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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 · Ken‑ichi 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. Kühl.

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 (n33°31′12″ W133°45′19″), during day-time (11:00–14:00) 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 μl of 2 X hexadecyltri-methylammonium bromide buffer and 20 μl 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.

PCr–rFlP

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 % Tris–acetate 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 [D1–D3 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 http://www.geneious.com/) 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). Kimura’s 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. KF383295–KF383298 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 μmol m−1 s−1) 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 level. The experiments were conducted at 23.3 °C (mean water temperature at Tei), at 31.8 °C (maximum water temperature at Tei), and at 35 °C (approximate high-est body temperature of the anemone recorded in this study, see result for details).

after the measurements, each anemone was cut through the central disk into four pieces, with wet weights used to obtain exact proportions. One was dried at 60 °C for 48 h to measure dry weight; one was homog-enized and used for the quantification of algae in a hemo-cytometer slide; one was used for the rFlP analysis to determine the Symbiodinium clade. The last piece was used for the measurement of chlorophyll a and chloro-phyll c2 content. For the chlorophyll measurement, the piece of the anemone was homogenized and extracted with acetone in the dark at 4 °C for 24 h. each extract was then centrifuged, and the absorbance of the superna-tant was measured at 630, 663, and 750 nm using Ultro-spec 2,000 UV/visible spectrophotometer (Pharmacia Biotech, UK).

Photosynthesis–irradiance (P–I) curves for each anem-one were modeled from a continuous exponential function equation of Platt et al. (1980): Pnet = Pgmax × (1 − exp(–I/Ik)) − R, where Pnet is net productivity, Pgmax is the asymptotic maximum gross productivity, I is irradiance, Ik is the irradiance at which the initial slope intersects the asymptote (saturation irradiance), and R is the dark respira-tion value. The raw data from the photosynthetic activity assays were fitted to the model using the general fit proce-dure of Kaleidagraph (Synergy Software, USa). We further determined three additional parameters derived from the model: Pnmax (=Pgmax − R), the maximum net productivity; Ic (=−Ik × ln(1– R/Pgmax)), the irradiance at zero net oxy-gen flux (compensation irradiance); and α (=Pgmax/Ik), the initial slope of the curve (photosynthetic efficiency). We used general linear models to evaluate the differences in photosynthetic parameters among anemones with distinct Symbiodinium clades.

Statistical platform and assumptions

all statistical analyses were performed using JMP version 9.0 (SaS Institute, USa).

We ensured approximate normality of the residuals and homogeneity of variance by inspecting normal quantile plots with lilliefors confidence limits (Sall et al. 2007).

Results

Anthopleura uchidai was abundant on intertidal rock surfaces. The intertidal height at which A. uchidai first occurred, i.e., this species upper limit, was about 100 cm below the extreme high tide line (eHT) and it became less abundant near the extreme low tide line (elT), about 200 cm below the eHT. Within its distribution range, there were often more than 10 individuals of A. uchidai within 1 m2. We measured the vertical height and body tempera-ture of 111 anemones. The genetic analysis of the anemo-nes showed that all of the sequences were mostly identical to the published A. uchidai sequence. Further, no cryptic species were detected from the analysis of the COIII gene since there were no genetic variations in these anemones irrespective of their vertical position.

genetic identification of Symbiodinium in A. uchidai

The rFlP patterns of Symbiodinium were discriminated into 5 types. Of the five, three rFlP patterns were consist-ent with the rFlP patterns of the clade F2, F3, and clade a or g, shown in Pochon et al. (2001). Two other rFlP patterns were comprised of the combination of these rFlP patterns (F2 + F3, F2 + a or g). To confirm the obtained rFlP patterns, we determined the 28S ribosomal Dna sequences of the representative rFlP patterns. The phylo-genetic relationship inferred by the 28S gene and by Ml search is shown in Fig. 1. The phylogeny showed that the sequences from the two rFlP patterns (supposedly F2, F3) were indeed included within clade F and that the sequences from the other rFlP pattern (supposedly a or g) were included within clade a. The phylogeny further showed that, within the clade F, the sequences from rFlP pattern F3 corresponded to the clade F3. However, the sequences from rFlP pattern F2 did not correspond to clade F2 and formed a unique group adjacent to clades F2 and F3. This clade was genetically well separated from any reported types in the clade F (6–13 % of sequence divergence). We tentatively refer to this new clade/type as F6. Only two studies have reported the appearance of clade F Symbiod-inium in cnidarians (rodriguez-lanetty et al. 2003; laJeu-nesse et al. 2010). The 28S sequence of the Symbiodinium from one of these cnidarians (Anthopleura sp. from Thai-land: the unpublished sequence was kindly provided by one of the reviewers for the preliminary analysis) was very sim-ilar to clade F6 in A. uchidai, suggesting this Symbiodinium associates the temperate and tropical intertidal anemones over a large geographical range. note that the genetic anal-ysis based on the 28S gene did not provide enough infor-mation to distinguish Symbiodinium at the species level, and thus there is a possibility that the taxonomic unit we used in this study (clade a, F3 and F6) may contain several

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Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Symbiodinium sp. from A.uchidai

Gymnodinium simplex (AF060900)

Clade C

Clade H

F6

F3

F2

F4

F5

Clade B

Clade I

Clade G

Clade D

Clade E

Clade A

Clade F

AJ620945

AJ308888

AJ308887

AF427463

AJ872075

AJ311943

AJ621128

AJ291516

AJ308892

AJ291515

AJ291513

AJ291520

AJ621129

AJ621148

AJ621131

AJ308895

AJ291525

AJ830916

AJ872076

AJ830908

AJ830911

AJ830912

AJ830914

AJ621146

AJ621147

AJ621135

AJ291527

AJ621145

AF427462

AJ872077

AJ291529

AJ291535

AF427457

AF427459

AF427458

AF427460

FN561559

FN561560

FN561561

FN561562

AJ291538

AJ291537

AJ291539

AF427464

AJ308900

AJ308902

AF396627

AJ311948

Symbiodinium voratum n. sp. (AF060899)

AF427456

AF427453

AF427454

AF427455

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10098

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85

55

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0.05

Fig. 1 Molecular phylogeny of endosymbiotic zooxanthellae in the genus Symbiodinium. a maximum likelihood tree was constructed based on 771 base pairs of the 28S ribosomal Dna. Samples from genBank were shown with their accession nos. Symbiodinium clades

obtained from A. uchidai were shaded. numbers near nodes are the support values for the major clades (values <50 % not shown). The scale bar represents the phylogenetic distances expressed as units of expected nucleotide substitutions per site

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distinct species. Further analyses based on high-resolu-tion genetic markers perhaps with detailed morphological inspections are necessary to identify these Symbiodinium at the species level.

Zonation pattern seen in the intertidal Symbiodinium

We found that these Symbiodinium clades were unevenly distributed along the vertical gradient of the intertidal shore (glM, F(4,106) = 88.2, P < 0.01). Clade F6 was dominant in the upper shore (Fig. 2). It appeared at 100 cm below the eHT and showed the highest abundance at 120–150 cm below the eHT and gradually decreased in abundance on the lower shore. Clade F3 was rare and exclusively occurred on the upper shore. In contrast, clade a was prev-alent on the lower shore, exhibiting the highest relative abundance near the lower limit of the distribution range of A. uchidai. The anemone individuals often harbored two Symbiodinium clades in the intermediate height where the distribution of the two Symbiodinium clades overlapped (Fig. 2).

The body temperature of the anemones was positively correlated with their intertidal height. The anemones in the lower shore had lower body temperatures (25–26 °C), which was only slightly higher than subtidal water tem-perature (23.2 °C). The body temperatures gradually increased to 32–35 °C in the middle shore and reached

an asymptotic level between the middle and upper shore (Fig. 3). Clade a Symbiodinium was predominantly observed in anemones in lower shore with lower body temperatures (<30 °C), while clades F3 and F6 were domi-nant on the upper shore where they are exposed to higher temperatures (>30 °C) (Fig. 3).

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tanc

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om th

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)

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Fig. 2 Distribution pattern of the genetically distinct clades of Sym-biodinium along the vertical gradient of the intertidal shore. eHT represents the extreme high tide line. Proportions of these Symbiod-inium clades were shown

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tanc

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(cm

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Fig. 3 Body temperatures of the anemones along the intertidal depth. eHT represents the extreme high tide line. Symbols represent the anemones with the single or pairs of the Symbiodinium clades

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Fig. 4 Measurement of photosynthetically active radiation (Par) along water depth at Tei port, June 2012. The points are the means of the five replicate runs, and error bars represent ± SD

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We also confirmed that the amount of light decreased with water depth (Fig. 4). Our results show that at a sea-water depth of 100 cm in the intertidal, Par is reduced by 25–35 %. Since the intertidal shore at the sampling site is often covered by white water due to wave action, the actual difference in light resources between the upper and lower shore is perhaps larger than our estimation. Thus, Symbiod-inium in anemones near the lower distribution limit are able to utilize at maximum (and probably less) 75 % of light resources compared to Symbiodinium in anemones near their upper limit when these anemones are submerged in seawater. These results confirm that temperature and light condition greatly vary along the intertidal shore.

Different photosynthetic rates among the Symbiodinium clades

We measured the photosynthetic rate of 4 individual anem-ones with clade a Symbiodinium, 8 individuals with clade F6, and 5 individuals with both clades. although we also measured the photosynthetic rate of anemones with clades F3 and F3 + F6, we excluded them from further analyses because of their small sample size (n = 1 for each). Since there were three different thermal conditions and 8 differ-ent light conditions, each individual has 24 measurements. Thus, a total of 408 data points were used for the estimation of photosynthetic activity. These anemones with clade a, F6, or both clades have similar densities of Symbiodinium (glM, F(2,16) = 1.14, P = 0.34). Further, the chlorophyll content (per one cell of alga) did not differ among the Sym-biodinium clades (glM, chl a, F(2,16) = 0.89, P = 0.43; chl c2, F(2,16) = 1.37, P = 0.29; total chl, F(2,16) = 0.18, P = 0.84, Table 1).

The anemones containing Symbiodinium in clade a, F6, and both clades exhibited similar photosynthetic activ-ity at 23.3 °C, as shown by the comparison of fitted curves (Fig. 5). Further, all photosynthetic parameters (Pnmax, Pgmax, R, α, Ic, and Ik) were also similar among the anemo-nes with distinct Symbiodinium clades (Table 2). Simi-lar to 23.3 °C, there were no significant differences in all photosynthetic parameters at 31.8 °C among the anemo-nes containing Symbiodinium in clade a, F6, and both clades (Table 2, see also Fig. 5). In contrast, at 35 °C, the maximum net productivity varied significantly among

anemones with different Symbiodinium clades (Table 2; glM, F(2,16) = 4.17, P < 0.05, see also Fig. 5). The anemo-nes containing clade a Symbiodinium exhibited low net productivity compared to the anemones containing clade F6 Symbiodinium at all irradiance levels. The values for Ic were significantly larger in the anemones with clade a Symbiodinium (Table 2; glM, F(2,16) = 8.40, P < 0.01). Other photosynthetic values (Pgmax, R, α, and Ik) were similar among the anemones with distinct clades at 35 °C (Table 2).

Table 1 Chlorophyll a and c2 content of Symbiodinium in different clades

Unit for chlorophyll content is pg cell−1 . all values are means (±Se)

Clade chl a chl c2 Total chl

Clade a 11.05 (1.15) 5.10 (0.51) 16.15 (1.43)

Clade F6 14.37 (2.07) 3.71 (0.69) 18.08 (2.72)

Clade a + F6 10.59 (2.95) 5.29 (0.97) 15.88 (3.81)

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Irradiance (umol m-2 s-1)

O2

flux

(mg

O2

mg

chl-1

h-1

)

23.3 oC

31.8 oC

35 oC

Fig. 5 Photosynthesis–irradiance curves of Symbiodinium clades at three different temperatures (23.3, 31.8, and 35 °C). The points are the means of the measurements, and error bars represent ± SD

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Discussion

We found that the endosymbiotic zooxanthellae in the genus Symbiodinium within the host sea anemone A. uchi-dai clearly shows a zonation pattern along the intertidal vertical gradient. Clade F Symbiodinium were more abun-dant on the upper shore, while clade a Symbiodinium appeared at high frequency on the lower shore. Our pho-tosynthesis study further demonstrated that the net pro-ductivity of clade a Symbiodinium is strongly inhibited by heating, while clade F Symbiodinium are highly tolerant of thermal stress. These results suggest that distinct tolerance to thermal stress is one of the major factors shaping the intertidal zonation of Symbiodinium clades along the inter-tidal shore, within a population of A. uchidai.

rowan and Knowlton (1995) first documented subtidal zonation of Symbiodinium clades in Caribbean corals. They found that the reef building corals Orbicella annularis and O. faveolata in shallow water (<6 m) contained clade a and clade B Symbiodinium while these corals exclu-sively contained clade C Symbiodinium at depths over 9 m (rowan and Knowlton 1995). a similar phenomenon is documented by laJeunesse (2002) and Finny et al. (2010) who analyzed the distribution of Symbiodinium in many species of corals in the Caribbean and found that clade a Symbiodinium was a major component of the shallow coral community while clade C Symbiodinium (particularly clade C3) dominated the coral community at deep habitats. These studies consistently exhibited that clade a is primar-ily found in hosts in shallow water. Consistent with these studies, we found that most of the sea anemones near the subtidal zone possessed clade a Symbiodinium (Fig. 2). However, clade a Symbiodinium decreased in proportion with increasing intertidal height and disappeared in upper intertidal anemones (Fig. 2). Instead, all of the anemones in the high intertidal zone contained clade F Symbiodinium (clades F3 and F6, see Fig. 2). although the composition of

Symbiodinium clades changes gradually across a subtidal depth gradient (rowan and Knowlton 1995; laJeunesse 2002; Finney et al. 2010), we observed a radical alteration of the composition within a dozen centimeters in the inter-tidal habitat (Fig. 2). This is perhaps because the environ-mental gradient in the intertidal is much more extreme than that in the subtidal. Our findings demonstrated that there is a clear zonation pattern of Symbiodinium in a population of the anemones at the scale of centimeters in the intertidal habitat.

Zonation patterns are often associated with physical constraints (Connell 1972). Heating and strong sunlight are the major factors setting the upper intertidal limit of marine organisms (Connell 1972). We found that the anemones at the upper shore exhibited an approximately 10 °C higher body temperature compared with those at lower heights during the low tide (Fig. 3). additionally, we found that the irradiance level decreased at least 25 % in 100 cm of seawater (Fig. 4). These striking differences in thermal and light conditions at the upper and lower shores can affect the distribution of Symbiodinium along the intertidal gradient. To evaluate the optimum environment for each Symbiodin-ium clade in the anemone, we conducted the photosynthe-sis activity assay under a variety of temperature and light conditions. We found that the net productivity of clade a Symbiodinium was significantly lower than clade F6 Sym-biodinium at 35 °C at all irradiance levels while they exhib-ited similar productivities at the lower temperatures (23.3 and 31.8 °C) (Fig. 5; Table 2). The anemones with both Symbiodinium clades exhibited intermediate productivity at 35 °C (Fig. 5; Table 2), probably because of counteracting effects of clade a and F6 Symbiodinium. These findings, taken together with environmental assays, suggest that photosynthesis of clade a Symbiodinium is suppressed in the upper shore where they are often exposed to high tem-perature (around 35 °C) and may suffer higher mortality. In contrast, clade F6 Symbiodinium can be well adapted to

Table 2 Parameters of photosynthesis–irradiance curves for the anemones with the distinct Symbiodinium clades

Units for Pnmax, Pgmax, and R are mg O2 mg chl−1 h−1, and unit of α is O2 flux irradiance−1, and units of Ic and Ik are umol photon m−2 s−1. all values are means (±Se). Boldface refers to a statistical significance among the clades

Temperature (°C) Pgmax Pnmax R α Ic Ik

23.3

Clade a 2.84 (0.28) 1.38 (0.19) 1.47 (0.11) 0.006 (0.0009) 340.6 (29.0) 463.4 (40.6)

Clade F6 3.40 (0.47) 2.00 (0.39) 1.40 (0.14) 0.005 (0.0006) 357.8 (33.9) 673.3 (112.8)

Clade a + F6 3.60 (0.32) 1.91 (0.28) 1.69 (0.09) 0.007 (0.0003) 331.0 (10.0) 519.1 (52.4)

31.8

Clade a 4.97 (0.73) 2.28 (0.53) 2.69 (0.21) 0.011 (0.0012) 358.5 (22.5) 449.6 (57.3)

Clade F6 5.19 (0.65) 2.54 (0.30) 2.65 (0.38) 0.010 (0.0062) 374.3 (18.3) 537.2 (39.9)

Clade a + F6 5.90 (0.58) 3.00 (0.40) 2.91 (0.28) 0.010 (0.0066) 421.6 (53.0) 621.8 (95.4)

35

Clade a 4.21 (0.35) 0.90 (0.18) 3.31 (0.31) 0.011 (0.0014) 613.5 (34.3) 398.3 (51.9)

Clade F6 5.40 (0.72) 2.32 (0.39) 3.08 (0.36) 0.012 (0.0013) 394.8 (33.5) 453.1 (40.0)

Clade a + F6 5.25 (0.36) 1.82 (0.09) 3.43 (0.40) 0.012 (0.0015) 465.4 (38.3) 453.8 (46.4)

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physical conditions in the upper shore since they are highly tolerant of heating in terms of photosynthetic productiv-ity (Fig. 5). These results demonstrate that heat stress is a major environmental factor setting the upper limit of the vertical distribution of Symbiodinium clades in the popula-tion of A. uchidai along the intertidal height gradient.

although clade a and F6 Symbiodinium exhibited simi-lar net productivities at the lower temperatures (Fig. 5), clade F6 Symbiodinium decreased in abundance at 150–200 cm below eHT where temperature is relatively low (Fig. 2). This distribution pattern suggests that there may be a cost to heat tolerance, which limits the distribution of F6 Symbiodinium at lower shore. For example, Somero (2002) demonstrated a trade-off between heat tolerance and growth rate in intertidal snails. Heat-tolerant snails inhabit-ing the upper shore exhibited a 2- to 3-fold lower growth rate compared to its congeners living the lower intertidal and subtidal zones (Somero 2002). although evidence for a cost to heat tolerance in Symbiodinium is preliminary, several studies demonstrated that corals with heat-tolerant Symbiodinium grew slower than those with heat-sensitive algae under ordinary temperature conditions (little et al. 2004; Sotka and Thacker 2005; Jones and Berkelmans 2010). We expect that clade a Symbiodinium grow faster and outcompete the heat-tolerant clade F6 Symbiodinium on the lower shore, determining the lower distribution limit of clade F6 Symbiodinium. Further field and laboratory experiments are needed to confirm this hypothesis and fully understand the factors shaping the lower distribution limit of the Symbiodinium clades in the intertidal zone.

Symbiotic and/or parasitic endogenous organisms are prevalent in the intertidal zone. Other than symbiotic algae studied here, trematodes, nematodes, cestodes, and acan-thocephalans use a variety of intertidal invertebrates as their intermediate or definitive hosts (Mouritsen and Poulin 2002). among those, trematodes are the most common endogenous organisms of intertidal animals (Mouritsen and Poulin 2002). Interestingly, some species of trematodes alter behavior of their host snails and change the intertidal distribution of the infected snails. The infected snails often prefer distinct envi-ronmental conditions than uninfected snails, causing them to occur at different intertidal heights than most uninfected individuals and creating zonation within the snail popula-tion (Curtis 1987; Miura et al. 2006). Further, infection by different trematode species can cause preferences for dif-ferent microhabitats among infected hosts, creating further spatial segregation within the zone of infected snails (Miura and Chiba 2007). Our findings, coupled with above preced-ing studies, suggest that intertidal zonation may be prevalent in endogenous organisms in their intertidal hosts. Our study contributes toward a better understanding of the pattern and mechanisms of the intertidal zonation of marine organisms, particularly microscopic ones within their intertidal hosts.

Acknowledgments We thank K. Fukami for his valuable comments on this study. We also thank Y. Kumekawa, K. Matsuyama, K. Ohga, n. Yokoyama for their field assistance, and C. Keogh for english edit-ing. Two anonymous reviewers provided useful comments. This study was performed through the Program to Disseminate Tenure Tracking System of the Ministry of education, Culture, Sports, Science and Technology, the Japanese government.

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