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Photophysiological consequences of vertical stratification of Symbiodinium in tissue of the coral Porites lutea.

Introduction

Corals vary in biomass and tissue thickness among seasons, depths, and species (Brown et al., 1999; Fitt et al., 2000), but it is unclear how this variation is achieved (Thornhill et al., 2011). Multiple mechanisms are probably involved and include the allocation of food reserves to cells and mesoglea (Winters et al., 2009; Thornhill et al., 2011), the addition of cells to tissue layers, variation in polyp size and density (Lasker, 1981), and changes in the extent to which tissue permeates the skeleton (Lough and Barnes, 2000). Changes in coral biomass reflect shifts in the size of food reserves (Grottoli et al., 2006) and perhaps in the capacity for autotrophy through the accommodation of additional Symbiodinium within holobiont tissues (Jones and Yellowlees, 1997; Thornhill et al., 2011). Variation in biomass might also affect the dimensions of polyps and tentacles, thereby affecting boundary layers around the coral (Patterson, 1992) and the capacity to capture particulate food.

Coral tissues and their interactions with the skeleton create microhabitats that differ in physical condition from the external environment (Kiihl et at., 1995; Al-Horani et al., 2003). Within the tissues, Symbiodinium can be shaded by fluorescent pigment granules (Salih et al., 2000) and by other Symbiodinium (Jones and Yellowlees, 1997); in addition, through variation in tissue thickness, biomass can affect the penetration of light into the skeleton (Highsmith, 1981; Enriquez et al., 2005; Kaniewska et at., 2011). Changes in biomass also have implications for the diffusion of metabolites between tissue and seawater, with increasing biomass lengthening diffusion pathways, reducing solute flux, and affecting gradients of metabolites (Patterson, 1992). During the night, for example, aerobic respiration creates an [0.sub.2] gradient into coral tissue, with intracellular [0.sub.2] concentration declining to less than 2% of air saturation. During the day, photosynthesis reverses the gradient, and [0.sub.2] concentration in the tissue rises to greater than 250% of air saturation (Kuhl et al., 1995).

In this study we investigate the functional consequences of thick coral tissues that characterize important genera including Porites, Montipora, Astreopora, and Acropora (Gladfelter, 1982; Barnes and Lough, 1992, 1996; Santos et al., 2009); and we specifically address the physiological implications in massive Porites spp. We focus on a massive Porites spp. because it represents a functional group of corals that is ecologically important on reefs in the Indo-Pacific (Done and Potts, 1992) and Caribbean (Green et al., 2008), and is relatively resistant to environmental assaults (Loya et al., 2001). Given evidence suggesting that massive Porites spp. may be more physiologically resilient than other corals to current conditions, it is timely to explore phenotypic traits that are unique to this taxon. Here we describe the outcome of experiments designed to test the hypothesis that Symbiodinium in Porites lutea are phenotypically and genetically homogeneous with regard to their location within the tissue and in their response to elevated temperature.

Materials and Methods

General approach

The Pacific coral Porites lutea Edwards and Haime, 1860, was used as a representative of the massive Porites spp. functional group, in which tissues penetrate 5-10 mm into a perforate skeleton (Barnes and Lough, 1992; Edmunds, 2008). The thick tissue of P. lutea allowed for characterization of an outer and inner layer of tissue, contrasting the outer--2 mm with the immediately underlying inner--2 mm layer. Symbiodinium were sampled for pho-tophysiology, density, chlorophyll a, and genetic identity in both layers after fracturing the colony perpendicular to the outer surface (see Fig. 1). The sampling resolution (-2 mm of tissue thickness) was determined by the full width of the tissue (-5-6 mm) that was conceptualized as two adjacent layers for the sake of this study. A pulse-amplitude modulation (PAM) fluorometer was used to assess photophysiol-ogy with a 2-mm-diameter probe on the outer and inner layers of tissue. Likewise, Symbiodinium densities and chlorophyll a content were compared between tissue layers that were either cut from decalcified tissue with a scalpel or from the coral skeleton using a 700-[micro]m-thick cutting wheel, respectively.

Analyses were conducted in two parts. First, the photo-physiology, density, chlorophyll a, and genetics of Symbio-dinium in outer and inner tissue were quantified on both the top and sides of colonies. Second, a manipulative experiment tested the hypothesis that Symbiodinium in outer and inner tissue on the top of colonies were affected differentially by 12-h exposures to 28 [degrees]C, 30 [degrees]C, and 32 [degrees]C at an irradiance of 700 it mol quanta [m.sup.-2] [s.sup.-1]. Our second hypothesis was contextualized by the positive synergy between high light intensity and elevated temperature in coral bleaching (Jones et al., 1998) which, together with the inferred shading of inner coral tissue by outer tissue, would suggest that Symbiodinium in hospite within inner tissue would be less susceptible to thermal stress. As some phylotypes of Symbiodinium are more susceptible in vitro to thermal stress when acclimated to low versus high light intensities (Robison and Warner, 2006), we employed a non-direction (two-tailed) test of the response of Symbio-dinium to thermal stress in inner versus outer tissue. All irradiances in this study were recorded as photosynthetically active radiation (PAR, 400-700 nm) using a spherical quantum sensor (LI-193 sensor fitted to a Li Cor LI 1400 datalogger; Li-Cor, Lincoln, NE).

Analyses were completed between 2009 and 2012 using juvenile Porites lutea from 2-m depth in the back reef of Moorea, French Polynesia. Mensurative experiments were completed between 17 and 22 February 2009, in April 2009, and in May 2012, and a manipulative experiment was conducted in April and May 2009. In 2009, 5-8 colonies for each analysis were obtained through temporal replication with 1-3 colonies collected daily. Corals were freshly collected in the afternoon and transported to the laboratory, where they were kept for less than 1 day in an aquarium receiving sunlight (at < 100 [micro]mol quanta [m.sup.-2] [s.sup.-1]) and flowing seawater at the ambient seawater temperature (28.1-29.8 [degrees]C in February and 27.7-29.8 [degrees]C in April and May). Chlorophyll fluorescence was assessed after dark acclimation on the day of collection, and Symbiodiniurn were accessed by fracturing the colony using a knife and hammer. The newly exposed coral tissue was sampled within 5 min, and fracturing took place under a dim red light so as not to compromise the dark-acclimated state. Following the analysis of photophysiology, Symbiodiniuni were sampled to measure densities and for genetic identification. In 2012, corals were collected from the same location in an identical way to analyze chlorophyll a content.

Mensurative experiment to characterize Symbiodinium

The mensurative experiments were completed on the day of collection in February 2009, and consisted of measuring the maximum photochemical efficiency of open reaction centers of Photosystem II (RCIIs) [F.sub.v]/[F.sub.m] at different positions within the coral tissue. [F.sub.v]/[F.sub.m] was calculated from ([F.sub.m] - [F.sub.o])/[F.sub.m], (Cosgrove and Borowitzka, 2010), where [F.sub.m] is maximum fluorescence yield and [F.sub.o] is minimum fluorescence yield. This metric is suited to testing for photophysiological similarity between Symbiodinium in outer and inner tissue because it provides a rapid, noninvasive determination of photophysiology that is sensitive to temperature (Warner et aL, 2010). In addition to [F.sub.v]/[F.sub.m], rapid light curves (RLCs, Ralph and Gademann, 2005) were prepared for outer and inner tissues to evaluate how Symbiodinium utilized low intensities of PAR. RLCs were used to examine the relationship between relative electron transport rate through PSII (rETR) as a function of irradiance, with the slope of the initial linear portion of this relationship used to evaluate the efficiency with which low intensities of light are utilized (Frade et al., 2008; Hennige et at., 2008).

A separate experiment was used to test the hypothesis that fracturing of the coral affected Symbiodinium within the tissue. In this analysis, [F.sub.v]/[F.sub.m], [F.sup.o], and [F.sub.m] were determined using the PAM fluorometer both in a conventional mode (i.e., on intact corals with the probe normal to the colony surface) and with the probe placed on a freshly fractured skeleton, perpendicular to the outer surface of the coral. Five corals were retained for 12 h in a tank receiving weak sunlight at 28 [degrees]C, dark-acclimated for 2 h, and then measured for [F.sub.v]/[F.sub.m], [F.sub.o] and [F.sub.m] on the upper surface of intact colonies, and on tissue exposed by fracturing. In the normal orientation, the PAM probe integrates fluorescence originating from Symbiodinium at multiple depths within the tissue, whereas when applied on the fractured surface, the probe integrates within the tissue and parallel to the surface of the colony. This sampling was initiated to evaluate the possibility that the tearing of tissue during fracturing would perturb Symbiodinium physiology.

To compare the photophysiology of Symbiodinium in adjacent tissue layers, dark-acclimation began at sunset (~1830 h), and 2 h later colonies were fractured and processed for photophysiology using a Diving PAM (Waltz, GmbH) fitted with a 2-mm-diameter probe. Separate experiments were used to (1) fix the duration of dark-acclimation at ~2 h, having demonstrated that [F.sub.o] and [F.sun.m] stabilized after 15-60 min of darkness; (2) adjust the gain and measuring light intensity on the Diving PAM to settings of 12 to bring the recordings within the range of the instrument using the small 2-mm-diameter probe; and (3) adjust the duration and intensity of the saturation pulse to settings of 0.8 s and 8, respectively, to ensure that [F.sub.m], was saturated. Outer and inner tissues were contrasted for [F.sub.v]/[F.sub.m] on the top and sides of colonies because the sides of corals receive less light than the upper surface (Brakel, 1979; Heikoop et at., 1998) and, therefore, position on the colony might interact with depth in tissue to affect Symbiodinium. The probe was hand-held perpendicular to the measurement surface with the tip lightly touching the coral. To prevent biases arising from the sequence in which outer and inner tissue (as well as top and sides of the colonies) were sampled, one half of each fractured colony was selected randomly for processing, and the other half was returned to darkened seawater and processed later. The first half was assigned randomly to a group for which either the outer or inner tissues of the top and side were sampled.

Following measurements of [F.sub.v]/[F.sub.m], RLCs were applied to approximately the same position on the fractured surfaces at the top of the coral using an identical protocol to that used for [F.sub.v]/[F.sub.m]; RLCs were not applied to tissue layers on the sides of colonies. RLCs were prepared using 30-s exposures to each of nine irradiances emitted by the Diving PAM as measured by the Li-Cor sensor, starting at 4 [mocro]mol photons [m.sup.-2] [s.sup.-1] and ending at 1235 [micro]mol(photons [m.sup.-2] [s.sup.-1]. At each irradiance, the effective photochemical efficiency of RCIIs in actinic light ([DELTA][F.sub.v]/[F.sub.m]') was measured using the relationship ([F.sub.m]' - F')/[F.sub.m]' where [F.sub.m]' is the maximum fluorescence in actinic light, F' the fluorescence yield in actinic light, and [DELTA]F the difference between [F.sub.m]' and F'; rETR was calculated from [DELTA][F.sub.v]/[F.sub.m]' X irradiance x 0.5 (Cosgrove and Borowitzka, 2010). The slope (in relative units) of the first portion of these relationships (i.e., for six irradiances between 0 and 353 iurnol quanta [m.sup.-1] [s.sup.-1])was determined by least squares linear regression and used as an indication of shade acclimation; values of this metric in corals generally increase with shade acclimation (Frade et al., 2008; Hennige et at., 2008).

To evaluate the extent to which the photophysiology of the green alga Ostreobium spp. (Fig. 1) might affect the analysis of Symbiodinium, [F.sub.v]/[F.sub.m] of the Ostreobium spp. was also measured. The measurements of Ostreobium spp. were completed on one coral (two determinations), and the instrument settings were reduced to 9 for measuring both the light intensity and gain.

To compare the densities of Symbiodinium between inner and outer tissues of juvenile Porites lutea, freshly collected colonies (three from 21 February 2009 and two from 10 April 2009) were preserved in 5% formalin in seawater and decalcified in 5% HC1 in fresh water. Decalcification resulted in a tissue tunic from which layers of outer and inner tissue (~2 mm thick) were sliced from the top of each colony using a razor blade. Tissue slices were homogenized with an ultrasonic dismembrator (Fisher 15-338-550) fitted with a 3.2-mm-diameter probe (Fisher 15-338-67) and the Symbiodinium quantified. Symbiodinium cells were counted using a hemocytometer (5-10 replicate counts) and an aliquot was dried to a constant weight at 60 [degrees]C so that the Symbiodinium could be expressed as cells per milligram.

A similar technique was used in April 2012 to evaluate the chlorophyll content of Symbiodinium. Juvenile colonies of Porites lutea were bisected with a 700-[micro]m-thick cutting wheel, and the same wheel was used to remove rectangular blocks of skeleton and tissue from the outer and inner layers of tissue. Blocks were ~5.0x 6.0 x 1.5 mm with their long axis parallel to the surface of the colony and their smallest dimension perpendicular to the surface. Blocks were measured with calipers ([+ or -] 100 [mocro]m) and then either extracted in 100% acetone (overnight at 4 [degrees]C in darkness) for chlorophyll a content, or processed for biomass by fixation (5% formalin in seawater) and decalcification (5% HC1 in fresh water). Acetone extracts were used to determine chlorophyll a content (nanograms per cubic millimeter) using the equations of Jeffrey and Humphrey (1975) for dinoflagellates, and the fixed and decalcified pieces were dried at 60 [degrees]C to obtain biomass (micrograms per cubic millimeter). The chlorophyll a and biomass content of the blocks were used to express chlorophyll a as a function of biomass (nanograms per microgram), and in turn, this was used to estimate the chlorophyll a content of the Symbiodinium (picograms per cell) on the basis of Symbiodinium densities (cells per milligram) measured in 2009.

Manipulative experiment to test the response of Symbiodinium to temperature

The response of Symbiodinium to temperature was determined in April and May 2009 using 12-h incubations with five trials, each utilizing one coral in each of three temperatures. Juvenile Porites lutea were retained in an aquarium at ambient seawater temperature (~28.9 [degrees]C), and natural light (<100 11 mo1 quanta [m.sup.-2] [s.sup.-1]). The following morning (0700 h) corals were allocated randomly to treatments of 28 [degrees]C, 30 [degrees]C, or 32 [degrees]C at~700 lusnol quanta [m.sup.-2] [s.sup.-1] supplied by 1000-W metal halide lamps. Temperatures spanned the upper range in the lagoon of Moorea (Putnam and Edmunds, 2011). A 12-h exposure to light provided a daily integrated irradiance that was similar to that occurring at the collection depth (~94% of mean integrated total irradiance) in the lagoon of Moorea in May (n = 3 days, R. C. Carpenter, California State University, Northridge; unpubl. data), and this intensity is likely to accentuate the consequences of thermal stress for Symbiodinium (Jones et al., 1998). After 12 h, the corals were dark-acclimated for 2 h at their treatment temperatures, fractured, and processed for the determination of [F.sub.v]/[F.sub.m], as described above. In this analysis, outer and inner tissue on the top of the colony was sampled to test the effects of temperature.

Symbiodinium genetic typing

To genetically identify the Symbiodinium within Porites lutea, small pieces (<2 [mm.sup.3]) were removed from the outer and inner tissues of colonies collected in February 2009 and used for the analysis of [F.sub.v]/[F.sub.m]. Samples for DNA analysis were removed from about the same locations where [F.sub.v]/[F.sub.m] was measured. Samples were collected with a sterile razor blade and placed in 600 [micro]1 of guanidinium extraction buffer consisting of 50% (w/v) guanidinium isothiocyanate, 50 [mmoll.sup.-1] Tris pH 7.6, 10 [[micro]mol1.sup.-1] ethylenediaminetetra-acetic acid (EDTA), and 4.2% (w/v) sarkosyl; 2.1% (v/v) [beta]-mercaptoethanol. To extract DNA, samples were incubated at 72 [degrees]C for 10 min, centrifuged at 15,700 x g for 5 min, and the supernatant mixed with an equal volume of 100% isopropanol and incubated at--20 [degrees]C overnight. DNA was gathered by centrifugation at 15,700 X g for 15 min, and the pellet washed in 70% ethanol and resuspended in deionized water.

Polymerase chain reaction (PCR) amplification of the nuclear ribosomal internal transcribed spacer 2 (ITS2) region was carried out using the forward primer ITS-D (5'-GTGAATTGCAGAACTCCGTG-3'; Pochon et al., 2001) and the reverse primer ITS2rev2 (5'-CCTCCGCTTACT-TATATGCTT-3'; Stat et al., 2009). Reactions were carried out in a 25-11.1 volume, and a touchdown PCR protocol was employed to minimize cross hybridization of the primers with the coral host as follows: (1) 95 [degrees]C for 10 min; (2) 25 cycles of 94 [degrees]C for 30 s, 65 [degrees]C for 30 s (decreasing the annealing temperature by 0.5 [degrees]C for every cycle after cycle 1), and 72 [degrees]C for 1 min; (3) 14 cycles of 94 [degrees]C for 30 s, 52 [degrees]C for 30 s, and 72 [degrees]C for 1 min; and (4) final extension of 72 [degrees]C for 10 min. PCR products were purified using the QIAquick PCR Purification kit (Qiagen, CA) and eluted into 40 [micro]1 of elution buffer. The sequence of interest was ligated into pGEM-T Easy vector (Promega, WI), transformed into a-select gold efficiency competent cells (Bio-line, MA), and grown overnight in SOC medium (Bioline, MA) on agar plates containing ampicillin, IPTG (isopropyl-P-D-thiogalactopyranoside, Fermentas UAB), and XGAL (EMD, NJ). White transformed colonies were selected and picked for PCR screening with M13 primer sequences forward (5'-GTAAAACGACGGCCAG-3') and reverse (5'-CAGGAAACAGCTATGAC-3'). Ten clones that contained the fragment of interest were selected from each of the samples for sequencing. Following purification of the samples by Exosap (Fennentas, OH) to remove any residual primers or dNTPs, the PCR products were sequenced using BigDye Terminator chemistry of Applied Biosystems (Perkin Elmer) on an automated sequencer.

Symbiodinium sequences were screened for polymorphisms using Sequencher ver. 4.7 (Gene Codes) and aligned using BioEdit ver. 5.0.9 (Hall, 1999). Specifically, a sequence type was included in the downstream analysis if found in three or more clone libraries (i.e., original samples). Any sequences not meeting the initial criteria were edited to reflect the closest dominant sequence type (Stat et al., 2009), thereby offering a conservative estimate of sequence diversity.

Statistical analysis

A Student's t-test was used to test the hypothesis that the photophysiology of Symbiodinium was unaffected by fracturing. Photophysiological performance was compared between tissue layers and positions on the colony using a three-way ANOVA in which position (top vs. side) and tissue layer were fixed, and colony was a blocking factor; [F.sub.v]/[F.sub.m] was used as response variables. Symbiodinium density was compared between tissue layers with a two-way ANOVA in which tissue layer was a fixed effect and colony was a blocking factor. The initial slope of the rETR versus irradiance relationship as well as chlorophyll a content were compared between tissue layers with a t-test. To test the effects of temperature on Symbiodinium, a split-plot ANOVA was employed in which treatment was a fixed factor, the corals served as plots within each treatment, and tissue layers served as a random, within-plot factor; the dependent variable was [F.sub.v]/[F.sub.m]. The normality and homoscedasticity assumptions of ANOVA were tested through graphical analyses of residuals, and statistical procedures were completed using Systat 11.0 on a Windows platform.

To assess the Symbiodinium types in the inner and outer tissues, haplotype networks of the unique sequence types were created using statistical parsimony, 95% connection limits, and gaps as a 5th state (TCS, ver. 1.21; Clement et al., 2000). To compare the Symbiodinium sequence assemblages between inner and outer tissues, a one-way ANOSIM (Primer 6.1.12, Primer E; Clarke and Warwick, 2001) of the Bray-Curtis similarity of Symbiodinium sequence assemblages between tissues was used.

Results

Mensurative experiment to characterize Symbiodinium in tissue layers

Following dark-acclimation, the corals were fractured to expose clean surfaces (Fig. 1). In the analyses to evaluate the effect of fracturing on Symbiodinium, [F.sub.v]/[F.sub.m] did not differ between the upper surface of intact corals and the outer tissue of freshly fractured corals (t = 0.918, df = 8, P = 0.385). Thus, assuming that integration of the fluorescence signal vertically through the tissue is functionally analogous to integrating the fluorescence signal horizontally across the tissue, fracturing per se did not affect the photo-physiology of Symbiodinium. In the contrast of outer and inner tissue, [F.sub.v]/[F.sub.m], ranged from 0.487 to 0.672 in the outer tissues, and from 0.548 to 0.651 in the inner tissues. [F.sub.v]/[F.sub.m] differed between the top and side of colonies in a pattern that varied between tissue layers (i.e., the interaction was significant: F(1,19) = 6.468, P = 0.020), but the main effects of position on the colony and tissue layer were not significant alone (P [greater than or equal to ]0.140). Mean [F.sub.v]/[F.sub.m], was 7% greater in inner compared to outer tissues on the top of colonies, but was 6% greater in outer compared to inner tissues on the side of the colonies (Fig. 2); exploratory post hoc analyses revealed that these effects were significant on the top of colonies (t = 2.182, df = 14, P = 0.047), but not on the side (t = 1.898, df = 12, P = 0.082). The analysis of Ostreobium spp. revealed that the mean [F.sub.v]/[F.sub.m], (0.639 [+ or -] 0.015 [[+ or -] SD]) was similar to that of Symbiodinium in the same corals.

The RLCs generated rETR versus irradiance relationships for tissue on the top of colonies that had statistically identical slopes for the initial portion of the relationship (t = 1.591, df = 12, P = 0.138). Although not significantly different, the mean values for this parameter tended to be lower for inner tissue (slope = 0.090 [+ or -] 0.028, mean [r.sup.2] = 0.88 [+ or -] 0.06 [[+ or -] SE, n = 6]) compared to outer tissue (slope = 0.140 [+ or -] 0.021, mean [r.sup.2] = 0.96 [+ or -] 0.02 [[+ or -] SE, n = 81).

Based on corals collected in February 2009 (n = 3) and April 2009 (n = 2) and pooled for a single analysis, Symbiodinium densities differed between tissue layers (F(1,4) = 10.767, P = 0.030), with 65 [+ or -] 17 x 103 cells [mg.sup.-1] in inner tissue, and 318 [+ or -] 61 X 103 cells [mg.sup.-1] in outer tissue. Chlorophyll a content in corals sampled in April 2012 was similar between layers when standardized by tissue volume (t = 0.109, df = 12, P = 0.915), but since sampling in 2009 demonstrated there was 4.9-fold more Symbiodinium in outer compared to inner tissue, the estimated chlorophyll a per Symbiodinium cell was greater for inner tissue (20 [+ or -] 3 pg [cell.sup.-1]) compared to outer tissue (3 [+ or -] 1 pg [cell.sup.-1]) (both [+ or -] SE, n = 7). The difference between tissue layers for estimated chlorophyll a per Symbiodinium cell was significant (t = 4.683, df = 12, P = 0.001). Chlorophyll content was not measured in 2009, but at that time F, was recorded with the Diving PAM fluorometer and used as a proxy for chlorophyll content (Jones, 2004; Fabricius, 2006). In 2009, the content of chlorophyll a per Symbiodinium cell, as evaluated by [F.sub.0], was greater for inner versus outer tissue. This trend is consistent with the measurements of chlorophyll a in 2012.

Manipulative experiment to test the response of Symbiodinium to temperature

Porites lutea tolerated the 12-h incubations at the three temperatures without conspicuous signs of stress such as paling or production of mucus. Mean values for Ffin, ranged from 0.507 [+ or -] 0.011 in inner tissues at 32 [degrees]C, to 0.591 [+ or -] 0.007 (both [+ or -] SE, n = 5) for inner tissues at 28 [degrees]C (Fig. 3). Despite a trend for depression of FifErn at high temperature, the effect was not significant (F(2,12) = 2.838, P = 0.098), as was the case with tissue depth (F(2,12) = 1.922, P = 0.189), and the interaction between the two (F(1,12) = 0.144, P = 0.711).

Symbiodinium genetic typing

Genetic analysis of the ITS2 region of the nuclear rDNA revealed three Symbiodinium sequence haplotypes in the 108 sequences recovered. The Symbiodinium sequences were dominated by ITS2 type C15 (AY239369, ~92%) and a minor proportion of two sequence haplotypes 1 and 2 bp from C15 (C15.10, FR823361; C15.11, FR823362; Fig. 4). A one-way ANOSIM of Bray-Curtis similarities of the Symbiodinium sequence assemblages between inner and outer tissues revealed no difference in sequence haplotypes between tissue layers (R =-0.082, P > 0.05).

Discussion

This study was motivated by the advantages of exploring the functional basis of the physiological resilience of massive Porites spp. in the face of contemporary environmental assaults (Loya et at., 2001; Green et al., 2008; Edmunds, 2011). Our experimental approach was strongly influenced by the dynamic relationship between biomass and skeleton in corals, as illustrated by the extent to which biomass differs among taxa, seasons (Brown et al., 1999; Fitt et al., 2000; Thornhill et al., 2011), and treatments (Grottoli et al., 2006; Edmunds, 2011), as well as by variation in the depth to which tissue penetrates the skeleton (Barnes and Lough, 1992). We reasoned that, together with evidence of vertically stratified physical and chemical microenvironments within coral tissue (Salih et al., 2000; Kiihl et al., 2008; Agostini et al., 2011; Kaniewska et al., 2011), it was compelling to test for physiological and genetic gradients in the Symbiodinium distributed throughout the tissue layer. Potentially, microenvironments within coral tissue could mediate the response of the holobiont to the environment, and because coral biomass varies among sites and times (Barnes and Lough, 1999; Brown et al., 1999; Fitt et al.,2000; Thornhill et al., 2011), these effects might differ over similar scales.

Our analyses demonstrate that despite being genetically indistinguishable, the photophysiological performance ([F.sub.v]/[F.sub.m]) of Symbiodinium in outer and inner tissues of Porites lutea differed significantly. There are several explanations for these differences. First, it is possible that they reflect microspatial heterogeneity in the distribution of genetic variants of Symbiodinium within colonies (Rowan and Knowlton, 1995). However, the 1TS2 sequence assemblages in the two tissue layers of P. lutea were dominated by Symbiodinium sub-type C15 (~92% of total sequences), and were statistically indistinguishable. C15 has high fidelity to massive Porites spp. and is dominant in this functional group of corals across the Pacific (LaJeunesse et at., 2004; Stat et at., 2008; Barshis et at., 2010). Although a small number of unique Symbiodinium haplotypes were detected (~8% of sequences), they differed by only 1-2 bases from C15, with no significant differences in the proportion in both tissue layers. As a result, the differences in photophysi-ology of Symbiodinium in outer and inner tissue did not reflect genetic differences, but instead represent phenotypic plasticity. This finding is similar to that described for Pocillopora damicornis, where Symbiodinium phenotypes vary within a colony but share a common genotype (Ulstrup et at., 2006). However, both P. lutea and P. damicornis contrast with Montastraea annularis, in which the distribution of Symbiodinium genotypes maps onto microspatial variation in the physical environment surrounding the coral (Rowan and Knowlton, 1995; Baker, 2003; see also Ulstrup et at., 2007).

Second, it is possible that endolithic algae (Ostreobium spp.) beneath the Porites lutea tissues influenced the analysis of Symbiodinium. In acquiring data, we reduced the potentially confounding effects of endoliths by positioning the PAM probe on the coral tissue and avoiding Ostreobium spp. This ensured that dense invasions of green filaments of this alga into the zone of the skeleton occupied by the coral tissue were avoided, but did not exclude the possibility of sampling coral tissues that were permeated by pale and invisible filaments of Ostreobium spp. (Le Campion-Alsumard et al., 1995). The potential for Ostreobium spp. to affect the fluorescent measurements obtained from coral tissue was also explored through an investigation of the photophysiology of the Ostreobium spp. With the Diving PAM set at a reduced sensitivity compared to that necessary for Symbiodinium spp., the analysis of Ostreobium spp. revealed that [F.sub.v]/[F.sub.m] was similar to that in Symbiodinium spp. Together, these observations suggest that measurements of [F.sub.v]/[F.sub.m] in P. lutea were not affected by Ostreobium spp.

With confirmation that the variation in Symbiodinium of Porites lutea was phenotypic plasticity and likely unaffected by Ostreobium spp., we hypothesized that it represented shade acclimatization (Falkowski and Dubinsky, 1981; Anthony and Hoegh-Guldberg, 2003) because tissue within the skeleton of Porites spp. absorbs light (Magnusson et at., 2007; see also Kaniewska et al., 2011). Further, if Symbiodinium are shade-acclimatized in inner tissue, they might be more resistant to thermal bleaching than those in outer tissue (Jones et al., 1998; but see Robison and Warner, 2006). Such a possibility is consistent with reports of preferential loss of Symbiodinium from well-illuminated portions of coral colonies at high temperature (Brown et at., 1995), the observation that massive Porites spp. are resistant to bleaching (Jones et al., 2000; Loya et at., 2001), and the finding that Symbiodinium in deeper tissue remain undamaged during thermal stress and hasten recovery following cessation of stress (Jones et at., 2000).

Classically, shade-aclimatization in corals is associated with increased chlorophyll a per Symbiodinium cell, little change in Symbiodinium density per coral area, an increased Symbiodinium density per coral biomass, increased [F.sub.v]/[F.sub.m], and an increased slope of the initial portion of the rETR versus irradiance relationship (Falkowski and Dubinsky, 1981; Jones and Hoegh-Guldberg, 2001; Anthony and Hoegh-Guldberg, 2003; Frade et at., 2008; Hennige et at., 2008). In contrast to these expectations, our results are equivocal with regard to demonstrating shade acclimatization. For Symbiodinium in inner versus outer tissue of Po-rites lutea, we indirectly showed that they contain more chlorophyll a, and directly showed a reduced biomass-specific population density, inconsistent variation in [F.sub.v]/[F.sub.m], (characterized by a location X tissue layer interaction), and no increase in the initial slope of the rETR versus irradiance relationship. While the increased chlorophyll a content of Symbiodinium in inner tissue supports the notion of shade acclimatization, this interpretation does not account for the complexity of our results.

Given the attenuation of light by Porites spp. tissue (Magnusson et al., 2007)--achieved through light absorption by Symbiodinium (Enriquez et al., 2005) as well as shading (Crossland and Barnes, 1977) and host pigments (Salih et al., 2000; Kaniewska et al., 2011)--it is surprising that Symbiodinium within inner and outer tissues of P. lutea were affected equally by 12 h at 32 [degrees]C. While the evidence for thermal damage at 32 [degrees]C is sparse, involving only a trend for a 6%-14% reduction in [F.sub.v]/[F.sub.m] relative to 28 [degrees]C, it is nonetheless inconsistent with the hypothesis of shade-induced protection from thermal stress for Symbiodinium deep in Porites spp. tissues. Although this outcome might have changed with a longer incubation or higher temperature, it is possible that inner tissue is shaded less than suggested by the <10% of incident PAR that passes through coral tissue (Magnusson et al., 2007; Kaniewska et al., 2011). Porites spp. tissue absorbs a large proportion of incident light (Magnusson et al., 2007), but the optical properties of the skeleton have the potential to counteract this effect (Enriquez et al., 2005). While this means it is easier for light to enter the tissue than it is to escape (Kiihl et al., 1995), measurements of light below the tissue of corals do not support the notion that light intensity within the tissue is augmented by the skeleton (Kaniewska et al., 2011).

Regardless of the light regime within the tissue of Porites lutea, and its potential role in modulating shade acclimatization in Symbiodinium, the photophysiological responses do not lend themselves to a hypothesis evoking a single causal mechanism (i.e., reduced irradiance). For instance, the proportional penetration of light into coral tissue should be relatively similar on the top and side of colonies (Kaniewska et al., 2011), yet the absolute response (as assessed by [F.sub.v]/[F.sub.m]) was reversed between tissue layers in these two positions, and disappeared when the experiment was conducted in April/May as compared to February. These findings suggest that additional aspects of the microenvironment within coral tissue are affecting the photo-physiology of Symbiodinium, and in this regard, chemical conditions might be important. Metabolic processes create conditions in the gastrovascular cavity and tissues of corals that differ from those on the open reef (Kuhl et at., 1995; Venn et al., 2009; Gordon and Leggat, 2010; Agostini et al., 2011), and aspects of these conditions affect algal photo-physiology. For example, hypoxia depresses [F.sub.v]/[F.sub.m] of Symbiodinium in corals (Ulstrup et al., 2005), p[CO.sub.2] increases the growth and photosynthesis of some (but not all) phylotypes of Symbiodinium (Brading et al., 2011), and ammonium favors increases in chlorophyll content of Symbiodinium, particularly at low cell densities (Dubinsky et al., 1990; Jones, 1997). As p[0.sub.2], p[CO.sub.2] and [[NH.sub.4].sup.+] are likely to differ in inner versus outer tissues of massive Ponies spp., it might be productive to investigate their roles in driving within-tissue phenotypic variation in Symbiodinium.

Acknowledgments

This research was supported by grants (OCE 04-17412, OCE 10-26852 to PJE, BIO-OCE 08-44785 to the University of Hawaii, and OCE-0752604 to RDG) from the National Science Foundation as well as gifts from the Gordon and Betty Moore Foundation; it was completed under a permit from the French Polynesian Ministry of Research. We thank N. Davies and the staff of the UC Berkeley and Richard B. Gump South Pacific Research Station for making our visits to Moorea productive, and we appreciate comments from two anonymous reviewers that improved an earlier draft of this paper. This is a product of the Moorea Coral Reef (MCR) LTER, and is contribution number 186 of the Marine Biology Program of California State University, Northridge, 1518 of the Hawaii Institute of Marine Biology, and 8749 of the School of Ocean and Earth Science and Technology, University of Hawaii.

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Received 12 January 2012; accepted 14 September 2012.

* To whom correspondence should be addressed. E-mail: peter.edmunds@csun.edu

PETER J. EDMUNDS (1) *, HOLLIE M. PUTNAM (2), AND RUTH D. GATES (2)

(1) Department of Biology, California State University, 18111 Nordhoff Street, Northridge, California 91330; and (2) University of Hawaii, Hawaii Institute of Marine Biology, PO Box 1346, Kaneohe, Hawaii 96744
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