DOI: 10.5061/dryad.00000009d
title: Coral community metabolism during year-long exposure to experimental elevated CO2 conditions

description: The implications of ocean acidification are acute for calcifying
 organisms, notably tropical reef corals, for which accretion generally is
 depressed and dissolution enhanced at reduced seawater pH. We describe
 year-long experiments in which back reef and fore reef (17-m depth)
 communities from Moorea, French Polynesia, were incubated outdoors under
 pCO2 regimes reflecting endpoints of representative concentration pathways
 (RCPs) expected by the end the century. Incubations were completed in 3–4
 flumes (5.0 × 0.3 m, 500 L) in which seawater was refreshed and circulated
 at 0.1 m s-1, and the response of the communities was evaluated monthly by
 measurements of net community calcification (NCC) and net community
 photosynthesis (NCP). For both communities, NCC (but not NCP) was affected
 by treatments and time, with NCC declining with increasing pCO2, and for
 the fore reef, becoming negative (i.e., dissolution was occurring) at the
 highest pCO2 (1067–1433 µatm, RCP8.5). There was scant evidence of
 community adjustment to reduce the negative effects of ocean
 acidification, and inhibition of NCC intensified in the back reef as the
 abundance of massive Porites spp. declined. These results highlight the
 risks of dissolution under ocean acidification for coral reefs and suggest
 these effects will be most acute in fore reef habitats. Without signs of
 amelioration of the negative effects of ocean acidification during
 year-long experiments, it is reasonable to expect that the future of coral
 reefs in acidic seas can be predicted from their current known
 susceptibility to ocean acidification.
description: <em>Common infrastructure</em>
 Experiments were conducted in flumes, consisting of a 5.0 × 0.3 m
 working section filled to ~ 0.3-m depth with 500L of seawater. Four flumes
 were used for both experiments, but equipment malfunction resulted in one
 flume being dropped from the analysis of the fore reef communities. See
 Supplementary Information in published article for more details.
 Communities were exposed to treatments targeting ambient (~ 400
 µatm), 700 µatm, 1000 µatm, and 1300 µatm pCO<sub>2</sub> that
 approximate atmospheric conditions expected by ~ 2140 under different
 RCPs. Experiments began in late Austral spring in 2015 (back reef) and
 2017 (fore reef), and extended 12 months with monthly measurements of
 community metabolism (this study) and community structure.
 The flumes were filled with seawater pumped from 14-m depth in
 Cook’s Bay and filtered through sand (~ 450–550 µm pore size), and was
 continuously added to the flumes at 300 L h<sup>-1</sup>.
 Seawater was circulated in each flume at 0.1 m s<sup>-1
 </sup>using a pump (W. Lim Wave II 373 J
 s<sup>-1</sup>), and flow speeds were measured in the working
 section using a Nortek (Boston, MA) Vectrino Acoustic Doppler Velocimeter.
 A flow speed of 0.1 m s<sup>-1 </sup>is ecologically relevant
 to the back reef, and the fore reef at 15-m depth. The
 seawater was temperature-controlled with chillers that matched the monthly
 temperatures to the ambient seawater in Cook’s Bay. Temperatures were
 increased from ~ 27 °C in November to ~ 29 °C around April and May, then
 back to ~ 27 °C the following November. Temperature was recorded using
 loggers (Onset Hobo Pro 2, ± 0.2 °C) and a certified thermometer (± 0.05
 °C, model 15-077, Fisher Scientific). Sunlight was shaded with neutral
 density mesh to approximate the light at 2-m depth in the back reef, or
 17-m depth on the fore reef, and for the fore reef, blue filters (LEE
 #183, Lee Filters, Andover, England) were used to simulate light at 17-m
 depth. Photosynthetically active radiation (PAR) was recorded using
 cosine-corrected loggers (Odyssey, Dataflow Systems, Ltd., Christchurch,
 New Zealand), calibrated against a certified Li-Cor cosine sensor (± 5%
 resolution, LI 192A, Li-Cor, Lincoln, NE, USA) in units of photon flux
 density (PFD, µmol photons m<sup>-2</sup>
 s<sup>-1</sup>) Flumes were covered with clear UV-transparent
 acrylic lids to prevent rain from entering and to reduce wind speed at the
 air-seawater interface. The 5-m working section was
 divided into a central 2.4-m portion and two flanking portions, each 1.3 m
 in length. The central portion included a 0.3-m deep sediment box that was
 filled with sediment for the back reef community, but was covered and
 sealed for the fore reef community where sand accumulations are rare. The
 sand in the sediment box for the back reef was arranged with a smooth
 surface, and it lacked the ripples in surface texture that can enhance
 advective exchange with seawater. Members of the benthic community from
 each habitat were scattered along the working section to create a
 community composition benchmarked against the empirical benthic community.
 Community members in the central portion of the flumes were attached to a
 metal grid and were not removed from the flume so that they could be
 quantified photographically. In the flanking portions, community members
 were removed monthly to measure buoyant weight that was used in the
 analysis of the ecological responses to treatments. The communities
 included a few invertebrates (e.g., coral ectosymbionts such as crabs and
 brittle stars) with herbivores (e.g., sea urchins) to aid in the control
 of algae. Routine cleaning (~ weekly) removed excess algal biomass on the
 walls and other exposed surfaces of the flumes.
 pCO<sub>2</sub> regimes were assigned randomly to the
 four flumes in each experiment, and the flumes were oriented with their
 long axis aligned north-south. Seawater carbonate chemistry was not
 controlled in the ambient flumes, but in the other three flumes was
 regulated with CO<sub>2</sub> gas to approach target values.
 CO<sub>2</sub> was added to the treatment flumes to alter
 seawater pH relative to a set point (controlled with AquaControllers,
 Neptune Systems, Morgan Hills, CA) that operated a solenoid to regulate
 the supply of CO<sub>2</sub>, except in the ambient flume;
 ambient air was bubbled into all flumes. The system was programmed to
 apply a nocturnal downward pH adjustment of ~ 0.1 unit to simulate
 conditions on the reefs of Moorea. Seawater pH on the
 total hydrogen scale (pH<sub>T</sub>) was measured daily using
 a hand-held meter (with a DG 115-SC electrode, Mettler Toledo, Columbus,
 OH, USA) that was calibrated with TRIS buffer (SOP 6a). These records were
 used together with temperature to adjust the pH set points of the
 AquaController to approach target values. Seawater carbonate chemistry was
 calculated weekly using pH<sub>T</sub> and the total
 alkalinity (A<sub>T</sub>) recorded once during the day and
 night. A<sub>T</sub> was measured using open-cell, acidimetric
 titrations (SOP 3b) with an automatic titrator (T50 Mettler Toledo)
 operated with a DG 115-SC probe and filled with certified acid (A.
 Dickson, Scripps Institute of Oceanography [SIO]). The accuracy and
 precision of the analyses were determined from reference materials (CRMs,
 batches 158 and 172, A. Dickson, SIO). Relative to CRMs, determinations of
 A<sub>T</sub> maintained an accuracy of 1.7 ± 0.3 µmol
 kg<sup>-1</sup> to 2.7 ± 0.4 µmol kg<sup>-1</sup>,
 and a precision of 1.8 ± 0.1 µmol kg<sup>-1</sup>
 (<em>n</em> = 475). Calculations to determine seawater
 carbonate chemistry were made using temperature, salinity, and
 pH<sub>T</sub>, in the R package Seacarb.
 <em>Calculations of benthic metabolism</em>
 Community metabolism was assessed using net community
 calcification (NCC) and net community productivity (NCP) that were
 calculated using the alkalinity anomaly method (Equation 1 in published
 paper), and changes in dissolved oxygen (DO see Supplementary Information)
 (for NCP, Equation 2 in published paper). NCC and NCP
 were measured under ambient conditions in all flumes at the start of the
 experiments, the pCO<sub>2</sub> treatments were initiated,
 and NCC and NCP again were measured. For the back reef community, NCC and
 NCP were measured biweekly or monthly for the first four months (to March
 2016) and then monthly until the end of the experiment. Single
 measurements every month revealed variance attributed to weather and,
 therefore for the fore reef community, NCC and NCP were recorded on three
 days every month, with the three days usually consecutive. Each day of
 measurements consisted of six determinations, two in the morning
 (06:00–09:00 hrs and 09:00–12:00 hrs), two in the afternoon (12:00–15:00
 hrs and 15:00–18:00 hrs), and two at night (18:00–24:00 hrs and
 24:00–06:00 hrs). These times were accurately recorded, but each period
 varied to accommodate logistical constraints. Each determination was
 completed with the flumes operating in closed-circuit mode, during which
 the inflow of fresh seawater was halted. The flumes were flushed for ~ 30
 minutes between each incubation by re-initiating the inflow of seawater at
 ~ 5 L min<sup>-1</sup>, thereby exchanging ~ 25–30% of the
 seawater between incubations. For the back reef
 community, for which a single day of measurements was obtained monthly,
 missing values were interpolated using a third order polynomial (for NCC)
 or a fourth order polynomial (NCP) for empirical values against time of
 day (Carpenter et al. 2018). NCC and NCP were calculated as hourly rates
 (mmol CaCO<sub>3</sub> m<sup>-2</sup>
 h<sup>-1</sup> and mmol O<sub>2</sub>
 m<sup>-2</sup> h<sup>-1</sup>, respectively) with
 positive values reflecting net CaCO<sub>3 </sub>accretion and
 a net release of photosynthetically-fixed O<sub>2</sub>, and
 negative values reflecting net dissolution of CaCO<sub>3</sub>
 and a net uptake of O<sub>2</sub>, all respectively. NCC was
 integrated over the day and night to gain insight into the relative
 effects of treatment on carbonate accretion (that occurs mostly during the
 day) versus dissolution (that occurs mostly at night), and NCP was
 integrated over the day to evaluate net photosynthesis. Local day length
 was used to calculate day and night rates, with day length varying from
 13.17 hrs in December to 11.08 hrs in June.
 <em>Back reef experiment, 2015-2017</em>
 The back reef experiment began in November 2015, and the
 communities were assembled to mimic the community found in the back reef
 of Moorea in 2013. Community members were collected from ~ 2-m depth on
 the north shore. Initially the communities had ~ 25% coral cover with 11%
 massive <em>Porites</em> spp., 7% <em>P</em>.
 <em>rus</em>, 4% <em>Montipora</em> spp., 3%
 <em>Pocillopora</em> spp., and ~7% crustose coralline algae
 (CCA) with 4% <em>Porolithon onkodes</em>, and 3%
 <em>Lithophyllum kotschyanum.</em> Corals and CCA were
 attached to plastic bases (using Z-Spar A788). Coral rubble (~ 1-cm
 diameter) was added to ~ 5% cover, and the remainder of the floor of the
 flume was sand. Communities were assembled on top of the sand and were
 augmented with a few holothurians (~ 8-cm long,
 <em>Holothuria</em> spp.) and macroalgae (<em>Turbinaria
 ornata</em> and <em>Halimeda minima</em>) that were
 added to ecologically relevant cover for the back reef in 2013 (~ 4–5%
 cover). The communities were established in the flumes by 12 November
 2015, and were maintained under ambient pCO<sub>2 </sub>for 5
 days until pCO<sub>2</sub> treatments were established in
 three flumes; pCO<sub>2</sub> was increased over 24 h to
 establish target values. <em>Fore reef
 experiment, 2017-2018</em> The fore reef
 experiment began in November 2017, and communities were assembled in the
 flumes to mimic the community found at 17-m depth on the fore reef of
 Moorea in 2006. An historic community was used as a target because it
 better reflected the long-term representative fore reef community in this
 location, and because it allowed a more direct comparison with previous
 short-term incubations completed with fore reef communities. Community
 members were collected from 17-m depth on the north shore fore reef.
 Initially, the communities had ~ 27% coral cover with ~ 11%
 <em>Pocillopora</em> spp., ~ 8% massive
 <em>Porites</em> spp., ~ 8% <em>Acropora</em> spp.
 and 53% reef rock. The <em>Pocillopora</em> conformed to
 <em>P. verrucosa</em>, but multiple species probably were
 present. Likewise, the <em>Acropora</em> spp. targeted
 <em>A. hyacinthus</em> and <em>A. retusa</em>,
 which are common on the fore reef of Moorea, but possibly included other
 species. Corals were attached to plastic bases (with Z-Spar A788). The
 reef rock consisted of rubble (~ 12-cm diameter) and the flora and fauna
 with which it was associated. The communities were established in the
 flumes by 27 October 2017 and were maintained under ambient
 pCO<sub>2</sub> for 7 days, when the
 pCO<sub>2</sub> treatments were initiated over 24 h to reach
 target values. Because of equipment malfunctions, the fore reef experiment
 was completed with 3 flumes that targeted ambient conditions (400 µatm),
 700 µatm, and 1300 µatm pCO<sub>2</sub>.
 <em>Statistical analysis</em> NCC
 and NCP were analyzed separately for each experiment, and in both cases,
 linear, mixed-effects models were fitted using restricted maximum
 likelihood (REML) methods, in which treatments
 (pCO<sub>2</sub> regimes) and time (i.e., measurement day)
 were fixed effects (covariates), and light, temperature, and coral cover
 were random covariates. Four analyses were conducted for each experiment,
 with each employing a different dependent variable (24h-NCC, day-NCC,
 night-NCC, and NCP). For the fore reef experiment, the 3 measurements
 month<sup>-1</sup> were averaged to provide a single value for
 each month to facilitate a contrast with the back reef experiment. To
 evaluate changes in metabolism over time, the average of the monthly
 measurements was registered against the second of the three-day
 measurement sequence. Light was integrated over a day (mol photons
 m<sup>-2</sup> d<sup>-1</sup>) on the day that
 community metabolism was measured (back reef), or was averaged over the 3
 measurement periods (fore reef). Temperature was the mean daily
 temperature over the month preceding the metabolism measurements, and
 coral cover was calculated from planar photographs. To
 understand the integrative balance between inorganic (NCC) and organic
 (NCP) carbon sequestration, the relationships between NCC and NCP (by
 hour) were explored using scatter plots, with associations tested using
 Pearson correlations. Linear relationships were displayed using Model I
 regressions. To test for variation in NCC–NCP relationships over time, the
 slopes and elevations of the NCC–NCP relationships were compared among
 seasons using ANCOVA. The seasonal contrast was created by segregating
 results from November–February (near Austral summer), March–May (near
 austral autumn), June–August (near Austral winter), and September–November
 (near Austral spring). To test for linear relationships
 between 24h-NCC and pCO<sub>2</sub>, Model I linear
 regressions were fit to the results by month (<em>n</em> = 3–4
 pCO<sub>2</sub> treatments) using the single measure
 month<sup>-1</sup> for the back reef experiment, and the 3
 measures month<sup>-1</sup> for the fore reef experiment. To
 test for variation over time (year) in the sensitivity of the 24h,
 NCC–pCO<sub>2</sub> relationship, Model I linear regression
 was used, in which the slopes of the 24h-NCC–pCO<sub>2</sub>
 relationships by time were the dependent variable, and the independent
 variable was time (days over the incubation year).
 Statistical analyses were completed using Systat 13.0 software
 (Inpixon, Palo Alto, USA), and the statistical assumptions of ANOVA were
 tested using graphical analysis of residuals.
description: # Coral community metabolism during year-long exposure to experimental
 elevated CO2 conditions 
 These data describe the metabolism of two coral community types (back reef
 and fore reef) each incubated for a year in 3-4 experimental flumes in
 which the partial pressure of carbon dioxide gas was modified to upto for
 levels. ## Description of the data and file structure COMMUNITY METABOLISM
 DATA SET This files has columns as described below. Community = Community
 type, fore reef or back reef Flume =  Flume number, F1, F2, F3 or F4
 Target = Target seawater pCO2 at which the flumes were targeted Period =
 Period of incubations each day. Six measurements were recorded each 24
 period of each determination: two in the afternoon (A1 and A2), two in the
 morning (M1 and M2) and two at night (N1 and N2). Note N1-2 indicates only
 one night incubation was completed. Incubation = Incubation number in
 series (0-15) Day/night hours = Number of hours in either the day (A1, A2,
 M1, M2) or night (N1, N2) as determined by day length as the sun position
 varied by season Date2 = Date of record for daily incubations. Incubations
 began in the morning, but the second night incubation often started in the
 early am of the following day; this value was used in calculating daily
 metabolism of the previous day Date1 = Calendar date on which measurements
 were made Time = Time at which incubation began on 24 h clock NCP = Net
 community photosynthesis in units of mmolO2/m^2/h NCC = Net community
 calcification in units of mmolCaCO3/m^2/h LINEAR MIXED MODEL ANALYSUS.
 This file has colum headings as described below: Habitat = Habitat type
 used in the experiment, Fore reef or back reef Date  = Date of
 measurements over the year long experiment month/day/year Day-NCC =
 Daytime net community calcification mmol CaCO3/m^2/day Night-NCC =
 Nightime net community calcification mmol CaCO3/m^2/day 24 h-NCC = Net
 community calcification over 24 hours mmol CaCO3/m^2/day Day-NCP = Daytime
 net community photosynthesis mmol O2/m2/day Time = Numbers of days into
 the experiment PCO2 Target = Target pCO2 treatment of the three flumes
 µatm Coral Cover = Percentage cover of corals in flumes Temperature =
 Seawater temperature (°C) at the time of measurement Integrated PFD =
 Light (photon flux density [PFD} in the photosynthetically active range
 [PAR])integrated over the say of measurement of community metabolism mol
 photons m-2 d-1