Sclerite calcification and reef-building in the fleshy octocoral genus Sinularia (Octocorallia: Alcyonacea)
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- Jeng, M., Huang, H., Dai, C. et al. Coral Reefs (2011) 30: 925. doi:10.1007/s00338-011-0765-z
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Alcyonacean octocorals in tropical reefs are usually not considered as reef builders. Some Sinularia species, however, are capable of consolidating sclerites at the colony base to form spiculite. Nanwan Bay, southern Taiwan, features both fossilized and recently formed boulders composed of spiculite, thus demonstrating the role of Sinularia in contributing to the reef structure. Section radiography of an 18.5 kg spiculite boulder demonstrated a regular density banding of 3–6-mm intervals. Core survey indicated spiculite coverage of 25–30% on the live reef and of 30–40% on the uplifted boulders. Cores taken from living Sinularia revealed a distinct transition from discrete sclerites to compact spiculite and amorphous calcium carbonate cementing the sclerites. In the widespread S. gibberosa, sclerite formation appeared to start intracellularly, followed by a prolonged extracellular calcification process. At the calcification site, multiple sclerocytes formed expanded pseudopod-like membranes that interconnected, forming multicellular vesicles (MCVs) around the sclerites. The MCVs and the pseudopods disappeared at sclerite maturation, followed by degradation of the sclerocytes around the mature sclerites. At the colony base, granular vesicles were distributed among the sclerites, indicating a cementing process in progress. These findings suggest that colonies of Sinularia are able to cement sclerites and consolidate them at their base into spiculite, thus making them reef builders.
It is well accepted that the carbonate skeletons of stony corals form the foundations of coral reefs. New skeleton, in the form of aragonite crystals, is deposited externally by the calicoblastic epithelium of these corals onto previously formed ones (Le Tissier 1991; Muscatine et al. 1997). Octocorallia produce calcite sclerites within fleshy tissue as internal support (Fabricius and Alderslade 2001) and are generally not considered to contribute to the reef structure. The discovery of rocks composed of octocoral sclerites, termed spiculite (Konishi 1981; Accordi et al. 1989), led to the suggestion that fleshy octocorals may contribute to reef construction and growth (Schuhmacher 1997). Most spiculites are made of consolidated sclerites from octocorals of the family Alcyoniidae (order Alcyonacea). Cary (1931) first focused attention on sclerite masses left by colonies of Alcyonium (= Sinularia) confertum in American Samoa. Later, below flourishing living colonies of the fleshy octocoral, Sinularia, cemented layers of sclerites were found on modern coastal reefs in the Ryukyu Islands (southern Japan) and Australia that had been accreting for thousands of years (Konishi 1981; Kleypas 1996). Fossil spiculites were found as distant as in Somalia (Accordi et al. 1989) and can be traced back to the Silurian period in fossils from the Island of Gotland in the Baltic Sea (Bengtson 1981). Sinularia spiculite has contributed to the basis of modern reefs in the central Great Barrier Reef since the end of the last Ice Age (Johnson and Risk 1987). Although spiculite rocks have been found in many regions, the mechanisms of sclerite cementation into solid calcareous rock layers are still not understood.
Among octocoral colonies, calcitic sclerites in the coenenchyme function as means of physical support and defense against predation (Alstyne et al. 1992) and may increase the ability of the organism to withstand shear forces caused by water flow (Grillo et al. 1993; Lin and Dai 1997). Sclerite formation could be initiated intracellularly in primary sclerocytes, followed by extracellular growth processes conducted by secondary sclerocytes (Goldberg and Benayahu 1987; Grillo et al. 1993). Another possible mechanism relates to the formation and growth within sclerocytes containing numerous sclerite-forming vacuoles (Kingsley and Watabe 1982; Kingsley and Dupree 1993). Konishi (1981) hypothesized that in Sinularia species, discrete sclerites may fuse and be incorporated into spiculite masses at the base of the colony. Thus, it was concluded that octocoral sclerites undergo cementation and fusion processes that imply persistent calcification, even after sclerite maturation.
In the present work, we studied spiculite of Sinularia spp. in southern Taiwan. Our findings demonstrate the extent of spiculite occurrence and, at the ultrastructural level, reveal the cellular activities that lead to sclerite cementation. We examined spiculite found in both living colonies and in uplifted (fossil) boulders and evaluate the roles and contribution of spiculite in building reef structures.
Materials and methods
Study site and sampling
Surveys of spiculite boulders and rock formation were conducted in uplifted reefs of the upper intertidal zone of Houpihu, at approximately 10 m above sea level. Spiculite found embedded in the reef boulders was photographed and tape measured; the abundance of spiculite in the area was estimated according to size and occurrence. Thirty-five loose spiculite boulders were collected at random for microscopic examination. In the laboratory, thick sections (2.5–3.0 mm) were sampled from 10 boulders with a recognizable growth direction. Sections were cut parallel to the growth direction using a diamond saw and then examined with x-radiography (SOFTEX CMB-2). To determine the abundance of spiculite in the uplifted reefs, cores of spiculite masses from six exposed reef boulders (one for each) were taken, using a pump drill equipped with 50–cm-long steel tubes (70 mm inner and 80 mm outer diameter).
In order to examine the deposition of spiculite beneath living Sinularia colonies, core samples were obtained from the reef at 10–15 m deep. For this purpose, coverage of living Sinularia was determined by surveying two 50 m parallel transect lines, parallel to the shoreline and 10 m apart. For core sampling, colonies with a diameter of >1 m were selected, and a core was removed from each one by hammering a steel tube (1 m long, and 45 mm inner and 49 mm outer diameter) through the center of the upper surface down to the base (~20 cm deep). The morphological features of spiculite associated with the living colonies were recorded by underwater photography using a Nikon RS camera. The morphology of the sclerites found in the cores was examined under a compound microscope, and all of the sampled Sinularia colonies were identified to species level following Verseveldt (1980). Six samples from living colonies, which included the transition zone between discrete sclerites of the living parts and solid spiculite, were carefully excised from the cores using a knife and chisel. They were then subjected to critical-point drying, gold-coated, and examined with a scanning electron microscope (Hitachi S-2500).
Tissue preparation of S. gibberosa and electron microscopy
The process of sclerite consolidation, the function of the adjacent sclerocytes, and the features of spiculite were studied in S. gibberosa Tixier-Durivault, 1970. Six cuttings, 15 × 15 cm in size, with a spiculite layer were removed from the edge of six random colonies >1 m in diameter (November 2000). In order to obtain active sclerocytes, four cutting samples were transferred to the laboratory, kept for 5 days in aerated seawater tanks (90 × 30 × 30 cm) at 22°C with 12:12 h fluorescent illumination (Philips 36 W/865 tube light). The other two samples were transported on dry ice in a cooler to the laboratory and then stored in a freezer at −70°C for further examination (see below).
In order to study the process of sclerite cementation in the spiculite, we examined the structural features of individual and fused sclerites in S. gibberosa. Twenty-four tissue samples, 0.5 cm3 in size, which included the surface layer and the interiors of the lobes and stalk, were removed from the six colony cuttings (four samples for each colony, see also Verseveldt 1980). They were individually placed in 5% sodium hypochloride for 24 h, and the remaining sclerites were repeatedly rinsed with distilled water and then air-dried. Six samples of fused sclerites, 0.25 cm3 in size, one for each colony, were removed with a knife and chisel from the spiculite of the colonies. All preparations were mounted on stubs, gold-coated, and examined by scanning electron microscopy (SEM).
In order to examine sclerite deposition by sclerocytes, 35 tissue samples, each 1–2 mm3 in size, were removed (8–9 samples per colony block) from the top and basal parts of the aforementioned four cuttings of S. gibberosa maintained in an aquarium. They were fixed for 1–2 h in a modified Karnovsky-type mixture containing 4% paraformaldehyde and 6.25% glutaraldehyde in 0.2 M phosphate buffer at pH 7.6–8.0. In order to improve fixation, 0.2% ruthenium red was added to the mixture (Goldberg and Benayahu 1987). Subsequent procedures included post-fixing samples in 1% OsO4 in 0.2 M phosphate buffer with 0.1% ruthenium red for 1 h. After fixation, all samples were decalcified in 5% EDTA (buffered to pH 7.6 with 0.2 M phosphate) for 2–7 days, followed by dehydration in a graded series of ethanol and infiltration with Spurr resin. All steps were carried out at room temperature. Infiltration for final embedding was conducted in a 70°C vacuum oven for 18–24 h. Sections of the decalcified tissue samples were obtained using glass knives and stained with ethanol–uranyl acetate followed by lead citrate and then examined with a transmission electron microscope (TEM; Hitachi H-700).
Distribution and morphology of spiculite
Shallow-water Sinularia species from Nanwan Bay cored for this study
Order ALCYONACEA Lamouroux, 1812
Family ALCYONIIDAE Lamouroux, 1812
Sinularia capillosa Tixier-Durivault, 1970
Sinularia crassa Tixier-Durivault, 1945
Sinularia cristata Tixier-Durivault, 1969
Sinularia cruciata Tixier-Durivault, 1970
Sinularia deformis Tixier-Durivault, 1969
Sinularia dissecta Tixier-Durivault, 1945
Sinularia exilis Tixier-Durivault, 1970
Sinularia fungoides Thomson & Henderson, 1906
Sinularia gibberosa Tixier-Durivault, 1970
Sinularia inelegans Tixier-Durivault, 1970
Sinularia inexplicita Tixier-Durivault, 1970
Sinularia leptoclados (Ehrenberg, 1834)
Sinularia lochmodes Kolonko, 1926
Sinularia minima Verseveldt, 1971
Sinularia muralis May, 1899
Sinularia nanolobata Verseveldt, 1977
Sinularia notanda Tixier-Durivault, 1966
Sinularia numerosa Tixier-Durivault, 1970
Sinularia parva Tixier-Durivault, 1970
Sinularia scabra Tixier-Durivault, 1970
Sinularia sp. A
Sinularia sp. B
Sclerocytes of S. gibberosa
The findings of the field surveys revealed a continuous (for the last 2800 years) spiculite-forming zone of the Sinularia colonies from the subtidal zone to the uplifted reefs of Nanwan Bay, southern Taiwan (Chen 1993). In the lower part of living Sinularia colonies, discrete coenenchymal sclerites were cemented together by calcium carbonate adhesive. The fused sclerites formed solid spiculite beneath the colony. The size and thickness of the spiculite layer in the boulders suggested that, in addition to deposition of spiculite layers onto the existing reef platform, Sinularia colonies generate three-dimensional reef structures (Schuhmacher 1997).
The pattern of layered growth bands in the spiculite boulders (Fig. 2e) corresponded with the previous findings of Schuhmacher (1997), indicating a distinct periodicity in spiculite deposition. Close ranges of band widths of the two study sites also indicated similar deposition rates between them. SEM showed that the surface of newly formed spiculite was covered with amorphous calcium carbonate, and thus, the microstructure of the sclerites could not be determined. This could have been caused by decalcification and re-deposition of calcium carbonate by octocoral colonies. Kingsley et al. (1996) reported seasonal changes in tissue characteristics of the gorgonian Leptogorgia virgulata. Its tissue becomes acidic in winter thus leading to sclerite decalcification; whereas in summer, sclerocytes redeposit calcium carbonate on the surface of the sclerites. Influenced by the warm Kuroshio current, the water temperature in Nanwan Bay ranges between 22.5–29°C (Dai 1991b). It is possible that similar deposition and re-absorption cycles may also occur in Sinularia in Nanwan Bay, causing the formation and cementation of sclerites and seasonal changes in the spiculite density.
Inter-cemented coenenchyme sclerites in the basal part of S. gibberosa colonies revealed where sclerite consolidation into spiculite is initiated. Johnson and Risk (1987) noted that aragonite is the main component of the adhesive for cementing calcite sclerites of Sinularia. During sclerite calcification, calcium carbonate crystals are integrated by means of organic matrix (Rahman and Omura 2009). The chemical condition in deeper tissue of colonies is more acidic and may favor the production of amorphous calcium carbonate (Tentori and Van Ofwegen 2011); at the colony base, there were few sclerocytes containing multicellular vesicles, which leads to the amorphous calcification products that was noted as adhesives among sclerites. Therefore, the adhesive calcium carbonate is produced by a calcification mode which differs from that responsible for sclerite formation and growth.
The abundance and location of the sclerocytes suggested that S. gibberosa may synthesize sclerite primordia intracellularly and then extrude them for extracellular calcification. The intracellular stage occurs in primary sclerocytes (Fig. 5a). We suggest that in these primary sclerocytes, primordia of calcite crystals are secreted onto an organic matrix within the vesicles and then transported outside the cell for subsequent extracellular calcification similarly to sclerite formation in the gorgonian Pseudoplexaura flagellosa (Goldberg and Benayahu 1987). In the present study, although active primary sclerocytes were rarely observed after five days of tissue regeneration, there were no sign that primary sclerocytes had transformed into secondary ones. On the other hand, degenerated sclerocytes were found in most parts of the colonies. These findings led us to speculate that the sclerocytes are short-lived, may deposit sclerite primordia, and subsequently degenerate within a short period.
In the extracellular stage of sclerite growth of S. gibberosa, the sclerite primordia begin to grow inside the overlapping cell membrane extensions (MCVs) by means of multiple secondary sclerocytes (Figs. 5b, 6). Around MCVs, electron-opaque bodies in secondary sclerocytes may contain high concentrations of calcium (Kingsley 1990), which may be transported into the MCVs, and then deposited on the organic matrix for calcium carbonate crystal growth. In scleractinians, the organic matrix in the subepithelial space plays the role of a template determining the crystal morphology of the coral skeleton (Johnston 1980; Isa 1986; Le Tissier 1991). We assumed that the organic matrix in MCVs of S. gibberosa may have a similar function. Based upon the TEM findings (Fig. 6), MCVs serve as an isolated compartment in which sclerocytes deposit calcium carbonate. The locations and shapes of MCVs may determine the shape and size of the sclerites.
The mesoglea of S. gibberosa is the site where congregations of MCVs and sclerite formation occur. At that site, dense mesoglea fibers were observed (Fig. 6). Mesoglea fibers appeared to facilitate bonding between the MCVs and the developing sclerites. The distribution and array of mesoglea fibers may also determine the location where calcium carbonate is deposited, as well as sclerite morphology (see also Goldberg and Benayahu 1987).
The calcification process causes cementation of the sclerites, which takes place at the base of the colony, and differs from that leading to sclerite formation. At the colony base of S. gibberosa, active sclerocytes and MCVs, which are usually associated with developing sclerites in the mesoglea, were rarely observed. Rather, multiple free granular vesicles and the organic matrix occurring in the spaces among mature sclerites (Fig. 8) could be the source and template of the adhesive calcium carbonate. During the process of their degeneration, sclerocytes may release calcareous organelles or other materials outside the cell; these extruded organelles may retain the function of calcification. In the absence of facilitation of MCVs, calcium carbonate adhesives were deposited as amorphous aragonite in contrast to the calcite crystalline of the sclerites. This revealed that the sclerocytes and MCVs may also play roles in determining the calcite structure of the calcification products.
Fleshy octocorals are able to inhabit turbid environments that are less suitable for scleractinian corals, allowing them access to different ecological niches (Dai 1991a). The bias in common knowledge is toward viewing reefs as products of stony corals. We suggest that octocorals such as Sinularia, however, are also capable of accreting material into significant reef structures over geological time scales. This may allow the development of reef structures by spiculite-producing fleshy octocorals in areas where stony corals are unable to develop as a reef base.
The authors thank Mr. T.-S. Chen and the Administration of Kenting National Park, Taiwan for assistance with the fieldwork. Y. Benayahu was partly supported by the Israel Cohen Chair in Environmental Zoology. We also acknowledge the funding and electron microscope support from the Research Center for Biodiversity, Academia Sinica, Taiwan.