DOI: 10.5061/dryad.qv9s4mwbw
title: Data from: Amelioration of ocean acidification and warming effects through physiological buffering of a macroalgae

description: Concurrent anthropogenic global climate change and ocean acidification is
 expected to have a negative impact on calcifying marine organisms. While
 knowledge of biological responses of organisms to oceanic stress has
 emerged from single species experiments, these do not capture ecologically
 relevant scenarios where the potential for multi-organism physiological
 interactions is assessed. Marine algae provide an interesting case study,
 as their photosynthetic activity elevates pH in the surrounding
 microenvironment, potentially buffering more acidic conditions for
 associated epiphytes. We present findings that indicate increased
 tolerance of an important epiphytic foraminifera, Marginopora vertebralis,
 to the effects of increased temperature (±3 °C) and pCO2 (~1000 µatm) when
 associated with its common algal host, Laurencia intricata. Specimens of
 M. vertebralis were incubated for 15 days in flow-through aquaria
 simulating current and end-of-century temperature and pH conditions.
 Physiological measures of growth (change in wet weight), calcification
 (measured change in total alkalinity in closed bottles), photochemical
 efficiency (Fv/Fm), total chlorophyll, photosynthesis (oxygen flux), and
 respiration, were determined. When incubated in isolation, M. vertebralis
 exhibited reduced growth in end-of-century projections of ocean
 acidification conditions, while calcification rates were lowest in the
 high-temperature, low-pH treatment. Interestingly, association with L.
 intricata ameliorated these stress effects with the growth and
 calcification rates of M. vertebralis being similar to those observed in
 ambient conditions. Total chlorophyll levels in M. vertebralis decreased
 when in association with L. intricata, while maximum photochemical
 efficiency increased in ambient conditions. Net production estimates
 remained similar between M. vertebralis in isolation and in association
 with L. intricata, although both production and respiration rates of M.
 vertebralis were significantly higher when associated with L. intricata.
 These results indicate that the association with L. intricata increases
 the resilience of M. vertebralis to stress, providing one of the first
 examples of physiological buffering by a marine alga that can ameliorate
 the negative effects of changing ocean conditions.
description: Collection and acclimation Specimens of
 <i>Marginopora vertebralis </i>(as identified by Renema 2018)
 and <i>Laurencia intricata</i> were collected from Coconut
 Beach (1-3 m depth), Lizard Island (014°40’08”S, 145°27’34”E) on the Great
 Barrier Reef, Australia, in October 2015 (Fig. 1). Samples were
 immediately transported back to Lizard Island Research Station and placed
 into flow-through ambient seawater conditions and light for 5 days to
 acclimate to laboratory conditions. Specimens of <i>L. intricata
 </i>were then separated into ~1 g (wet weight) replicates, and all
 visible epiphytes (<i>M. vertebralis</i> and other LBFs such
 as <i>Calcarina hispida, Amphistegina lobifera</i>, and
 <i>Baculogypsina sphaerulata</i>) removed. The <i>M.
 vertebralis</i> were separated into experimental replicates in which
 6 <i>M. vertebralis</i> (~0.5 g wet weight, all approximately
 similar size of ~5 mm diameter) were placed into 60 mL jars with 40 mL of
 seawater, similar to densities found <i>in situ </i>(Doo,
 pers. obs.). The experimental treatment groups of <i>M.
 vertebralis</i> only, and <i>L. intricata
 </i>with<i> M. vertebralis </i>were established prior to
 the start of the experiment, and acclimated 3 days before the initiation
 of the experiment. Specimens were incubated in polypropylene jars with a
 hole cut from the side to prevent overflow over the top. A 462 µm plankton
 mesh was glued to the side of the jar to allow for overflow of water
 through the mesh, while maintaining flow-through conditions, and resulted
 in a total of ~40 mL water in each container. Light was provided using LED
 cool white lights (LED Type 3528) to an intensity of ~100 µmol photos
 m<sup>-2</sup> s<sup>-1</sup> for the duration of
 the experiment (2 weeks) in a 12hr:12hr, day-night cycle. A flow-through
 dripper tap system was used for experimental water delivery (~40 mL
 min<sup>-1</sup>). Experimental conditions were gradually
 reached over a three-day period, with an increase of 1°C, and decrease of
 0.1 pH unit each day prior to the start of the experiment when all
 replicates were incubated in ambient temperature (~25.5°C and
 ~pH<sub>Total</sub> 7.95 conditions). Experimental water was
 collected from Lizard Island lagoon and filtered with a 5 µm filter bag,
 and delivered into 60L header tanks, from which all treatment groups were
 supplied incubation water. Two temperatures (ambient
 [26°C] and high [29°C]), and two pH’s (ambient [8.0] and low [7.7]
 pH<sub>Total</sub> units) fully orthogonal treatment groups
 were used. To determine the effect of symbiosis of <i>L.
 intricata</i> and <i>M. vertebralis</i>, 10 replicates
 each of <i>M. vertebralis </i>only, and<i> L.
 intricata</i> with <i>M. vertebralis</i> were incubated
 in each of the pH/temperature treatment groups for a total of 80
 replicates. Incubation parameters and seawater
 chemistry The seawater pH and temperature conditions
 were controlled using a Neptune Apex system dosing pure
 CO<sub>2</sub> to regulate pH. In the experiment, a total of 4
 sumps were used, one for each manipulated seawater condition (see above).
 This water was pumped into individual jars (see above), maintaining
 independence between replicates. Total alkalinity, pH, and temperature of
 the header tanks were measured on a daily basis, from randomly selected
 drippers. Total alkalinity samples were filtered with a 0.22 µm filter
 prior to analysis to eliminate possible contamination of calcium carbonate
 in the sample and measured using open-cell potentiometric titrations (DOE
 1994). Seawater pH was monitored using m-cresol spectrophotometric
 measurements on an Ocean Optics USB4000+ spectrophotometer, and
 pH<sub>Total</sub> calculated based using standard protocols
 (DOE 1994). These were referenced to seawater Certified Reference Material
 (CRM), Batch 161, prepared by A. Dickson in the Scripps School of
 Oceanography. Temperature and salinity measurements were collected with
 Vernier TMP-BTA and CON-BTA probes, respectively. Seawater parameters
 remained stable throughout the experimental incubation (Table
 1). Growth measurements The wet
 weight of <i>M. vertebralis </i>across individual replicates
 were pooled within replicate jars and measured prior to the start of the
 experiment, and at the termination using a Mettler-Toledo ML240 balance to
 10<sup>-4</sup>g resolution. Measurements were then converted
 into a percentage daily change in weight. In treatments of both
 <i>L. intricata </i>and <i>M. vertebralis</i>,
 only the pooled <i>M. vertebralis </i>from each replicate were
 weighed at the initiation and termination of the experiment. 
 Instantaneous calcification measurements
 After a 2-week incubation, alkalinity anomaly measurements were
 made using close bottle experiments as a proxy of instantaneous
 calcification. Organisms were carefully sealed in ~20mL glass
 scintillation vials with their chosen treatment group water, and in the
 case of the algal associated groups, with the algal hosts. The sealed
 vials were immersed in the appropriate flow-through water system to
 maintain treatment temperature. Treatment groups were incubated for 8h in
 light conditions. Analyses of water samples for total
 alkalinity were as above, and calcification (G) was calculated using Eqn.
 1: TA is total alkalinity (µmol kg<sup>-1</sup>), p is
 seawater density, V is chamber volume (mL), w.w. is wet weight (mg), and T
 is incubation time (h). All calculated values were normalized to final wet
 weight.  
 
 <i>G</i><i>(µmolCaC</i><i>O</i><sub><i>3</i></sub><i>)=-0.5×∆TA×ρ×V×</i><i>w.w.</i><sup><i>-1</i></sup><i>×</i><i>T</i><sup><i>-1</i></sup> <b>(Eqn. 1)</b> Photochemical efficiency measurements At the termination of the experiment, maximum photochemical efficiency (<i>Fv/Fm</i>) data were measured using WALZ DIVING-PAM underwater fluorometer (similar to Schmidt et al. 2014a). Measurements were recorded 4 hours after sunset in dark conditions, and individual <i>M. vertebralis</i>, and averaged across pseudoreplicate <i>M. vertebralis </i>within individual treatments. Total chlorophyll measurements Following measurement of wet weight, samples were immediately frozen (-20°C) in dark conditions and stored for chlorophyll analyses. Samples were placed in 15 mL polypropylene plastic tubes with 10mL of 90% acetone, and subsequently mechanically ground with a hard metal rod. Samples were then incubated in 4°C overnight in the dark. Absorbance measurements were then taken from the supernatant using an Ocean Optics usb4000+ spectrophotometer, and wavelengths of 630nm, 647nm, 664nm, and 691nm were recorded. Total chlorophyll was calculated based on universal equations developed by (Ritchie 2008). In treatments of both <i>L. intricata </i>and <i>M. vertebralis</i>, <i>M. vertebralis</i> were pooled within the replicate sample jar, and measured separately. All measurements were normalized to final wet weight of the corresponding <i>M. vertebralis</i> replicate. Oxygen flux measurements Oxygen flux measurements were made with a Presens Oxy-10 mini 10-channel optical sensor. At the end of the 15-day incubation period, oxygen flux measurements were taken in 30 mL glass scintillation vials that were gently stirred. Replicate samples (including <i>L. intricata </i>in association treatments) were gently placed in the glass jars with corresponding pH and temperature conditions, and allowed to acclimate for at least 5 minutes before measurements were recorded. For oxygen production measurements, light conditions in replicates were ~100 µmol photons m<sup>-2</sup> s<sup>-1</sup> during measurements (similar to incubation levels) and measured for a total of ~30 min in light conditions first. Subsequently, respiration was measured in dark conditions for ~30 min, allowing for 5 min of acclimation, and rate of oxygen consumption measured after the acclimation period. All analyses were performed using standard protocols for LBFs outlined in (Uthicke and Fabricius 2012). As the association treatment of <i>M. vertebralis</i> was incubated with <i>L. intricata</i>, an additional set of experiments was performed to separate the effect of LBF from macroalgae by independently measuring oxygen flux rates of algae in isolation. A total of 10 replicates were measured using similar incubations protocols described above, and the average of the four pH and temperature treatment groups were subtracted from the <i>L. intricata </i>with <i>M. vertebralis </i>replicates to obtain oxygen flux measurements of <i>M. vertebralis </i>in association treatment groups (Table 2). Statistical analyses For growth rate, instantaneous calcification, maximum photochemical efficiency (<i>Fv/Fm</i>), total chlorophyll, and oxygen flux measurement data, a three-way ANOVA was performed using pH (amb, -0.3 pH units), temperature (amb, and +3°C), and association (no association—treatments of <i>M. vertebralis </i>only<i>, </i>and with association--treatments of <i>M. vertebralis</i> and <i>L. intricata</i>) as fixed factors<i>. </i>Assumptions of ANOVA (homogeneity of variance and normality) were tested and met. All analyses were performed in R Tukey HSD test analyses conducted with the <b>agricolae</b> package.
description: Metadata are provided on the first tab of the excel file.