intensity change is more favorable than two-digit (on/off) controls of light intensity laboratory culturing is usually equipped with. Degree of light reduction (16%) by the plastic tank is much smaller than natural variation between sunny and cloudy days. Water flow was depleted, but the reef water also naturally becomes stagnant during the low tide at Ishigaki island (see the environmental settings of Shiraho Reef in Appendix A). Thus, light depletion and reduced water flow conditions of our experiment are well within any natural variation typically experienced by these corals. Detailed evaluation of possible stress is provided in Appendix B. Both Sr-enrichment (this study) and alizarin red staining (Sandeman, 2008) demonstrated similar diurnal patterns of ultrastructural deposition, even though the latter method is considered to

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be toxic for corals (Dodge et al., 1984; Gaetani et al., 2011). FerrierPages et al. (2002) reported that the incorporation of Sr2+ versus external Sr2+ concentration into Stylophora postillata skeleton shows a linear relationship up to a concentration 37.5 times higher than normal seawater. They also observed that this linear incorporation continues for longer periods (more than 3 days), indicating that high Sr does not overstress corals or affect growth at a daily scale. Since all methods have both advantages and limitations, further comparative experiments will provide definitive criteria for choosing between methods, depending upon the data that is sought. 5. Conclusion The diurnal patterns of skeletal growth demonstrated in this study have revealed a sub-daily time scale of skeletal deposition that corresponds to discreet ultrastructural features. From this, past climate reconstructions from coral skeletons can potentially move toward a daily scale, as the mechanisms of elemental incorporation into different regions of the skeleton are clarified and understood. Insight into daily coral growth patterns will improve our understanding of factors affecting skeletal deposition and growth processes and the formation of coral reefs. Further experiments with improved marking intervals will help to reveal whether a short time lag between the night/day cycles and growth-line/fibrous-layer deposition exists. Acknowledgments This work was supported as part of The Researchers Exchange Program between Japan and Australia by the Australian Academy of Sciences (AAS) and the Japan Society for the Promotion of Science (JSPS) and by Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Coral reef science for symbiosis and coexistence of human and ecosystem under combined stresses’’ (No. 20121004) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. KS was funded by a JSPS Research Fellowships for Young Scientists. Comments from reviewers greatly improved the manuscript. We are grateful to Yasuaki Tanaka for the preliminary experiment and T. Sagawa, WWF Japan for providing the SST data. Appendix A. Environmental settings of Shiraho Reef The Shiraho Reef is a well-developed fringing reef. The reef flat, which is 800 m wide, can be divided into four subunits: a moat (shallow lagoon), an inner reef flat, a reef crest, and an outer reef flat (Nakamura and Nakamori, 2007, 2009). The Shiraho Reef has both a high abundance and diversity of coral species (Veron, 1992), indicating that the local environment is optimal for corals. Various detailed observations have been undertaken at Shiraho Reef, including hydrodynamics (Tamura et al., 2007), water chemistry, and carbon dynamics (Suzuki et al., 1995; Hata et al., 2002; Suzuki and Kawahata, 2003; Nakamura and Nakamori, 2007, 2009). The seawater temperature generally fluctuates on a diurnal cycle with highest temperature in the afternoon and lowest temperatures before sunrise, but the fluctuation patterns can also be affected by the tidal cycle. At low tide the reef crest is exposed, with seawater on the reef flat becoming isolated from the outer ocean and remaining this way for up to 5 h of slack water, especially during spring tides (e.g. Tamura et al., 2007). When this slack water occurs during the day time in summer, water temperature and dissolved oxygen levels increase while total alkalinity and total inorganic carbon decrease significantly (Suzuki et al., 1995; Hata et al., 2002). When the slack water period is during the night

time, the opposite changes occur (except for alkalinity). As the high tide returns, the stagnated water is flushed away and water properties return to those of the outer ocean. Such variations are significantly large during spring tides, while the variation is relatively small at neap tides. Hata, H., Kudo, S., Yamano, H., Kurano, N., Kayanne, H., 2002. Organic carbon flux in Shiraho coral reef (Ishigaki Island, Japan). Mar. Ecol. Prog. Ser. 232, 129–140. Tamura, H., Nadaoka, K., Paringit, E.C., 2007. Hydrodynamic characteristics of a fringing coral reef on the east coast of Ishigaki Island, southwest Japan. Coral Reefs 26, 17–34. Veron, J.E.N., 1992. Conservation of biodiversity – a critical time for the hermatypic corals of Japan. Coral Reefs 11, 13–21.

Appendix B. Calculation for changes of water chemistry during culture experiment and evaluation of the possible stress Possible limitations of the culture method in this study, and in general any enclosed-incubation method, include potential changes in seawater composition such as pH, oxygen and carbonate ion concentrations due to the calcification, photosynthetic and respiratory activities of the coral over the course of the experiment. Unfortunately we could not measure the water chemistry during the experiment, however we have calculated the predicted changes of seawater composition to estimate the possible impacts upon coral calcification using the following parameters. (1) amount of the coral used for the experiment, (2) linear extension rate of massive Porites australiensis (mm/year, Mitsuguchi et al., 2003), and respiration and photosynthetic activity of massive Porites lutea (lmol O2 cm 2 h 1, Alution et al., 2010). The coral surface area was measured by two methods, the planar projection method (PP) and advanced geometry method (AG), because reported linear extension rate based on PP area, while reported metabolic parameters are normalized to AG area. The advanced geometry method regards the coral surface as an aggregation of a triangle and quadrangle taking into account the concavity and convexity of the skeleton, while the planar projection method only considers the overall surface coverage (Naumann et al., 2009). The surface areas of corals used for incubation were: DT: 98 cm2 (AG), 51 cm2 (PP); NT: 90 cm2 (AG), 52 cm2 (PP); FD: 41 cm2 (AG), 31 cm2 (PP). As expected, the flatter-surfaced samples showed less deviation in the area calculated by each of the two different methods, while more irregular wavy coral blocks showed much larger deviations between the calculated results. Possible changes in seawater chemistry due to coral metabolism during the course of the experiments can be readily calculated from calcium and carbonate consumption by calcification, from consumption and production of oxygen by respiration and photosynthesis respectively, and generation of CO2 through respiration. Based on the annual linear extension rate of massive Porites australiensis from Ishigaki Island in summer (18–21.3 mm yr 1, Mitsuguchi et al., 2003), the daily linear extension rate is estimated to be 49–58 lm d 1. With this, the linear extension rate of a massive Porites coral corresponds to approximately 0.9–1.1 lm h 1 in the dark and 2.7–3.2 lm h 1 in the light. This assumes that linear extension rates are three times higher during the day compared to night (Gattuso et al., 1999) and that day light is from 6:00 to 21:00. If we consider the density of a massive Porites skeleton as 1.1– 1.2 g cm 3 (Mitsuguchi et al., 2003), then the coral can be predicted to have deposited carbonate at an approximate rate of 0.30–0.39 mg cm 2 h 1 in the light and 0.10–0.13 mg cm 2 h 1 in the dark. Multiplying this hourly calcification rate with the surface area estimated by the planar projection method (as calcification rate was estimated by linear extension rate), we calculated the consumption of CaCO3 from the seawater by experimental coral

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samples, approximately 145–187 mg CaCO3 in the day-time, 47– 60 mg CaCO3 in the night-time, and 125–162 mg CaCO3 in the full-day experiments. Since more corals were the day-time and night-time experiment than full-day experiment, consumption/ deposition of CaCO3 was estimated to be larger in the day-time than full-day experiments. Day time net photosynthetic rate and night respiration rate of massive Porites lutea were estimated at 1.1 lmol O2 cm 2 h 1 and 0.67 lmol O2 cm 2 h 1, respectively (Alutoin et al., 2001). To include the effect of photosynthesis and respiration in our calculations, we used the surface area obtained by the advanced geometry method because the reported metabolic parameters of Alutoin et al. (2001) were estimated based on surface area obtained by the aluminium foil method (i.e. both methods consider 3-dimensional surface area including the curving of concavity and convexity). Photosynthesis and calcification alter total dissolved inorganic carbon (CT) and total alkalinity (AT), such that when organic matter containing 1 mol C is produced, CT decreases by 1 mol, whereas AT hardly changes. When 1 mol of CaCO3 is produced, AT decreases by 2 mol and CT decreases by 1 mol. Values of pH and other CO2 system parameters (such as aragonite saturation state Xarg) could be calculated from AT, CT, temperature and salinity. Using the above mentioned physiological parameters, we calculated the compositional variation based on Nakamura and Nakamori (2009). For the calculation, we assumed that the initial conditions were as follows: seawater temperature: 29.1 °C (mean value during the experiment), salinity: 34.5 psu, AT: 2150 lmol kg 1 and CT: 1850 lmol kg 1. This calculation predicts that pH (total hydrogen ion concentration scale) varies from the initial condition of 8.0 to 8.0 in the day-time, to 7.8 in the night-time and to 7.8 in the full-day, and Xarg varies from initial condition of 3.4 to 2.6 in the day-time, to 2.2 in the night-time and to 2.0 in the full-day. We also calculated the dissolved oxygen (DO) change. The oxygen change was calculated as  + 1025 lmol in the day-time,  537 lmol in the night-time, and  + 227 lmol in the full-day, with the 10 L of seawater containing 2000–2500 lmol O2 at the beginning of experiment. Thus 30% net oxygen was consumed during the night-time incubation. At the slack water period in Ishigaki Island, pH and DO of the seawater on the inner reef flat can naturally increase up to 8.8 and 450 lmol kg 1 during day time and decrease down to 7.8 and 50 lmol kg 1 during night time (Suzuki et al., 1995). Considering that the corals’ natural habitat also extends to more isolated water environments, such as tide pools or inter tidal regions, where these variations would be even greater, then our estimated variations that occurred during incubation are no greater than any natural variation experienced routinely by these corals. Further, the use of large volumes of seawater during incubation helps to limit the extent of changes in seawater composition and to reduce any impact upon calcification. Since corals must actively modulate pH at the calcification site, species-specific ultrastructure is probably regulated and controlled more by internal micro-environmental changes, and less by macroenvironmental changes. This idea is supported by the skeletal ultrastructural data reported by Cohen et al. (2009). In their images, typical coral skeletal ultrastructures, such as ordered acicular crystals, can be observed even in corals exposed to the second lowest aragonite saturation (Xarg = 1.03). While the morphological change in the crystals themselves is significant, the collapse of the overall skeletal ultrastructure is only observed in corals exposed to the lowest aragonite saturation (Xarg = 0.22), suggesting that the ability of the coral to adequately maintain and regulate conditions within the local internal environment is high. Cohen et al. (2009) also demonstrated that depletion of aragonite saturation decrease Mg/Ca ratio while increas Sr/Ca ratio in the skeleton. In the present

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study, Mg and S distribution (Fig. S5) showed no significant variation between labeled (in tank) and non-labeled (natural condition) skeleton, and variation is well within the range of natural variation observed in non-labeled area. This indicates that stress artifact due to the culture experiment are smaller than the natural variability. Janiszewska et al. (2011) also suggest strong biological control on ultrastructure based on the species-specific ultrastructure of solitary deep-sea Micrabaciids corals, even though their habitats range from 49 to 5000 m depth. Taking all of this into account we conclude that the observed day and night time growth patterns result from regulated physiological changes occurring naturally along diurnal time frames. Cohen, A.L., McCorkle, D.C., de Putron, S., Gaetani, G.A., Rose, K.A., 2009. Morphological and compositional changes in the skeletons of new coral recruits reared in acidified seawater: insights into the biomineralization response to ocean acidification. Geochem. Geophys. Geosyst. 10. Alutoin, S., Boberg, J., Nystrom, M., Tedengren, M., 2001. Effects of the multiple stressors copper and reduced salinity on the metabolism of the hermatypic coral Porites lutea. Mar. Env. Res. 52, 289– 299. Mitsuguchi, T., Matsumoto, E., Uchida, T., 2003. Mg/Ca and Sr/Ca ratios of Porites coral skeleton: Evaluation of the effect of skeletal growth rate. Coral Reefs 22, 381–388. Naumann, M.S., Niggl, W., Laforsch, C., Glaser, C., Wild, C., 2009. Coral surface area quantification-evaluation of established techniques by comparison with computer tomography. Coral Reefs 28, 109–117. Suzuki, A., Nakamori, T., Kayanne, H., 1995. The mechanism of production enhancement in coral-reef carbonate systems – model and empirical results. Sed. Geol. 99, 259–280. Appendix C. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2012.05.017. References Al-Horani, F.A., Al-Moghrabi, S.M., de Beer, D., 2003. Microsensor study of photosynthesis and calcification in the scleractinian coral, Galaxea fascicularis: active internal carbon cycle. Mar. Ecol. Prog. Ser. 194, 75–85. Allison, N., Finch, A.A., 2004. High-resolution Sr/Ca records in modern Porites lobata corals: effects of skeletal extension rate and architecture. Geochem. Geophys. Geosyst. 5. http://dx.doi.org/10.1029/2004GC000696. Allison, N., Finch, A.A., 2007. High temporal resolution Mg/Ca and Ba/Ca records in modern Porites lobata corals. Geochem. Geophys. Geosyst. 8. http://dx.doi.org/ 10.1029/2006GC001477. Allison, N., Finch, A.A., EIMF., 2010. delta B-11, Sr, Mg and B in a modern Porites coral: the relationship between calcification site pH and skeletal chemistry. Geochim. Cosmochim. Acta 74, 1790–1800. Anthony, K.R.N., Kline, D.I., Diaz-Pulido, G., Dove, S., Hoegh-Guldberg, O., 2008. Ocean acidification causes bleaching and productivity loss in coral reef builders. Proc. Natl. Acad. Sci. 105, 17442–17446. Barnes, D.J., 1970. Coral skeletons: an explanation of their growth and structure. Science 170, 1305–1308. Barnes, D.J., 1972. Structure and formation of growth-ridges in scleractinian coral skeletons. Proc. R. Soc. London, B. 182, 331. Barnes, D.J., Crossland, C.J., 1980. Diurnal and seasonal-variations in the growth of a staghorn coral measured by time-laps photography. Limnol. Oceanogr. 25, 1113–1117. Böhm, F., Gussone, N., Eisenhauer, A., Dullo, W.C., Reynaud, S., et al., 2006. Calcium isotope fractionation in modern scleractinian corals. Geochim. Cosmochim. Acta 70, 4454–4462. Bryan, W.H., Hill, D., 1941. Spherulitic crystallization as a mechanism of skeletal growth in the hexacorals. Proc. R. Soc. Queensland 52, 78–91. Clode, P.L., Marshall, A.T., 2002. Low temperature FESEM of the calcifying interface of a scleractinian coral. Tissue Cell 34 (3), 187–198. Clode, P.L., Marshall, A.T., 2003a. Skeletal microstructure of Galaxea fascicularis exsert septa: A high-resolution SEM study. Biol. Bull. 204, 146–154. Clode, P.L., Marshall, A.T., 2003b. Variation in skeletal microstructure of the coral Galaxea fascicularis: effects of an aquarium environment and preparatory techniques. Biol. Bull. 204, 138–145.

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