A Stable Isotope Study of the Relationship between Coral Tissues and Zooxanthellae in a Seasonal Tropical Environment of East Kalimantan, Indonesia  

Handoko Adi Susanto1 , Makoto Komoda2 , Masaaki Yoneda2 , Akihiro Kano3 , Mutsunori Tokeshi4 , Hiroko Koike3
1. Marine Protected Areas Governance, Jl. Wolter Monginsidi No. 63B. Kebayoran Baru, Jakarta Selatan, 12180. Indonesia
2. Japan Wildlife Research Center, Taitoku, Tokyo, Japan
3. Department of Environmental Changes, SCS, Kyushu University, Motooka, Nishiku, Fukuoka, Japan
4. Amakusa Marine Biology Laboratory, Department of Biology, Faculty of Science, Kyushu University, Japan
Author    Correspondence author
International Journal of Marine Science, 2013, Vol. 3, No. 36   doi: 10.5376/ijms.2013.03.0036
Received: 26 May, 2013    Accepted: 23 Jun., 2013    Published: 02 Jul., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Susanto et al, 2013, Effect of Sedimentation to Branching Corals Detected Using Nitrogen and Carbon Isotopes, International Journal of Marine Science, Vol.3, No.36 285-294 (doi: 10.5376/ijms.2013.03.0036)


This study assessed variations in the relationship between zooxanthellae and their coral hosts seasonally and spatially. Carbon and nitrogen stable isotopes (d13C and d15N) were used to analyze the patterns of nutritional transport between coral and zooxanthellae. Studies of temporal differences were conducted during two dry seasons and one rainy season in the Berau Marine Conservation Area, East Kalimantan, Indonesia. To assess spatial variations, coral samples from three genera (Porites, Seriatopora and Stylophora) were collected from three localities, designated as nearest, medium, and farthest from the Berau River mouth. The lower d13C of Seriatopora compared with the other two genera suggested that corals in this genus were more actively utilizing particulate organic matter and adopting a heterotrophic feeding mode. The d13C values of coral tissue and zooxanthellae also were affected by differences in seasons, localities, and depth, whereas the d15N values varied significantly with seasons alone. Differences in d15N values between coral tissue and zooxanthellae were always positive in the first and second dry seasons, but negative in the rainy season. This finding indicated that zooxanthellae were the main source of coral nutrients during the dry season, while host coral may support zooxanthellae nutrition during rainy season of low turbidity. Spatially, sedimentation does not show significant impact to the relationship between coral and zooxanthellae.

Coral; Zooxanthellae; East Kalimantan; Indonesia; Nitrogen and carbon isotopes

Coral reefs are one of the most productive marine ecosystems (Birkeland, 1997) and they harbor over 4000 different fish species, 700 coral species, and thousands of other plants and animals (McAllister et al., 1994; Hinrichsen, 1997). Such systems typically have high photosynthetic production reaching 5–20 g m–2 day–1 of organic carbon or 50–200 g m–2 day–1 of wet biomass (Sorokin, 1993). However, coral reefs are declining in many areas of the world as a result of steadily increasing threats from direct human pressure (Chansang et al., 1981; Pastorok and Bilyard, 1985; Fabricius, 2005; Marionet al., 2005) and the indirect effects of global climate change (Hoegh-Guldberg, 1999; Hughes et al., 2003; Hoegh-Guldberg et al., 2007).

The role of reef construction is mostly performed by reef-building (or hermatypic) corals. Many hermatypic corals have a symbiotic relationship with algae called zooxanthellae (Sorokin, 1993). These algae live inside coral polyps and perform photosynthesis, producing carbon compounds that are shared with the coral. In turn, the coral provides the algae with protection and access to light. Zooxanthellae utilize nitrates, phosphates, and carbon dioxide produced in the polyp and also lend their color to their coral symbionts. Coral bleaching occurs when corals lose their zooxanthellae, thereby exposing the white color of the calcium carbonate skeleton through the transparent coral body.
Variations in the relationship between corals and their zooxanthellae can be assessed by analyzing carbon and nitrogen isotopes. Pioneering work by Johannes and Wiebe (1970) on the nutritional relationship between hermatypic scleractinian corals and zooxanthellae determined the coral tissue biomass and composition using a water-pick method to separate zooxanthellae and coral tissue from the coral skeleton. This successful separation was followed by isotopic analyses of zooxanthellae and coral tissue. Carbon isotope analyses indicated that the majority of coral tissues had higher δ13C values than zooxanthellae (Land et al., 1977).
The relationship between coral tissue and zooxanthellae was studied by isotopic analysis, and focused on resource partitioning in Jamaican reef corals using δ13C (Muscatine et al., 1989) and δ15N (Muscatine and Kaplan, 1994). These studies concluded that the δ13C value of zooxanthellae was high in shallow water, and became lower as depth increased. These investigations illustrate depth-related spatial differences in nutrient use by corals.
Risk et al (1994) studied scleractinian corals from the central region of the Great Barrier Reefs to determine the degree to which corals utilize terrigenous carbon as an ultimate food source. δ13C values of coral tissue and zooxanthellae increased linearly with distance from shore. The study implied that inshore corals derived much of their nutrients from terrigenous sources, and these varied spatially.
Swart et al (2005) analyzed temporal and spatial variation of Montastrea faveolata from the Florida reef tract. This research revealed that the δ13C and δ15N values of the zooxanthellae were less than in the coral tissue. The study also discovered that there was no significant difference between nearshore and offshore coral in either δ13C or δ15N.
Heikoop et al (2000a) noted that the δ13C of coral tissue can be used to determine whether their nutrition is primarily autotrophic or heterotrophic. Autotrophic corals typically have similar δ13C values in zooxanthellae and coral tissue. Heterotrophic corals have lower δ13C that closely reflect the δ13C values of their diets (Muscatine et al.,1989; Heikoop et al., 2000a). An experimental study on the effect of heterotrophy conditions under varying turbidity levels with two coral species showed that Porites was affected less than Goniastrea (Anthony and Fabricius, 2000). Porites corals were able to utilize suspended particulate matter as part of its energy budget. An investigation of Acropora coral using 13C and 15N isotope tracers showed that algal photosynthetic products were transferred to the host (Tanaka et al., 2006).
Heikoop et al (1998) suggested that light was a factor influencing the nitrogen isotopic composition of coral containing symbiotic zooxanthellae. Under conditions of internal nutrient limitation due to light and nutrient concentration, corals might exhibit minimal fractionation of nitrogen, and δ15N could prove to be a useful indicator during the dietary analysis of seawater-dissolved inorganic nitrogen (DIN).
The δ13C and δ15N values of organic tissues have been successfully used to trace the input of primary producers by assuming that the δ13C of consumers reflects the δ13C in their diet and that the δ15N values of consumers are higher than the δ15N values in their diet (Peterson and Fry, 1987; Yamamuro et al., 1995). The δ13C composition of animals reflects their diet with a small enrichment of 0.5–1‰ (Michener and Kaufman, 2007), especially in shallow water of depth 1~10 m (Muscatine et al. 1989), while the δ15N values of consumers are about 3‰~5‰ higher than their diet (Peterson and Fry, 1987).
The current study investigated variations in the biosynthetic relationship between corals and zooxanthellae. Under normal conditions where zooxanthellae produce sufficient photosynthetic products, corals tend to function autotrophically and obtain most of their nutrients from the zooxanthellae, which results in the δ15N values of corals being higher than zooxanthellae. However, zooxanthellae might fail to provide sufficient nutrients to corals under turbid conditions due to reduced light intensity and limited photosynthesis production, which results in differences in the δ13C and δ15N composition of coral tissues and zooxanthellae.Thus, the objective of this study was to identify where these differences occurred temporally (rainy versus dry seasons) and spatially (distance from a river mouth) based on a carbon and nitrogen stable isotopes analysis of coral and zooxanthellae. The river and rainy season are discussed as likely sources of increased turbidity which may affect the observed patterns in the coral reef ecosystem.
2 Material and Method
The study was conducted in Berau, East Kalimantan, Indonesia, which is known as part of the Coral Triangle in the West Pacific (Green and Mous, 2004). Spatially, the study area was divided into three localities, as shown in Figure 1. Locality 1 was the nearest to the Berau River mouth, at Rabu-rabu and Panjang Islands. Locality 2 was an intermediate distance from the river mouth, at Derawan Island, Tababinga reefs, and Masimbung reefs. Locality 3 was the farthest from the river mouth, at Semama and Sangalaki Islands. Samples were generally collected on the western and eastern sides of the islands at two different depths, 3 m and 10 m.

Figure 1 Map of sampling points in Localities 1, 2 and 3 in the Berau, Marine Conservation Area, East Kalimantan, Indonesia

Coral samples were collected in August 2006 (designated as the 1st dry season), November 2007 (the rainy season) and August 2008 (the 2nd dry season).
Average rainfall at those times were 83.7 mm, 235.8 mm and 141 mm, respectively.
In August 2006, a total of 70 coral samples were collectedat 14 dive sites in the three localities, which consisted of 34 Porites samples, 17 Seriatopora samples and 19 Stylophora samples. In November 2007, a total of 62 coral samples were collected at eight dive sites in the three localities, and these were composed of 23 Porites samples, 23 Seriatopora samples and 16 Stylophora samples. In August 2008, a total of 30 coral samples were collected at five dive sites in the three localities and these were composed of 10 Porites samples, 12 Seriatopora samples and 8 Stylophora samples. Only genus names are referred to in this paper.
The three branching coral genera, Porites, Seriatopora and Stylophora were relatively easy to find in clear and turbid waters. All these corals are categorized as small polyp stony (SPS) corals and possess short tentacles. During three field seasons, most of these three genera were present at the same dive sites in the three localities. However, Stylophora was not found at Locality 1 during the 2nd dry season and it may have disappeared from this area.
Samples were collected using pliers to cut the apices of individual branches from coral colonies. Coral nubbins were placed in a clear plastic bag, and after collection a field number was assigned to samples before scientific identification. After identification, a picture was taken of every sample before placing each sample in a separate plastic bottle and adding ethanol until the sample was totally immersed. Samples were then transported to the laboratory for further analysis.
Zooxanthellae were separated from their host coral in the laboratory using a standard ethanol and sonication method (Piniak and Lipschultz, 2004). Coral samples were treated with an ultrasonic generator for 10 min and centrifuged at 3000 rpm for 10 min. After sonication, the supernatant was separated from the bulk coral samples and placed into a Falcon tube. The supernatant was then centrifuged at 2500 rpm for 10 min to separate the zooxanthellae (pellet) from the solution. The zooxanthellae pellet was decalcified using 0.05 N HCl to remove any carbonates, before rinsing twice with distilled and deionized water (DDW). The pellet was kept in the deep freezer for c. 10 min until it was frozen, and it was then freeze-dried overnight in an Eyela freeze-dryer (Tokyo-Rika, FDU 506). Sample aliquots of 0.8 ± 0.05 mg were placed in tin capsules (two capsules per sample) for isotopic measurement, following the method of Chisholm et al (1982) and Chisholm and Koike (1996).
Coral tissue was obtained using the decalcification method. Each sample was ground to coarse fragments using a ball mill and placed in a cellulose tube. The tube was placed in a beaker containing 0.2 N HCl, which was then put on a stirrer (Horai et al., 1989). The HCl solution was changed every day for seven days until the decalcification of the tissue samples was complete. Decalcified tissues were then centrifuged and freeze-dried overnight.
Twenty-six samples of particulate organic matter (POM) were also collected using a 100 mm mesh plankton net from coral reefs in the area of Locality 1 (eight samples), Locality 2 (eight samples) and Locality 3 (10 samples). The samples were then centrifuged at 3000 rpm for 10 min to remove the supernatant. Samples were decalcified using 0.05 N HCl to remove any inorganic carbonate, before rinsing twice with DDW. After decalcification, samples were prefrozened and then freeze-dried overnight using an Eyela freeze-dryer. Eleven zooplankton samples (one sample of Locality 1, five samples of Locality 2 and five samples of Locality 3) were disaggregated from POM samples under a binocular microscope.
Sample weights of 0.80 ± 0.05 mg were placed in tin capsules for isotope measurements. The δ13C and δ15N values were analyzed in duplicate using a continuous flow stable isotope ratio mass-spectrometer (ANCA-mass 20-20, Europe Scientific Instruments, UK), where glycine and/or citric acid were run as standards. Measurement errors were within 0.1‰ for δ13C and 0.3‰ for δ15N. Samples were re-analyzed if the difference between the two sample analyses exceeded the measurement error.
Data were statistically analyzed using SPSS software. Analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) were conducted with the following dependent factors: δ13C of coral tissue; δ13C of zooxanthellae; Δδ13C, which indicated the differences in δ13C between the coral tissue and zooxanthellae; δ15N of coral tissue; δ15N of zooxanthellae; and Δδ15N between the coral tissue and zooxanthellae. Differences in species, seasons, localities and depths were subjected to a factor analysis. An alpha of 0.05 was taken to indicate statistical significance.
3 Results
Coral samples for this study were collected in August 2006 (1st dry season), November 2007 (rainy season) and August 2008 (2nd dry season). Average rainfall during survey were 83.7 mm, 235.8 mm and 141 mm respectively. In the study area dry season usually falls between July and September and rainy season occurs from October to May (Wiryawan et al., 2005).
3.1 Comparison among genera
Table 1 shows the distribution of the carbon content, nitrogen content, C-N ratios, and the δ13C and δ15N values of coral tissue and zooxanthellae from the three branching. The mean carbon content in the coral tissues, as well as zooxanthellae, from Porites was lower than that in Seriatopora and Stylophora, although these differences were not significant. The only mean nitrogen content of zooxanthellae from Seriatopora and Stylophora were significantly higher than Porites.The mean C-N ratio of coral tissues from Porites was higher than those of Seriatopora and Stylophora, but the differences were not significant.

Table 1 Mean %C, %N, C/N, δ13C and δ15N values of coral tissues and zooxanthellae for Porites, Seriatopora and Stylophora. P values are presented for ANOVA results

When the
δ13C values of corals were examined, it was found that there were significant differences of coral tissues and zooxanthellae among genera. Seriatopora had lower values of δ13C in both the coral tissue and zooxanthellae when compared with Porites and Stylophora.
3.2 Comparison by depth
The mean values δ13C of coral tissues and zooxanthellae collected from 3 m depth were significantly different (ANOVA, n=162, p<0.05) compared to those of samples collected from 10 m. Those collected from 3 m (–13.6 ± 1.8‰ and –14.2 ± 1.8‰ for coral tissues and zooxanthellae, respectively) had higher δ13C values than those from 10 m (–14.3 ± 1.5‰ and –14.7 ± 1.4‰, respectively). The mean δ15N values of coral tissues and zooxanthellae from 3 m depth (state values) were lower than those from 10 m (4.5±0.9‰ and 4.1 ±0.7‰, respectively). However, there were no significant statistical differences in the δ15N values of samples from –3 m and –10 m.
3.3 Comparison of localities and seasons
Locality 1 was situated in the northern coral area and it was the closest to the main river route, while Locality 3 was pure marine water; Localitly 2 was intermediate. Table 2 shows the mean δ13C values for coral tissue and zooxanthellae during the 1st dry season, the rainy season, and the 2nd dry season. The δ13C values of coral tissue were always higher than those of zooxanthellae in all seasons. The δ15N values of coral tissue were higher during the 1st and 2nd dry season compared with zooxanthellae. However, during the rainy season, the δ15N values of coral tissues and zooxanthellae were very similar, with no significant differences.

Table 2 Mean δ13C and δ15N values (±SD) of coral tissue, zooxanthellae, POM and Zooplankton for the different localities and seasons

At Locality 2, the mean
δ13C values of coral tissue were higher than zooxanthellae during the 1st dry and rainy seasons, while they were similar during the 2nd dry season (Table 2). During the 1st and 2nd dry seasons, the mean δ15N values of coral tissues were higher than zooxanthellae, whereas they were lower during the rainy season. However, only δ15N values of coral tissue of 2nd dry season were significantly different compared to the rainy season.
At Locality 3, the mean δ13C values of coral tissues were higher than zooxanthellae during the 1st and 2nd dry seasons, but they were similar during the rainy season. However, there were no significant differences among them. The mean δ15N values of coral tissues were higher than zooxanthellae during the 1st and 2nd dry seasons, but were significantly lower during the rainy season.
3.4 Comparison with POMs and zooplanktons
Twenty-six particulate organic matter (POM) and eleven zooplankton samples collected during the dry season were analyzed for δ13C and δ15N. POM samples collected during the rainy season had widely ranging carbon contents (2.3l–24.0%; mean = 9.3 ± 7.0%). The mean δ13C values of the POMs from Locality 1 (–19.1±1.2‰) were lower than those from Locality 3 (–15.9±4.3‰), but not significantly different. The mean δ13C values of POMs during the dry season (–18.9±1.1‰) and the rainy season(19.0±4.3‰) were relatively similar.
During the 1st dry season, the mean δ15N values of POMs (5.4±1.0‰) were slightly higher than corals. The mean δ15N values of zooplankton and POMs were similar or higher than corals during the 2nd dry season. However, during the rainy season, the mean δ15N values of POMs were significantly lower than corals, especially in Localities 1 and 3, suggesting that corals tended to utilize POMs as nutrient sources.
Mean δ13C values of zooplankton ranged from –22.3‰ to –18.0‰, while δ15N values ranged from +4.1‰ to +9.8‰. δ13C values of zooplankton collected from the estuary were lower than those collected from the marine area. The mean δ15N value of zooplankton was lower during the rainy
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