Supplementary Information: Ocean acidification and elevated temperature negatively affect recruitment, oxygen consumption and calcification of the reef-building Dendropoma cristatum early life stages: evidence from a manipulative field study

Abstract

Figure S1: Vermetid cores were transplanted during low tide in Baia di Levante along pCO2 gradient. As said in the manuscript, cores were fixed to a bakelite slab and 14 cm diameter PVC tubing, both colored in white and black to manipulate the temperature in situ.

Figure S2: Embryos of Dendropoma cristatum. A) Egg capsule of D. cristatum released from the mother right after thermic shock and H2O2 treatment; in the other photos have been reported measurement on embryos of D. cristatum, B) length of the shell (um) taken through optical microscope connected to a camera (Leica MC 170 HD) C) wide (um), and D) shell surface in um 2. All photos were analyzed through image J.

Figure S3: Recruits of Dendropoma cristatum. A) red arrows indicate the recruitment of D. cristatum after 30 days of experiment in Baia di Levante, Isola di Vulcano. White line represents 1 cm scale. B) Recruits settled during experimental conditions and photographed under 20X magnification. The blue arrow indicates the white protruding edge that constitutes the limit between the protoconch and the teleoconch in D. cristatum recruits. White line represents 1 mm scale.

Figure S4: Investigation on the chemical composition of recruits shells, looking at Ca +2 , Mg +2 and Sr +2 concentration, by an energy dispersive spectrometer (EDS-LINK ISIS) integrated to the S.E.M.. Repetitive measurements were taken in six points of the shell (1 to 6). Points 1 and 2 are located in the Teloconch with the goal to analyze Ca, Mg and Sr concentration in the new portion of the shell grown under different environmental conditions. Points 3 to 6 were chosen with the purpose to investigate the oldest part of the shell, which

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Supplementary Information
Ocean acidification and elevated temperature negatively affect recruitment,
oxygen consumption and calcification of the reef-building Dendropoma
cristatum early life stages: evidence from a manipulative field study
Cinzia Alessia*, Folco Giomia, Francesco Furnaria, Gianluca Saràa, Renato Chemelloa,b, Marco
Milazzoa,b
aDepartment of Earth and Marine Sciences (DiSTeM), University of Palermo, via Archirafi 20-22, 90123 Palermo,
Italy
bConsorzio Nazionale Interuniversitario per le Scienze del Mare (CoNISMa), Rome, Italy
*Corresponding author: Cinzia Alessi, email: cinzia.alessi01@community.unipa.it
Supplementary table:
Table S1: Full statistical analyses (PERMANOVAs) performed in this study. Significant
effects are in bold.
Supplementary figures:
Figure S1: Vermetid cores were transplanted during low tide in Baia di Levante along
pCO2 gradient. As said in the manuscript, cores were fixed to a bakelite slab and 14 cm
diameter PVC tubing, both colored in white and black to manipulate the temperature in situ.
Figure S2: Embryos of Dendropoma cristatum. A) Egg capsule of D. cristatum released from
the mother right after thermic shock and H2O2 treatment; in the other photos have been reported
measurement on embryos of D. cristatum, B) length of the shell (um) taken through optical
microscope connected to a camera (Leica MC 170 HD) C) wide (um), and D) shell surface in
um2. All photos were analyzed through image J.
Figure S3: Recruits of Dendropoma cristatum. A) red arrows indicate the recruitment of D.
cristatum after 30 days of experiment in Baia di Levante, Isola di Vulcano. White line
represents 1 cm scale. B) Recruits settled during experimental conditions and photographed
under 20X magnification. The blue arrow indicates the white protruding edge that constitutes
the limit between the protoconch and the teleoconch in D. cristatum recruits. White line
represents 1 mm scale.
Figure S4: Investigation on the chemical composition of recruits shells, looking at Ca+2,
Mg+2 and Sr+2 concentration, by an energy dispersive spectrometer (EDS - LINK ISIS)
integrated to the S.E.M.. Repetitive measurements were taken in six points of the shell (1 to
6). Points 1 and 2 are located in the Teloconch with the goal to analyze Ca, Mg and Sr
concentration in the new portion of the shell grown under different environmental conditions.
Points 3 to 6 were chosen with the purpose to investigate the oldest part of the shell, which

developed inside eggs capsule and experimented also the settlement. We collected repetitive
measurements (three) in each point (1 to 6) and analyzed the average for shell portion
(Teloconch and Protoconch).
Figure S5: Percentage dissolution in the shell of Dendropoma cristatum recruits. A) Photos
were taken and analyzed using Image J with the goal to quantify dissolution of the organic
periostracum (dissolved area/ total area * 100). Recruit of D. cristatum collected from control
pH sites (left); recruit of D. cristatum settled in 7.8 pH sites (right). B) average percentage
dissolution of recruits settled and grown in different pH and T conditions.
Figure S6: The chemical composition of the D. cristatum embryos shell (MgO, CaO, SrO
%) was affected by pH and temperature despite their development inside egg capsules.
Mg was affected by both factors (PhXT Pseudo-F1-15= 7.2125; P(perm) = 0.0204), showing its
maximum present under elevated temperature and experimental pH. Sr was not affected by any
of the experimental factors, showing a normal distribution across replications. Even in this case
Ca shows higher concentration in the 8.1 pH sites (Pseudo-F1-15= 7.1784; P(perm) = 0.0205).
These results show a strong correlation between the chemical composition of the recruit shells
and embryo shells, suggesting that climate change could affect calcification in D. cristatum in
its more vulnerable stage such as embryos development and recruitment.
Supplementary video:
Video S1: Once the larvae are ready to hatch, the mother releases them and a new phase
begins. Crawling larval stage only takes a few hours and allows the larva to find a suitable
spot to recruit on in order to start a sessile life.

Table S1
Adult density
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
13507
13507
2.120
0.1767
8815
T
1
5297.2
5297.2
0.831
0.4043
8814
pHXT
1
28107
28107
4.413
0.0622
8806
Res
8
50950
6368.8
Total
11
97860
n. of egg capsules
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
5.3333
5.3333
0.310
0.6197
849
T
1
48
48
2.796
0.1279
858
pHXT
1
5.3333
5.3333
0.310
0.626
842
Res
8
137.33
17.167
Total
11
196
n. of embryos
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
2.288
2.288
0.345
0.5757
7113
T
1
37.145
37.145
5.607
0.0429
7014
pHXT
1
23.073
23.073
3.483
0.1007
7075
Res
8
52.995
6.6243
Total
11
115.5
Length embryos
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
0.12046
0.12046
46.54
0.0001
9826
T
1
5.39E-02
5.39E-02
20.80
0.0001
9831
pHxT
1
3.92E-02
3.92E-02
15.14
0.0001
9858
Res
640
1.6564
2.59E-03
Total
643
1.7887
Recruitment success
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
9130.1
9130.1
12.932
0.0097
7769
T
1
374.08
374.08
0.529
0.4867
7809
pHXT
1
1656.8
1656.8
2.346
0.1591
7840
Res
8
5648
706
Total
11
16809
Mortality Percentage
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
138.44
138.44
0.677
0.4343
8852
T
1
284.65
284.65
1.393
0.2778
8839
pHxT
1
28.281
28.281
0.138
0.7131
8807
Res
8
1633.7
204.21
Total
11
2085
Teloconch Surface area
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
8.38E-02
8.38E-02
5.437
0.0215
9836
T
1
7.90E-04
7.90E-04
5.12E-02
0.8154
9822
pHxT
1
3.51E-02
3.51E-02
2.274
0.1364
9849
Res
477
7.3516
1.54E-02
Total
480
7.4666
Recruits Shell % dissolution
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
1871
1871
14.391
0.0001
9852

T
1
28.272
28.272
0.21744
0.66
9844
pHxT
1
23.721
23.721
0.18245
0.6775
9832
Res
56
7281
130.02
Total
59
9204
Chemical composition of the recruit shells
df
SS
MS
PseudoF
P(perm)
Unique
perms
Mg2+ in
protoconch
pH
1
1.9525
1.9525
10.071
0.0088
9821
T
1
4.04E-04
4.04E-04
2.08E-03
0.9686
9839
pHxT
1
2.29E-02
2.29E-02
0.11796
0.7631
9852
Res
12
2.3263
0.19386
Total
15
4.3021
Mg2+ in
teloconch
pH
1
30.28
30.28
32.243
0.0001
9836
T
1
2.6671
2.6671
2.84
0.1099
9836
pHxT
1
4.1728
4.1728
4.443
0.0608
9855
Res
12
11.27
0.93913
Total
15
48.39
Sr2+ in
Protoconch
pH
1
3.11E-02
3.11E-02
1.094
0.3153
9828
T
1
6.34E-03
6.34E-03
0.223
0.6448
9833
pHxT
1
7.61E-02
7.61E-02
2.678
0.1285
9851
Res
12
0.34091
2.84E-02
Total
15
0.45445
Sr2+ in
Teloconch
pH
1
3.55E-02
3.55E-02
0.387
0.5431
9825
T
1
0.18652
0.18652
2.036
0.1816
9836
pHxT
1
1.0802
1.0802
11.79
0.006
9809
Res
12
1.0992
9.16E-02
Total
15
2.4014
Ca2+ in
Protoconch
pH
1
34.211
34.211
7.631
0.0193
9830
T
1
0.24092
0.24092
5.37E-02
0.8248
9844
pHxT
1
3.7411
3.7411
0.834
0.3758
9809
Res
12
53.794
4.4828
Total
15
91.987
Ca2+ in
Teloconch
pH
1
387.86
387.86
23.83
0.0009
9832
T
1
0.15242
0.15242
9.37E-03
0.9257
9848
pHxT
1
7.2168
7.2168
0.443
0.5117
9842
Res
12
195.24
16.27
Total
15
590.47
Chemical composition in the embryo shells
df
SS
MS
PseudoF
P(perm)
Unique
perms
Mg2+
pH
1
2.06E-02
2.06E-02
3.578
0.0828
9848
T
1
1.95E-02
1.95E-02
3.388
0.093
9803
pHxT
1
4.15E-02
4.15E-02
7.212
0.0204
9792
Res
12
6.91E-02
5.76E-03
Total
15
0.15067
Sr2+
pH
1
2.68E-03
2.68E-03
0.460
0.5097
9846
T
1
1.75E-02
1.75E-02
3.010
0.1049
9817
pHxT
1
2.78E-02
2.78E-02
4.774
0.053
9813
Res
12
6.99E-02
5.83E-03
Total
15
0.11801
Ca2+
pH
1
2.1165
2.1165
7.178
0.0205
9850
T
1
0.81531
0.81531
2.765
0.1232
9837
pHxT
1
0.46424
0.46424
1.574
0.2389
9846
Res
12
3.5381
0.29484
Total
15
6.9341
Recruits Oxygen Consumption
df
SS
MS
PseudoF
P(perm)
Unique
perms
pH
1
1894.4
1894.4
8.8093
0.0032
9814

Te
1
13136
13136
61.086
0.0001
9845
Tp
1
25.93
25.93
0.12058
0.7323
9837
pHxTe
1
1181.5
1181.5
5.4941
0.0222
9843
pHxTp
1
0.3351
0.3351
1.56E-03
0.9721
9853
T exTp
1
20.479
20.479
9.52E-02
0.7572
9830
pHxTexTp
1
1.7593
1.7593
8.18E-03
0.9269
9805
Res
56
12043
215.05
Total
63
28303

Figure S1

Figure S2

Figure S3

Figure S4
1
2
3
4
5
6

Figure S5
0
25
50
75
100
Ta T+ Ta T +
7.8pH 8.1pH
Disso l uti on
B

Figure S6
0
0.2
0.4
0.6
0.8
1
Ta T+ Ta T+
7.8pH 8 .1pH
MgO %concentrationinthe
embryosshel l
*
0
0.2
0.4
0.6
0.8
1
Ta T+ Ta T+
7.8pH 8 .1pH
SrO %conce ntrationinthe 
embryosshel l
80
85
90
95
100
Ta T+ Ta T+
7.8pH 8 .1pH
CaO %conc entration in
theEmbryosshell
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