J. Phycol. 44, 1126–1135 (2008) Ó 2008 Phycological Society of America DOI: 10.1111/j.1529-8817.2008.00567.x

CORRESPONDENCEBETWEENCOLDTOLERANCEANDTEMPERATE BIOGEOGRAPHYINAWESTERNATLANTIC SYMBIODINIUM (DINOPHYTA)LINEAGE1

Daniel J. Thornhill2 Odum School of Ecology, University of Georgia, Athens, Georgia 30602, USA Department of Biological Sciences, 101 Rouse Life Sciences Building, Auburn University, Auburn, Alabama 36849, USA Dustin W. Kemp Odum School of Ecology, University of Georgia, Athens, Georgia 30602, USA Brigitte U. Bruns Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA William K. Fitt Odum School of Ecology, University of Georgia, Athens, Georgia 30602, USA and Gregory W. Schmidt Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA

Many corals form obligate symbioses with photo- climates correspond significantly with the photosyn- synthetic dinoflagellates of the genus Symbiodinium thetic cold tolerance of these symbiotic algae. Freudenthal (1962). These symbionts vary geno- Key index words: Astrangia poculata; biogeogra- typically, with their geographical distribution and phy; cold temperature stress; coral; molecular abundance dependent upon host specificity and tol- diversity; arbuscula; Symbiodinium; zoo- erance to temperature and light variation. Despite xanthellae the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. Abbreviations: ITS2, internal transcribed spacer remain poorly characterized. Here, we report that region 2; PAM, pulse amplitude modulated rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbus- cula from northerly habitats of the western Atlantic. The structural and trophic foundations for coral Using pulse-amplitude-modulated (PAM) fluoro- reef ecosystems are dependent upon the obligate metry, we compared maximum photochemical effi- mutualisms between reef-building corals and their ciency of PSII of type B2 to that of common dinoflagellate algal symbionts (genus Symbiodinium). tropical Symbiodinium lineages (types A3, B1, and Optimally, intracellular Symbiodinium provides the C2) under cold-stress conditions. Symbiont cultures coral host with 90% or more of its energy needs were gradually cooled from 26°C to 10°C to simu- through photosynthesis (Muscatine 1990). In the late seasonal temperature declines. Cold stress past few decades, however, episodes of increased decreased the maximum photochemical efficiency ocean temperatures have led to disruptions of these of PSII and likely the photosynthetic potential for important symbiotic associations as a result of ther- all Symbiodinium clades tested. Cultures were then mal stress (Hoegh-Guldberg 1999, Hughes et al. maintained at 10°C for a 2-week period and gradu- 2003). Therefore, considerable attention has been ally returned to initial conditions. Subsequent to low given to the possibility that thermal acclimatization temperature stress, only type B2 displayed rapid of corals may occur via acquisition of heat-tolerant and full recovery of PSII photochemical efficiency, dinoflagellate symbiont complements (e.g., Bud- whereas other symbiont phylotypes remained demeier and Fautin 1993, Baker et al. 2004, Berkel- nonfunctional. These findings indicate that the mans and van Oppen 2006, Goulet 2006, Thornhill distribution and abundance of Symbiodinium spp., et al. 2006a,b). An alternative conjecture is that and by extension their cnidarian hosts, in temperate corals might undergo selective adaptation including latitudinal shifts in distribution. This possibility would require host and symbiont resilience to 1Received 20 August 2007. Accepted 10 March 2008. considerable seasonal changes in temperature and 2Author for correspondence: e-mail [email protected].

1126 COLD-TOLERANT SYMBIODINIUM 1127 diurnal light exposure compared to the more con- Carrington 2007). Seasonal fluxes in temperature, stant environment in the tropics (Muller-Parker and light, and nutrients are considerably more pro- Davy 2001). nounced in temperate versus tropical marine habi- Symbiodinium is a genetically diverse group, con- tats (e.g., Muller-Parker and Davy 2001). Despite sisting of eight divergent lineages or clades (desig- this, zooxanthellae densities in symbiotic anemones, nated A through H; Pochon et al. 2006) and for example, are apparently more stable and less numerous more taxonomically specific types seasonal in temperate than in tropical hosts (Muller- (reviewed in Baker 2003, Coffroth and Santos 2005, Parker and Davy 2001 and references within), raising Stat et al. 2006). However, relatively little is known questions about how these temperate symbioses about the physiological properties that distinguish function physiologically. the various types. Differential tolerance of some Tropical and subtropical coral reefs contain com- symbiont species to high temperature stress has plex communities of numerous Symbiodinium species been the subject of many recent reports (e.g., War- (e.g., Rowan and Knowlton 1995, van Oppen et al. ner et al. 1996, 1999, Rowan 2004, Tchernov et al. 2001, LaJeunesse 2002, LaJeunesse et al. 2003, 2004, Berkelmans and van Oppen 2006). Exposure Thornhill et al. 2006a,b). The diversity of Symbiodini- to high temperature stress is now recognized to gen- um types from temperate habitats is apparently erally culminate in chronic photoinhibition in the lower than that occurring in the tropics (LaJeunesse symbionts (Warner et al. 1999). Such thermally and Trench 2000, LaJeunesse 2001, Rodriguez- induced perturbations are likely triggered by Lanetty et al. 2001, 2003, Savage et al. 2002, Visram decreased photochemical and nonphotochemical et al. 2006). However, symbiotic diversity in many quenching of PSII excitation energy, which can be temperate regions and the physiological properties exacerbated by disabled repair of photodamaged of these symbionts remain largely unexplored. Here, PSII in some symbiont types (Warner et al. 1999). we examine the biogeography and photosynthetic Symbiotic dinoflagellates exposed to cold tempera- physiology of Symbiodinium spp. associated with two ture stress may also undergo chronic photoinhibi- scleractinian hosts from the temperate western tion (Saxby et al. 2003), corresponding to decreased Atlantic. The identities of Symbiodinium types from photosynthetic membrane fluidity, as has been doc- the corals A. poculata and O. arbuscula were charac- umented for many vascular plant species (Smillie terized using denaturing gradient gel electrophore- et al. 1988, Aro et al. 1990, Greer 1990, Krause sis (DGGE) and sequencing of the ITS2 region of 1992). Furthermore, low temperature stress might the rDNA. disrupt the host–symbiont association by mecha- It has been hypothesized that temperate zooxan- nisms akin to high-temperature-induced coral thellae should be able to withstand greater ranges bleaching (Steen and Muscatine 1987, Hoegh- of temperatures than their tropical counterparts Guldberg et al. 2005). (Muller-Parker and Davy 2001). Therefore, we tested The distribution of coral reefs appears to be pri- this hypothesis using PAM fluorometry to character- marily limited by a lower thermal threshold of ize the maximum quantum efficiency of PSII 18°C, light limitation, and decreased aragonite (Fv ⁄ Fm) and relative electron transport rates (ETR) saturation state in high-nutrient temperate waters of cultured temperate and tropical Symbiodinium (Dana 1843, Vaughan 1919, Birkeland 1988, Cross- types under cold-stress conditions. Such investiga- land 1988, Kleypas et al. 1999), although biotic tions of the relationship between Symbiodinium physi- interactions have also been implicated (Johannes ology and biogeography provide further insights et al. 1983, Miller and Hay 1996). While diversity of into the ecology and evolution of this important symbiotic cnidarians is low outside the tropics, dinoflagellate group. several species of symbiotic corals, anemones, and other cnidarians occur in temperate habitats MATERIALSANDMETHODS (Schuhmacher and Zibrowius 1985). Similar to trop- Sample collection and Symbiodinium identification. In 2005, ical corals, temperate cnidarians hosting Symbi- the symbiotic hosts A. poculata (= A. danae,=A. astreiformis, odinium benefit from enhanced calcification and Peters et al. 1988) and O. arbuscula were surveyed from three growth rates (Jacques and Pilson 1980, Jacques et al. coastal locations along the eastern seaboard of the U.S., 1983, Miller 1995, Dimond and Carrington 2007), including a limestone and live-bottom reef near Gray’s Reef reduced nitrogen loss (Szmant-Froelich and Pilson National Marine Sanctuary (J Reef, Georgia, USA; 31.601° N, 80.791° W); an open bay site in Fort Wetherill State Park, 1984), and translocation of photosynthetically fixed Narragansett Bay (Rhode Island, USA; 41.478° N, 71.357° W); carbon (Szmant-Froelich 1981, Schiller 1993, Miller and a tidal river site in the lower Pettaquamscutt River, 1995). However, unlike the obligate associations Narragansett Bay (Rhode Island, USA; 41.449° N, 71.449° W; occurring in most tropical hosts, temperate symbi- Table 1). A map of the estimated distributions of these otic cnidarians typically associate facultatively with scleractinian species and sampling locations for this study is Symbiodinium and often survive without symbionts provided in Figure 1. Temperatures at these noncoral reef sites are always <18°C during winter months (Table 1; for more via heterotrophic feeding alone (Jacques et al. 1983, details on the temperature and other conditions in Narragan- Szmant-Froelich and Pilson 1984, Schuhmacher and sett Bay see Dimond and Carrington 2007), the purported Zibrowius 1985, Farrant et al. 1987, Dimond and 1128 DANIELJ.THORNHILLETAL.

Table 1. Symbiodinium ITS2 types from symbiotic corals along the U.S. East Coast.

Min. temp. Max. temp. Mean Depth Date Symbiodinium GenBank Host species Location (°C)a (°C)a temp. (°C)a n (m) (month ⁄ year) ITS2 type accession no. Oculina arbuscula J Reef, GA 10.5 32.6 21.2 11 18.3–21.3 07 ⁄ 2005 B2 EU315253 Astrangia poculata J Reef, GA 10.5 32.6 21.2 3 18.3–21.3 07 ⁄ 2005 B2 EU315253 Astrangia poculata Ft. Wetherill, RI )0.9 24.4 10.7 8 3.0–7.0 01 ⁄ 2005 B2 EU315253 Astrangia poculata Lower Pettaquamscutt )0.9 24.4 10.7 6 3.0 01 ⁄ 2005 B2 EU315253 River, RI aTemperature data obtained from nearest adjacent site from NOAA National Buoy Data Center 2005 data (http:// www.ndbc.noaa.gov/). n = number of samples.

identify Symbiodinium ITS2 types (LaJeunesse 2001, 2002). The ITS2 rDNA was amplified from each extract using the primer set ‘‘ITS2clamp’’ and ‘‘ITSintfor2’’ (LaJeunesse and Trench 2000) with touch-down thermal cycle as described in LaJeu- nesse et al. (2003). Products from these PCR reactions were electrophoresed for 1,500 volt-hours at 60°C on 8% polyacryl- amide denaturing gradient gels (45%–80% formamide ⁄ urea wherein 100% of the denaturants are 40% formamide and 7 M urea) using a CBS Scientific DGGE system (Del Mar, CA, USA). The diagnostic bands (including dominant and minor bands) in each profile were then excised, reamplified, and bidirec- tionally sequenced using Genome LabTM Quick Start Mix (Beckman Coulter, Fullerton, CA, USA) on a Beckman CEQ 8000 Genetic Analysis System (Beckman Coulter) according to the protocol of LaJeunesse (2002). Sequences were deposited in GenBank (accession no. EU315253; Table 1). As a means of validating the Symbiodinium ITS2 sequences as diagnostic markers for a particular symbiont type, secondary structural analysis was performed by superimposing individual ITS2 sequences onto the secondary structure of the most similar Symbiodinium type reported in Hunter et al. (2007). Areas of high sequence conservation with other Symbiodinium types served as reference points. Less conserved regions were folded by eye to conserve general secondary structure charac- teristics. Additional details of this method can be found in Hunter et al. (2007) and Thornhill et al. (2007). Cold-stress experiment and fluorescence measurements. To inves- Fig. 1. Map of the eastern coast of North America including tigate the physiological properties of Symbiodinium type B2 the estimated distributions of Astrangia poculata (light gray) and relative to other types of symbionts, four clonal Symbiodinium Oculina arbuscula (dark gray) as well as collection locations for cultures representing Symbiodinium ITS2 types A3, B1, B2, and this study (indicated by star symbols; GA: J reef; RI: Narragansett C2 (sensu LaJeunesse 2001; Fig. 1b) were grown in ASP-8A medium at 26°C and 60–80 lmol photons Æ m)2 Æ s)1 under Bay sites). Distributions based on McCloskey (1970), Ruppert and  Fox (1988), and Veron (2000). cool-white fluorescent lamps at a 10:14 light:dark (L:D) cycle under otherwise standard culturing conditions (Blank 1987; see Table 2 for culture details). Culture stocks have been lower temperature threshold for coral reef accretion (Dana maintained under these conditions for many years and gener- 1843, Vaughan 1919, Kleypas et al. 1999). Coral fragments ations, with bimonthly refreshments of culture media. Exper- (<5 cm3) were collected by SCUBA using a hammer and chisel. imental cultures (one flask per ITS2 type) were grown from Fragments were placed into 70% ethanol to preserve DNA small stock cultures of 50 mL to a volume of 2 L over 50 during transport back to the laboratory. generations. Cultures were selected based on available isolates Genomic DNA was extracted with the WizardÒ (Promega, as representatives of the predominantly temperate ITS2 type Madison, WI, USA) DNA preparation protocol following the B2 (see results below) and several common tropical symbiont methods of LaJeunesse et al. (2003). PCR-DGGE fingerprint- types that associate with a range of symbiotic hosts (LaJeunesse ing of the ITS2 region of nuclear ribosomal DNA was used to 2002, LaJeunesse et al. 2003). Batch cultures were refreshed

Table 2. Geographic and host origin of the cultures used for this experiment.

Isolate Symbiodinium number ITS2 type Geographic origin Host origin 64 B1 Jamaica, Caribbean Cassiopea xamachana (Rhizostomeae) 141 B2 Bermuda, W. Atlantic Oculina diffusa (Scleractinaria) 203 C2 Palau, W. Pacific Hippopus hippopus (Bivalvia) 292 A3 Palau, W. Pacific Tridacnia maxima (Bivalvia) COLD-TOLERANT SYMBIODINIUM 1129 with new medium (one part fresh medium to two parts Symbiodinium ITS2 type B2 was the only symbiont medium) 2 d before the beginning of the experiment. detected in A. poculata and O. arbuscula coral colo- To examine the effect temperature decline has on the nies from the selected temperate habitats. Excision photophysiological properties of representative Symbiodinium types, an experiment was devised for three phases: graduated and direct sequencing of dominant and faint minor temperature decline from 26°C to 10°C, extended low temper- bands from each DGGE profile recovered only the ature at 10°C, and gradual recovery from 10°C back to 26°C. type B2 sequence, indicating that no other symbiont The cold-stress condition of 10°C was selected as an approx- types were present at the detection limits of this imation of the annual minimum temperature experienced at technique (see Thornhill et al. 2006b). Gray’s Reef National Marine Sanctuary and the mean temper- The ITS2 rRNA secondary structure of the ature in Narragansett Bay, RI (Table 1). Additionally, temper- sequence recovered for type B2 was also assessed atures of 9°C to 12°C have been reported as the minimum experienced by several other temperate zooxanthellate cnida- (Fig. 3) to validate this sequence as relevant for bio- rians (Kevin and Hudson 1979, Farrant et al. 1987, Schiller diversity estimates and not indicative of a nonfunc- 1993). Cultures were first subjected to gradual cooling by tional rDNA copy or pseudogene (see Thornhill decreasing the incubation temperature 2°C per day to a final et al. 2007). The deduced secondary structure pre- temperature of 10°C. Cultures were maintained at 10°C for a sented in Figure 3 is consistent with that of other 2-week period and finally warmed by 2°C per day until the clade B Symbiodinium (Hunter et al. 2007) and temperature returned to 26°C. Control cultures of each symbiont type were maintained at 26°C and 60–80 lmol pho- lacked mutations at any positions that would poten- tons Æ m)2 Æ s)1 under otherwise identical conditions. Six hours tially disrupt the proper folding and processing to after the onset of light, repeated measurements of maximum generate mature rRNA products. Therefore, we quantum yields of PSII (Fv ⁄ Fm) were obtained using a Walz DIVING-PAM fluorometer (Walz, Eichenring, Germany) throughout the 31 d experiment (Warner et al. 1996). Fv ⁄ Fm is a measure of the percentage of absorbed light that is converted into biochemical energy, nonphotochemically quenched by thermal decay of excitation energy, or emitted as fluorescence. Five replicate measurements were taken on each Symbiodinium culture (measurements were made on different areas of the flask to confirm physiological homoge- neity throughout the culture) daily as temperatures were decreased, every other day as temperatures were maintained at 10°C, and daily again as temperatures were restored to 26°C. Cultures were dark acclimated for 30 min and allowed to settle, and immobile dinoflagellates were analyzed by affixing the fiber optic probe to random regions of the bottoms of culture flasks. This method eliminates stirring artifacts in fluorescence measurements that have been recently noted with the stir- ring ⁄ illumination ⁄ detection array of the PHYTO-PAM and WATER–PAM instruments (Cosgrove and Borowitzka 2006). Furthermore, results obtained via this protocol are statistically indistinguishable from those obtained by direct measurement of homogenized coccoid and motile cells (J. McCabe-Reynolds and G. Schmidt, unpublished data), indicating that these results are not biased by preferential measurement of coccoid cell stages at the flask bottom. In addition, relative ETR versus irradiance measurements were conducted using the DIVING-PAM fluorometer according to the manufacturer’s protocol (Walz, DIVING-PAM Manual, http://www.walz.com). Subsamples of homogenized Symbiodi- nium cultures were collected and preserved with 1% formalin, and cell densities were calculated from replicate (n = 6 per culture) hemocytometer counts. Chl a levels were also mea- sured (from 1 to 2 replicates per culture) by centrifuging 10–15 mL of homogenized culture at 8,000g, removing the supernatant and extracting chl in 1 mL of N,N¢-dimethyl- Fig. 2. Example PCR-DGGE profiles of the Symbiodinium ITS2 formamide (DMF). Samples were vortexed and centrifuged region, presented as reverse images. (a) Profiles of representative again at 8,000g, and absorbance of the resulting supernatant colonies of Astrangia poculata and Oculina arbuscula collected off was measured at wavelengths of 664 and 647 nm on a BioRad the coast of Georgia, USA (GA), or Rhode Island, USA (RI). A SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, cultured standard of type B2 is also included for reference. Pri- Richmond, CA, USA). Chl a concentration was calculated mary diagnostic band is indicated for type B2. Fainter bands in according to Moran (1982). each profile were also excised and sequenced; the resulting sequences were all identical to type B2, indicating no detectable mixed symbiont populations in these corals. (b) Profiles of the RESULTS Symbiodinium cultures used for the physiological portion of this experiment. Diagnostic bands are labeled for types B1, B2, C2, Genotyping from the Symbiodinium biogeography and A3. DGGE, denaturing gradient gel electrophoresis; ITS2, surveys is presented in Figure 2 and Table 1. internal transcribed spacer region 2. 1130 DANIELJ.THORNHILLETAL.

steady declines in photosynthesis upon cooling, with minimal function at 10°C (Fig. 5b), and remained minimally functional throughout the 2- week cold-stress treatment at 10°C. However, once gradual warming to 26°C was initiated, only Symbio- dinium type B2 returned to prestress levels of photosynthetic function, whereas the other Symbio- dinium types remained nonfunctional (Fig. 5c). At the end of the 31 d experimental period, Symbi- odinium B2 had returned to prestress levels of pho- tosynthesis, while the other types remained photosynthetically inactive. There was little differ- ence in the overall curves between pretemperature stress experimental cultures (Fig. 5a) and cultures kept at control illumination and temperature for an equivalent 31 d (Fig. 5d). The sustained photo- synthetic efficiency for all four symbiont types kept at prestress conditions excludes the possibility that Fig. 3. Proposed ITS2 secondary structure of Symbiodinium nutrient depletion or aging of the cultures was type B2. ITS2, internal transcribed spacer region 2. causative in the differential cold responses of the Symbiodinium types. Symbiodinium cell densities throughout the experi- interpret this B2 sequence as representative of the ment are provided for each culture in Figure 6a. dominant rDNA copy for this symbiont type and Although cell density varied somewhat between cul- consequently relevant as a marker for ecological tures and time points, densities remained approxi- diversity estimates (Thornhill et al. 2007). It should mately stable or underwent modest declines. Thus, be noted that this structure is different from that the cultures were in stationary phase through most presented for type B2 in the supplemental materials of the experiment, although cultures of tropical of Hunter et al. (2007). Hunter et al. used an symbionts likely became moribund in response to unpublished clade B sequence instead of type B2 in the cold treatment. Differences in cell density their proposed secondary structure resulting in a between cultures likely relates to cell size and type- slightly different estimate of folding of the molecule. specific nutrient requirements (data not shown). The biogeography of symbionts from temperate For instance, type B1 cells were the smallest, and and tropical communities led to a supposition that this culture averaged the highest overall cell density, Symbiodinium ITS2 type B2 represents a cold-tolerant whereas type C2 cells were larger and maintained lineage able to survive conditions inhospitable to lower densities overall (see also LaJeunesse 2001). most other Symbiodinium spp. To test this idea, four The greatest increases in cell density were observed clonal Symbiodinium cultures, representing the tem- in the early part of the experiment for type A3, perate type B2 and three tropical Symbiodinium types although cell density subsequently decreased during (Fig. 2b and Table 2), were subjected gradual cool- the temperature decline. This initial growth possibly ing, followed by a prolonged period of cold stress. relates to the low starting concentrations of type A3 As the cultures were cooled, maximum photochemi- relative to the other cultures. Type B2 experienced cal efficiency of PSII (Fv ⁄ Fm) decreased for all four modest declines in cell density through the cold- types concomitant with decreasing temperature stress phase of the experiment, indicating that this (Fig. 4), indicating diminished electron transport temperate symbiont type was affected to a limited and ⁄ or increased photoinhibition (Schreiber and extent by exposure to low temperatures. Bilger 1987). Interestingly, during the cooling phase Chl a levels for the four cultures are also pro- of the experiment, Symbiodinium type C2 from a vided in Figure 6b. From these measurements, we tropical host responded more slowly. At 10°C, Symbi- innovated extraction of Symbiodinium cultured cells odinium type B2 F ⁄ F values diminished to 0.1; with DMF instead of the more conventional acetone v m  Fv ⁄ Fm values for other Symbiodinium types steadily solvent: with the latter reagent, precipitates of cell decreased to 0.0. When restoration to 26°C was debris remained visibly green, whereas DMF yielded gradually imposed, type B2 readily returned to pre- white pellets. Cultures all showed a general trend of stress Fv ⁄ Fm values. However, types A1, B1, and C2 increasing chl a concentration during the first 5 d did not recover in the subsequent phase of the of the experiment. Maximum chl a levels varied by experiment (Fig. 4). Symbiodinium type: culture C2 reached the highest Relative ETR versus irradiance curves are pre- chl concentration by day 5, whereas culture B2 sented in Figure 5. Each Symbiodinium type dis- reached the lowest relative concentration. Sub- played a different, type-specific curve under initial sequently, chl a levels decreased for types A3, B1, conditions (Fig 5a). All four types experienced and C2. Type B2 maintained relatively stable COLD-TOLERANT SYMBIODINIUM 1131

Fig. 4. (a) Maximum photochemical efficiency of PSII (Fv/Fm) of clonal cultures of Symbiodinium types A3 ()), B1 (h), B2 (d), and C2 ( ) throughout the 31 d experiment (five replicates per Symbiodinium type). Data are presented as mean ± 95% confidence intervals. (b) Temperature4 (°C) levels experienced by cultures for the duration of the experiment. concentrations of chl a as other cultures declined, (Savage et al. 2002). Furthermore, no Symbiodinium further suggesting photosynthetic cold tolerance in types commonly occurring in the tropics were recov- this symbiont type. However, the low chl sample ered at any of the temperate habitats (Table 1; D. J. number and some variance in these data prevented Thornhill and D. W. Kemp, unpublished data). robust statistical comparisons between cultures or Annually, the environmental conditions at these time points. It should be noted that Symbiodinium temperate locations fluctuate considerably; conse- B2 regained photosynthetic efficiency during warm- quently, type B2 must be able to physiologically ing of cold-treated cells without a substantial tolerate a wide range of temperature, light, and increase in cellular chl levels. This finding reflects other conditions. Here we tested this hypothesis on restoration of reaction centers through repair pro- cold-stressed B2 Symbiodinium and three tropical cesses that could include recruitment of chl a from Symbiodinium types using PAM fluorometry. Our residual light-harvesting complexes. results indicated that type B2 is physiologically cold tolerant and able to rapidly recover from prolonged exposure to low temperatures. Unlike type B2, the DISCUSSION tropical Symbiodinium spp. exposed to a prolonged Symbiodinium type B2 was the only symbiont period of cold stress demonstrated no signs of pho- detected in any of the A. poculata or O. arbuscula cor- tosynthetic recovery when returned to prestress con- als from temperate western Atlantic habitats. In con- ditions. Although it is possible that some unknown trast to the dominance of type B2 in these temperate factor, such as growth phase of the cultures, influ- corals, B2 is a relatively rare symbiont in tropical and enced these data, results from the nontemperature subtropical ecosystems (LaJeunesse 2002, LaJeunesse stressed control cultures diminish this possibility. et al. 2003, Thornhill et al. 2006a,b). Previously, Therefore it seems likely that the cold-tolerant physi- ITS2 type B2 has been reported only from northerly ology demonstrated by type B2 contributes signifi- coral reef habitats, including the Florida Keys cantly to the distribution of this symbiont in (LaJeunesse 2001, Santos et al. 2001) and Bermuda temperate versus tropical habitats and hosts. 1132 DANIELJ.THORNHILLETAL.

Fig. 5. Relative electron transport rate (ETR) versus PAR (lmol quanta Æ m)2 Æ s)1) curves for four clonal cultures of Symbiodinium types A3 ()), B1 (h), B2 (d), and C2 ( ) (five replicates per Symbiodinium type). Data are presented as mean ± 95% confidence inter- vals. (a) Day 1, 26°C. (b) Day 18, 10°C. (c)4 Day 31, after return to 26°C. (d) Control cultures day 31, 26°C.

In a report on the effects of abrupt, short-term unsaturated lipid membranes could enable certain cold exposure on symbiotic corals from tropical symbionts to remain photosynthetically functional environments, chronically depressed Fv ⁄ Fm values under moderate to severe cold conditions. However, persisted several hours after temperatures were type B2 exhibits a cold-induced decrease in photo- returned to the original state (Saxby et al. 2003). synthetic efficiency that largely parallels that of Irreversible photodamage to the symbionts was con- other Symbiodinium clade representatives. In fact, the cluded to have been inflicted as a consequence of type that sustains the highest level of photosynthetic the adverse irradiance and temperature conditions activity during gradual decreasing temperature (Saxby et al. 2003). While similar phenomena likely exposure is type C2 isolated from a tropical host. occurred for Symbiodinium types A3, B1, and C2 in Consequently, we cannot attribute type B2’s cold this study, photoinhibition and its consequences are tolerance to unique fluidity properties of its photo- clearly reversible or repairable in type B2. Recovery synthetic thylakoid membranes. However, there is a of photosynthetic capacity in B2 was detected as possibility that other membranous components in temperatures warmed to 12°C and substantial recov- this symbiont type are endowed with lipid bilayers ery was observed at 18°C. Recovery was sustained with a larger proportion of unsaturated fatty acids during each day of warming until prestress levels of or sterols, contributing to the cold-stress recovery photosynthetic function were achieved at 22°C process. and above.  Type B2 is the first Symbiodinium ITS2 type to be Symbiodinium type B2 presumably has either identified in temperate environments of the western photodamage repair and ⁄ or protective mechanisms Atlantic. The cold thermal tolerance apparently pro- to enable its ability to withstand prolonged periods vides this symbiont with a survival advantage in tem- of cold. Recent work by Tchernov et al. (2004) perate habitats over that of other Symbiodinium documented that reduced membrane fluidity, species. It should be noted, however, that other correlating with higher unsaturated fatty acid com- types of Symbiodinium, such as types A4, B4 (S. mus- position, partially correlates with high temperature catenei), E1 (S. californium) (LaJeunesse and Trench tolerance for some Symbiodinium isolates. A recipro- 2000, LaJeunesse 2001, Muller-Parker et al. 2007), cal phenomenon could contribute to low tempera- and an additional type of ‘‘temperate’’ clade A ture tolerance, since a high proportion of (Savage et al. 2002, Visram et al. 2006), have been COLD-TOLERANT SYMBIODINIUM 1133

Fig. 6. (a) Cell density (cells · 105 Æ mL)1) from clonal cultures of Symbiodinium types A3 ()), B1 (h), B2 (d), and C2 ( ) through- out the experiment (six replicated counts per cultured isolate). Data are presented as mean ± 95% confidence intervals. (b)4 Chl a levels (lg Æ mL)1) for each Symbiodinium culture throughout the experiment (one to two replicates per cultured isolate). Raw data are presented as a scatter plot fitted with a B-spline curve (de Boor 1978). (c) Temperature (°C) levels experienced by cultures for the duration of the experiment. documented to occur in other hosts of temperate throughout the colder months of the year, persist- marine climates. Thus, temperate Symbiodinium ITS2 ing without making a major contribution to the types likely share a similar underlying basis for cold nutrition of the host. In fact, Jacques et al. (1983) tolerance of subclade B2, described here as com- found no contribution by Symbiodinium sp. to plete photosynthetic recovery after cold thermal A. poculata growth or calcification at temperatures stress. below 15°C. During the warm season, as simulated The unusual capacity of Symbiodinium type B2 to in the recovery phase of this experiment, Symbiodini- rapidly recover photosynthetic efficiency after low um B2 likely increases its photosynthetic function temperature exposure possibly contributes to the and therefore would contribute more actively to its biogeography of its hosts. Although symbiosis is fac- host’s calcification and growth (Jacques et al. 1983, ultative in A. poculata and O. arbuscula, harboring Dimond and Carrington 2007). It is noteworthy that Symbiodinium leads to enhanced calcification and A. poculata and Oculina spp. are known to survive growth (Jacques et al. 1983, Miller 1995, Dimond indefinitely without symbionts via heterotrophic and Carrington 2007). Therefore, the physiological feeding (e.g., Jacques et al. 1983, Farrant et al. tolerance of Symbiodinium spp. may contribute to 1987, Dimond and Carrington 2007). Therefore, their hosts’ fitness and distribution, particularly in the symbioses between Symbiodinium B2 and these habitats where coral growth is otherwise limited hosts may shift seasonally between mutualism in the (i.e., by a lack of available prey, low temperatures summer months to commensalism or parasitism throughout much of the year, etc.). during winter when type B2 is photosynthetically While Symbiodinium type B2 remained only mini- inactive. Furthermore, in contrast, the marked mally functional at temperatures as low as 10°C, it seasonal fluctuations recorded in tropical hosts rapidly recovered upon warming. Assuming a similar (Muller-Parker 1987, Fagoonee et al. 1999, Fitt et al. pattern probably occurs in hospite, this symbiont 2000), temperate symbiotic cnidarians are known probably can remain photosynthetically inactive to maintain stable zooxanthellae densities through 1134 DANIELJ.THORNHILLETAL. spatial gradients (Bythell et al. 1997) and seasonal anemone Anemonia viridis. Proc. R. Soc. Lond. B Biol. Sci. fluctuations (Dykens and Schick 1984, Farrant et al. 264:1277–82. Coffroth, M. A. & Santos, S. R. 2005. Genetic diversity of symbiotic 1987) of light and temperature. For instance, dinoflagellates in the genus Symbiodinium. Protist 156:19–34. zooxanthellae density in temperate scleractinian Cosgrove, J. & Borowitzka, M. 2006. Applying pulse amplitude Plesiastrea urvillei and mitotic index in the temperate modulation (PAM) fluorometry to microalgae suspensions: anemone Anemonia ciridis did not decline when sub- stirring potentially impacts fluorescence. Photosynth. Res. jected to prolonged periods (48 and 66 d, respec- 88:343–50. Crossland, C. J. 1988. Latitudinal comparisons of coral reef struc- tively) of total darkness (Kevin and Hudson 1979, ture and function. Proc. 6th Int. Coral Reef Symp. 1:221–6. Beaver 1996), whereas tropical hosts quickly lose Dana, D. J. 1843. On the temperature limiting the distribution of their symbionts under such conditions (Steen and corals. Am. J. Sci. 45:130–1. Muscatine 1987). Therefore, seasonal shifts in the Dimond, J. & Carrington, E. 2007. Temporal variation in the sym- biosis and growth of the temperate scleractinian coral Astran- nature of symbioses seem likely in many temperate gia poculata. Mar. Ecol. Prog. Ser. 348:161–72. cnidarian–Symbiodinium associations. Dykens, J. A. & Schick, J. M. 1984. Photobiology of the symbiotic sea In summary, A. poculata and O. arbuscula are two anemone Anthopleura elegantissima: defenses against photody- symbiotic cnidarians that commonly occur in the namic effects, and seasonal photoacclimatization. Biol. Bull. temperate western Atlantic. Both of these corals har- 167:683–97. Fagoonee, I., Wilson, H. B., Hassell, M. P. & Turner, J. R. 1999. The bor Symbiodinium type B2, an extremely rare symbi- dynamics of zooxanthellae populations: a long-term study in ont type in tropical environments. The ability of the field. Science 283:843–5. these symbionts to survive extended periods of cold Farrant, P. A., Borowizka, M. A., Hinde, R. & King, R. J. 1987. temperature stress and rapidly return to normal Nutrition of the temperate Australian soft coral Capnella gaboensis. I. Photosynthesis and carbon fixation. Mar. Biol. photosynthetic function when permissive conditions 95:565–74. recur is undoubtedly critical for their survival in Fitt, W. K., McFarland, F. K., Warner, M. E. & Chilcoat, G. C. 2000. temperate environments and may play a key role Seasonal patterns of tissue biomass and densities of symbiotic shaping temperate coral communities. dinoflagellates in reef corals and relation to coral bleaching. Limnol. Oceanogr. 45:677–85. This work was funded by NSF (9906976 and 0137007). We Goulet, T. L. 2006. Most corals may not change their symbionts. would like to thank Daniel F. Gleason, R. Robert Ruzicka, Mar. Ecol. Prog. Ser. 321:1–7. and Keith Goldman for assistance in coral sampling at Gray’s Greer, D. H. 1990. The combined effects of chilling and light stress Reef National Marine Sanctuary. We are also grateful for the on photoinhibition of photosynthesis and its subsequent help of James L. Dimond who contributed samples from recovery. Plant Physiol. Biochem. 28:447–55. Hoegh-Guldberg, O. 1999. Climate change, coral bleaching and Rhode Island to the biogeography portion of this study. Ken- the future of the world’s coral reefs. Mar. Freshw. Res. 50:839– neth M. Halaynch graciously provided use of his laboratory 66. and NSF grant support for sequencing of these samples. Hoegh-Guldberg, O., Fine, M., Skirving, W., Johnstone, R., Dove, S. Finally, we would like to thank Scott R. Santos, Todd C. La- & Strong, A. E. 2005. Coral bleaching following wintry Jeunesse, Clay B. Cook, and two anonymous reviewers for weather. Limnol. Oceanogr. 50:265–71. their insightful comments on this manuscript. This is Auburn Hughes, T. P., Baird, A. H., Bellwood, D. R., Card, M., Connolly, S. University Marine Biology Program contribution number 33. R., Folke, C., Grosberg, R., et al. 2003. Climate change, human impacts, and the resilience of coral reefs. Science Aro, E. M., Hundal, T., Carlberg, I. & Andersson, B. 1990. In vitro 301:929–33. studies on light-induced inhibition of photosystem II and D-1 Hunter, R. L., LaJeunesse, T. C. & Santos, S. R. 2007. Structure and protein degradation at low temperatures. Biochim. Biophys. Acta evolution of the rDNA internal transcribed spacer (ITS) re- 1019:269–75. gion 2 in the symbiotic dinoflagellates (Symbiodinium, Dino- Baker, A. C. 2003. Flexibility and specificity in coral-algal symbiosis: phyta). J. Phycol. 43:120–8. diversity, ecology, and biogeography of Symbiodinium. Annu. Jacques, T. G., Marshall, N. & Pilson, M. E. Q. 1983. Experimental Rev. Ecol. Evol. Syst. 34:661–89. ecology of the temperate scleractinian Astrangia danae. II. Ef- Baker, A. C., Starger, C. J., McClanahan, T. R. & Glynn, P. W. 2004. fect of temperature, light intensity and symbiosis with zoo- Corals’ adaptive response to climate change. Nature 430:741. xanthellae on metabolic rate and calcification. Mar. Biol. Beaver, R. 1996. The regulation of the population of symbionts 76:135–48. in Anemonia viridis. PhD thesis, University of Glasgow, UK, Jacques, T. G. & Pilson, M. E. Q. 1980. Experimental ecology of the 168 pp. temperate scleractinian coral Astrangia danae. 1. Partition of Berkelmans, R. & van Oppen, M. J. H. 2006. The role of zooxan- respiration, photosynthesis and calcification between hosts thellae in the thermal tolerance of corals: a ‘nugget of hope’ and symbionts. Mar. Biol. 60:167–78. for coral reefs in an era of climate change. Proc. R. Soc. Lond. B Johannes, R. E., Weibe, W. J., Crossland, D., Rimmer, D. W. & Biol. Sci. 273:2305–12. Smith, S. V. 1983. Latitudinal limits to coral reef growth. Mar. Birkeland, C. 1988. Geographic comparisons of coral–reef com- Ecol. Prog. Ser. 11:105–11. munity processes. Proc. 6th Int. Coral Reef Symp. 1:211–20. Kevin, K. M. & Hudson, R. C. L. 1979. The role of zooxanthellae in Blank, R. J. 1987. Presumed gametes of Symbiodinium – feintings by the hermatypic coral Plesiastrea urvillei (Milne Edwards and a fungal parasite. Endocytobiosis Cell Res. 4:297–304. Haime) from cold waters. J. Exp. Mar. Biol. Ecol. 36:157–70. de Boor, C. 1978. A Practical Guide to Splines. Springer-Verlag, Kleypas, J. A., McManus, J. W. & Lambert, A. B. 1999. Environ- Berlin, 392 pp. mental limits to coral reef development: where do we draw the Buddemeier, R. W. & Fautin, D. G. 1993. Coral bleaching as an line? Am. Zool. 39:146–59. adaptive mechanism – a testable hypothesis. Bioscience 43: Krause, G. H. 1992. Effects of temperature on energy-dependant 320–6. fluorescence quenching in chloroplasts. Photosynthetica 27: Bythell, J. C., Douglas, A. E., Sharp, V. A., Searle, J. B. & Brown, B. 249–52. E. 1997. Algal genotype and photoacclimatory response of the LaJeunesse, T. C. 2001. Investigating the biodiversity, ecology, and symbiotic alga Symbiodinium in natural populations of the sea phylogeny of endosymbiotic dinoflagellates in the genus COLD-TOLERANT SYMBIODINIUM 1135

Symbiodinium using the ITS region: in search of a ‘‘species’’ Savage, A. M., Goodson, M. S., Visram, S., Trapido-Rosenthal, H., level marker. J. Phycol. 37:866–80. Wiedenmann, J. & Douglas, A. E. 2002. Molecular diversity of LaJeunesse, T. C. 2002. Diversity and community structure of symbiotic algae at the latitudinal margins of their distribution: symbiotic dinoflagellates from Caribbean coral reefs. Mar. Biol. dinoflagellates of the genus Symbiodinium in corals and sea 141:387–400. anemones. Mar. Ecol. Prog. Ser. 244:17–26. LaJeunesse, T. C., Loh, W. K. W., van Woesik, R., Hoegh-Guldberg, Saxby, T., Dennison, W. C. & Hoegh-Guldberg, O. 2003. Photo- O., Schmidt, G. W. & Fitt, W. K. 2003. Low symbiont diversity synthetic responses of the coral Montipora digitata to cold in southern Great Barrier Reef corals, relative to those of the temperature stress. Mar. Ecol. Prog. Ser. 248:85–97. Caribbean. Limnol. Oceanogr. 48:2046–54. Schiller, C. 1993. Ecology of the symbiotic coral Cladocera caespitosa LaJeunesse, T. C. & Trench, R. K. 2000. Biogeography of two spe- (L.) (Faviidae, ) in the Bay of Piran (Adriatic Sea): cies of Symbiodinium (Freudenthal) inhabiting the intertidal ii, Energy budget. P.S.Z.N.I. Mar. Ecol. 14:221–38. sea anemone Anthopleura elegantissima (Brandt). Biol. Bull. Schreiber, U. & Bilger, W. 1987. Continuous recordings of photo- 199:126–34. chemical and non-photochemical chlorophyll fluorescence McCloskey, L. R. 1970. The dynamics of the community associated quenching with a new type of modulation fluorometry. In with a marine scleractinian coral. Int. Rev. Ges. Hydrobiol. Tenhunen, J., Catarino, F. M., Lange, O. L. & Oechel, W. C. 55:13–81. [Eds.] Plant Response to Stress. Springer-Verlag, Berlin, pp. 27– Miller, M. W. 1995. Growth of a temperate coral: effects of tem- 53. perature, light, depth, and heterotrophy. Mar. Ecol. Prog. Ser. Schuhmacher, H. & Zibrowius, H. 1985. What is hermatypic? 122:217–25. A redefinition of ecological groups in corals and other Miller, M. W. & Hay, M. E. 1996. Coral-seaweed-grazer-nutrient organisms. Coral Reefs 4:1–9. interactions on temperate reefs. Ecol. Monogr. 66:323–44. Smillie, R. G., Hetherington, S. E., He, J. & Nott, R. 1988. Pho- Moran, R. 1982. Formulae for determination of chlorophyllous toinhibition at chilling temperatures. Aust. J. Plant Physiol. pigments extracted with N,N¢–Dimethylformamide. Plant 15:207–22. Physiol. 68:1376–81. Stat, M., Carter, D. & Hoegh-Guldberg, O. 2006. The evolutionary Muller-Parker, G. 1987. Seasonal variation in light-shade adaptation history of Symbiodinium and scleractinian hosts – symbiosis, of natural populations of the symbiotic sea anemone Aiptasia diversity, and the effect of climate change. Perspect. Plant Ecol. pulchella (Carlgren, 1943) in Hawaii. J. Exp. Mar. Biol. Ecol. 8:23–43. 112:165–83. Steen, R. G. & Muscatine, L. 1987. Low temperature evokes rapid Muller-Parker, G. & Davy, S. K. 2001. Temperate and tropical algal- exocytosis of symbiotic algae by a sea anemone. Biol. Bull. sea anemone symbioses. Invertebr. Biol. 120:104–23. 172:246–63. Muller-Parker, G., Pierce-Cravens, J. & Bingham, B. L. 2007. Broad Szmant-Froelich, A. 1981. Coral nutrition: comparison of the fate of thermal tolerance of the symbiotic dinoflagellate Symbiodinium 14C from ingested labeled brine shrimp and from the uptake 14 muscatinei (Dinophyta) in the sea anemone Anthopleura elegan- of NaH CO3 by its zooxanthellae. J. Exp. Mar. Biol. Ecol. tissima () from northern latitudes. J. Phycol. 43:25–31. 55:133–44. Muscatine, L. 1990. The role of symbiotic algae in carbon and Szmant-Froelich, A. & Pilson, M. E. Q. 1984. Effects of feeding energy flux in reef corals. In Dubinski, Z. [Ed.] Ecosystems of the frequency and symbiosis with zooxanthellae on nitrogen World: Coral Reefs, Vol. 24. Elsevier, Amsterdam, pp. 75–87. metabolism and respiration of the coral Astrangia danae. Mar. van Oppen, M. J. H., Palstra, F. P., Piquet, A. M. T. & Miller, D. J. Biol. 81:153–62. 2001. Patterns of coral-dinoflagellate associations in Acropora: Tchernov, D., Gorbunov, M. Y., de Vargas, C., Yadav, S. N., significance of local availability and physiology of Symbiodinium Milligan, A. J., Haggblom, M. & Falkowski, P. G. 2004. strains and host-symbiont selectivity. Proc. R. Soc. Lond. B Biol. Membrane lipids of symbiotic algae are diagnostic of sensi- Sci. 268:1759–67. tivity to thermal bleaching in corals. Proc. Natl. Acad. Sci. Peters, E. C., Cairns, S. D., Pilson, M. E. Q., Wells, J. W., Jaap, W. C., U. S. A. 101:13531–5. Lang, J. C., Vasleski, C. E. & St. Pierre Gollahon, L. 1988. Thornhill, D. J., Fitt, W. K. & Schmidt, G. W. 2006a. Highly stable Nomenclature and biology of Astrangia poculata (= A. dannae, symbioses among western Atlantic brooding corals. Coral Reefs = A. astreiformis) (Cnidaria: ). Proc. Biol. Soc. Wash. 25:515–9. 101:234–50. Thornhill, D. J., LaJeunesse, T. C., Kemp, D. W., Fitt, W. K. & Pochon, X., Montoya-Burgos, J. I., Stadelmann, B. & Pawlowski, J. Schmidt, G. W. 2006b. Multi-year, seasonal genotypic surveys 2006. Molecular phylogeny, evolutionary rates, and divergence of coral-algal symbioses reveal prevalent stability or post- timing of the symbiotic dinoflagellate genus Symbiodinium. Mol. bleaching reversion. Mar. Biol. 148:711–22. Phylogenet. Evol. 38:20–30. Thornhill, D. J., LaJeunesse, T. C. & Santos, S. R. 2007. Measuring Rodriguez-Lanetty, M., Chang, S. J. & Song, J. I. 2003. Specificity of rDNA diversity in eukaryotic microbial systems: how intrage- two temperate dinoflagellate-anthozoan associations from the nomic variation, pseudogenes, and PCR artifacts confound north-western Pacific Ocean. Mar. Biol. 143:1193–9. biodiversity estimates. Mol. Ecol. 16:5326–40. Rodriguez-Lanetty, M., Loh, W., Carter, D. & Hoegh-Guldberg, O. Vaughan, T. W. 1919. Corals and the formation of coral reefs. 2001. Latitudinal variability in symbiont specificity within the Annu. Rep. Smithson. Inst. 17:189–238. widespread scleractinian coral Plesiastrea versipora. Mar. Biol. Veron, J. E. N. 2000. Corals of the World. Australian Institute of 138:1175–81. Marine Science, Townsville, Australia, 1382 pp. Rowan, R. 2004. Thermal adaptation in reef coral symbionts. Nature Visram, S., Wiedenmann, J. & Douglas, A. E. 2006. Molecular 430:742. diversity of symbiotic algae of the genus Symbiodinium (Zoo- Rowan, R. & Knowlton, N. 1995. Intraspecific diversity and eco- xanthellae) in cnidarians of the Mediterranean Sea. J. Mar. logical zonation in coral-algal symbiosis. Proc. Natl. Acad. Sci. Biol. Assoc. U. K. 86:1281–3. U. S. A. 92:2850–3. Warner, M. E., Fitt, W. K. & Schmidt, G. W. 1996. The effects of Ruppert, E. E. & Fox, R. S. 1988. Seashore of the Southeast. elevated temperature on the photosynthetic efficiency of University of South Carolina Press, Columbia, 429 pp. zooxanthellae in hospite from four different species of reef Santos, S. R., Taylor, D. J. & Coffroth, M. A. 2001. Genetic com- coral: a novel approach. Plant Cell Environ. 19:291–9. parisons of freshly isolated vs. cultured symbiotic dinoflagel- Warner, M. E., Fitt, W. K. & Schmidt, G. W. 1999. Damage to lates: implications for extrapolating to the intact symbiosis. photosystem II in symbiotic dinoflagellates: a determinant of J. Phycol. 37:900–12. coral bleaching. Proc. Natl. Acad. Sci. U. S. A. 96:8007–12.