Vertical zonation of endosymbiotic zooxanthellae within a population of the intertidal sea anemone, Anthopleura uchidai

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    Mar BiolDOI 10.1007/s00227-014-2456-0

    OrIgInal PaPer

    Vertical zonation of endosymbiotic zooxanthellae within a population of the intertidal sea anemone, Anthopleura uchidai

    Osamu Miura Teeyaporn Keawtawee Nobuko Sato Kenichi Onodera

    received: 5 September 2013 / accepted: 2 May 2014 Springer-Verlag Berlin Heidelberg 2014

    gradient across tidal height is a major factor shaping the zonation pattern of Symbiodinium clades in A. uchidai.


    In the intertidal zone, there are clear zonation bands of marine organisms along a vertical gradient. For example, barnacles are often attached at the highest area of the inter-tidal shore, mussels appear between the middle and lower shore, and seaweeds appear at the lower intertidal shore and subtidal (Stephenson and Stephenson 1949; luckens 1975). Many other intertidal organisms such as sea anem-ones, snails, and sponges also occupy their own positions along the vertical gradient in the intertidal zone (Stephen-son and Stephenson 1949; Ottaway 1973; Underwood 1973; luckens 1975; Underwood 1975; Yamada and Boul-ding 1996; Chavanich and Wilson 2000). These zonation patterns have historically provided a major focus for many intertidal works, both in terms of the analysis of static pat-terns (e.g., Stephenson and Stephenson 1949; lewis 1978) and dynamic processes (Connell 1972). The upper limits of each band are generally maintained by physical constraints such as resistance to desiccation, high temperature, and/or strong solar radiation, while lower limits are often set by biological interactions such as competition and predation (Connell 1972).

    These environmental stressors may also affect the dis-tribution patterns of symbiotic organisms associated with marine organisms. This possibility is well examined in the symbiotic algae within corals. Corals generally con-tain within their tissue endosymbiotic zooxanthellae in the genus Symbiodinium, which are capable of photosyn-thesis. Symbiodinium are small single-celled algae with restricted morphology and have a number of cryptic species

    Abstract Intertidal organisms commonly form zonation bands along the shore. environmental stressors often deter-mine the vertical position of each zonation band. These stressors may similarly affect the distribution pattern of endogenous species in their intertidal hosts. To evaluate this possibility, we investigated the distribution pattern of endosymbiotic zooxanthellae in the genus Symbiodinium in a population of the intertidal sea anemone Anthopleura uchidai. We used molecular genetics to identify the Symbi-odinium clades and found that A. uchidai has two clades of Symbiodinium, clades a and F. These Symbiodinium clades were disproportionally distributed along the vertical gradi-ent of the intertidal shore. anemones on the upper shore exclusively possessed clade F Symbiodinium while clade a Symbiodinium became dominant in the sea anemones on the lower shore. Photosynthesis activity assays showed that these Symbiodinium clades had similar net productivities at 23.3 and 31.8 C at all irradiance levels. at 35 C, how-ever, clade a Symbiodinium exhibited substantially lower net productivities than clade F Symbiodinium, demonstrat-ing that these Symbiodinium clades have distinct tolerances to thermal stress. These results suggest that the thermal

    Communicated by M. Khl.

    O. Miura (*) n. Sato Oceanography Section, Science research Center, Kochi University, 200 Monobe, nankoku, Kochi 783-8502, Japane-mail:

    T. Keawtawee Department of aquatic Science, Faculty of natural resources, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

    K. Onodera Oceanography Section, Science research Center, Kochi University, Oko, nankoku, Kochi 783-8505, Japan

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    that are indistinguishable without genetic analysis (lajeu-nesse et al. 2012). Using molecular genetics, rowan and Knowlton (1995) documented that Symbiodinium within a population of Caribbean corals in the genus Orbicella exhibited zonation along the depth gradient of the subtidal area. after this landmark study, several researchers found similar zonation patterns of Symbiodinium in other subtidal coral species (laJeunesse 2002; Finney et al. 2010) and in a subtidal sea anemone (Venn et al. 2008). Further, Kemp et al. (2008) demonstrated that some coral colonies have genetically distinct Symbiodinium, which were unevenly distributed on a microscale. These zonation patterns are in part formed due to host specificity (Finney et al. 2010) but are also shaped by physical stressors such as temperature and solar radiance (rowan and Knowlton 1995; Finney et al. 2010) that are also the major environmental factors determining the vertical position of intertidal sessile and semi-sessile organisms (Connell 1972). Symbiodinium genetic clades often have a distinct optimum temperature and irradiance for photosynthesis (goulet et al. 2005; robi-son and Warner 2006; Warner et al. 2006; Suggett et al. 2008), which may affect the zonation pattern among Sym-biodinium clades.

    Despite several studies focused on the zonation pattern of Symbiodinium in the subtidal area (rowan and Knowl-ton 1995; laJeunesse 2002; Finney et al. 2010), their dis-tribution pattern in the intertidal zone has scarcely been explored (but see Bates 2000; Secord and augustine 2000). In subtidal areas, both temperature and light intensity grad-ually change with water depth. In contrast, in the intertidal zone, these factors steeply change only within a dozen cen-timeters because intertidal habitats are often exposed to air and direct sunlight. Because of the steep environmen-tal gradient, Symbiodinium may form a clearer zonation at the scale of centimeters along the depth gradient of the intertidal zone. The east asian sea anemone, Anthopleura uchidai (=A. japonica), is one of the common organisms on the intertidal rocky shore in Japan. A. uchidai harbors Symbiodinium within their tissue (geller and Walton 2001) and is distributed along a wide range of the intertidal zone (Uchida and Soyama 2001), providing an ideal system to evaluate the distribution pattern of Symbiodinium along the intertidal shore. In this study, we investigated the dis-tribution pattern of A. uchidai and Symbiodinium across an intertidal depth gradient to examine whether Symbiodinium form a zonation pattern along a stress gradient. Because most Symbiodinium are indistinguishable by morphological inspections (lajeunesse et al. 2012), we analyzed the 28S ribosomal Dna sequences to identify Symbiodinium at the clade level. Finally, we conducted photosynthesis activity assays to evaluate which environmental factors determine the distribution pattern of the Symbiodinium clades in the intertidal zone.

    Materials and methods

    Sample collection

    Anthopleura uchidai were collected at Tei in Kochi Pre-fecture, Japan (n333112 W1334519), during day-time (11:0014:00) at low tide on June 4, 2012. For each individual sea anemone, we measured the vertical distance from the extreme high tide line (eHT) using SPrInTer 150 Digital level (leica geosystems, Switzerland). addi-tionally, we measured the body temperature of each anem-one using MF-500 digital thermometer (Chino, Japan) by inserting the apical sensor into the oral part of the anemone. The anemones were brought to the laboratory and stored at 20 C for further molecular analyses of their endosym-bionts. We also measured photosynthetically active radia-tion (Par) along each depth of the intertidal zone (at 10 cm intervals) using lI-192Sa connected to lI-250 quantum radiometer (lI-COr, USa) around noon on cloudless day. The measurement was repeated 5 times. Our sampling site was exposed to wave action that interrupts the Par meas-urements along the depth gradient due to the turbulence of the sea surface. Therefore, to prevent disruption by wave action, we conducted this measurement at Tei port, about 1 km north from the sampling site.

    Dna extraction

    Anthopleura uchidai were dissected under a stereomicro-scope to obtain Symbiodinium spp. numerous Symbiodin-ium cells were observed in the tentacles and oral disk of the anemones. From each anemone, a single tentacle contain-ing Symbiodinium was removed with forceps and Symbiod-inium Dna was isolated using a modification of the CTaB method described in Doyle and Doyle (1987). The tentacle was soaked in a solution of 300 l of 2 X hexadecyltri-methylammonium bromide buffer and 20 l of 10 mg/ml proteinase K, incubated at 60 C overnight, extracted once with phenol/chloroform (v:v, 1:1) and precipitated with two volumes of ethanol. The Dna pellets were briefly washed in 75 % ethanol, air-dried, and dissolved in 50 ml of deionized distilled water.


    To identify the genetically distinct Symbiodinium clades, we performed PCr-based rFlP analyses following Pochon et al. (2001). We used the dinoflagellate-specific primers, ITS-DInO and l_O (Pochon et al. 2001), to amplify a nuclear Dna region of Symbiodinium, contain-ing partial 5.8S ribosomal rna gene (5.8S), whole inter-nal transcribed spacer 2 region (ITS2), and partial 28S ribosomal rna gene (28S). PCr was performed with

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    ex Taq polymerase (Takara, Japan) and run for 35 cycles under the following conditions: denaturing at 94 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 120 s. The 35 cycles were preceded by an initial denaturing at 94 C for 1 min followed by a final extension of 72 C for 7 min. Ten microliters of the unpurified PCr products were digested overnight by the six-base cutting restriction enzyme, endonuclease Hind III (new england Biolabs, USa). The restricted fragments were separated using elec-trophoresis in 2 % Trisacetate eDTa (Tae) agarose gels at 50 V for 1 h. Bands were visualized by staining with gelgreen (Biotium, USa). The Symbiodinium clades were identified based on the rFlP fragment patterns following Pochon et al. (2001).

    Dna sequencing and estimation of phylogenetic relationship

    Samples possessing a particular rFlP pattern were selected and used for subsequent sequence analyses. The original PCr products of the samples were purified, and the partial 28S ribosomal Dna sequences [D1D3 domains (Pochon et al. 2001)] were determined using the internal sequence primers from both directions (forward: symb-F 5-gaaTTTaagCaTaTaagTaagCgg-3, reverse: symb-r 5-CCCaCgTaTgaCgaaCgaTTTgCaCg-3; both primers were developed in this study) and BigDye Terminator version 3.1 (applied Biosystems, USa). We Sanger sequenced the partial 28S gene using an automated sequencer (aBI 3130-avant). The obtained sequences were aligned with the published Symbiodinium 28S ribosomal Dna sequences (Pochon and gates 2010). The sequence alignment of these samples was obtained using ClustalW (Thompson et al. 1994), implemented in geneious ver-sion 6.1.6 (created by Biomatters, available from for further phylogenetic analyses. all insertions and deletions (indels) were removed from the alignment to standardize the number of nucleotides for the phylogenetic analysis. a phylogenetic tree was con-structed using maximum likelihood (Ml) algorithms. We used the Bayesian information criterion to select the best evolutionary model using Mega5 (Tamura et al. 2011). Kimuras two-parameter model with gamma distribution was selected for the tree construction. The Ml analysis was conducted by Mega5 using an automatically generated initial tree and nnI heuristic search. node robustness was assessed using bootstrapping and 1,000 replicates.

    The taxonomy of the anemones is still confusing, and they often have cryptic species within a single morphos-pecies (Haylor et al. 1984; Monteiro et al. 1997; Schama et al. 2005). Thus, we also analyzed the mitochondrial cytochrome oxidase c subunit III (COIII) gene of the anem-ones to ensure that the anemones in the upper and lower

    shore were the same species. We haphazardly selected 40 individuals from across the vertical range of their distribu-tion, including extremely high and low positions, for this analysis. We used a combination of the anemone-specific forward primer (anthCOIIIF) and the universal reverse primer (COIIIr) for the amplification of the COIII gene (approximately 500 bp) of the anemones (geller and Wal-ton 2001). PCr was run for 35 cycles under the following conditions: denaturing at 95 C for 30 s, annealing at 45 C for 30 s and extension at 72 C for 60 s. The 35 cycles were preceded by an initial denaturing at 95 C for 2 min fol-lowed by a final extension of 72 C for 5 min. The PCr products were purified, and the sequences were determined using an automated sequencer (aBI 3130-avant). The sequence alignment of these samples was obtained using ClustalW, and the sequences were compared with pub-lished COIII sequences of A. uchidai (aF375795) (geller and Walton 2001).

    all sequences analyzed here were deposited in genBank (accession nos. KF383295KF383298 for Symbiodinium, and KF383294 for A. uchidai).

    Vertical distribution of the Symbiodinium clades

    after genetic identification of Symbiodinium clades, we analyzed the distribution pattern of the genetically distinct Symbiodinium along the depth gradient in the intertidal zone. We used a general linear model to evaluate whether these Symbiodinium clades were disproportionally distrib-uted along the intertidal height gradient.

    Photosynthetic activity assay

    Two of the major environmental factors that control pho-tosynthetic activity are temperature and light (e.g., Mor-tain-Bertrand et al. 1988). To evaluate how these factors affected the productivity of each Symbiodinium clade, we conducted photosynthetic activity assays under a variety of temperature and light conditions. We collected nine-teen intact A. uchidai individuals from Tei. The anemones were brought to the laboratory and kept in an incubator at 20 C (12 h light and 12 h dark). Dark respiration and net photosynthesis were measured in a 200-ml BOD bot-tle. The anemones were placed in bottles filled with filtered seawater and allowed to attach on the bottom for several hours. right before the measurement, we exchanged the fil-tered seawater and placed the bottle in a constant-temper-ature water bath in a dark room and allowed the anemone to acclimate to the experimental water temperature for half an hour. a Clark-type polarographic self-stirring DO probe (200-BOD, Yellow Spring Instruments, USa) was inserted into the bottle. The DO meter was calibrated in an air of 100 % relative humidity before the measurements.

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    The assay began with 10 min of dark condition followed by measurements in the light at increasing light intensi-ties. a 100-watt halogen light provided stepwise increase in light intensities (30, 60, 120, 240, 480, 960, 1,500, and 1,920 mol m1 s1) of Par measured with lI-192Sa connected to lI-250 quantum radiometer (lI-COr, USa). The oxygen flux was measured for 10 min at each irradi-ance lev...


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