1
Schoepf et al.: Annual coral bleaching and the long-term recovery capacity of coral
2
3
Supplementary material
4
5
1. Additional methods
6
7
(a) Lipid, protein, and carbohydrate
8
9
For all energy reserve measurements, only branch tips or samples with a growing
10
edge were used. Soluble lipids were extracted from a whole, ground coral sample
11
(skeleton + animal tissue + algal endosymbiont) in a 2:1 chloroform:methanol solution
12
for 1 hour [1, 2], washed in 0.88% KCl followed by 100% chloroform and another wash
13
with 0.88% KCl. The extract was dried to constant weight under a stream of pure
14
nitrogen (UPH grade 5.0) and standardized to the ash-free dry weight.
15
Animal soluble protein and carbohydrate were extracted from grinding a whole
16
second branch tip of the same fragment [2]. Briefly, Milli-Q water was added to the
17
ground coral sample and the resulting slurry was sonicated (5 min) and then centrifuged
18
twice (5000 rpm, 10 min) to separate the animal tissue from the skeleton and
19
endosymbiotic algae. Protein and carbohydrate was extracted from the animal tissue only.
20
One subsample of this animal tissue slurry was used for protein extraction using the
21
bicinchoninic acid method [3] with bovine serum albumin as a standard (Pierce BCA
22
Protein Assay Kit). A second subsample was used for carbohydrate quantification using
23
the phenol-sulfuric acid method [4] with glucose as a standard. Soluble animal protein
24
and carbohydrate concentrations were standardized to the ash-free dry weight.
25
The energetic content of lipid, protein, and carbohydrates is better assessed from an
26
energetic point of view [5], as specific enthalpies of combustion differ significantly
27
among energy reserve pools: -39.5 kJ g−1 for lipid, −23.9 kJ g−1 for protein, and −17.5 kJ
28
g−1 for carbohydrate [6]. Nevertheless, lipid, protein, and carbohydrate values are also
1
29
shown for each species in g per g ash-free dry weight in Figure S4 to facilitate
30
comparison with other studies.
31
32
(b) Statistical analyses
33
34
Multivariate analysis of similarity (ANOSIM), SIMPER and non-metric
35
multidimensional scaling (NMDS) were computed using Euclidean distances. All
36
variables were normalised. For ANOSIMs, 999 permutations were used. In addition to
37
the significance level, R-values give an absolute measure of separation for all groups. R-
38
values range from 0 (indistinguishable) to 1 (all compositional dissimilarities between
39
groups are larger than any dissimilarity among samples within groups). R-values >0.75
40
are interpreted as “well-separated groups”, R-values >0.5 as “overlapping but clearly
41
different groups”, R-values >0.25 as “strongly overlapping groups”, and R-values <0.25
42
as “barely separable groups” [7]. Since both ANOSIM and NMDS are based on rank
43
dissimilarities, they are non-parametric, complementary statistical procedures. For
44
NMDS, two dimensions were used for the ordination and a stress value of 0.2 was
45
obtained. Multivariate analyses were performed using PRIMER software, version 6.1.16.
46
Univariate three-way ANOVAs were run for each species separately.
47
Temperature was fixed with two levels (control and treatment), time was fixed with three
48
levels (0, 1.5, and 11 months), and genotype was a random factor with 9 levels. The
49
purpose of including genotype in the ANOVA model was to determine if any single
50
genotype was systematically different from all others for a given variable. In cases where
51
significant genotype effects were detected, Tukey post hoc tests revealed that the
52
distribution of the genotype average values completely overlapped such that no one
53
genotype was completely different from all of the others. As such, we concluded that the
54
selected colonies represented the natural variation in the population well as no single or
55
group of genotypes were consistently different from the others. This is reassuring since
56
full exploration of any genotype interaction terms was not possible because genotype was
2
57
not replicated within cells. Thus, interaction terms involving genotype were not included
58
in the ANOVAs.
59
For chlorophyll a concentration and calcification rates, values at 0 and 1.5 months
60
on the reef were taken from Schoepf et al. [8] and Grottoli et al. [9], respectively, and
61
combined with the values at 11 months on the reef to run the full ANOVA model. All
62
data were tested for normality using Shapiro-Wilk’s test. Outliers defined after Hoaglin et
63
al. [10] were removed from the entire data set when transformations alone did not
64
achieve normality. Homogeneity of variance was assessed with plots of expected versus
65
residual values. Post hoc slice tests [e.g., tests of simple effects, 11] determined if the
66
control and treatment averages significantly differed within each time interval and
67
species.
68
All 9 fragments for each species, treatment and recovery interval were collected
69
from each parent colony at the beginning of the study and had their own history from that
70
point forward. They were thus considered independent fragments, though with genetic
71
commonality with their respective parent colonies. ANOVAs without repeated-measures
72
were therefore regarded as the most appropriate statistical method. Since all fragments
73
were exposed to identical conditions except temperature during the single and repeat
74
bleaching treatment, any differences in the observed responses were due to temperature
75
effects alone and independent of seasonal variation. Bonferroni corrections were not
76
applied due to increased likelihood of false negatives [12, 13]. P-values ≤0.05 were
77
considered significant. Univariate analyses were performed using SAS software, Version
78
9.2 of the SAS System for Windows.
79
80
2. Additional results
81
82
(a) Health status
83
84
85
At the start of the repeat bleaching treatment, all coral fragments of all species
appeared healthy and non-bleached (i.e., dark brown or dark yellow in colour). After 17
3
86
days of exposure to elevated seawater temperature (= 0 months on the reef), 43%, 100%,
87
and 44% of treatment P. divaricata, P. astreoides, and O. faveolata, respectively, were
88
partially bleached (Fig. S4). Only one fragment of O. faveolata died during the repeat
89
bleaching treatment (Fig. S4C) but no mortality occurred in the other two species. After
90
1.5 months on the reef, the percentage of visibly bleached corals decreased to 22%, 84%,
91
and 6% in P. divaricata, P. astreoides, and O. faveolata, respectively, and the percentage
92
of dead fragments and/or fragments with partial mortality increased in both P. astreoides
93
and O. faveolata (Fig. S4). Except for P. astreoides, no coral fragments remained visibly
94
bleached after 11 months on the reef, but 14%, 33%, and 63% of all P. divaricata, P.
95
astreoides, and O. faveolata, respectively, showed partial mortality (Fig. S4).
96
97
(b) Tissue isotopes following repeat bleaching
98
99
P. divaricata. δ13Ch and δ13Ce did not differ between treatment and control corals
100
at any time point (Fig. 2A, B; Table S4). The average δ13Ch - δ13Ce value was greater in
101
treatment than in control corals after 0 months on the reef, but was the same for the
102
remainder of the year (Fig. 2C, Table S4). δ15Nh did not differ between treatment and
103
control corals following repeat bleaching but varied seasonally with maximum values
104
observed after 1.5 months on the reef (Fig. 2D, Table S4). However, δ15Ne of treatment
105
corals was more enriched than controls immediately after repeat bleaching, but had fully
106
recovered after 1.5 months (Fig. 2E, Table S4).
107
P. astreoides. δ13Ch of treatment corals did not differ from controls after 0 months
108
on the reef, but was more enriched than controls after 1.5 months and had fully recovered
109
after 11 months (Fig. 2F, Table S4). At the same time, δ13Ce did not differ between
110
treatment and control corals, but increased steadily over the 11 months following repeat
111
bleaching (Fig. 2G, Table S4). The average δ13Ch - δ13Ce values were the same in
112
treatment and control corals at 0 and 1.5 months on the reef but was significantly lower in
113
treatment corals compared to controls after 11 months on the reef (Fig. 2H, Table S4).
114
δ15Nh of treatment corals did not differ from controls after 0 months on the reef, but was
4
115
more enriched than controls after 1.5 months and had fully recovered by 11 months (Fig.
116
2I, Table S4). δ15Ne of treatment corals was more enriched than controls for the first 1.5
117
months on the reef, but had fully recovered by 11 months (Fig. 2J, Table S4).
118
O. faveolata. Treatment δ13Ch did not differ from controls after 0 months on the
119
reef, but was more depleted relative to controls after 1. 5 months on the reef and had fully
120
recovered after 11 months (Fig. 2K). δ13Ce of treatment corals was more depleted than in
121
controls throughout the study, particularly after 1.5 months on the reef (Fig. 2L, Table
122
S4). Average δ13Ch - δ13Ce was greater in treatment than in control corals after 0 months
123
on the reef, but did not differ between treatment and controls for the remainder of the
124
study (Fig. 2M). Though δ15Nh did not differ between treatment and control corals at any
125
given time point, δ15Nh was more enriched in treatment corals than in controls overall and
126
significantly declined after 11 months on the reef (Fig. 2N, Table S4). δ15Ne did not differ
127
between treatment and control corals at any point during the study but also significantly
128
declined after 11 months on the reef (Fig. 2O, Table S4).
129
130
5
131
References
132
133
134
135
1.
Grottoli AG, Rodrigues LJ, Juarez C. 2004 Lipids and stable carbon isotopes in
two species of Hawaiian corals, Porites compressa and Montipora verrucosa, following a
bleaching event. Mar. Biol. 145, 621-631.
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Rodrigues LJ, Grottoli AG. 2007 Energy reserves and metabolism as indicators of
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Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD,
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Clarke KR, Gorley RN. 2001 Primer v5: User Manual/Tutorial. (Plymouth,
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Grottoli AG, Warner M, Levas SJ, Aschaffenburg M, Schoepf V, McGinley M,
Baumann J, Matsui Y. 2014 The cumulative impact of annual coral bleaching can turn
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Hoaglin E, Musteller F, Tukey J. 1983 Understanding Robust and Exploratory
Data Analysis. New York, John Wiley & Sons; 447 p.
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11.
Winer BJ. 1971 Statistical Principles in Experimental Design. New York,
McGraw-Hill.
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12.
Quinn GP, Keough MJ. 2002 Experimental Design and Data Analysis for
Biologists. New York, Cambridge University Press.
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13.
Moran MD. 2003 Arguments for rejecting the sequential Bonferroni in ecological
studies. Oikos 100, 403-405. (doi:10.1034/j.1600-0706.2003.12010.x)
167
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168
Supplementary tables
169
170
171
172
173
174
175
176
177
178
179
180
Table S1. Results from SIMPER analyses for Porites divaricata, Porites astreoides
and Orbicella faveolata. Only variables contributing most towards dissimilarity due to
factor levels (i.e., >50% cumulative contribution) are listed. Av. Sq. Dist. = average
squared distance, Sq. Dist./SD = squared distance/standard deviation, Contrib. =
contribution, Cum. Contrib. = cumulative contribution, Mo = months, Calc =
calcification rate in mg day-1 cm-2, LipidJ = lipid content in J gdw-1, ProtJ = protein
content in J gdw-1, CarbJ = carbohydrate content in J gdw-1, Chla = chlorophyll a
concentration in μg cm-2, δ15Ne = nitrogen isotopic composition of the endosymbiont
fraction in ‰, δ13Ch-e = the difference between the carbon isotopic composition of the
host minus that of the endosymbiont in ‰. All data were normalised.
Factor
Variable
Av. Sq.
Dist.
Sq. Dist.
/SD
Contrib.
(%)
Cum.
Contrib. (%)
δ13Ch-e
ProtJ
Calc
Chla
ProtJ
δ13Ch-e
δ15Ne
CarbJ
δ13Ch-e
LipidJ
δ15Ne
2.45
1.84
1.78
4.12
2.12
2.04
3.85
3.65
3.31
4.42
3.64
0.50
0.82
0.66
0.98
0.65
0.68
1.25
0.93
0.70
1.34
1.29
21.79
16.34
15.80
28.16
14.47
13.96
19.15
18.15
16.43
29.27
24.15
21.79
38.13
53.93
28.16
42.63
56.59
19.15
37.30
53.73
29.27
53.42
Chla
δ15Ne
Calc
ProtJ
CarbJ
Calc
CarbJ
ProtJ
δ13Ch-e
LipidJ
δ15Ne
δ13Ch-e
3.47
2.45
2.35
2.73
2.61
2.32
3.28
2.70
1.96
2.28
2.26
2.05
0.98
0.49
0.66
0.64
0.67
0.75
1.05
0.97
0.48
0.98
0.49
0.51
21.23
15.01
14.35
19.78
18.95
16.82
23.74
19.55
14.22
19.68
19.47
17.65
21.23
36.25
50.60
19.78
38.73
55.55
23.74
43.29
57.51
19.68
39.15
56.80
P. divaricata
Temperature
Time: 0 vs. 2 mo
0 vs. 11 mo
2 vs. 11 mo
P. astreoides
Temperature
Time: 0 vs. 2 mo
0 vs. 11 mo
2 vs. 11 mo
8
O. faveolata
Temperature
Time: 0 vs. 2 mo
0 vs. 11 mo
2 vs. 11 mo
Chla
δ13Ch-e
LipidJ
Calc
δ13Ch-e
Chla
Calc
δ13Ch-e
δ15Ne
CarbJ
δ15Ne
2.26
2.15
1.90
2.50
2.42
2.42
3.80
2.82
2.74
4.90
3.85
0.87
0.56
0.71
0.81
0.58
0.58
0.84
0.62
1.11
1.13
0.95
181
182
9
20.24
19.30
17.03
18.75
18.19
18.16
23.63
17.54
17.07
28.66
22.49
20.24
39.54
56.57
18.75
36.94
55.10
23.63
41.18
58.24
28.66
51.15
183
184
185
186
187
188
189
Table S2. Vector correlations for each variable used in non-metric multidimensional
scaling (NMDS). Calc = calcification rate in mg day-1 cm-2, LipidJ = lipid content in J
gdw-1, ProtJ = protein content in J gdw-1, CarbJ = carbohydrate content in J gdw-1, Chla =
chlorophyll a concentration in μg cm-2, δ15Ne = nitrogen isotopic composition of the
endosymbiont fraction in ‰, δ13Ch-e = the difference between the carbon isotopic
composition of the host minus that of the endosymbiont in ‰. All data were normalised.
MDS 1
MDS 2
Calc
0.14894
-0.69089
LipidJ
0.39843
0.48579
ProtJ
0.79383
0.00478
CarbJ
0.77127
0.18672
190
10
Chla
0.37938
-0.55497
δ15Ne
-0.00189
0.51840
δ13Ch-e
-0.71964
-0.00775
191
192
193
194
195
196
197
Table S3. Results of three-way ANOVAs for chlorophyll a (Chl a), lipid, protein,
carbohydrate (carbs) concentration, and calcification (calc) rates of Porites
divaricata, Porites astreoides, and Orbicella faveolata. The effect of temperature
(Temp.) was fixed and fully crossed with two levels (control, treatment). Time was fixed
and fully crossed with 3 levels (0, 1.5, and 11 months). Genotype (Geno) was a random
factor with 9 levels (1-9). Significant p-values (p ≤ 0.05) are highlighted in bold.
Variable
P. divaricata
Chl a
Lipid
Protein
Carbs
Calc.
Effect
df
SS
F-statistic
p-value
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
13, 40
1
2
8
2
13, 39
1
2
8
2
13, 39
1
2
8
2
13, 39
1
2
8
1
13, 39
1
2
8
2
199.478
0.2038
98.1069
30.5531
52.6506
311177222.4
899469.2
164835833.1
46160587.2
50073665.5
3534981.2
2256.4
1802784.1
1672292.6
648.431
6747146.2
285680.4
4651028.9
1145273.6
234708.6
0.5093
0.0393
0.0840
0.4242
0.0082
3.63
0.05
11.59
0.90
6.22
5.43
0.20
18.71
1.31
5.68
0.41
0.00
1.37
0.32
0.00
1.75
0.96
7.83
0.48
0.40
1.12
1.12
1.20
1.52
0.12
0.0023
0.8280
0.0002
0.5286
0.0060
0.0001
0.6551
<0.0001
0.2820
0.0090
0.9513
0.9538
0.2721
0.9521
0.9995
0.1093
0.3356
0.0022
0.8575
0.6774
0.3863
0.2988
0.3169
0.1996
0.8896
Model
Temp.
Time
Geno
Temp. x Time
Model
13, 50
1
2
8
2
13, 51
509.98
278.02
7.9396
35.119
176.36
76540516.2
11
14.59
103.43
1.48
1.63
32.80
1.51
<0.0001
<0.0001
0.2415
0.1488
<0.0001
0.1578
P. astreoides
Chl a
Lipid
Protein
Carbs
Calc.
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
G
Temp. x Time
1
2
8
2
13, 52
1
2
8
2
13, 52
1
2
8
2
13, 47
1
2
8
2
1327495.8
4500687.5
54747857.7
14241105.3
49536946.3
12311820.7
11458755.9
8188692.0
15936155.8
8393328.1
345012.4
1399568.7
3014098.4
3433371.9
1.3173
0.3843
0.4149
0.1731
0.2798
0.34
0.58
1.76
1.83
6.86
22.18
10.32
1.84
14.35
4.07
2.17
4.41
2.37
10.82
5.76
21.83
11.79
1.23
7.95
0.5627
0.5659
0.1166
0.1745
<0.0001
<0.0001
0.0003
0.0980
<0.0001
0.0003
0.1483
0.0188
0.0345
0.0002
<0.0001
<0.0001
0.0001
0.3125
0.0015
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
13, 47
1
2
8
2
13, 45
1
2
8
2
13, 46
1
2
8
2
13, 46
1
2
8
2
13, 46
1
235.28
90.146
106.01
12.337
16.545
2.7056
0.1293
0.3878
1.4611
0.7504
11082849.9
4153.7
579949.3
9575975.6
554370.7
16.1426
0.0024
12.3004
2.2360
0.3875
4.8861
0.3245
12
3.56
17.72
10.42
0.30
1.63
1.52
0.95
1.42
1.34
2.74
2.72
0.01
0.93
3.82
0.88
8.33
0.02
41.28
1.88
1.30
3.49
3.02
0.0015
0.0002
0.0003
0.9597
0.2117
0.1631
0.3382
0.2571
0.2622
0.0795
0.0101
0.9090
0.4065
0.0028
0.4225
<0.0001
0.8990
<0.0001
0.0978
0.2860
0.0018
0.0917
O. faveolata
Chl a
Lipid
Protein
Carbs
Calc.
198
199
200
Time
2
2.7474
Geno
8
1.4813
Temp. x Time
2
0.4561
df, degrees of freedom; SS, sum of squares of the effect;
13
12.76
1.73
2.12
<0.0001
0.1279
0.1361
201
202
203
204
205
206
207
208
Table S4. Results of three-way ANOVAs for δ13Ch, δ13Ce, δ13Ch-δ13Ce, δ15Nh, and
δ15Ne of Porites divaricata, Porites astreoides, and Orbicella faveolata. The effect of
temperature (Temp.) was fixed and fully crossed with two levels (control, treatment).
Time was fixed and fully crossed with 3 levels (0, 1.5, and 11 months). Genotype (Geno)
was a random factor with 9 levels (1-9). Significant p-values (p≤0.05) are highlighted in
bold.
Variable
P. divaricata
δ13Ch
δ13Ce
δ13Ch - δ13Ce
δ15Nh
δ15Ne
Effect
df
SS
F-statistic
p-value
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
13, 31
1
2
8
2
13, 31
1
2
8
2
13, 31
1
2
8
2
13, 31
1
2
8
2
13, 31
1
2
8
2
8.8066
0.8290
0.0286
7.0898
0.4791
12.1400
0.1249
0.5134
9.8585
1.7399
4.0121
0.3104
0.4782
0.7805
1.3400
8.4067
0.4368
2.3564
3.3117
0.3331
4.2960
0.3398
2.4058
0.3257
0.1292
1.69
2.07
0.04
2.21
0.60
2.87
0.38
0.79
3.79
2.67
2.47
2.48
1.91
0.78
5.36
2.62
1.77
4.77
1.68
0.67
4.44
4.57
16.17
0.55
0.87
0.1492
0.1675
0.9651
0.0773
0.5606
0.0199
0.5433
0.4694
0.0090
0.0962
0.0385
0.1325
0.1765
0.6252
0.0149
0.0300
0.2000
0.0217
0.1724
0.5217
0.0021
0.0466
<0.0001
0.8060
0.4365
Model
Temp.
Time
Geno
Temp. x Time
Model
13, 50
7.2590
1
0.0342
2
2.1075
8
3.4347
2
1.2661
13, 50
35.3205
14
2.09
0.13
3.95
1.61
2.37
2.56
0.0392
0.7222
0.0278
0.1552
0.1071
0.0125
P. astreoides
δ13Ch
δ13Ce
δ13Ch - δ13Ce
δ15Nh
δ15Ne
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
1
2
8
2
13, 50
1
2
8
2
13, 49
1
2
8
2
13, 50
1
2
8
2
0.7803
20.7324
14.1237
2.0659
16.4006
1.6308
9.1049
3.8810
4.8776
22.0190
3.4501
13.5470
3.0233
2.9674
12.5850
4.5010
2.2163
3.2946
1.6751
0.74
9.77
1.66
0.97
4.06
5.24
14.64
1.56
7.84
6.07
12.37
24.29
1.36
5.32
6.77
31.55
7.75
2.88
5.86
0.3966
0.0004
0.1403
0.3871
0.0004
0.0278
<0.0001
0.1705
0.0014
<0.0001
0.0012
<0.0001
0.2492
0.0095
<0.0001
<0.0001
0.0015
0.0134
0.0062
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
Geno
Temp. x Time
Model
Temp.
Time
13, 47
1
2
8
2
13, 47
1
2
8
2
13, 47
1
2
8
2
13, 46
1
2
8
2
13, 47
1
2
15
19.9135
0.3334
3.4247
13.9428
2.2763
20.7655
4.3534
3.2393
12.3950
0.4332
9.0652
2.2774
1.9827
3.7909
1.0378
0.1744
0.0156
0.1061
0.0373
0.0005
11.7354
0.0948
7.1388
6.38
1.39
7.14
7.26
4.74
5.97
16.28
6.06
5.80
0.81
4.54
14.82
6.45
3.08
3.38
6.70
7.80
26.48
2.33
0.13
8.67
0.91
34.29
<0.0001
0.2467
0.0026
<0.0001
0.0153
<0.0001
0.0003
0.0056
0.0001
0.4532
0.0002
0.0005
0.0042
0.0100
0.0459
<0.0001
0.0086
<0.0001
0.0418
0.8779
<0.0001
0.3466
<0.0001
O. faveolata
δ13Ch
δ13Ce
δ13Ch - δ13Ce
δ15Nh
δ15Ne
209
210
Geno
8
3.3887
Temp. x Time
2
0.1639
df, degrees of freedom; SS, sum of squares of the effect.
16
4.07
0.79
0.0018
0.4631
6 fragments/colony
x 9 colonies
27
27
Treatment
Control
30.6 C
Summer
2010
30.4 C
°
°
15 days
31.5 C
17 days
31.6 C
0 month
9
reef
Single
bleaching
°
9
9
9
°
9
1.5 months
9
11 months
Repeat
bleaching
reef
Summer
2009
reef
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
Supplementary figures
reef
211
Figure S1. Flow diagram of experimental design and coral fragments collected in
the single and repeat bleaching experiment of 2009–2010. Orbicella faveolata
pictured. This schematic also applies to Porites divaricata and Porites astreoides. days =
days in the tanks, reef = time spent on the reef at ambient temperatures. Modified from
Grottoli et al. [9].
17
32
32
30
30
B
C
Reef
32
Control
o
Seawater temperature ( C)
Single bleaching
.
Repeat beaching
30
28
26
A
24
240
241
242
243
244
245
J
A
S
O
N
D
J F
2010
M
A
M
J
J
A
S
O
N
D
J F
2011
M
A
M
J
Date
Figure S2. Average daily seawater temperature records (A) throughout the study.
Inset boxes show details of average daily temperature profiles of the treatment and
control tanks during (B) the 2009 single bleaching and (C) the 2010 repeat bleaching.
Months are indicated by their first letter. Modified from Grottoli et al. [9].
246
247
248
18
249
250
251
252
253
254
255
256
257
258
259
260
261
262
Figure S3. NMDS plot representing the graphical relationship between response
variables (vectors) and the overall response of each species to repeat bleaching. Dark
and light green circles represent non-bleached control (MF2NB) and treatment (MF2BL)
Orbicella faveolata, respectively. Dark and light blue squares represent non-bleached
control (PA2NB) and treatment (PA2BL) Porites astreoides, respectively. Purple and
pink triangles represent non-bleached control (PD2NB) and treatment (PD2BL) Porites
divaricata, respectively. Calc = calcification rate in mg day-1 cm-2, chla = chlorophyll a
concentration in μg cm-2, lipidJ = lipid content in J gdw-1, protJ = protein content in J
gdw-1, carbJ = carbohydrate content in J gdw-1, δ15Ne = nitrogen isotopic composition of
the endosymbiont fraction in ‰, δ13Ch-e = the difference between the carbon isotopic
composition of the host minus that of the endosymbiont in ‰. All data were normalised.
Vector correlations for each variable are given in Table S2.
263
19
% of
treatment corals
P. divaricata
100
80
60
40
20
P. astreoides
B
A
0
1.5
11
O. faveolata
C
0
1.5
11
0
1.5
11
Months on the reef following repeat bleaching
264
non-bleached
partially bleached
bleached
partially dead non-bleached
partially dead bleached
dead
265
266
267
268
269
270
271
272
273
274
275
276
277
278
Figure S4. Health status of treatment (A) Porites divaricata, (B) Porites astreoides,
and (C) Orbicella faveolata after 0, 1.5, and 11 months on the reef following repeat
bleaching. Corals designated as non-bleached were dark brown in colour and completely
covered by living tissue. Partially bleached fragments were either entirely pale (but not
white) or some of the tissue was bleached and some healthy, and they were completely
covered by live tissue. Bleached fragments were either 100% white in colour or >50%
white and the rest pale, and they were completely covered by living tissue. Partially dead
non-bleached fragments were partially covered by filamentous or encrusting algae (or
both), and partially covered by patches of living tissue that was brown in colour. Partially
dead bleached fragments were partially covered by filamentous or encrusting algae (or
both), and partially covered by patches of living tissue that were pale in colour. Dead
fragments were completely covered by filamentous or encrusting algae (or both), with no
living tissue remaining.
279
20
P. astreoides
P. divaricata
Carbohydrate
-1
(g gdw )
Protein
-1
(g gdw )
Lipid
-1
(g gdw )
0.4
D
A
0.3
282
283
284
285
286
G
0.2
0.1
B
E
C
F
*
0.15
*
H
0.10
0.05
0.15
I
*
*
*
0
1.5
11
0.10
0.05
0
280
281
control
treatment
*
O. faveolata
1.5
11
0
1.5
11
Months on the reef following repeat bleaching
Figure S5. Average lipid, protein, and carbohydrate in g per g ash-free dry weight
of (A-C) Porites divaricata, (D-F) Porites astreoides, and (G-I) Orbicella faveolata
after 0, 1.5, and 11 months on the reef following repeat bleaching. Averages are
shown ± 1 SE. Asterisks indicate significant differences between treatment and control
corals within a time point and species. Sample size per average ranges from 5-9.
287
21