Biol. Bull. 215: 243-252. (December 2008)
© 2008 Marine Biological Laboratory
Importance of Time and Place: Patterns in Abundance of Symbiodinium Clades A and B in the Tropical Sea Anemone Condylactis gigantea
A. A. Venn1,2,*,
,
J. E. Loram1,2,
H. G. Trapido-Rosenthal2,,
D. A. Joyce3 and
A. E. Douglas1
1 Department of Biology, University of York, York, YO10 5YW, United Kingdom
2 Bermuda Institute of Ocean Sciences, Ferry Reach, Bermuda, GE01
3 Department of Biological Sciences, University of Hull, Hull, HU6 7RX, United Kingdom
* To whom correspondence should be addressed. E-mail: Alexandervenn{at}yahoo.com
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Abstract
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The capacity of some corals and other cnidarians to form symbioses with multiple algae (Symbiodinium) is a candidate route by which these symbioses tolerate variable environmental conditions. On Bermuda, the coral reef dwelling anemone Condylactis gigantea bears Symbiodinium of clades A and B. At thermally variable inshore and nearshore sites, clade A predominates (as sole symbiont or in mixed infection with clade B), whereas animals at offshore sites with more uniform temperatures bear only clade B or mixed infections. Individual animals at one nearshore site monitored over a year by sampling tentacles showed increased prevalence of clade A in March-November, when sea waters were warm (average 26 °C), and increased clade B in November-March when cool waters prevailed (average 18.5 °C). In laboratory analyses of excised tentacles, the symbiosis with clade B, but not clade A, bleached at elevated temperature (32 °C), suggesting that thermal tolerance may contribute to the higher prevalence of clade A at inshore/nearshore sites and in the summer. The temporal changes in the algal complement were not accompanied by bleaching, and Symbiodinium density fluctuated in hosts with stable Symbiodinium composition but not in hosts with variable composition. This suggests that changes in the relative abundance of Symbiodinium clades do not require bleaching and may even protect the symbiosis from large fluctuations in algal density.
Abbreviations: AFLP, amplified fragment length polymorphism • q-PCR, real-time quantitative PCR • LSU, large subunit
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Introduction
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Virtually all corals and other cnidarians on coral reefs in the photic zone are infected by mutualistic dinoflagellate algae of the genus Symbiodinium. These symbioses have a nutritional basis, with evidence that the algal cells release substantial amounts of photosynthate to the host and may also recycle nitrogen (Muscatine, 1990; Wang and Douglas, 1998). The ecological importance of the symbiosis is indicated by the deleterious consequences of bleaching episodes, comprising the rapid reduction of algal populations, generally in response to elevated temperature associated with climate change (Hoegh-Guldberg, 1999). Many bleached corals recover their algal populations but display depressed growth and reproduction and increased susceptibility to mechanical damage and disease; some bleaching events result in mass mortality (Szmant and Gassman, 1990; Hoegh-Guldberg, 1999; Baird and Marshall, 2002).
Symbiodinium is a diverse genus at both the molecular and functional levels, comprising eight clades (A–H) (Pochon et al., 2006) and including genotypes with different thermal and irradiance responses and nutritional traits (Savage et al., 2002a; Tchernov et al., 2004; Loram et al., 2007a). This variation has been suggested as a basis for coral tolerance of bleaching stressors, including elevated temperature. Specifically, hosts are proposed to enhance bleaching resistance by their association with thermally tolerant Symbiodinium genotypes (Buddemeier and Fautin, 1993; Fautin and Buddemeier, 2004). Field observations of changes in the Symbiodinium complement of corals after bleaching provide supportive evidence for this proposal (Rowan et al., 1997; Berkelmans and van Oppen, 2006; Jones et al., 2008). More generally, these issues have heightened research interest in the incidence and properties of symbioses with mixed infections of Symbiodinium. Methods to identify and quantify the algae have improved progressively (see discussion in Loram et al., 2007b), and 23% of scleractinian coral species have been estimated to include mixed infections (Goulet, 2006).
The purpose of this study was to explore the impact of natural variation in temperature on a symbiosis with a mixed infection of Symbiodinium. Our system was the tropical sea anemone Condylactis gigantea at the high-latitude site of Bermuda (32°N). The temperature range in Bermudian waters is high and varies reliably along the offshore-to-inshore gradient, from 18–28 °C on offshore reefs buffered by oceanic water, to 15–32 °C in the relatively enclosed inshore locations (Morris et al., 1977; Cook et al., 1990). C. gigantea is a common and conspicuous species in many locations along the inshore-offshore temperature gradient, including shallow semi-enclosed inshore bays and offshore rim reefs at the edge of the 665-km2 Bermuda platform (Venn and Loram, pers. obs.). C. gigantea is ideally suited to our purpose for three reasons. First, it bears Symbiodinium of two clades, A and B, as either single or mixed infections (Savage et al., 2002b). Second, tentacles of C. gigantea can be sampled, allowing repeated sampling of individual anemones. The cladal identity of Symbiodinium is uniform both along individual tentacles (from base to tip) and among the tentacles of one anemone, and the population in the tentacles accounts for the great majority of the total algal complement, with algal cells sparsely distributed or undetectable in the oral disc, body column, and pedal disc (Loram, 2004; Venn, 2005). Finally, the symbiosis in C. gigantea is broadly representative of Symbiodinium-cnidarian associations. The 24S rRNA gene sequences of the algae in C. gigantea are not distinctively different from those in other host species, including corals (Savage et al., 2002b), and as with various other tropical and temperate symbioses, the algae in C. gigantea release 30%–40% of photosynthetically fixed carbon to the host (Loram et al., 2007a) and contribute to the lipid nutrition of the anemone (Battey and Patton, 1984). However, linked to the large size of the anemones and their capacity to exploit live prey of a great range in size, the food sources available to C. gigantea are predicted to differ from those utilized by cnidarians with small polyps, including some sea anemone species and all colonial corals.
The core objectives of this study were to quantify the composition of Symbiodinium clades in C. gigantea across the inshore-to-offshore thermal gradient and over time at one site. Supplementary experiments investigated the contribution of temperature as a determinant of the patterns obtained. This research was facilitated by the development of a real-time PCR assay for the precise quantification of clades A and B; this assay has been validated by the PCR-independent method of fluorescence in situ hybridization of individual algal cells (Loram et al., 2007b).
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Materials and Methods
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Sampling
Sampling of Condylactis gigantea (Weinland, 1860) was conducted on the reefs of Bermuda's northern platform, a 665-km2 lagoon with a mean depth of 8.5 m. Two sampling strategies were used: first, random sampling to investigate spatial variation of Symbiodinium clades across the platform; second, repetitive sampling for temporal analysis of Symbiodinium clades at one location on the platform. To investigate spatial variation of Symbiodinium clades, anemones were sampled at 19 locations that included inshore, nearshore, and offshore reefs during 2002 and 2003 (Fig. 1). Inshore and nearshore reef sites were sampled year-round, with most sites visited in both the summer and winter, but unfavourable weather conditions in the winter restricted sampling of offshore sites to June-November. Each sampled anemone was tagged with a rubber cow ear tag fixed to the reef with a 6'' nail, to prevent re-sampling of the same animal. For the analysis of temporal variation in the symbiosis, 31 anemones at 6-m depth at the reef site off Tyne's Bay, Bermuda (32°18.44.6N, 64°46.58W) (Fig. 1) were tagged as above and sampled on 29–30 March, 15–17 August, and 5 November 2003, and 29 January and 28–29 March 2004. In total, 25 of the 31 anemones persisted for the full sampling period, and the distinctive characteristics of each animal (e.g., size, tentacle color) did not change, consistent with natural history observations that this species is not highly mobile. The seawater temperature at this site was recorded continuously by data loggers (In Situ Onset Stowaway) installed on the reef.
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Figure 1. Distribution of Condylactis gigantea on the Bermuda platform sampled for the spatial analysis of Symbiodinium clades. Pie charts indicate frequency of C. gigantea bearing Symbiodinium clades A (black), A + B (grey) and B (white) at sampling sites at inshore (open circles), nearshore (open squares), and offshore (closed squares) sites, with number of replicates (n=) indicated. TB = the location of the Tyne's Bay study site for temporal analysis of Symbiodinium complement.
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Anemones were sampled by excision of five tentacles from each animal and transferred in ambient seawater to plastic collection tubes for transport to the laboratory. For the analysis of temporal variation, each sample was homogenized in zooxanthella isolation buffer (ZIB) (Rowan and Powers, 1991) and an aliquot taken for protein quantification using the micro-assay of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hemel Hempstead, UK) according to the manufacturer's instructions, with bovine serum albumin in 50 mmol l–1 Tris-HCl, pH 7.5, as standard. An enriched algal preparation was obtained from the remaining homogenate by three washes in ZIB with centrifugation (325 x g, 15 min, 4 °C) and divided into two aliquots for determination of algal density by hemacytometer and for extraction of Symbiodinium DNA. For the analysis of spatial variation, algal cells were not separated from animal tissue prior to DNA extraction. For both the spatial and temporal analysis, DNA was extracted using the DNeasy plant kit (Qiagen), following the manufacturer's instructions. After preliminary experiments that confirmed that the algal complement was uniform across different tentacles of individual animals (Loram, 2004), the results were considered as representative of the symbiosis in each animal. Tissue from the body column and pedal disc of the animal consistently gave negative PCR results for Symbiodinium (Loram, 2004; Venn, 2005).
Quantification of Symbiodinium by real-time quantitative PCR
The relative abundance of the algae of clades A and B was determined by real-time quantitative PCR (q-PCR) using absolute quantification assays based on TaqMan chemistry (Applied Biosystems) for determination of the absolute number of rRNA gene copies of each clade. The primers and probes were designed to provide exact matches to the previously documented sequences of clade A (GenBank accession number AY074949) and clade B (AY074950) algae in C. gigantea without amplifying sequences from Symbiodinium of different clades. An alignment of large subunit (LSU) sequences from Symbiodinium of clades A–C in Bermudian host species and clade D from Caribbean hosts (clade D is not known on Bermuda) generated with MegAlign (DNASTAR Inc., Madison, WI), was used as input to the primer/probe design software, Primer Express ver. 1.0 (Applied Biosystems). The primers/probe sets were as follows: clade A: forward primer (AY074949. SymbiodiniumA-LSU.492): 5'- CAGCTTGAACGTCAACTTCTATGC-3'; reverse primer (AY074949. SymbiodiniumA-LSU.568): 5'- ACAAGAGTATCGCCACAGCAAC-3'; probe (AY074949.SymbiodiniumA-LSU.523): FAM-AGGATGGCCCTTGGCTCATGCTG-TAMRA; clade B forward primer (AY074950.SymbiodiniumB.-LSU.533) 5'-TTCAGCTCACATGGTACACTTGTTAG-3'; reverse primer (AY074950. SymbiodiniumB.-LSU.620): 5'-CGAAAA AAACCACTGGGTCAA-3'; probe (AY074950.SymbiodiniumB.-LSU.567) FAM-TGTGCGGTGTGGGACTCAGA CAGG-TAMRA. The specificity of the primers and probes was checked using nucleotide-nucleotide BLAST (Altschul et al., 1990) and a local database of LSU rRNA gene sequences of Bermudian Symbiodinium (Savage et al., 2002b). The clade B primer-probe set exactly matched the target sequences of isolates from 12 of 13 Bermudian host species (Savage et al., 2002b). (The single mismatch between the forward primer and the target site of AY074969 [from the coral Siderastrea radians] was probably due to a sequencing error for AY074969, since the base at this position is highly conserved across all other clade B, C, and D sequences from Bermudian, Caribbean, and Pacific hosts.) For clade A, the reverse primer matched all Bermudian clade A sequences perfectly, the probe had a single mismatch with target sequence AY074941 (Bartholomea annulata), and the forward primer had one mismatch and three mismatches with target sequences AY074943 (Cassiopiea xamachana) and AY074941 (B. annulata), respectively, none of which were in the critical 3' regions of the primers/probes. Each PCR reaction comprised 1x TaqMan Buffer A; 5.5 mmol l–1 MgCl2; 200 nmol l–1 each dATP, dCTP, and dGTP and dTTP; 0.625 i.u. AmpliTaq Gold DNA polymerase; 300 nmol l–1 reverse and forward primers; 100 nmol l–1 probe; and 5 µl of template in a 25-µl volume. Thermal cycling conditions were 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and then 1 min at 60 °C, using an ABI Prism 7700 sequence detection system (PE Biosystems) for amplification and fluorescence measurement with real-time data collection. Gene copy number was estimated from a standard curve generated with serial dilutions of linearized LSU rRNA gene fragments cloned from C. gigantea. All samples and standards were run in duplicate with template-free controls.
The protocols were checked by control q-PCR experiments and parallel analysis of the algal complement by fluorescence in situ hybridization (Loram et al., 2007b). In brief, amplification over 40 cycles was undetectable with clade B template and clade A primer/probe set, and vice versa; the efficiency (10–1/slope) of amplifications was confirmed to be within the range required for optimal quantification (1.8–2.0), for both assays; and the slopes of dilution curves for standards and samples also did not differ significantly, confirming uniform amplification efficiency between standards and samples for both assays. The last analysis was conducted with Symbiodinium samples from a range of Bermudian species, providing assurance that any mismatches between the template and the primer/probe sets (both known and unknown) did not interfere with the assay.
Response of the C. gigantea tentacles to temperature
Experiments were conducted on freshly excised tentacles taken from anemones at Tyne's Bay site (Fig. 1) bearing single infections of clade A or B. Treatments were administered over 24 h, following pilot experiments confirming that the algal density in tentacles incubated at ambient (control) temperatures are stable over this period. The impacts of high (32 °C) and low (15 °C) temperature were conducted in August and April with control temperatures of 28 °C and 21 °C, respectively. Tentacles from five replicate anemones bearing either clade A or B were incubated individually in 1 ml of filtered seawater under a light regime of 12L:12D with 200 µmol m–2 s–1 photosynthetically active radiation, and the algal density per tentacle was determined as above.
Amplified fragment length polymorphism analysis of C. gigantea
Genomic DNA was extracted from anemone pedal discs. Of the 31 tagged animals, just 7 were amenable to retrieval of the pedal disc without unacceptable damage to corals surrounding the reef crevices where the anemones were located. Samples were stored in ethanol, and DNA extraction was performed by the method of Pinto et al. (2000), with a sample of the European snakelocks anemone Anemonia viridis as an outgroup for the phylogenetic analysis.
The amplified fragment length polymorphism (AFLP) analysis was modified from Vos et al. (1995) with a combined restriction and ligation step. Briefly, pre-amplification used a 1:10 dilution of restriction-ligation reaction as template, plus 0.2 mmol l–1 dNTPs and 10 pmol of Mse I primer (with C overhang) and Eco RI primer (with A overhang) in a 10-µl volume. The thermal cycling conditions comprised 72 °C for 2 min; 20 cycles of 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 2 min; and 60 °C for 30 min. The selective amplification with eight primer combinations used 4 µl of template (diluted 1:19) in 10 µl with 0.3 mmol l–1 MgCl2, 0.2 mmol l–1 dNTPs, 25 pmol of Mse I primer (with overhangs CTT, CTC, CTA, or CAT) and 5 pmol of 5'-labeled Eco RI primer (with overhangs ACA and AAG, labeled with Cy5 and Cy5.5 for multiplexing on a Beckman CEQ8000 capillary sequencer). Traces were scored using the Beckman fragment analysis software, and each peak was verified by visual inspection. Fragment data from all eight primer pair combinations were pooled, and genetic distance between samples was calculated using Treecon software ver. 1.3 (Link et al., 1995), with the neighbor-joining tree constructed using Phylip 3.5 (Felsenstein, 1989).
Analysis
The relative abundance of clades A and B in anemone tentacles was determined from parallel quantification of the LSU rRNA gene copies of each clade in DNA extracts, such that %A = A/(A + B), where A and B refer to the mean number of LSU copies of clade A and B, respectively, in the sample (Loram et al., 2007b). As in previous research (Loram et al., 2007a), symbioses with >90% of the dominant clade were identified as single infections to avoid the misidentification of surface contamination of a single infection as a mixed infection. Minimal differences in algal composition that can be detected reliably throughout the range of %A are up to 12% for clade A and 19% for clade B (Loram et al. 2007b). Therefore, a change in %A 
20% was scored as a shift in population composition in an individual sea anemone. Statistical analysis was performed with SPSS ver. 12 software. Spatial data (the frequency of the A, B, A + B groups across the Bermuda platform) were examined by chi-square analysis. The temporal variation of the Symbiodinium clades was analyzed by a chi-square test of association. Algal density data from anemones analyzed in the field were analyzed by Kruskal-Wallis with Mann-Whitney U tests performed between months as post hoc analysis. Data from the laboratory bleaching experiments were analyzed by comparing algal density in treatment and controls by paired Student's t-tests. Algal density data were plotted as the log2 of the ratio of treatment temperatures over controls for each clade.
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Results
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Distribution of Symbiodinium clades in C. gigantea on the Bermuda platform
Consistent with previous studies (Savage et al., 2002b; Loram et al., 2007b), Symbiodinium of clades A and B were identified in Condylactis gigantea on Bermuda. The distribution of the two clades on the Bermuda platform is shown in Figure 1. Animals at offshore sites bore either exclusively clade B or mixed infections of A and B, while most animals at inshore sites had single infections of A or mixed A + B infections, with B-only infections in just three animals at one site. Animals at both of the two nearshore sites included both A-only and B-only symbioses. The prevalence of the three types of symbioses varied significantly among the three zones (
2 = 83.70, P < 0.001).
Temporal variation in the C. gigantea algal complement
At Tyne's Bay, the site selected to explore temporal variation of the C. gigantea symbiosis, the mean daily seawater temperature varied between 17 °C and 31 °C during the sampling period of March 2003–March 2004 (Fig. 2a). Over this time, the relative abundance of the Symbiodinium clades varied by 20% or more in the tentacles of 17 (55%) of the 31 tagged C. gigantea; and in 16 of these 17 animals, the Symbiodinium complement changed by 20% or more between two consecutive sampling dates at least once (Table 1). A total of 30 changes in algal composition were recorded; 14/17 (82%) of the changes occurring during the warm period (March–November) were toward clade A, and 9/13 (69%) of changes in the cool period (November–March) were toward clade B (Fig. 2b). The frequency of changes toward clade A versus clade B differed significantly between the two periods (Fisher's test: 2 = 8.167, P < 0.01).
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Figure 2. (a) Daily mean temperature at study site (Tyne's Bay) recorded at about 6 m depth (arrows: sampling dates for the temporal study). (b) Frequency of changes in relative abundance of algal clades A and B in Condylactis gigantea tentacles (black bars = change toward A, white bars = change toward B).
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Table 1 Relative abundance of Symbiodinium clade A (%) in the 16 (of 31) tagged specimens of Condylactis gigantea with changes in algal complement 20% between consecutive sampling dates
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The total density of algal cells in tentacles of the tagged C. gigantea was quantified in parallel with the cladal analysis. For the 15 anemones that did not display 20% change in algal composition between two consecutive dates, algal density varied significantly with time (Kruskal Wallis: H4 = 22.312, P < 0.00l), with algal densities significantly increasing from August to November and decreasing between November and January (Fig. 3). The algal density in the 16 animals that displayed algal changes did not vary significantly over the study period (H4 = 8.21, P > 0.05).
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Figure 3. Mean algal density (±SE) in Condylactis gigantea tentacles with algal complements that changed by 20% between consecutive sampling dates (closed circles) and were stable (open circles). Letters indicate significantly different groups identified by post hoc analysis.
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Response of C. gigantea tentacles to temperature
When excised tentacles bearing clade B were exposed to high temperature (32 °C), packets of algal cells, readily visible at 10x magnification, were expelled and the algal density was reduced 4-fold over 24 h relative to the controls at 28 °C (t4 = 2.936, 0.01 < P < 0.05) (Fig. 4a). The density of algal cells in tentacles with clade A algae was not significantly altered by the elevated temperature (t4 = 1.109, P > 0.05), and expelled algal pellets were not observed in either this treatment or the controls. The low temperature treatment at 15 °C had no significant effect on the algal density of tentacles with either clade A (t4 = 0.507, P> 0.05) or clade B (t4 = 1.310, P > 0.05) (Fig. 4b).
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Figure 4. Impact of temperatures on algal density in Condylactis gigantea tentacles bearing Symbiodinium of clade A or clade B. Algal density is expressed as the log2 ratio of treatment vs. controls in each clade (number of cells mg-1 protein). (a) 32 °C relative to 28 °C (control), and (b) 15 °C relative to 21 °C (control). * indicates significant difference between treatment and control.
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Amplified fragment length polymorphism analysis of tagged C. gigantea at Tyne's Bay
Genetic variation among the seven anemones for which samples could be obtained (see Methods) was detected by AFLP. The neighbor-joining distance tree (Fig. 5) revealed no genetic partitioning among the animals either according to their algal complement at the time of analysis or between animals that changed their algal complement relative to those with a stable algal complement. The analysis provides no evidence to suggest that individuals of C. gigantea with either different algal complements or stable/variable algal complements are genetically distinct.
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Figure 5. Genetic diversity of Condylactis gigantea. The neighbor-joining tree based on genetic distance used 591 polymorphic AFLP loci, with the sea anemone Anemonia viridis as the outgroup and 1000 bootstrap replicates shown as percentages. Labels indicate the algal complement.
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Discussion
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This study on the spatiotemporal pattern in the abundance and distribution of Symbiodinium clades in Condylactis gigantea on the Bermuda platform complements a number of published studies on the ecology of Symbiodinium at different scales. Global longitudinal and latitudinal patterns of the distribution of different clades have been studied intensively (e.g., Savage et al., 2002b; Baker, 2003; LaJeunesse et al., 2004; LaJeunesse, 2005). Algal identity can also vary at single sites with environmental gradients. For example, the Caribbean coral Montastraea annularis (sensu lato) is dominated by Symbiodinium clades A and B in shallow, well-illuminated sites, and by clade C in deep and poorly illuminated locations (Rowan and Knowlton, 1995; Rowan et al., 1997); and the temperate anemone Anthopleura elegantissima bears chlorophyte algae in low intertidal habitats and the shaded midsections of caves, but Symbiodinium on the upper reaches of the intertidal zone and in exposed cave entrances (Secord and Augustine, 2000; Secord and Muller-Parker, 2005). The pattern for Bermudian C. gigantea revealed here comprises dominance by Symbiodinium clade A at inshore sites and clade B at offshore sites, and increase in prevalence of clade A in summer 2003 and clade B in winter 2003–2004 at one intensively studied nearshore site, Tyne's Bay. Further research is required to establish whether the temporal variation observed at Tyne's Bay occurs generally across the Bermudian platform and whether this variation is a regular seasonal pattern. Although the inshore and nearshore sites other than Tyne's Bay were sampled through the year, the data sets had insufficient statistical power to discriminate whether the prevalence of the two Symbiodinium clades varied seasonally at multiple sites. If the temporal variation is general across the platform, then the spatial data in Figure 1 would tend to underestimate the prevalence of clade B at the offshore sites, which could not be sampled in the winter (when the prevalence of clade B is expected to be high).
Parallel experimental studies provide insight into the ecological determinants of the spatiotemporal patterns in the distribution of Symbiodinium. Specifically, tentacles of C. gigantea with clade B, but not clade A, bleached at high temperature (Fig. 4a). Consistent with this result, the photosynthetic performance of clade B in C. gigantea is depressed in short-term (<2 h) incubations at 30 °C (Loram et al., 2007a). Translating these findings to field conditions, clade A is predicted to be more tolerant than clade B of the relatively high temperatures in inshore and nearshore waters during the summer (Fig. 2a). In surveys of coral bleaching on the Bermuda platform, Cook et al. (1990) noted that the most severe bleaching took place on the outer reefs and suggested that differences in bleaching between inner and outer patch reefs reflected the local adaptation of inshore corals to higher temperatures. The data in the current study raise the possibility that other symbiotic cnidarians may also harbor thermally tolerant varieties of Symbiodinium at inshore locations. Further research is required to address this issue systematically, but all mixed symbioses are not necessarily expected to display the same cladal patterns as C. gigantea. Physiological characteristics vary greatly within and between Symbiodinium clades (Savage, 2002a; Tchernov et al., 2004; Venn et al., 2006) and may also depend on the host identity.
The increased prevalence of clade B in winter at Tyne's Bay (Fig. 2b and Table 1) could be interpreted to suggest that clade B is more cold-tolerant than clade A. Other data, however, do not support low temperature as a determinant of the spatiotemporal distribution of the Symbiodinium clades. Although low-temperature bleaching of corals has been reported during Bermudian winters (Trapido-Rosenthal et al., 2005), exposure to 15 °C did not trigger bleaching in C. gigantea tentacles bearing either clade A or clade B (Fig. 4b). The high prevalence of clade A in C. gigantea at inshore sites throughout the year (Fig. 1) also indicates that clade A is tolerant of low temperatures. These data suggest that factors other than low-temperature tolerance contribute to the relative abundance of clade B in the association. One known difference between clades A and B in C. gigantea (in addition to their responses to high temperature) relates to the nutritional interaction with the host. Clade B releases less photosynthate of lower apparent quality than clade A, with photosynthetic carbon recovered principally from organic acids in symbiosis with clade B and from amino acids in symbioses with clade A (Loram et al., 2007a). These data raise the possibility that clade B invests less in the symbiosis (i.e., retains more nutrients of higher value for its own growth) than clade A and so may be at a competitive advantage and proliferate more rapidly when not constrained by abiotic factors such as high temperature. The impact of temperature on the symbiosis in field populations of C. gigantea might be compounded by other abiotic factors that were not investigated experimentally in this study; these factors include sedimentation and nutrient concentrations.
Algal densities have been observed to fluctuate seasonally in both corals and anemones mostly in association with abiotic factors (Fagoonee et al., 1999; Fitt et al., 2000). Because the Bermuda platform experiences wider seasonal variation in environmental factors than most coral reefs, temperature and other abiotic factors are expected to have an important influence on the fluctuations in algal density observed in C. gigantea. Changes in algal density (either an increase associated with elevated nutrients or a reduction at bleaching) may be costly for the host, resulting in the reduced supply of alga-derived nutrients from small algal populations and increased consumption of host nutrients by large populations.
In the present study, although significant variation in algal density was recorded in anemones that retained a stable algal complement, algal densities did not vary significantly in anemones that changed algal complement. One possible explanation is that the anemones that changed algal complement and those that retained a stable complement were two different strains of C. gigantea. The genetic analysis in Figure 5 does not support this interpretation. An alternative perspective is that shifts in the algal composition buffer the association from fluctuations in algal density driven by environmental factors. This raises a number of issues for future investigation, including identification of the factors determining whether the composition of Symbiodinium changes or is stable in a symbiosis and the relative fitness of animals with stable and variable symbioses. Although firm conclusions on the role of symbiont change in maintaining stable algal densities cannot be made, shifts in algal complement occurred in the present study without bleaching. These data, together with reports of changes in the complement of Symbiodinium without bleaching in corals (Chen et al., 2005), are important because they contrast with the interpretations made in other systems that bleaching is necessary for symbiont change in both anemones (Secord and Muller-Parker, 2005) and corals (Baker, 2001; Toller et al., 2001).
We recognize in these considerations that the classification of the algae in C. gigantea as either clade A or clade B may mask important aspects of the patterns in the spatiotemporal variation of the symbiosis and underlying processes. Although there are limitations in the use of subcladal markers (see the discussions of Appril and Gates, 2007; Thornhill et al., 2007), some data indicate that subcladal types correlate well with ecological characteristics (e.g., LaJeunesse, 2002; Sampayo et al., 2007). The continued development of these markers offers additional insight into the distribution of Symbiodinium, the composition of Symbiodinium in hosts with stable and variable symbioses, and the functional traits of the symbiosis. Even so, our analysis illustrates that the Symbiodinium symbioses can be highly dynamic. Analysis of the patterns in abundance and distribution of the algae offers the basis to understand the interactions that shape the composition and function of this symbiosis.
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Acknowledgments
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We thank Sandra Zielke for invaluable help with underwater fieldwork, and Daniela Malandruccolo, Jaret Bilewitch, Lyndsey Holland, Samantha de Putron, and Thaddeus Murdoch for fieldwork assistance. This research was supported by Natural Environmental Research Council (NERC) and UK Associates of BIOS. This is research contribution 1710 from the Bermuda Institute of Ocean Sciences.
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Footnotes
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Received 13 February 2008; accepted 28 June 2008.
Present addresses: A. A. Venn: Centre Scientifique de Monaco, Avenue Saint-Martin, 98000, Principality of Monaco; J. E. Loram: Bermuda Institute of Ocean Sciences, Ferry Reach, Bermuda, GE01; H. G. Trapido-Rosenthal: Center for Marine Microbial Ecology and Diversity, University of Hawaii at Manoa, Honolulu, Hawaii 96822-2327, USA. 
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Literature Cited
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Abstract
Introduction
