# Stable isotopes reveal limitations in C and N assimilation in the Caribbean reef corals Madracis auretenra, M. carmabi and M. formosa

C. Maier, Markus G Weinbauer, J. Paetzold

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C. Maier, Markus G Weinbauer, J. Paetzold. Stable isotopes reveal limitations in C and N assimilation in the Caribbean reef corals Madracis auretenra, M. carmabi and M. formosa. Marine Ecology Progress Series, 2010, 412, pp.103-112. 10.3354/meps08674. hal-03502082

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# Stable isotopes reveal limitations in C and N assimilation in the Caribbean reef corals *Madracis auretenra, M. carmabi and M. formosa*

C. Maier<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,*, M. G. Weinbauer<fnref n="2" />,<fnref n="3" />, J. Pätzold<fnref n="4" />

**ABSTRACT:** We used tissue δ¹⁵N and δ¹³C to elucidate nitrogen and carbon utilization in 3 branching coral species of the genus *Madracis*. Coral branches were sampled between 5 and 47 m water depth, and tissue was sub-sampled from the tips and sides of branches. Photosynthetically active radiation (PAR) ranged between 1 and 71 % of surface PAR. There was a good correlation between δ¹⁵N and δ¹³C, and these had similar ranges of 5.6 and 5.2‰, respectively, suggesting that light-driven photosynthesis is a strong factor in isotope fractionation. However, at shallow depths, δ¹³C decreased more strongly while δ¹⁵N remained constant, while at deeper depths δ¹⁵N decreased while δ¹³C remained constant. Comparisons of δ¹³C in polyps and zooxanthellae revealed that prey capture at the tips of branches was more pronounced than at the corresponding sideward-facing positions and that corals at deeper depths did not increase their food uptake to compensate for a decrease in photosynthetic carbon assimilation. This indicates that foraging in branching *Madracis* depends on current regimes and prey encounter rather than selective food capture. The systematic analysis of tissue δ¹⁵N along a steep depth gradient revealed that the shallow-water coral *M. auretenra* was nitrogen limited throughout its entire depth range.

**KEY WORDS:** Stable isotopes · Nitrogen limitation · Carbon · Heterotrophy · Scleractinian coral

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## INTRODUCTION

Tropical scleractinian reef corals are extremely well adapted to oligotrophic conditions through their harbouring of endosymbiotic dinoflagellates (zooxanthellae) of the genus *Symbiodinium*. The algal symbionts contribute significantly to the energy demand of their host and translocate up to 95 % of photosynthetic products to the coral animal (Muscatine 1990). In shallow-water corals, this mechanism is considered sufficient to meet the daily carbon demand for animal respiration and growth (Davies 1984, McCloskey & Muscatine 1984). In deeper depths, zooxanthellae adapt to lower light by increasing their relative photosynthetic efficiency (Kaiser et al. 1993, Lesser et al. 2000, Titlyanov

et al. 2001). Less photosynthetically fixed carbon is supplied to the animal part of the coral, but the reduced contribution of photosynthetic carbon at increasing water depth can partially be compensated by increasing the allochthonous nutrient supply (McCloskey & Muscatine 1984, Ferrier-Pagès et al. 1998, Titlyanov et al. 2000).

We used tissue δ¹⁵N and δ¹³C to investigate how 3 branching coral species of the genus *Madracis* at reefs of the Caribbean island Curaçao (Netherlands Antilles) cope with decreasing light regimes. On the reefs of Curaçao, each of the 3 species shows a strict zonation pattern and one species successively replaces the other at increasing water depth (Vermeij & Bak 2003). *Madracis auretenra* (Locke, Weil and Coates 2007),

<footnote n="1">¹Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands</footnote>

<footnote n="2">²CNRS-INSU, Laboratoire d'Océanographie de Villefranche, BP28, 06234 Villefranche-sur-Mer Cedex, France</footnote>

<footnote n="3">³Université Pierre et Marie Curie-Paris6, Laboratoire d'Océanographie de Villefranche, 06230 Villefranche-sur-Mer, France</footnote>

<footnote n="4">⁴MARUM - Center for Marine Environmental Sciences, University of Bremen, PO Box 330440, 28334 Bremen, Germany</footnote>
also described as *Madracis mirabilis* sensu Wells, 1973, is abundant at shallow depths between 2 and 25 m, *M. carmabi* Vermeij et al., 2003 at intermediate depths of 20 to 40 m, and *M. formosa* Wells, 1973 is confined to depths between 30 and 60 m. Photoadaptation has its limits, and absolute photosynthetic rates are lower in deep corals than in shallow ones. We therefore investigate, by means of tissue δ¹³C and δ¹⁵N analyses, whether deep corals indeed compensate for the inevitable energy loss by increasing their heterotrophic food supply. Tissue δ¹⁵N and δ¹³C are good indicators for trophic relationships and metabolic functioning (DeNiro & Epstein 1978, DeNiro & Epstein 1981, Gannes et al. 1998, Alamaru et al. 2009a,b). The separate analysis of δ¹³C of endosymbionts and the host organism can indicate the shared portion of metabolic energy (Muscatine et al. 1989). Also, relatively low values of δ¹⁵N in primary producers can hint at nitrogen fixation (Yamamuro et al. 1995, France et al. 1998). The application of δ¹⁵N analysis has considerably increased our knowledge of food webs. With each trophic level, heterotrophic organisms are enriched in δ¹⁵N by an average of 3.4 ± 1.1‰ (Minagawa & Wada 1984). We systematically sampled tissue δ¹³C and δ¹⁵N over a steep depth and light gradient to elucidate patterns of carbon and nitrogen assimilation, heterotrophic feeding and resource partitioning between zooxanthellae and cnidarian hosts of branching *Madracis* spp. We further propose that combining and extending existing models of C and N isotope fractionation can reveal a coral's 'strategy' of investing in either enhanced tissue or skeletal growth.

# MATERIALS AND METHODS

## Study site, sampling and sample treatment

A total of 42 colonies of *Madracis* spp. were sub-sampled in June 2002 on the leeward coast of Curaçao, Netherlands Antilles (12° 12′ N, 68° 56′ W). Pairs of tissue samples were taken from the upward facing tip (0°) and the sideward-facing (90°) position of a branch from each colony (see Table 1). Coral tissue was removed with a water-pik, using about 200 ml of filtered seawater (Millipore, pore size 0.22 µm). The tissue slurry was passed through a 20 µm mesh gauze to remove skeleton parts and other debris. Comparisons of δ¹³C analyses with acidified and non-acidified aliquots proved that the 20 µm net was sufficient to remove all carbonate (data not shown), and acid-treatment prior to stable isotope analyses was consequently omitted. Half of the slurry was filtered on pre-combusted Whatman GF/F glass-fibre filters using low pressure. The remaining part was homogenised (Ultra-

Turrax® T50) for 5 to 10 min to release zooxanthellae from the animal tissue. The homogenate was repeatedly centrifuged (2000 × g) and washed to separate the zooxanthellae from animal tissue (Muscatine et al. 1989). Random microscopic inspections revealed high separation efficiency for the 2 types of material. The supernatant containing animal tissue was filtered on GF/F filters. The pellet containing zooxanthellae was re-suspended in filtered seawater and also filtered on GF/F filters. The GF/F filters were dried for at least 24 h in an oven at 60°C and stored for later analyses of tissue stable isotopes.

δ¹⁵N and δ¹³C of particulate organic matter (POM) in seawater from intermediate depths (about 25 m) was filtered onto a GF/F filter and processed in a similar manner to the coral samples. To measure δ¹³C of dissolved inorganic carbon (DIC) in seawater we took water samples (30 ml each) at 4 depths (5, 15, 25, 40 m) between April 16 and June 26, 2001 at at least bi-weekly intervals (N = 9 at each depth). Directly after sampling, HgCl₂ was injected, and bottles were sealed (air-tight) and stored at 4°C until analysis of δ¹³C.

## Photosynthetically active radiation

At the study site, photosynthetically active radiation (PAR) with a wavelength of 400 to 700 nm was determined during 16 days in May 2001 using a LI-192SA underwater quantum sensor (LI-COR) connected to a LI-1000 data logger (LI-COR). PAR of upward-facing, horizontal positions (0°) was determined at intervals of 1 m to a maximum depth of 40 m. For PAR of the sideward-facing, vertical position (90°), we repeatedly measured at 4 distinct depths (10, 15, 25 and 35 m) and used the average PAR relative to the upward-facing position (see Table 1).

## Isotopic analyses

Isotopic data are presented as δ¹⁵N and δ¹³C in ‰ notation with δX (‰) = [(R$_{sample}$ × R$_{standard}$) – 1] × 1000, where X = ¹⁵N or ¹³C, and R = ¹⁵N/¹⁴N or ¹³C/¹²C, respectively. The δ¹⁵N and δ¹³C of coral holobiont and POM were analysed in parallel and in triplicate with a Finnigan MAT Delta Plus mass spectrometer after being combusted at 1050°C in a NC 2500 Elemental Analyser (CE Instruments). δ¹⁵N is reported relative to air N₂. We used pure N₂ (99.996‰) as a laboratory working standard gas and IAEA standards N-1 and N-2. δ¹³C is reported relative to Vienna PDB (VPDB). Sucrose and polyethylene foil (PEF) were used as standards for δ¹³C. The analytical precision for both δ¹⁵N and δ¹³C based on repeated measurements of a labora-
tory sediment standard (WST2) was <0.26‰ ± 1 SD (N = 143). Samples of separate cnidarian host and zooxanthellae fractions were analysed for δ¹³C with benzoic acid as the δ¹³C standard. The analytical precision for δ¹³C was <0.15‰ ± 1 SD (N = 27) based on repeated measurements of a laboratory standard (acetalalide). Sampled material was insufficient to also analyse δ¹⁵N of separate cnidarian host and zooxanthellae.

## Statistical analyses

Statistical analyses were conducted using the Windows software package Statistica 7. For each coral colony, the tip and side positions were sampled separately thus resulting in a pair of samples from the tip and side for a single branch. Also, the δ¹³C of polyp and zooxanthellae was derived from corresponding samples. For corresponding sample pairs, we used the paired t-test for the statistical comparison of the bulk δ¹⁵N and δ¹³C of the tip and side position or δ¹³C of polyp and zooxanthellae. We also tested the mean differences of sample pairs for the 6 depths against zero. This provided information on whether or not the differences between paired samples are consistently (either negatively or positively) offset from each other throughout sampling depths.

To test the combined effects of depth and colony position, a 2-way ANOVA was conducted for each data set with bulk δ¹⁵N, bulk δ¹³C, δ¹³C of polyp and δ¹³C of zooxanthellae. For all tests, the chosen significance level was p < 0.05.

# RESULTS

## Light, seawater DIC and POM at the study site

Dependent on depth and colony position, PAR varied between 1 and 71% of surface PAR (Table 1). The average mid-day surface PAR was 817 µE m⁻² s⁻¹. Changes in insulation and cloud cover between sampling days accounted for a highly variable PAR with an average coefficient of variance (CV) of 34.7% ± 0.75 SE. This variability was independent of depth or colony position (r² = 0.033, p = 0.265, n = 40). Exponential regression functions for PAR at upward (PAR[0°]) and sideward (PAR[90°]) positions as a function of depth were PAR(0°) = 817.1 µE m⁻² s⁻¹ × e⁻⁰.⁰⁶⁸ × depth (r = 0.985, p << 0.001) and PAR(90°) = 93.7 µE m⁻² s⁻¹ × e⁻⁰.⁰⁵² × depth (r = 0.918, p = 0.041). This means that the attenuation coefficient k' for the upward and sideward positions was -0.068 and -0.052, respectively. Stable isotope signatures of POM sampled from the intermediate depth ranged between 3.9 and 4.8‰ and 20.1 and 20.3‰ for δ¹⁵N and δ¹³C (N = 3, each), respectively. Dissolved in-organic carbon in seawater at this site was on average 1.32‰ ± 0.02 (N = 36) and was independent of water depth or season (Maier 2004).

## Holobiont tissue δ¹³C and δ¹⁵N

The stable isotope values from the coral holobiont ranged from 0.4 to 6.0‰ for δ¹⁵N and from -22.1 to

<table><caption>Table 1. *Madracis auretenra*, *M. carmabi*, *M. formosa*. Sampling regimes, light characteristics and tissue isotope mean and SE values. Samples taken from the tip of a colony were horizontal (angle of 0°) and the side positions were vertical (90°). Depth is the sampling depth and the numbers in brackets give the theoretical depth for side positions at which same light intensity would occur relative to tip position. δ¹⁵N: holobiont δ¹⁵N; H-δ¹³C: holobiont δ¹³C; P-δ¹³C: host polyp δ¹³C; Z-δ¹³C: zooxanthellae δ¹³C; N: number of colonies investigated</caption><thead><tr><th>Species</th><th>N</th><th>Depth (m)</th><th>Branch position</th><th>PAR (µE m<sup>-2</sup> s<sup>-1</sup>)</th><th>δ<sup>15</sup>N (‰)</th><th>H-δ<sup>13</sup>C (‰)</th><th>P-δ<sup>13</sup>C (‰)</th><th>Z-δ<sup>13</sup>C (‰)</th></tr></thead><tbody><tr><td rowspan="6">M. auretenra</td><td>6</td><td>5</td><td>Tip</td><td>581.0</td><td>71.1</td><td>5.1 ± 0.1</td><td>-17.1 ± 0.1</td><td>-17.1 ± 0.1</td><td>-16.7 ± 0.2</td></tr><tr><td>6</td><td>5 (35)</td><td>Side</td><td>72.4</td><td>8.9</td><td>4.5 ± 0.2</td><td>-17.7 ± 0.2</td><td>-17.4 ± 0.1</td><td>-17.3 ± 0.1</td></tr><tr><td>8</td><td>10</td><td>Tip</td><td>413.1</td><td>50.6</td><td>4.7 ± 0.2</td><td>-17.7 ± 0.2</td><td>-17.8 ± 0.1</td><td>-17.1 ± 0.2</td></tr><tr><td>8</td><td>10 (39)</td><td>Side</td><td>55.9</td><td>6.8</td><td>4.3 ± 0.3</td><td>-18.0 ± 0.3</td><td>-17.7 ± 0.2</td><td>-17.5 ± 0.2</td></tr><tr><td>7</td><td>20</td><td>Tip</td><td>208.9</td><td>25.6</td><td>4.9 ± 0.1</td><td>-18.5 ± 0.3</td><td>-18.5 ± 0.2</td><td>-18.0 ± 0.2</td></tr><tr><td>7</td><td>20 (47)</td><td>Side</td><td>33.3</td><td>4.1</td><td>4.4 ± 0.2</td><td>-19.5 ± 0.4</td><td>-19.2 ± 0.3</td><td>-19.6 ± 0.3</td></tr><tr><td rowspan="2">M. carmabi</td><td>6</td><td>30</td><td>Tip</td><td>105.6</td><td>12.9</td><td>3.4 ± 0.3</td><td>-19.6 ± 0.3</td><td>-19.1 ± 0.2</td><td>-19.0 ± 0.2</td></tr><tr><td>6</td><td>30 (55)</td><td>Side</td><td>19.9</td><td>2.4</td><td>3.0 ± 0.4</td><td>-20.2 ± 0.2</td><td>-20.0 ± 0.3</td><td>-20.0 ± 0.3</td></tr><tr><td rowspan="4">M. formosa</td><td>10</td><td>40</td><td>Tip</td><td>53.4</td><td>6.5</td><td>3.3 ± 0.1</td><td>-19.7 ± 0.2</td><td>-19.6 ± 0.2</td><td>-19.1 ± 0.2</td></tr><tr><td>10</td><td>40 (62)</td><td>Side</td><td>11.8</td><td>1.4</td><td>2.2 ± 0.2</td><td>-20.8 ± 0.3</td><td>-20.2 ± 0.3</td><td>-20.1 ± 0.3</td></tr><tr><td>5</td><td>47</td><td>Tip</td><td>33.1</td><td>4.1</td><td>2.9 ± 0.2</td><td>-20.1 ± 0.4</td><td>-19.7 ± 0.3</td><td>-19.2 ± 0.3</td></tr><tr><td>5</td><td>47 (67)</td><td>Side</td><td>8.2</td><td>1.0</td><td>1.6 ± 0.4</td><td>-20.4 ± 0.2</td><td>-20.0 ± 0.2</td><td>-20.2 ± 0.2</td></tr><tr><td>All samples</td><td>94</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3.7 ± 0.1</td><td>-19.1 ± 0.2</td><td>-18.9 ± 0.2</td><td>-1.7 ± 0.2</td></tr></tbody></table>
-16.9‰ for δ¹³C. The δ¹⁵N and δ¹³C values had an almost similar isotopic range with 5.6‰ and 5.2‰, respectively. Furthermore, δ¹⁵N and δ¹³C were significantly and positively correlated to each other, (R = 0.760, p << 0.001, N = 82) and each decreased significantly with water depth (R = -0.782 for δ¹⁵N and R = -0.816 for δ¹³C; with each N = 84 and p << 0.001). Despite the good overall correlation between δ¹⁵N and δ¹³C, correlations were weaker if corals from shallow (5 to 20 m, R = 0.327, p = 0.033, N = 43) or deep (30 to 47 m, R = 0.520, p = 0.001, N = 39) were considered separately (Fig. 1).

## δ¹³C in zooxanthellae and cnidarian host

The average δ¹³C values of polyp tissue and zooxanthellae were -18.9‰ ± 0.1 and -18.6‰ ± 0.2, respectively. The difference between polyp and zooxanthellae averaged 0.24‰ and was highly significant (paired t-test, *t* = 5.08, p << 0.001, N = 83). Paired t-tests between zooxanthellae and cnidarian δ¹³C at respective depths and colony positions revealed significant differences at all depths except at 30 m for the colony tips, whereas at side position zooxanthellae and host tissue at 10 and 20 m depth were significantly different (Fig. 2). At the colony tips, δ¹³C values of zooxanthellae were consistently heavier than corresponding values of the polyp tissue. This difference was consistent and

significant at tips (test of means against zero, p = 0.002, N = 6), while there was no consistent difference between zooxanthellae and host δ¹³C at side positions (test of means against zero, p = 0.721, N = 6). The difference between zooxanthellae and polyp δ¹³C was more pronounced for δ¹³C of tips of the shallow water coral *Madracis auretenra* with a significant mean difference of 0.58‰ ± 0.06 (t = 10.5, p << 0.001, N = 20) than for the tip position of the deeper colonies with a difference of 0.37‰ ± 0.09 (t = 3.9, p < 0.001, N = 21). However, the differences between zooxanthellae and polyp δ¹³C at the tip of a colony showed no significant depth effect (1-way ANOVA, F(5,35) = 1.815, p = 0.135).

## Effect of depth and colony position

Depth and colony position had a significant effect on stable isotope fractionation (2-way ANOVA, always p << 0.001, N = 42). Paired sample t-tests of tissue isotopes from tip and side position at distinct depths were also performed. At each depth, the δ¹³C values of zooxanthellae were significantly higher in tip than in side positions (p < 0.05), whereas δ¹³C of cnidarian host tissue was only significantly different at 20, 30 and 40 m (Fig. 2). For holobiont tissue, the paired t-test revealed significant differences between colony positions for δ¹³C at 5, 20, 30 and 40 m and for δ¹⁵N at 20, 40 and 47 m (Fig. 3). Generally, the tissue stable isotope signals were heavier at the tip (0°) than at the side (90°) of a colony. The mean differences between tip and side were significant for δ¹⁵N and δ¹³C of holobiont and zooxanthellae (test of means against zero, p < 0.01, N = 6), but not for δ¹³C of polyp host (test of means against zero, p = 0.07, N = 6).

# DISCUSSION

## Stable isotope models

In general, tissue stable isotopes were more depleted at deeper water depths regardless of tissue type or colony position, a pattern which generally substantiates earlier findings on photosynthetically driven isotope fractionation for both δ¹⁵N (Muscatine & Kaplan 1994, Heikoop et al. 1998) and δ¹³C (Land et al. 1975, Muscatine et al. 1989). Our data further indicate dissolved inorganic nitrogen (DIN) limitation for shallow water corals and a consistently more pronounced heterotrophic signature in the tips of branches compared to the sides. Contrary to earlier studies (McCloskey & Muscatine 1984, Ferrier-Pagès et al. 1998, Titlyanov et al. 2000), our results do not show that lower photosyn-

<figure><img src="image_2.png" /><figcaption>Fig. 1. *Madracis* spp. Correlation between δ¹⁵N and δ¹³C in holobiont tissue with δ¹⁵N = 16.7 + 0.69 × δ¹³C (r = 0.769, p < 0.0001, N = 84). (O) shallow (5 to 20 m), (●) deeper (30 to 47 m) water corals</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig. 2. *Madracis* spp. δ¹³C of zooxanthellae and host polyp for (a) tip and (b) side of colony positions versus water depth. Error bars are SE of mean values. Vertical dashed line gives estimate for δ¹³C of POM. \*p < 0.05 and \*\*p < 0.005 (paired t-test for tips and side of colony positions)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. *Madracis* spp. (a) Mean δ¹⁵N and (b) δ¹³C of holobiont tissue at the side and tip of branches versus water depth. Error bars are SE of mean values. Vertical dashed line gives estimate for δ¹⁵N and δ¹³C of POM. \*p < 0.05 and \*\*p < 0.005 (paired t-test for tips and side of colony positions)</figcaption></figure>

thetic performance at deeper depths is compensated for by an increase in heterotrophy.

We combined several models on carbon acquisition (Furla et al. 2000a), carbon isotope fractionation (Muscatine et al. 1989, Reynaud et al. 2002), nitrogen flux and isotope fractionation (Heikoop et al. 1998) to elucidate physiological constraints in the branching coral *Madracis* spp. (Fig. 4). Stable isotope fractionation oc-

curs during chemical reactions where lighter isotopes usually form weaker chemical bonds and react faster than the heavier isotopes. The isotopic signature in biological systems depends on the isotope ratio of source molecules, enzymes involved in reactions, reaction kinetics and physical constraints that control the replenishment of source molecules at the site of (bio-) chemical reactions (e.g. Urey 1947, Owens 1988, Laj-
<figure><img src="image_5.png" /></figure>

Fig. 4. *Madracis* spp. Model of pathways and fluxes of carbon and nitrogen contributing to stable isotope fractionation and com-
position of total organic carbon and nitrogen (TC<sub>org</sub> and TN<sub>org</sub>) as measured in the tissues of branching *Madracis* spp. The model
was combined and modified from models for carbon acquisition (Furla et al. 2000a), carbon isotope fractionation (Muscatine et al.
1989) and nitrogen flux and isotope fractionation (Heikoop et al. 1998). Numbers with letters in superscript are estimates for δ<sup>15</sup>N
or δ<sup>13</sup>C (‰). The values were approximated from various sources as follows: (a) this study with average δ<sup>13</sup>C seawater DIC
(δ<sup>13</sup>C<sub>DIC</sub>), seawater bicarbonate (HCO<sub>3</sub><sup>-</sup><sub>sw</sub>), metabolic CO<sub>2</sub> (CO<sub>2met</sub>), which is similar to δ<sup>13</sup>C of animal tissue (Muscatine et al.
1989), while metabolic bicarbonate (HCO<sub>3</sub><sup>-</sup><sub>met</sub>) is enriched by ~7‰ relative to CO<sub>2met</sub> and vice versa. The same fractionation fac-
tor of 7‰ was applied between δ<sup>13</sup>C<sub>DIC</sub> and inorganic CO<sub>2</sub> (CO<sub>2l</sub>), which is taken up by zooxanthellae (zoox). (b) δ<sup>13</sup>C of organic
carbon (C<sub>org</sub>) is about 1‰ enriched to prey (DeNiro & Epstein 1978). In our study, we assume that POM is the major allochthonous
food source. (c) Enrichment of organic δ<sup>15</sup>N (δ<sup>15</sup>N<sub>org</sub>) by heterotrophic feeding (N<sub>org</sub>) by an average 3.4‰ (Minagawa & Wada
1984). (d) Likely range of δ<sup>15</sup>N of seawater DIN (δ<sup>15</sup>N<sub>DIN</sub>) (Heikoop et al. 1998). (e) Maximum of 20% of assimilated prey nitrogen
has been shown to appear in endosymbiotic zooxanthellae (Piniak et al. 2003). (f) Range in skeletal δ<sup>13</sup>C (δ<sup>13</sup>C<sub>skel</sub>) for branching
*Madracis* spp. with a depth range similar to colonies of this study (Maier et al. 2003). (g) Equilibrium <sup>13</sup>C fractionation (δ<sup>13</sup>C) be-
tween aragonite and bicarbonate (Romanek et al. 1992). (h) Suggested contribution to skeletal carbon of 75 to 80% of metabolic
carbon and 20 to 25% of seawater bicarbonate (Erez 1978, Furla et al. 2000a). (i) Nitrogen fixers, such as endolithic algae, have an
δ<sup>15</sup>N value of ~2‰ (France et al. 1998). A transfer of photoassimilates from endolithic algae to coral tissue (dashed arrows) may be
of significance for coral metabolism in extreme situations such as in very low light environments or in bleached corals (Schlichter
et al. 1995, Fine & Loya 2002). The '?' indicates that it is uncertain if endolithic algae utilize CO<sub>2</sub> respired by corals. Dotted line
indicates optional contributions/pathways

tha & Michener 1994). A reef-building coral consists of
several micro-environments with an oral and aboral
layer of an ecto- and endoderm, which is separated by
the mesoglea and the coelenteron. The gastroderm
contains photosynthetic algal symbionts and the aboral

ectodermal layer forms the calicoblast, where the arag-
onite exoskeleton is excreted. The oral side is exposed
to surrounding seawater, while at the aboral side the
calicoblast layer is shielded from ambient seawater by
the skeleton (Fig. 4). Consequently, tissue isotope frac-
tionation depends on reactions taking place within
these micro-environments, the replenishment with
source C and N, as well as the translocation of C and N
from and to respective sites for biochemical reactions.

At the study site, the δ¹³C of external seawater DIC averaged 1.3‰ and temperature was 26.5°C (299.5 K) (Maier et al. 2003). The cellular CO₂, which is derived from ambient seawater DIC, would thus have a δ¹³C of -7.45‰ according to the equation δ¹³Cₐ₅₂ = δ¹³Cₐ₃₄ + 23.644 × 9701.5/Tₖ (Rau et al. 1996). The δ¹³C of metabolic CO₂ has an expected isotopic range between -17‰ for shallow and -20‰ for deep corals, which actually corresponds to our findings (Table 1). It has also been hypothesized that the internal DIC pool actually increases in size as a consequence of a light-dependent CO₂ concentrating mechanism to avoid limitation in CO₂ supply to photosynthesis (Weis et al. 1989, Al-Moghrabi et al. 1996, De Beer et al. 2000, Furla et al. 2000a).

Diffusion constraints

The maxima of shallow *Madracis auretenra* zooxanthellae δ¹³C were around -16‰. These values are relatively low compared to other zooxanthellate coral species, which mostly range between -12 and -14‰ at the same depths (Muscatine et al. 1989, Swart et al. 2005, Alamaru et al. 2009a), but similar values have been previously reported for *M. auretenra* (Muscatine et al. 1989). Lower δ¹³C values can hint at lower photosynthesis and/or at faster replenishment of internal DIC from ambient seawater DIC due to reduced diffusion distances. The genus *Madracis* indeed has several characteristics that could account for reduced diffusion distances and thus allow for faster exchange rates between internal and seawater DIC: (1) The tissue of *M. auretenra* is removed very easily by water-picking indicating that it is relatively superficially bound to the skeleton and does not extend deeply into the skeleton. Other corals often have either big corallites, as found in *Montastraea*, or a porous skeleton as in *Porites*, and in both cases the polyp tissue reaches several millimetres deep into the skeleton. This can increase diffusion distances or create stagnant regions on a small scale, and thus slow down DIC exchange between seawater and calicoblast layer. (2) The genus *Madracis* hardly produces any mucus (C. Maier pers. obs.) that may decelerate diffusion of DIC between ambient seawater and coral tissue. This means that a relatively thin tissue layer and the lack of mucus production, as observed in the branching *Madracis* species investigated here, would allow for rapid replenishment of DIC from ambient seawater into the coral tissue, and this would favour stronger isotopic discrimination. This could be

an explanation for the relatively low isotope values ob-
served in *Madracis* spp. (Muscatine et al. 1989, this
study).

For the calcification of carbon, 75 to 80% is suppos-
edly supplied by metabolic (respired) DIC, while the
remaining 20 to 25% originate from external seawater
DIC (Erez 1978, Furla et al. 2000b). If indeed the lighter
metabolic DIC is used for calcification, the internal
carbon pool becomes heavier, and light-enhanced cal-
cification further augments the enrichment of tissue
δ¹³C, specifically in rapidly calcifying corals (McCon-
naughey 1989a,b, McConnaughey & Whelan 1997).
This very likely explains the higher tissue δ¹³C of the
shallow-water coral *Madracis auretenra* in relation to
its deeper congeners.

N-limitation and saturation levels

Similar fractionation regimes and diffusion-limited N-
supply can influence the nitrogen isotope fractionation.
The 'depletion-diffusion' and 'host assimilation' models
for zooxanthellate corals are thoroughly discussed in
Heikoop et al. (1998). DIN is taken up from the external
seawater into the cytoplasm of the host, and from this in-
ternal DIN pool, nitrogen is taken up by the zooxanthel-
lae, assimilated into organic nitrogen and finally trans-
located back to the host (Fig. 4). Also, nitrogen from host
metabolism is released to the internal DIN pool.

The δ¹⁵N values of shallow-water *Madracis aure-
tenra* remained constant at their maxima between 5
and 20 m water depth, while δ¹³C remained fairly con-
stant at minimum values for deep corals between 30
and 47 m (Fig. 2). These constant maxima or minima at
either shallow or deep depths indicate a saturation
state for the processes involved in the respective iso-
tope fractionation. The constant δ¹⁵N maxima between
5 and 20 m water depth show that *M. auretenra* is actu-
ally DIN limited almost throughout its entire depth
range at the reefs of Curaçao. This consequently sug-
gests that *M. auretenra* has a high photosynthetic rate
that causes DIN limitation even at a depth of 20 m. At
the same time, tissue δ¹³C of *M. auretenra* decreased
from 5 to 20 m, signifying that more carbon is fixed by
photosynthesis and/or calcification at shallow depths.
An increase in photosynthetic carbon fixation at shal-
low depths accompanied by a decrease in nitrogen fix-
ation as a consequence of very high photosynthesis has
been suggested before, but not to the extent of DIN
limitation (Muscatine & Kaplan 1994). However, it has
been suggested that shallow water corals under high
light can become nitrogen-restricted (Rees 1991, Jokiel
et al. 1994, Muscatine & Kaplan 1994) and the data
presented in our study provide the first record of DIN
limitation actually being indicated by tissue δ¹⁵N.
## Heterotrophy and resource partitioning

The $\delta^{13}C$ and $\delta^{15}N$ values for POM are bordering corresponding minimum and maximum plateaus of tissue isotopes from deep and shallow colonies, respectively (Figs. 2 & 3). Both $\delta^{13}C$ and $\delta^{15}N$ were lower at the side than at the upward-facing tips of colony branches (Fig. 3). Also, at the colony tip there was a consistent and significant offset in $\delta^{13}C$ between zooxanthellae and cnidarian host tissue, which was lacking for side positions (Fig. 2). The lack in clear differences between zooxanthellae and cnidarian $\delta^{13}C$ at the side indicates an almost complete recycling of carbon between zooxanthellae and the animal host. This suggests that prey is captured only sporadically at the side position of a branch, which is actually contrary to our initial idea. We expected a more pronounced heterotrophic isotope signature at the side position to compensate for lower light intensity and consequently for less photosynthetic energy being available to the polyps.

Because photosynthetic DIN assimilation was limited and a saturation plateau for isotope fractionation was reached in shallow *Madracis auretenra*, it is possible to estimate heterotrophic nitrogen uptake from the observed difference in $\delta^{15}N$ at tip and side positions. As indicated by similar polyp and zooxanthellae $\delta^{13}C$, food capture is negligible at side positions. We can also assume that photosynthetic DIN assimilation rates at the tip and side positions were similar because they were saturated at both positions. As a consequence, the observed difference between $\delta^{15}N$ at the tip and side has to be attributed to heterotrophic feeding. If POM is the main heterotrophic food source and an average enrichment factor of 3.4‰ is assumed (Minagawa & Wada 1984), the average difference of 0.5‰ between the tip and side suggests that about 15% of the nitrogen was derived from POM. This is a conservative estimate because it is based on the assumption that no particulate organic nitrogen is taken up at the side position via prey capture. In any case, this estimate is well within the range of earlier estimates on uptake of organic matter (Anthony 1999).

We further suggest that the depth for saturation states for processes involved in stable isotope fractionation is indicated by a change in direction of the isotope curves: A change occurs for $\delta^{15}N$ between 20 and 30 m water depth and for $\delta^{13}C$ between 30 and 40 m (Fig. 3). This indicates that seawater DIN supply would be sufficient at, or below, this depth to meet levels of the actual photosynthetic capacity of the *Madracis* species investigated. The $\delta^{13}C$ values of POM—as an allochthonous food source—are similar to $\delta^{13}C$ values expected for enzymatically driven photosynthetic isotope fractionation by RubisCO, form II, known for dinoflagellates (Leggat et al. 1999, Robinson et al. 2003). This ambigu-

ity limits our ability to further interpret the trophic state of deep-water specimens by means of stable isotopes. However, differences in $\delta^{13}C$ between zooxanthellae and polyp remained constant with depth, which is in contrast to previous observations (Muscatine et al. 1989). This suggests that over the entire depth range from 5 to 47 m the branching *Madracis* species investigated do not necessarily increase prey capture and, thus, may not compensate for any inevitable reduction in photosynthetic carbon assimilation. Our findings that heterotrophy in branching *Madracis* appears to be the result of food availability as indicated by $\delta^{15}N$ at the tip and side positions also support this theory. Food availability is controlled by current regimes around and between colony branches. It has been shown that prey encounter and prey capture depend on flow speed and coral colony morphology (Sebens et al. 1997) as well as on the concentration of prey items (Anthony 1999). At the outer boundaries of branching colonies, i.e. at the outward-facing tip, faster flow regimes favour particle encounter and the possibility to capture prey. The sideward-facing positions are farther 'inside' of a branching colony, and water flow can reach a stagnant phase reducing particle encounter and thus, prey encounter (Kaandorp et al. 2003).

## Growth strategy of *Madracis auretenra*

The shallow water species *Madracis auretenra* has a remarkable growth strategy and skeletal growth is very fast at the tip of a branch. Skeletal growth rates approximate 0.5% d⁻¹ (Bak 1977) and a mean linear skeletal extension of 16 mm yr⁻¹ (Nagelkerken et al. 2000). Often only upper parts of branches are covered with live tissue while lower parts are void of tissue. Bare skeleton allows burrowing organisms to penetrate and destabilise the skeleton. This generates skeletal breaking points that facilitate reproduction by fragmentation (Highsmith 1982). On the shallow reefs of Curaçao, *M. auretenra* indeed forms large meadows (Bak 1977, van Duyl 1985) that are likely a result of spreading by colony fragmentation. Such a life strategy, with fast skeletal growth at branch tips and reproduction by fragmentation, is actually a good solution if tissue growth is nitrogen-limited. This seems to be the case for *M. auretenra*, for which tissue $\delta^{15}N$ indicates nitrogen-limitation, while at the same time excess carbon still facilitates rapid calcification rates under highlight conditions. This may explain why *M. auretenra* is not abundant below a certain depth, where photosynthetic carbon assimilation might be light-restricted, and a resultant reduction in calcification rates may not be able to sustain a life strategy that is based on fast skeletal growth and reproduction by fragmentation.
## CONCLUSIONS

By the combined and systematic comparison throughout a large depth and light range of the δ¹⁵N and δ¹³C signals of branching *Madracis* species we showed a very distinct pattern with respect to carbon and nitrogen assimilation and resource partitioning between coral polyp and algal symbionts. We showed DIN limitation for shallow water *M. auretenra* while DIN and DIC assimilation of corals at intermediate depths seemed neither carbon nor nitrogen restricted. Stable isotope signals revealed that food capture was more pronounced at colony tips than at side positions throughout all depths. This indicates that in the branching species of *Madracis*, heterotrophy is a function of food availability. Ultimately, we conjecture that tissue δ¹⁵N and δ¹³C can indicate a species' potential life strategy with respect to either investing in tissue growth and maintenance, or investing in faster skeletal growth at the expense of individual, older coral polyps.

*Acknowledgements.* We thank the staff of Carmabi for assistance during field work in Curaçao. J. M. Janssen conducted underwater light measurements. R. P. M. Bak and G. Nieuwland (MEE, Royal NIOZ) provided help in many ways. For assistance in isotopic analyses, we thank S. Schouten and M. Kienhuis (MBT, Royal NIOZ), G. Lavrik (MPI, Bremen), M. Segl and her team (Geoscience Department, University of Bremen). We thank J. -P. Labat for statistical advice, 3 anonymous referees for critical comments and E. Goldstein for revision of the English. C.M. acknowledges support from the Schure-Beijerinck-Popping Fonds (SBP/JK/2002-30), the EC Human Potential—Access to Research Infrastructures activity, project Paleostudies (HPRI-CT-2001-00124) and the Dutch-German cooperation program NWO-DFG (DN 76-230). The work is in partial fulfilment of the requirements for a PhD at the University of Amsterdam by C.M.

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