Supplementary Methods.
Collection and maintenance of corals. Analyses of variable chlorophyll fluorescence
(Fv/Fm; see below) utilised Pocillopora verrucosa because the relatively broad branch tips
of these corals provided samples exposed to a relatively uniform environment (primarily,
downward irradiance) among subjects, both under experimental conditions and with
respect to previous acclimatisation in nature. The spatial scale of these tissue samples also
approximated that of fluorometer fiberoptics.
In April 2002, two adjacent branches (clone-mates) were collected from the
approximate centres of medium-to-large size (15-20 cm diameter) colonies of P. verrucosa
living at ~ 1.5 m depth on upper, exposed surfaces of the fringing reef adjacent to Anae
Island, Guam (Site C in ref. 1). Identities of Symbiodinium in these corals could be
inferred from previous observations of bleaching and recovery (personal observations). To
obtain corals hosting different Symbiodinium from identical habitat, colonies were sampled
in pairs. Each pair comprised two adjacent (< 30 cm apart; same depth) corals, one hosting
Symbiodinium C and the other Symbiodinium D. Different colony pairs were separated by
> 5 m. Donor corals appeared healthy and analyses of neighbouring corals showed that
corals hosting Symbiodinium C versus D had similar contents of chlorophyll a.
Oxygen fluxes (Pmaxnet and R; see below) were measured using P. damicornis. The
more-finely branching morphology of P. damicornis allowed well-matched clone-mates
(paired samples, see below) to be obtained from one colony with relatively little physical
trauma, which was considered important when measuring whole-coral physiological
parameters. Also potentially relevant to the fact that oxygen fluxes were determined from
whole corals, the morphology of P. damicornis results in slightly more uniform exposure to
irradiance than in the case of P. verrucosa, and also made whole-coral illumination during
analyses less dissimilar to natural illumination. However, the irregular morphology of P.
damicornis provided poor control (among samples) for environmental history and
experimental irradiance at the scale of fluorometer fiberoptics, making these corals less
suitable for analyses of Fv/Fm. Evidence implies that Symbiodinium C and D in P.
damicornis are identical to Symbiodinium C and D (respectively) in P. verrucosa 1.
Branches of P. damicornis were collected in July 2002 from a narrow, fringing reef
platform at Double Reef, Guam at 1 m depth (between Sites C and E in ref. 1). The
collection site was known from previous surveys 1 to have P. damicornis hosting
Symbiodinium C interspersed with P. damicornis hosting Symbiodinium D. Specific corals
were chosen based on their size (15-20 cm diameter); appearance (typical ramose colony
morphology and no evidence of recent trauma); full exposure to sunlight; and the absence
of endolithic symbionts (algae or sponges). One branch (6-8 cm long) was chiseled from
the approximate centre of each colony; these branches were chosen for the potential to
obtain two replicate (i.e., similar in size, shape, original exposure, and colour) terminal
sub-branches from each one of them.
Collected coral branches were transported alive to the laboratory and placed in
individual clear glass bowls (325 ml volume) supplied with atmosphere-equilibrated
flowing seawater (~ 1.5 L  min-1 into each bowl). In April (P. verrucosa), this seawater
was pumped from the ocean and ranged in temperature between 28.3 C and 29.6 C; in July
(P. damicornis), seawater was pumped from a subterranean well (temperature, 28.1-28.4 C;
salinity, 33.7-34.1 ‰). Irradiance was sunlight attenuated (by ~ 85%) by a high roof of
translucent white polyvinyl chloride (PVC) sheet.
The following day, coral branches were pruned using a wire cutter (underwater, in a
large tank of flowing seawater) to create experimental subjects. Branch tips of P.
verrucosa (terminal ~ 1.5 cm) were mounted on the sides of 4-cm long, square sticks of
acrylic plastic using epoxy putty (A-788 Splash-Zone Compound) applied directly to the
broken branch surface. Branch tips were oriented vertically when the plastic sticks, which
served as stands, were horizontal. Each branch of P. damicornis was divided into two
replicate sub-branches of size 1.0-1.8 ml (measured by displacement); these sub-branches
were set on platforms of polypropylene mesh (~ 4 mm mesh size) mounted on 1.5-cm high
plastic stands. Survival of experimental subjects was 100%. For every coral, one left-over
piece was used to determine Symbiodinium identity by examining genes encoding large
ribosomal subunit RNA, as described 1. These identities were verified for every
experimental subject after experiments were completed.
Pruned and mounted corals were returned to glass bowls of flowing seawater,
keeping any clone-mates (branches from the same colony) together and branches from
different colonies separate. The following day, corals were transferred to colourless,
translucent polypropylene cups (230 ml volume) and then placed under experimental
conditions (see below). Cups, plastic coral stands, and mesh platforms were fouled by
algae, which were controlled in part using small snails. Additionally, cups were changed
for clean ones every other day. When cups were changed, coral stands were cleaned using
forceps and a small spatula; mesh platforms were changed. These operations were done
around sunset in a large tank of flowing, aerated seawater (corals remained submerged at
all times).
Beginning the night of the day they were collected, and on every night thereafter,
corals were fed freshly-hatched nauplii of Artemia sp. Seawater flow was shut off, and
nauplii were added to a density of 10-15  ml-1. Twenty to 30 min later, seawater flow was
resumed for 30-60 min, after which feeding was repeated twice.
Measurement of environmental temperature and irradiance. All thermometric devices
were cross-calibrated (to 0.1C) to one, high-resolution (0.05 C) mercury thermometer.
Irradiance was measured as photosynthetically-available radiation (PAR, 400-700 nm).
Irradiance experienced by corals in their natural environment was measured using the 2π
cosine-corrected fiberoptic sensor of a submersible, pulse-amplitude modulated
fluorometer (DIVING-PAM, Heinz Walz GmbH, Effeltrich, Germany). Downward
irradiance was measured by placing the sensor directly on top of a coral; within-colony
irradiance was measured by threading the fiberoptic filament among branches, from one
side, with the sensor directed perpendicular to branch axes (to approximate the side of a
colony branch).
Experimental conditions. Corals in plastic cups (above) were kept outdoors in a four-row
grid pattern and supplied with seawater pumped from one of two insulated, polyethylene
reservoirs, each of which held ~ 180 L. Seawater in each reservoir was mixed vigorously
and atmosphere-equilibrated by bubbling air, and temperature was recorded at five-minute
intervals (Stowaway TidBit temperature loggers, Onset Computer Corp.); frequent
comparisons of temperatures within reservoirs versus coral-containing cups, using a digital
thermometer, revealed differences of ≤ 0.1 C. One seawater reservoir was supplied with
seawater pumped from the ocean (P. verrucosa) or from a subterranean well (P.
damicornis). Oceanic seawater was first passed over the titanium heat-exchangers of two
mechanical chillers that were controlled by an on/off controller regulated by a thermistor,
to keep seawater in the reservoir between 28.2 C and 28.7 C. Chillers were not used with
subterranean seawater, which remained cool naturally.
The second seawater reservoir was demand-supplied from the first reservoir by
gravity flow. It contained three sets of aquarium heaters, each controlled by an on/off
timer. These were used to create daily temperature changes (temperature "spikes") similar
to those observed in nature 1, by having the heater sets turn on and off sequentially. To
regulate maximum daily temperature, the final heater set was controlled additionally by a
thermistor in the reservoir.
Each seawater reservoir supplied one plastic delivery manifold, which in turn
supplied seawater to one-half of the grid of experimental corals. One manifold supplied
rows 1 and 3; the other supplied rows 2 and 4. The rate of seawater flow into each cup was
adjusted to 1.2 – 1.5 L  min-1 by milling apertures and by adjusting manifold pressure with
a gate-valve at the end of each manifold. Apertures were cleaned daily, and flow rates
were measured every other day. In the double paired-sample design (P. verrucosa), paired
cups were placed next to one another, in adjacent (treatment versus control) rows, and had
inflow rates within 10% of each other. To limit the possibility of a “row” or “manifold”
effect, the positions of the heated (treatment) and the non-heated (control) rows were
switched every two days. To obviate pseudoreplication 2 of treatment, seawater
overflowing cups was not re-circulated.
Corals were exposed to sunlight diffused by an overhead basin of semi-clear,
colourless polypropylene containing ~ 12 cm depth of clean water, through which air was
constantly bubbled from a submerged grid of small diffusers to create a dynamic light
environment 3 for the corals below. The entire grid of corals was fenced with white PVC
sheet (~ 90% light attenuation) so that corals received sunlight primarily from above. That
sunlight was adjusted with one or more layers of neutral-density shade cloth (~ 20% or ~
50% attenuation) suspended above the polypropylene basin. Downward irradiance at one
fixed location within the grid of experimental corals was monitored and recorded as 15minute averages using a cosine-corrected PAR sensor (International Light Model
SUD033), a radiometer (International Light model IL1700), and a digital data-logger (LiCor Model LI1400). Experimental irradiance data presented in Fig. 1b and quoted below
were obtained from this sensor.
Experimental designs. Branch tips of P. verrucosa were paired randomly, one hosting
Symbiodinium C and one hosting Symbiodinium D, in one cup. For each of these pairs, a
set of clone-mates was pared identically in a separate cup. The 14 cups of coral were
placed under experimental conditions at 28.5  0.2 C (mean  one standard deviation,
calculated from temperatures recorded continuously at five-minute intervals; hereafter,
28.5 C) and irradiance of no more than ~ 50% full sunlight (889  208 µE · m-2 · sec-1
downward PAR between 12:30 and 13:30 hr; mean of all days  one standard deviation).
For comparison, downward PAR on colonies of P. verrucosa between 12:30 and 13:30 hr
at the collection site was 540  120 µE · m-2 · sec-1 (n = 40 corals) on a moderatelyovercast day and 1090  188 µE · m-2 · sec-1 (n = 35 corals) on a mostly-sunny day. Corals
were positioned haphazardly in the experimental grid, except that cups containing clonemates (paired cups of coral) were kept side-by-side in rows 1 and 2 or in rows 3 and 4.
After 14 days of acclimation at 28.5 C, paired cups of coral were assigned to treatment
versus control groups by coin-toss.
Control-group corals remained at 28.5 C. On days 15 through 20, corals in the
treatment group received temperature spikes beginning at 08:00 hr and rising to and
holding at 31.3  0.2 C (= 31.3 C) between 11:30 and 16:00 hr, after which temperature
decreased gradually back to 28.5 C at 20:30 hr. On days 21 through 25, the spikes were
increased to 32.0  0.2 C (= 32.0 C) between 11:30 and 16:00 hr. During treatment,
temperature between spikes (i.e., 20:30 to 08:00 hr) was 28.5 C. Treatment stopped after
day 25, and all corals remained at 28.5 C thereafter.
On four days of each treatment period, Fv/Fm was determined in every coral
between 12:30 and 16:00 hr in the laboratory, as follows. Two cups (paired treatment and
control) were placed in two dark boxes with flowing seawater for 45 min. Then, working
in the dark, one of the cups of coral (chosen by coin-toss) was transferred to, and
submerged in, a flowing seawater bath that held a submerged acrylic jig holding the
fluorometer’s fiberoptic probe opposite a slot that received the acrylic stick on which each
coral was mounted. By placing the stick in the jig (by feel, in the dark), a coral was placed
precisely beneath the fiberoptics, at a distance of 3-4 mm. Once the coral was so placed,
Fo and Fm were measured (see below). After the other dark-acclimated coral in the same
cup was measured the same way, the seawater bath was flushed; the corals in the second
dark-acclimated cup were measured the same way; and then all four corals were returned to
the outdoor experiment. In this way, corals paired within cups (Symbiodinium C versus
Symbiodinium D) were measured within < 5 min of each other; corals in paired cups
(clone-mates under control versus treatment conditions) were measured within 10 min of
each other. Measurements were ordered so as to obtain replicate data from each coral
throughout the 3.5-hr measurement period, on different days.
The four measurements obtained from each coral during each test period were
averaged, and those averages were used to represent each subject for each test period (data
in Fig 1a). Corals were compared using two-tailed Wilcoxon paired-sample tests, pairing
clone-mates to test the effect of treatment, pairing different corals in the same cup to
compare Symbiodinium C versus Symbiodinium D, or pairing repeated measures (i.e.,
averages for the two test periods) on the same coral to test for changes. As there was no
difference in Fv/Fm of control (28.5 C) corals between the first (31.3 C) and second (32.0
C) treatment periods (P > 0.5), averages for the entire experiment (i.e., eight measurements
on each coral) are given in Fig 1a.
The data shown in Fig. 1b are the eight daily averages for the seven corals hosting
Symbiodinium D at constant 28.5 C (controls), and also data obtained from the same corals,
using the same methods (but omitting the dark-acclimation step), on three days before
dawn (between 04:00 and 05:30 hr). The latter, pre-dawn values of Fv/Fm were averaged
across the three days and represent corals exposed to "no irradiance" in Fig. 1b. Also in
Fig. 1b are afternoon (as above) data obtained on a day of especially low irradiance during
a tropical storm; data from this ninth day were not included in the analysis reported in Fig.
1a.
Branch ends of P. damicornis were kept at 28.2  0.1 C (= 28.2 C) under shading
that produced 292  111 µE · m-2 · sec-1 PAR between 12:30 and 13:30 hr. This relatively
low irradiance (compare with P. verrucosa, above) was maintained throughout the
experiment and exposed many Symbiodinium to lower-than-average PAR, but also avoided
exposing any Symbiodinium to unnaturally-high PAR. For comparison, PAR
measurements made within colonies of P. damicornis at the collection site (n = 33 corals,
measured between 11:30 and 13:00 hr) indicated that more-shaded parts of experimental
branches would normally experience 270  180 µE · m-2 · sec-1 PAR on a mostly-sunny
day. Clone-mates were kept together in one cup prior to temperature treatment, and every
evening each coral was repositioned on its supportive plastic mesh to obviate prolonged
(days) self-shading of any one part of the colony.
Starting on day 8 (after corals were placed under experimental conditions) and
continuing for nine consecutive days thereafter, one pair of clone-mates of each type
(Symbiodinium C versus D) was chosen randomly and each pair of clone-mates was split
between two cups, one of which was assigned to the treatment group by a coin-toss (the
other remained at 28.2 C). Treatment was a daily temperature spike beginning at 08:00 hr
and ending at 20:00 hr and holding at 32.0  0.2 C (= 32.0 C) between 11:30 and 16:00 hr.
On the second day of treatment, between 12:30 and 16:00 hr, corals were brought
into the laboratory, one-at-a-time, for measurement of oxygen fluxes (see below). Four
corals were measured each day to produce one comparison of Symbiodinium C versus D.
On the first day of measurements, measurement order was determined by coin-tosses: the
first coral by two tosses (one for Symbiodinium C versus D; one for control versus
treatment); the second coral was the clone-mate of the first; and the third- and fourthmeasured corals were the other zooxanthellar genotype, in an order (control versus
treatment) determined by a coin-toss. On following days, the order was permuted to ensure
that measurements on different genotypes, controls, and treatments were not clustered 2
temporally. Clone-mates were compared using two-tailed Wilcoxon paired-sample tests to
test the effect of treatment. Corals hosting Symbiodinium C versus D were compared using
the two-tailed Mann-Whitney U-test.
Measurements of variable chlorophyll fluorescence. Variable chlorophyll fluorescence
was measured using a pulse-amplitude modulated fluorometer (DIVING-PAM, Heinz
Walz GmbH). The fluorometer's battery was changed as needed to maintain consistent
illumination, which was calibrated using the fluorometer’s PAR sensor. After darkacclimation and the positioning of a coral beneath fluorometer fiberoptics (see above),
minimum chlorophyll fluorescence (Fo) was measured using measuring light setting 6 in
“burst mode.” This was followed by a pulse of saturating actinic light (2 sec of ~ 2,800 µE
· m-2 · sec-1 PAR) and the measurement of maximal fluorescence (Fm), from which the
maximum quantum yield of PSII was calculated [Fv/Fm = (Fm – Fo)/Fm] 4.
Measurements of net photosynthesis and respiration. Using plastic forceps, a coral was
removed from its cup and placed in an upright position within a clear polycarbonate, waterjacketed respirometry chamber (volume, 28.8 ml) containing filtered (0.45 µm pore) and
autoclaved seawater that had been pre-equilibrated with ambient air and adjusted to 28.3 C
(for corals maintained at 28.2 C) or 32.0 C (for corals treated at 32.0 C). Seawater within
the chamber was mixed continuously using a magnetic stirrer on the bottom, and held at
28.3 C or at 32.0 C by pumping thermoregulated water through the jacket. After flushing
the chamber several times with additional sterile, pre-equilibrated seawater during ~ 5 min,
the chamber was sealed from above with a silicone stopper into which a microcathode
oxygen electrode (Strathkelvin Instruments, Model 1302 and electrode holder Model RC
300) was mounted. For making measurements at different temperatures, one electrode was
calibrated and held at 28.3 C and a second electrode was calibrated and held at 32.0 C.
Electrode outputs were measured (Strathkelvin Instruments, Model 781) and recorded
using a digital interface (Sable Systems Universal Interface) and data acquisition software
(Sable Systems Datacan).
Corals in the chamber were illuminated from one side with light produced by a 150W tungsten-halogen bulb and delivered by a terminally-splayed fiberoptic bundle (Walz
GmbH, Fiber-Illuminator FL-440). The opposing two-thirds of the chamber jacket were
surrounded by a curved, chrome-plated reflector; measurements taken within the chamber
using a cosine-corrected PAR sensor indicated that surfaces facing away from the light
source received 55% of the PAR received by surfaces facing the light source. Different
light intensities (13, 30, 65, 120, 220, 410, 740, and 1400 µE · m-2 · sec-1 PAR; measured
within the chamber, facing the light source) were produced by placing neutral-density
optical filters in the fiberoptic light path.
In preliminary tests, three corals of each type (Symbiodinium C versus D) were
tested at 28.3 C at all eight levels of illumination (and in the dark) to determine overall
photosynthesis-irradiance relationships (data not presented). Net photosynthetic rate was
light-saturated in all six corals at 410, 740, and 1400 µE · m-2 · sec-1 PAR, and estimates of
Ek, the irradiance at which photosynthesis begins to saturate 5, were similar for corals
hosting Symbiodinium C versus Symbiodinium D (136  23 versus 134  18 µE · m-2 · sec-1
PAR, respectively). Based on these observations, 740 µE · m-2 · sec-1 PAR was used to
measure photosynthetic capacity (maximum net photosynthetic rate, Pmaxnet) of all
experimental corals. Respiration (R) was measured in the dark.
During measurements, oxygen was first allowed to drop to 8-10% below saturation
with low photosynthetic rate (room lights only). Then the chamber was illuminated to
measure Pmaxnet until oxygen reached 8-10% above saturation; and then all light was
extinguished to measure R. This was done once per coral, and rates were calculated from
linear rates of change observed within  5% oxygen saturation. Immediately after
measurements were made, corals were frozen at –80 C. Later and under dim lighting,
tissue was removed from coral skeletons by blasting, using an artist's airbrush and ice-cold
phosphate-buffered saline (Sigma Chemical Company), and then homogenised in glass
dounce-type homogenizer at 0-5 C 1. One subsample of each tissue homogenate was fixed
with formalin and used to enumerate cells of Symbiodinium by haemacytometry; other
subsamples were sonicated and analysed for chorophyll a 6 and for total soluble protein 7
by fluorometry. Effect of treatment was tested using two-tailed Wilcoxon paired-sample
tests (pairing clone-mates); corals hosting Symbiodinium C versus Symbiodinium D were
compared using two-tailed Mann-Whitney U-tests.
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