Coral Reefs (2008) 27:17–25 DOI 10.1007/s00338-007-0283-1
REPORT
Effects of temperature, light and heterotrophy on the growth rate and budding of the temperate coral Cladocora caespitosa R. Rodolfo-Metalpa Æ A. Peirano Æ F. Houlbre`que Æ M. Abbate Æ C. Ferrier-Page`s
Received: 9 April 2007 / Accepted: 13 July 2007 / Published online: 4 August 2007 Springer-Verlag 2007
Abstract Recent investigations have shown the temperate scleractinian coral Cladocora caespitosa to be a new potential climate archive for the Mediterranean Sea. Whilst earlier studies have demonstrated a seasonal variation in growth rates, they were unable to distinguish which environmental parameter (light, temperature, or food) was influencing growth. In this study, the effect of these three factors on the coral physiology and calcification rate was characterized to aid the correct interpretation of skeletal trace element variations. Two temperatures (13 and 23C), irradiances (50 and 120 lmol m2 s1), and feeding regimes (unfed and fed with nauplii of Artemia salina) were tested under controlled laboratory conditions on the growth, zooxanthellae density, chlorophyll (chl) content, and asexual reproduction (budding) of C. caespitosa during a 7-week factorial experiment. Unlike irradiance, which had no effect, high temperature and food supply increased the growth rates of C. caespitosa. The effect of feeding was however higher for corals maintained at low temperature, suggesting that heterotrophy is especially important during the cold season, and that temperature is the predominant factor affecting the coral’s growth. At low temperature, fed
Communicated by Biology Editor M. P. Lesser. R. Rodolfo-Metalpa (&) C. Ferrier-Page`s Centre Scientifique de Monaco, Av. Saint Martin, Principality of Monaco 98000, Monaco e-mail:
[email protected] A. Peirano M. Abbate Marine Environmental Research Centre, ENEA Santa Teresa, PO Box 224, La Spezia 19100, Italy F. Houlbre`que Geological and Environmental Sciences, Stanford University, 450 Serra Mall, Bldg 320, Stanford, CA 94305-2115, USA
samples had higher zooxanthellae density and chl content, possibly for maximizing photosynthetic efficiency. Sexual reproduction investment of C. caespitosa was higher during favourable conditions characterised by high temperatures and zooplankton availability. Keywords Mediterranean corals Growth Temperature Light Feeding Asexual reproduction
Introduction Cladocora caespitosa is the main endemic Mediterranean coral, abundant both in past and recent times (Zibrowius 1980; Peirano et al. 1998). It is the sole zooxanthellate, constructional ahermatypic coral that forms structures comparable to tropical reefs (Schuhmacher and Zibrowius 1985; Kruzˇic and Pozˇar-Domac 2003). This species seems well adapted to the marked seasonality of the Mediterranean Sea, and experiences large changes in temperature, of up to 15C between winter and summer, and also rapid temperature increases above the normal seasonal mean (Rodolfo-Metalpa et al. 2006a). Cladocora caespitosa is most abundant in turbid waters at a depth of ca. 7–15 m (Peirano et al. 2005), but can also be found both in well-lit shallow waters (Schiller 1993a; Bitar and Zibrowius 1997) and down to 40 m depth (Morri et al. 1994) where irradiance is greatly reduced. Despite this wide range of environmental habitats, the effect of light, temperature and feeding on the physiology of C. caespitosa has never been thoroughly investigated, with the exception of one experimental study which demonstrated an upper thermal limit of 24C (Rodolfo-Metalpa et al. 2006b). Earlier studies have also shown a clear skeletal banding pattern, with high- and low-density bands
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deposited in winter and summer, respectively (Peirano et al. 1999). However this banding could not be attributed solely to the effect of temperature, since light may also have an impact for shallow water corals, and feeding for deep organisms (Peirano et al. 1999). It has also been suggested that feeding, important in winter, could explain the high-density band deposition. In two subsequent studies (Peirano et al. 2005; Montagna et al. 2007), linear growth was found to be higher during summer, resulting in longer but less dense calyx walls. By analysing the distribution of the large fossil samples dating from the Pleistocene, it was also hypothesized that the development of C. caespitosa reefs was favoured in the warmest climate phase of the last interglacial time in coastal environments with alluvial inputs (Fornos et al. 1996; Aguirre 1998; Peirano et al. 2004). Recently, skeletal isotopic analyses have shown that C. caespitosa could be used as a climate proxy for the Mediterranean Sea (Silenzi et al. 2005; Montagna et al. 2007). While all the above studies have demonstrated a seasonal variation in growth rates, they have not been able to distinguish which environmental parameter, (light, temperature and food) was influencing growth. In order to be able to make reliable climate reconstructions from coral skeletons it is essential to understand how these factors affect skeletal growth (Lough and Barnes 1990). Previous studies have considered the effect of temperature, light and heterotrophy on coral growth rates but with each factor considered independently from the others. These studies have also concentrated on tropical corals, which have been found to have a general, positive correlation between seawater temperature and growth rates (Lough and Barnes 2000; Carricart-Garnivet 2004) within each species’ normal temperature range. Light has also been demonstrated to stimulate coral growth rates (Goreau 1959; Buddemeier and Kinzie 1976; Wellington 1982; Barnes and Chalker 1990), except in one study (Grottoli 2002) where skeletal extension rate decreased with increasing irradiance. Heterotrophy also has been found to enhance skeletal growth (Wellington 1982; Lasker et al. 1983; Anthony and Fabricius 2000; Ferrier-Page`s et al. 2003; Houlbreque et al. 2003, 2004b), particularly for tropical corals living in turbid water (Anthony and Fabricius 2000). Grottoli et al (2006) showed that tropical corals were more resilient to bleaching by increasing their food ingestion. Two studies, testing the combined effect of light and heterotrophy (Wellington 1982; Grottoli 2002) however found controversial results, with a small (Wellington 1982) or a negative (Grottoli 2002) effect of heterotrophy on coral growth, suggesting that there are substantial differences in the degree of dependency on light and zooplankton among tropical coral species. In contrast, little is known about temperate corals, except a positive effect of temperature on growth rate
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(Jacques et al. 1983; Miller 1995), and of feeding on both rates of growth (Kevin and Hudson 1979; Miller 1995) and photosynthesis (Szmant-Froelich and Pilson 1984; Piniak 2002). It might be concluded, however, from studies on temperate gorgonian, sponges and benthic suspension feeders in general (Ribes et al. 1999; Coma et al. 2000, 2002; Bavestrello et al. 2006), that temperature, feeding, and light should play an important role in their physiology. Additionally, for both tropical and temperate corals, few studies have investigated the formation of new polyps by budding (Fadlallah 1982; Titlyanov et al. 2001a; Gilmour 2002; Gaten˜o and Rinkevich 2003). One part of the energy budget (stored energy, photosynthates and energy brought by heterotrophic feeding), could be used for this asexual reproduction mode that, in turn, would be limited to periods of ideal environmental conditions for corals. Finally, of the few studies, which have focused on the effect of environmental parameters on temperate corals (Jacques et al. 1983; Miller 1995; Howe and Marshall 2002), only one involved Mediterranean scleractinian corals (Peirano et al. 2005). This study, therefore, was designed to experimentally investigate the effects of temperature, light and feeding regimes on the growth rates, zooxanthellae, pigment contents and asexual reproduction of the main scleractinian coral of the Mediterranean Sea, C. caespitosa.
Materials and methods Sample collection Five colonies of C. caespitosa were collected in the Gulf of La Spezia (Ligurian Sea, 44030 N, 9550 E) at 7–9 m depth. They were transported under reduced light and continuous aeration to the nearby laboratory of ENEA Santa Teresa where they were placed in a 250-l continuous-flow tank under the same temperature and light conditions (18 ± 0.5C, mean ± range; ca. 70 lmol m2 s1) as measured in situ. After 1-week acclimation, 80 nubbins (7– 18 polyps) and 80 single corallites (1 polyp) were detached from the colonies, mixed and considered as independent samples. They were carefully cleaned of epiphytes, associated fauna, and sediment using a brush and placed on labelled PVC supports.
Experimental design To assess the cross-effect of temperature, light and feeding on the growth rates, zooxanthellae, chlorophyll (chl) contents and budding of C. caespitosa a three-factor orthogonal experiment (2 · 2 · 2) was designed. The
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factor temperature had two levels: high (HT, 23 ± 0.2C, mean ± range) and low (LT, 13 ± 0.2C), which were representative of the temperatures experienced by the corals in the Ligurian Sea (means: 13–24C) during winter and summer. The factor light had two levels: high (HL, ca. 120 lmol m2 s1) and low (LL, ca. 50 lmol m2 s1), which corresponds to the months of March–April (LL) and August–September (HL) at the collection site for 10 m depth, respectively (Peirano et al. 1999). Each light and temperature treatment was again divided into two groups for two feeding levels: unfed nubbins (U), and fed twice a week with Artemia salina nauplii (F) (ca. 150 mg dry weight per replicate). For each of the eight treatments, ten 3 l transparent plastic beakers (independent replicates) were prepared. One nubbin, and one single polyp, for growth rate, zooxanthellae and chl measurements, respectively, were randomly assigned to one of the 80 tank replicates. Each beaker was filled with filtered (0.6 lm, Fluxa, Hunter) seawater that was changed almost completely every day at the final salinity of 38 ± 0.8 psu. The ten beakers were positioned in a water bath (ca. 100 l), equipped with heaters or with a refrigerating system connected to an electronic controller (± 0.2C sensitivity). A small submersible pump circulated the seawater to minimise temperature fluctuations. Light was provided by fluorescent tubes (True-Lite and Sunlux) placed at a varying distance from the beakers according to the required irradiance. Light was measured using a Li-Cor 4p spherical underwater quantum sensor (LI-193SA). Photoperiod was 12:12 h light/dark. To minimise variations in irradiance, the position of beakers were changed within each water bath every 4 days. Each beaker was provided with an air stone to provide water movement. Salinity and temperatures were measured each day using a Idronaut 401 probe. At the beginning of the experiment, temperature was gradually changed (0.5C per day) and maintained for 2 weeks at the experimental conditions before the start of the experiment for a total length of 7 weeks.
Zooxanthellae and chlorophyll content At the end of the incubation, one single polyp per beaker (n = 10 for each treatment) was collected and frozen (80C) for zooxanthellae and chl measurements. Tissue was removed from the coral skeleton using a water pick in 0.45 lm filtered seawater (Whatman GF/F). The slurry was homogenized using a hand-held potter homogeniser and the volume of homogenate recorded. Zooxanthellae densities were determined on 5 ml of the slurry immediately after extraction, using a haemocytometer (Bu¨rker-Tu¨rk). Five replicates were counted from each sub-sample and the mean was calculated. Sub-samples for chl analysis (using 10 ml
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of the slurry) were filtered onto Whatman GF/F glass fibre filters and kept frozen. Chl was then extracted in 90% acetone at 4C for 24 h, concentrations were determined, using the method of Jeffrey and Humphrey (1975), and normalized per surface area of polyps (see below).
Growth rates and asexual reproduction At the end of the acclimation period and after a total of 7 weeks, nubbin weight (n = 10 for each treatment) was measured using the buoyant weight technique (Davies 1989). Skeletal density of C. caespitosa was 1.86 ± 0.09 g cm3 (mean ± standard deviation (SD); n = 10) as measured using the method of Davies (1989). Growth rates were calculated as the daily change in dry weight between the initial and the final weight, and normalised to the surface area of polyps. Surface area was calculated at the end of the experiment as proposed by Rodolfo-Metalpa et al. (2006b) using the equation: PS = (2pR) H + pR2, where R represents the polyp radius, and H is the exosarc extension, measured with a calliper. Mean (± SD) surface area of the polyps was 0.85 ± 0.13 cm2 (n = 831). At the beginning and at the end of the experiment (for a total of 9 weeks), occurrence of buds on polyp tissue was recorded for each nubbin, using a stereomicroscope. The total number of buds in each beaker was normalised to the number of polyps and the mean percentage was compared between treatments. Statistical analysis All data were tested for assumptions of normality and homoscedasticity by the Cochran’ test and were log transformed if necessary. When three-way ANOVAs showed significant differences (P < 0.05) on an interaction term, Tukey honest test (HSD) was used to attribute differences between specific factors. One-way ANOVAs were used to investigate differences within single factors. Statistical analyses were performed using STATISTICA1 software (StatSoft). All the data were expressed as mean ± standard deviation (SD). Results Zooxanthellae and chlorophyll content Zooxanthellae density and chl content on the collected samples were 2.85 ± 1.02 cells cm2, and 11.7 ± 1.75 lg chl a + c2 cm2, respectively. At the end of the experiment, the zooxanthellae density differed according to the feeding regime, the temperature (P < 0.01, Fig. 1a,
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When chl contents were normalized per zooxanthellae, significant differences were found for the effect of temperature, light and all the interactions between factors (Table 1), likely due to a unique high value measured in fed corals maintained at 23C and low light (HT LL F, Fig 1c). However, it is important to note that at the low temperature, the chl (a + c2) zoox1 did not vary between light and feeding (two-way ANOVA, P < 0.05).
Table 1) and the combination of both parameters. The highest density per surface area was obtained at 13C (LT) in fed corals (mean including light factor 4.1 ± 0.9 (SD); Fig. 1a), whereas the lowest density was measured at 23C (HT), always in fed corals (mean including light factor 1.4 ± 0.4 zoox cm2) (Tukey HSD: LT F > LT U = HT U > HT F). The chl concentration per unit surface area followed the zooxanthellae density pattern. It was similarly affected by the feeding regime, the temperature, and the combination of both parameters (Fig. 1b, Table 1). The highest concentration was measured in fed corals maintained at 13C (mean 14.2 ± 3.5 lg chl a + c2 cm2), and was twice that in unfed corals, independent of the light regime (Fig. 1b). However, there was a significant interaction between light, temperature and feeding (Table 1). Except for the treatment at 23C and 50 lmol m2 s1 (HT LL), one-way ANOVAs always showed significant differences between fed and unfed corals (P < 0.05) for zooxanthellae density and chl content (Fig. 1a, b).
(x106) zooxanthellae cm-2
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Temperature and feeding significantly influenced mean growth rates, measured after 7 weeks of incubation (P < 0.001, Table 1), without any interaction between the two parameters (Table 1). For a given food regime, growth rates measured at 23C were twice (mean 0.33 mg cm2 day1) those obtained at 13C (mean 0.15 mg cm2 day1) (Fig. 2). This temperature enhancement was three times in unfed corals vs 1.8 times in fed ones (Fig. 3). For a
a
Fed Unfed
5 4 3 2 1 0 25
b
20 chl a + c2 (µg cm-2)
Fig. 1 Zooxanthellae density a, chlorophyll (a + c2) content normalized per surface area b and per zooxanthellae c, measured at the end of the experiment on polyps of C. caespitosa maintained at low and high temperature (LT = 13C and HT = 23C), low and high light intensity (LL = 50 lmol m2 s1 and HL = 120 lmol m2 s1), and feeding regime (F, fed and U, unfed). Values are mean ± SD (n = 10)
Growth rates
15 10 5 0
pg chl (a + c2) zooxanthellae-1
9
c
8 7 6 5 4 3 2 1 0 LT LL F LT LL U 50 µE
LT HL F
13°C
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LT HL U HT LL F HT LL U HT HL F HT HL U 120 µE 50 µE
120 µE
23°C
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Growth Zoox
Chl cm2 Chl zoox1 Bud (%)
0.6 Growth rates (mg cm-2 day-1)
Table 1 Summary of the three-way ANOVA testing the effect of temperature (13 and 23C), light (50 and 120 lmol m2 s1) and feeding on the growth, zooxanthellae density (zoox), chlorophyll (chl a + c2) content normalised per surface area (chl cm2) and per zooxanthellae (chl zoox1), and percentage of budding (bud)
0.5 0.4
