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SUPPLEMENTARY METHODS
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Water Chemistry
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At all sampling sites, a 556 YSI (Yellow Springs, USA) probe was used on multiple visits for
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rapid assessment of pH, salinity and temperature (Table S1). Two measurements in triplicate were
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performed daily (a.m. and p.m.) from 5th-17th May 2013 in both High and Mid sites (n=26 each). Since
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previous studies revealed very low pH variability in the Low site (e.g.[1]), only seven random
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measurements were performed at this site within the sampling period. The pH sensor was calibrated
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using NBS scale standard buffers and then soaked in seawater for one hour. For each site, average pH
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was calculated from hydrogen ion concentrations before reconverting back to pH values.
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Four water samples for Total Alkalinity (TA) were collected at each site (n=3 in Mid site) on
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15th May 2013 from a 100 ml water sample passed through 0.2 µm pore size filters, poisoned with 0.05
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ml of 50% HgCl2 to avoid biological alteration, and then stored in the dark at 4° C. Three replicate sub-
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samples were analyzed at 25° C using a titration system. The pH was measured at 0.02 ml increments
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of 0.1 N HCl. Total alkalinity was calculated from the Gran function applied to pH variations from 4.2
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to 3.0, as mEq Kg-1 from the slope of the curve HCl volume versus pH. Total alkalinity measurements
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were corrected using standards provided by A.G. Dickson (batch 99 and 102).
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Parameters of the carbonate system (pCO2, CO3--, HCO3-) and saturation state of calcite and
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aragonite were calculated from mean pH, TA, temperature and salinity using the free-access CO2
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SYStat package [2]. To accurately capture the variation in water conditions at the three sites for all
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parameters of the carbonate system, we report minimum and maximum values of the carbonate
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parameters calculated from minimum and maximum pH. Due to measurement uncertainty when
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measuring pH on the NBS scale, we estimated an uncertainly error of ± 0.05 pH units. For total
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alkalinity, we assume a measurement uncertainty of 10 µmol kg-1. We consider this uncertainty when
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calculating and reporting pCO2, CO3--, HCO3- and saturation state of calcite and aragonite following the
1
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methods in the Guide to Best Practices [3] and using the sensitivity coefficients in [4] (Table S1).
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Dissociation constants of H2CO3 and HCO3- were from [5] while that of HSO4- was from [6].
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Field Surveys
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We sampled three sites along this gradient ranging from present day conditions (~600 m from
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the main seeping area, hereafter referred to as the ‘Low’ site with a mean CO2 concentration of 418
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µatm, Ωarag 3.56), a ‘Mid’ site (~400 m from the main seeping area, 638 µatm CO2, Ωarag 2.62) and a
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‘High’ site (~300 m from the main seeping area, 2283 µatm CO2, Ωarag 0.96) (Figure S1, Table S1).
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Snorkel surveys were undertaken in March, May, September 2012, May 2013, and May 2014 to assess
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the presence and appearance of A. acetabulum along the gradient. During the May 2014 survey, percent
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cover was measured at three transects at each site (1-3 m depth), with 10 random 0.5 by 0.5 m photo-
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quadrats in each transect. Acetabularia acetabulum samples were collected from the three sites in May
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2013 for materials testing.
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Mechanics: Beam Bending
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We used static cantilever beam theory to quantify the mechanical performance of both the stem,
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(a structure) and the material of which it is made. This technique has been applied to a wide variety of
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marine organisms previously, from corals to algae to sea anemones, to better understand how these
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organisms bend in response to flow (see [7] for example). In general, a beam will bend more under a
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given load if is it longer, thinner, and/or made of less stiff material. The beam (a stem in this study) is
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first clamped at one end and extends horizontally. A known force is applied at a known length from the
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fixed end and the beam deflects downward. The degree to which the beam deflects for a given force
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and length is known as its flexural stiffness, a mechanical property of the entire structure that depends
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on beam cross sectional shape and material composition. Dividing flexural stiffness by second moment
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of area gives the stiffness of the stem material, regardless of the beam shape.
2
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Beam bending was quantified by hanging a weight (mass measured to the 10-4 g) on the stem to
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exert a force (F, mass times gravitational acceleration in N) to deflect the algal beam 10-15% of its
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length. Photographs were taken at a fixed distance with a scale before and after the weight was hung to
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measure beam length (L, m) and vertical deflection of the point on the beam where the weight was
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hung from its original position (y, m) to the nearest 10-6 m.
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Mechanics: Second Moment of Area
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The second moment of area, I (m4), is a measure of the distribution of area around a bending
axis, in this case the minimum possible I for a hollow elliptical beam [8]:
I = (π/4) (RM*Rm3)-(rM*rm3).
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RM and Rm are the outer major and minor radii, respectively, and rM and rm are the inner major and
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minor radii, respectively. Radii were measured from cross sections of each stem to the nearest 0.0001
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mm using a scanning electron microscope. Stiffness (E), a material property, was calculated by
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dividing flexural stiffness (EI) by second moment of area (I).
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Statistics
Percent cover was square root transformed and compared among sites with a one-way ANOVA.
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The proportion of calcified tissue was compared among sites with a one-way ANOVA. When data
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were not normally distributed, a Kruskal-Wallis test was used. When applicable, pairwise differences
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between groups were determined using a Tukey’s HSD or Dunn test for parametric or non-parametric
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post-tests, respectively. Flexural stiffness and stiffness did not meet the assumptions of normality and
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were compared among sites with a Wilcoxon Signed-Rank test. Regression analysis was conducted to
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describe the relationship between algal calcification and stiffness using linear, exponential and
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polynomial models. The exponential curve was generated using a linear model on log-transformed data.
3
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Standardized major axis (SMA) regression was used for the linear models. The most parsimonious
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model with the lowest AIC (Akaike’s Information Criterion) was selected to describe the data (Table
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S2). The raw data for this analysis is shown in Table S3.
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4
75
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Figure S1. Map of study site in Levante Bay (Baia levante) off of Vulcano Island, Sicily, Italy.
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5
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Table S1. Seawater carbonate chemistry along a pCO2 gradient off Vulcano (mean ± SD for salinity,
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temperature, alkalinity; mean ± uncertainty for pH, pCO2, HCO3-, CO3--, Ω Aragonite
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, Ω Calcite). From 5-17 May 2013, three sites at increasing distance from the main seeping area were
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sampled along the gradient: High (n=26), Mid (n=26), and Low (i.e., ambient) CO2 levels (n=7).
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Samples for total alkalinity analyses (n=4) were collected at each site on 15 May 2013.
High
Mid
Low
280
390
640
Salinity
38.13 (±0.06)
38.1 (±0.06)
38.11 (±0.07)
Temperature (˚C)
19.36 (±0.51)
19.35 (±0.45)
19.40 (±0.44)
pHNBS
7.53 (±0.05)
8.03 (±0.05)
8.19 (±0.05)
min – max
6.81 – 8.07
7.59 – 8.22
8.13 – 8.23
Total Alkalinity (µ mol kg-1)
2552 (±43)
2519 (±49)
2549 (±61)
pCO2 (µatm)
2283 (±118)
638 (±37)
418 (±29)
547 (±28) – 12638 (±665)
378 (±22) – 1977 (±116)
370 (±26) – 490 (±34)
62 (±2)
172 (±7)
234 (±11)
12 (±1) – 189 (±7)
69 (±3) – 243 (±10)
211 (±10) – 252 (±12)
2400 (±36)
2102 (±35)
1981 (±40)
2092 (±31) – 2521 (±38)
1926 (±32) – 2350 (±39)
1937 (±39) – 2038 (±41)
0.96 (±0.03)
2.62 (±0.10)
3.56 (±0.17)
1.06 (±0.04) – 3.71(±0.14)
3.21 (±0.15) – 3.84 (±0.18)
4.02 (±0.16)
5.47 (±0.26)
1.62 (±0.06) – 5.70 (±0.22)
4.92 (±0.23) – 5.90 (±0.28)
Distance from the vents (m)
min - max
CO3-- (µmol kg-1)
min - max
HCO3- (µmol kg-1)
min - max
Ω Aragonite
min - max 0.19 (±0.01) – 2.89 (±0.10)
Ω Calcite
1.47 (±0.05)
min - max 0.29 (±0.01) – 4.43 (±0.15)
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Table S2. Summary of regression analyses used to describe the relationship between stiffness, E, and
proportion calcified, C (Figure 2C). See supplementary methods for details. The exponential curve
was chosen because it had the lowest AIC.
R2
P
AIC
E = 1.26 e 9.18 * C
0.51
< 0.001
2
2
E = 5738.2C-3009.9
0.16
0.03
391
3
E = 1113.6C2 +993.8C-315.4
0.16
0.11
461
Regression
Parameters
Equation
Exponential
2
Linear
Polynomial
F
90
91
7
2.41
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Table S3. Raw data for Figure 2C, stiffness (E, MPa) as a function of proportion calcified of the stem.
Proportion
Calcified
0.27174
0.43353
0.51948
0.57432
0.59091
0.59242
0.61017
0.63362
0.66292
0.67052
0.67279
0.71739
0.72353
0.72934
0.73057
0.73077
0.73444
0.73770
0.73929
0.74011
0.74346
0.74477
0.74914
0.76981
0.77236
0.77465
0.77500
0.77725
0.77846
E
(MPa)
7.39
567.49
210.75
647.63
1131.39
337.08
208.15
1081.31
675.45
1443.84
343.64
777.51
993.66
945.03
2112.31
636.86
908
516.35
667.57
1040.4
1695.21
1165.71
864.43
3351.93
1448.04
607.82
422.89
433.36
515.69
93
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95
Supplemental References
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1. Boatta F, D'Alessandro W, Gagliano AL, Liotta M, Milazzo M, Rodolfo-Metalpa R, Hall-Spencer
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JM, Parello F. 2013 Geochemical survey of Levante Bay, Vulcano Island (Italy), a natural laboratory
98
for the study of ocean acidification. Mar. Pollut. Bull. 73, 485–494.
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(doi:10.1016/j.marpolbul.2013.01.029)
100
101
2. Pierrot D, Lewis E, Wallace DWR. 2006 MS Excel program developed for CO2 system calculations.
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ORNL/CDIAC-105a. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory,
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U.S. Department of Energy, Oak Ridge, Tennessee.
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(doi:10.3334/CDIAC/otg.CO2SYS_XLS_CDIAC105a)
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3. Riebesell U, Fabry VJ, Hansson L, and Gattuso J-P. (Eds.), 2010. Guide to best practices for ocean
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acidification research and data reporting, 260 p. Luxembourg: Publications Office of the European
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Union
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4. Dickson AG, Riley JP. 1978. The effect of analytical error on the evaluation of the components of
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the aquatic carbon-dioxide system. Mar. Chem. 6, 77-85.
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5. Roy RN, Roy LN, Vogel KM, Porter-Moore C. 1993 The dissociation constants of carbonic acid in
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seawater at salinities 5 to 45 and temperatures 0 to 45 C. Mar. Chem. 44, 249–267. (doi:10.1016/0304-
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4203(93)90207-5)
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6. Dickson AG. 1990 Standard potential of the reaction: AgCl(s) + ½ H2(g) = Ag(s) + HCl(aq), and the
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standard acidity constant of the ion HSO4− in synthetic sea water from 273.15 to 318.15 K. J. Chem.
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Thermodyn. 22, 113–127. (doi:10.1016/0021-9614(90)90074-Z)
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101, 963–970. (doi:10.1111/1365-2745.12099)
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8. Myers, JA. 1962 Handbook of Equations for Mass and Area Properties of Various Geometrical
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Shapes. U.S. Naval Ordnance Test Station, Publication 2838. China Lake, CA: Publishing Division
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