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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.

    Introduction

    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: miurao@kochi-u.ac.jp

    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.

    PCrrFlP

    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 gelg

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