DOI: 10.5061/dryad.gb5mkkx09
title: Data from: Shifting seagrass-oyster interactions alter species response to ocean warming and acidification

description: A major challenge in biodiversity research is the incorporation of species
 interactions into frameworks describing population and community response
 to global environmental change (GEC). Mutualisms are a type of species
 interaction especially sensitive to changing environmental conditions, and
 the breakdown of facilitative species interactions could amplify the
 negative impacts of novel climate regimes on focal species. Here, we
 investigate how reciprocal interactions between two coastal foundation
 species, the eastern oyster (Crassostrea virginica) and eelgrass (Zostera
 marina) shift in sign and magnitude in response to ocean warming (+1.5C)
 and acidification (-0.4 pH) via a manipulative co-culture experiment in
 mesocosms. Under ambient environmental conditions, oysters facilitated
 eelgrass leaf growth and clonal reproduction by 35% and 38%, respectively.
 Simultaneously, eelgrass increased oyster condition index (the ratio of
 tissue to shell biomass) by 35%, indicating greater allocation of energy
 to production of soft tissues instead of shell at ambient conditions.
 Varying sensitivity of each species to ocean warming and/or acidification
 treatments led to complex shifts in species interactions that were trait
 dependent. As such, community outcomes under future conditions were
 influenced by species interactions that amplified and mitigated species
 response to environmental change. Synthesis: Given that species
 interaction effect sizes were similar in magnitude to effect sizes of
 warming or pH treatments, our results underscore the need to identify key
 species and interaction types that strongly influence community response
 to GEC. Specifically, for macrophyte-bivalve interactions, understanding
 how physiological limitations on growth are impacted by environmental
 heterogeneity and co-culture will support successful restoration of
 natural populations and the rapid expansion of aquaculture.
description: <strong>Species Collections and Preparation:
 </strong>We haphazardly collected 600 eelgrass (<em>Zostera
 marina</em>) ramets (non-reproductive shoots with<strong>
 </strong>rhizomes and roots) from a 200 m<sup>2</sup>
 area encompassing the low intertidal and shallow subtidal<strong>
 </strong>zones in Brewer Cove, Orr’s Island, ME (43º 47’ 33.5” N,
 69º 57’ 14.8’’ W) on June 27th, 2021.<strong>
 </strong>Simultaneously, we collected sediment from the adjacent
 mudflat, which we sieved through 5<strong> </strong>mm mesh
 and homogenized. We standardized eelgrass ramets to one terminal shoot
 (length ± SD, 90 ± 23 cm) with 3 cm<strong> </strong>of
 rhizome and held standardized shoots in a cooler with seawater until
 planting them in<strong> </strong>mesocosms on the same
 day. We obtained 480 triploid eastern oysters
 (<em>Crassostrea virginica</em>)<strong>
 </strong>from a local oyster aquaculture facility, the Quahog Bay
 Conservancy, on June 30th, 2021.  Oysters were approximately the same size
 (mean ± SD, 5.9 ± 2.2 cm<sup>2</sup>) and drawn at random from
 hatchery provided bags. We used Gluemasters Industrial
 Strength<strong> </strong>Cyanoacrylate Adhesive to attach six
 oysters equally spaced on to a 15 x 15 cm PVC plate (80<strong>
 </strong>plates total).  Each plate was numbered and photographed
 with a camera facing down at a fixed<strong> </strong>height
 to measure initial projected surface area for each oyster using the
 software ImageJ.<strong> </strong>Oyster plates were
 transferred to mesocosms within a few hours of being
 photographed. <strong>Mesocosm Experiment:
 </strong>Our goal was to understand how reciprocal interactions
 between oysters and seagrass changed<strong> </strong>across
 warming and pH treatments. We built a system of 60 outdoor
 flowthrough-seawater<strong> </strong>mesocosms to culture
 “oyster monoculture”, “seagrass monoculture”, and “oysters + seagrass
 co-culture” under ambient<strong> </strong>and three future
 ocean treatments consisting of warming-only, low pH (elevated
 pCO<sub>2</sub>)-only, as<strong> </strong>well as
 warming and low pH combined. Therefore, our fully factorial
 experimental<strong> </strong>design consisted of 12 unique
 culture and ocean treatment combinations, with five<strong>
 </strong>mesocosm replicates (N=5) for each of the 12 combinations.
 Spatially, we arranged mesocosms in a 5 x 12 grid. Each column of 5
 mesocosms was fed by a single seawater manifold, therefore the same
 temperature and pH treatment combinations were applied to all mesocosms
 with a column.  We systematically alternated temperature and pH treatments
 across the 12 columns.  Along each manifold, we systematically alternated
 the position of “oyster monoculture”, “seagrass monoculture”, and “oysters
 + seagrass co-culture” mesocosms.  Thus, despite mesocosms experiencing
 temporary shading from nearby trees or buildings throughout the day,
 replicates for each of the 12 treatment combinations were spread evenly
 throughout the grid of mesocosms. Mesocosms were 27 L
 plastic<strong> </strong>buckets that measured 30 cm diameter
 x 50 cm high. We added approximately 10 cm of sieved<strong>
 </strong>and homogenized Brewer Cove sediment to each mesocosm. We
 planted 15 standardized<strong> </strong>eelgrass shoots (see
 Species Collection and Preparation) within each “seagrass monoculture”
 and<strong> </strong>“oysters + seagrass co-culture” mesocosm
 (equivalent to 205 shoots m<sup>-2</sup>, average shoot
 density in Brewer<strong> </strong>Cove during Summer 2021 was
 145 shoots m<sup>-2</sup> in the intertidal zone to 250 shoots
 m<sup>-2</sup> in the<strong> </strong>subtidal
 zone). We suspended two oyster plates (or 12 oysters total, see Species
 Collection and<strong> </strong>Preparation) back-to-back in
 the water column (fully submerged) within the center of each<strong>
 </strong>“oysters monoculture” and “oysters + seagrass co-culture”
 mesocosm.  Plates without any oysters attached were suspended in “seagrass
 monoculture” mesocosms to control for any potential shading effects of the
 plates.  We ran unfiltered seawater into all<strong>
 </strong>mesocosms at a rate of approximately 1.2 L
 min<sup>-1</sup> (seawater turnover rate was
 approximately<strong> </strong>three times per hour).  We
 checked seawater flow rates and removed epiphytes from seagrass and oyster
 surfaces approximately once a day. We allowed seagrass
 and oysters to acclimate to mesocosms for approximately a week before
 beginning warming and pH treatments on July 6th, 2021. We plumbed ambient
 seawater directly into ambient ocean treatment mesocosms.  For future
 ocean treatments, we used three 200 L sump tanks which we either heated
 and/or bubbled with CO<sub>2</sub>. In both the warming only
 and the warming and low pH sumps we installed two Process Technologies
 1000 W titanium aquaculture heaters.  We<strong>
 </strong>installed twelve Onset Temperature Pendants within the
 mesocosms, one in each of the twelve<strong> </strong>species
 monoculture or co-culture, temperature, and pH combinations.  We
 continuously controlled pH in both the low pH-only and the warming and low
 pH combined sumps using an APEX Aquacontroller system to bubble
 CO<sub>2</sub>, which was outfitted with APEX lab-grade pH
 probes (precision ±0.1 pH units), APEX temperature probes (precision ±
 0.5°C) and the APEX Fusion software. We set a goal treatment pH of 7.6,
 turning on a CO<sub>2</sub> exchanger to bubble
 CO<sub>2</sub> into sumps when pH was above 7.6.  Temperature
 treatments averaged 19.06 ± 0.01°C<strong> </strong>in warmed
 mesocosms compared to 17.78 ± 0.01°C in ambient mesocosms (Fig 1 a).  For
 comparison, the long-term average summer sea surface temperature for the
 Gulf of Maine is 15°C, with Summer 2021 being the hottest on record with
 an average sea surface temperature of 17.23°C (Kemberling 2023). Solar
 heating and<strong> </strong>tidal cycles caused daily
 temperature fluctuations within mesocosms; for example, in
 early<strong> </strong>August the warmed mesocosms ranged
 daily from 17.02 - 21.61°C and ambient mesocosms<strong>
 </strong>ranged daily from 16.53 - 19.38°C (Fig. 1 b). Throughout
 the summer, average temperatures<strong> </strong>increased by
 approximately 2°C. Daytime pH within the mesocosms differed between
 our<strong> </strong>ambient and low pH treatments (7.91 ±
 0.01 and 7.53 ± 0.04, respectively, Fig. 1 c). However, the 24-hour pH
 profile of a single mesocosm from our warm and low pH<strong>
 </strong>treatment containing both eelgrass and oysters demonstrates
 that pH fluctuated greatly within the<strong>
 </strong>mesocosms, with pH ranging from a maximum of 8.17 during
 the day and a minimum of 7.47 at<strong> </strong>night (Fig.
 1 d). There was no impact of environment or species co-culture on daytime
 total<strong> </strong>alkalinity (average TA range: 2095 –
 2118, see<strong> </strong>supplemental Fig. S1). Because of
 tidal cycles and daily fluctuations, treatments are more accurately
 described relative to ambient conditions (+1.5°C and -0.4 pH units
 relative to ambient, see Fig. 1). We ran the experiment for 34
 days. One week prior to the end of the experiment we
 tagged three eelgrass shoots in each “seagrass monoculture” and “oysters +
 seagrass co-culture” mesocosm with flagging tape and we used a hypodermic
 needle to mark the leaves of these shoots to determine leaf growth rates
 (see “hole-punch method” Dennison 1987). At the end of the experiment, we
 collected all eelgrass from each “seagrass monoculture” and “oysters +
 seagrass co-culture” mesocosm. We measured leaf growth rates on the tagged
 shoots. To assess changes in eelgrass clonal reproduction (addition of new
 clonal side shoots), aboveground and belowground biomass, we first rinsed
 all eelgrass from each mesocosm in freshwater, counted the total number of
 shoots, and then divided the eelgrass into aboveground tissue (shoots) and
 belowground tissue (rhizomes + roots). We dried eelgrass tissue at 60°C
 and weighed dry biomass for both tissue types. We rephotographed all
 oyster plates and then calculated total shell growth over the experimental
 period as initial projected shell area minus final projected shell area
 using the software ImageJ (Ricart et al. 2021a). We froze all oysters at
 -80°C.  To determine changes in oyster tissue biomass and condition index,
 we dissected the remaining oysters and dried oyster tissue and shells
 separately at 60°C. We calculated oyster condition index, a common proxy
 for energy allocation and stress response in bivalves, using the following
 equation (Rainer and Mann 1992, Ricart et al. 2021a).
 CI = Tissue Dry Mass / Shell Dry Mass * 100
 <strong>Additional seawater chemistry monitoring:
 </strong>We monitored pH and temperature continuously in all
 treatment sumps and in one mesocosm<strong> </strong>(oysters
 + seagrass co-culture, warmed and low pH combined) with APEX
 temperature<strong> </strong>and lab-grade pH probes. We also
 assessed daytime water chemistry within<strong>
 </strong>mesocosms by taking discrete water samples and spot
 measurements using a YSI sonde (YSI 556<strong>
 </strong>multiparameter handheld). Once a week, we took water
 samples from twelve haphazardly<strong> </strong>selected
 mesocosm (representing each of the twelve culture/co-culture, temperature,
 and pH<strong> </strong>combinations). We filtered all water
 samples through 0.2 µm GF/F filters into borosilicate glass<strong>
 </strong>containers and fixed them with 100 µL of HgCL2 (Dickson et
 al. 2007). Two bottles were<strong> </strong>collected for
 each mesocosm at each sampling interval, one for pH and one for Total
 Alkalinity<strong> </strong>(TA). We partitioned each bottle
 sample into two cuvettes, which were each<strong>
 </strong>analyzed in triplicate. We analyzed water sample pH
 spectrophotometrically (Cary 300 UV-Vis<strong>
 </strong>Spectrophotometer) using the m-cresol purple sodium salt
 (mCP: 10mM) method (Dickson et al.<strong> </strong>2007,
 Dickson 2010). We quantified total alkalinity via titration using a
 Metrohm<strong> </strong>905 Titrando (Dickson et al. 2007).
 In-situ pH was calculated using the ‘pHinsi’ function in the
 seacarb<strong> </strong>package in R (Gattuso et al. 2015).
 We took pH measurements within all 60 mesocosms using<strong>
 </strong>the YSI during one mid-July water sampling session. For
 this sampling date, YSI pH readings<strong> </strong>(mV) were
 regressed against in-situ pH values as a means of confirming the efficacy
 of our YSI<strong> </strong>and APEX probe-based pH
 measurements (R<sup>2</sup> = 0.893, p &lt; 0.001).
 Because our sampling design for water chemistry did<strong>
 </strong>not replicate measurements across treatment combinations
 within a<strong> </strong>sampling timepoint and there were
 rapid changes in water chemistry over the 4-hour sampling<strong>
 </strong>period (from 10:00 AM – 2:00 PM), these measurements are
 only sufficient for confirming the<strong> </strong>efficacy
 of the pCO<sub>2</sub> treatments and not for assessing
 species effects on daytime pH within<strong>
 </strong>mesocosms.
description: # Data from: Shifting seagrass-oyster interactions alter species response
 to ocean warming and acidification
 [https://doi.org/10.5061/dryad.gb5mkkx09](https://doi.org/10.5061/dryad.gb5mkkx09) ## Description of the data and file structure Shifting seagrass-oyster interactions alter species response to ocean warming and acidification, DuBois et al.  Journal of Ecology, 2024.   ### Files and variables #### File: JEcol0837\_sampling\_log\_meso\_final.csv **Description:** Data from YSI and bottle sampling on subset of mesocosms to determine pH treatments. ##### Variables * date: Date and time of sampling * YSI_temp_C: Temperature in Celsius read by YSI sonde  * YSI_salinity_psu: Salinity in PSU read by YSI sonde * YSI_pH: pH read by YSI sonde * YSI_pH_mV: pH in mV read by YSI sonde * Bucket_Number: Bucket ID number, 1-60 mesocosms * Apex.pH: pH read by Apex probe * TA: TA bottle sample, via titration  * TA_sd: Standard deviation of TA bottle sample, via titration * pHp_insitu: pH calculated insitu temperature * pHp_insitu_sd: Standard deviation of pH calculated at insitu temperature * YSI_pHT: YSI pH corrected at insitu temperature * YSI_calc_pH_minus_measured_pH: Difference between YSI and bottle sample pH measurements * rsquared: R2 values between replicates for YSI and bottle sample comparison #### File: JEcol0837\_OysterDeath.txt **Description:** Calculated in R script - number of oysters that died during the experiment in each treatment.  ##### Variables * Treatment: temperature and pH treatments listed jointly * Species_Condition: Eelgrass is present or oysters are grown in monoculture * Bucket_Number: Mesocosm ID 1-60 * Plate_Side: Oysters attached to both sides of plate, A or B * Rep: Oyster position on each side of plate, 1-6 * Isotope: Was oyster tissue used for isotope analyses, unpublished * Dead: did oyster die during the experiment? 1 - dead, 0- alive #### File: JEcol0837\_OysterCondition.txt **Description:** Data on oyster tissue and shell dry mass ##### Variables * OysterID: BucketID (1-60) - Rep (1-6) Plate Side (A/B) * Bucket_Number: Mesocosm ID 1-60 * Plate_Side: Oysters attached to both sides of plate, A or B * Rep: Oyster position on each side of plate, 1-6 * Vial_g: Mass of scintillation vial used to dry oyster tissue, grams * Length_mm: longest length of oyster in mm * Width_mm: width of oyster perpendicular to length in mm * Height_mm: height on oyster in mm * WetShell_g: wet shell mass in grams, after shucking * WetBodyVial_g: wet body tissue mass in vial in grams * DryShell_g: dry shell mass in grams * WetMass: calculated wet mass of tissue after vial weight subtraction in grams * DryMass: calculated dry mass of tissue after vial weight subtraction in grams #### File: JEcol0837\_EelgrassGrowthRates.csv **Description:** Growth of all leaves (measured using hole punch method) for eelgrass. ##### Variables * Bucket: Mesocosm ID 1-60 * Tray: Tray ID 1-6, 10 buckets placed in each tray. * Shoot ID: Up to three shoots (subreplication 1-3) tagged and punched in each mesocosm * Shoot Count: Total number of shoots in each mesocosm * Growth 1 (cm): Growth distance between punch mark, leaf 1 in cm * Growth 2 (cm): Growth distance between punch mark, leaf 2 in cm * Growth 3 (cm): Growth distance between punch mark, leaf 3 in cm * Growth 4 (cm): Growth distance between punch mark, leaf 4 in cm * Rhiz. Length (cm): Total rhizome length of tagged subreplicate shoot in cm  * Days_Grown: Number of days between marking and measurement * Growth_D: growth rate calculated base on sum of growth across all leaved divided by number of days growing.  #### File: JEcol0837\_EelgrassBiomass.csv **Description:** Data for total eelgrass biomass in each mesocosom ##### Variables * Bucket: Mesocosm ID 1-60 * Shoot: ALL (total for entire mesocosm), 1-3 (dry mass data for three subreplicate shoots that were tagged for growth rate measurements. * Tissue: Tissue type, above (leaves) or below (roots and rhizomes) * Dry Mass (g): The dry mass of each tissue type in grams #### File: JEcol0837\_BucketID.csv **Description:** An standardized ID data frame to attached to each data set in R to make analyses and visualization easier.  ##### Variables * Bucket_Number: Mesocosm ID 1-60 * Tray_Number: Tray ID 1-6, 10 buckets placed in each tray. * Header: Which header sump tank (1-4) supplied water to tray, i.e. block of mesocosoms.  * Treatment: All four pH/Temp treatment listed out in one column (combined) useful for graphing * Species*Condition: Seagrass*_oyster (coculture) or seagrass (monoculture) or oyster (monoculture) * pH: pH treatment, low or ambient * Temp: Temperature treatment, high or ambient #### File: JEcol0837\_OysterGrowth.csv **Description:** Oyster growth rate data calculated from ImageJ analyses.  ##### Variables * Alive_Dead: Was the oyster alive or dead at the end of the experiment, low confidence in this data collection * Death_Date: When was oyster recorded/noted as dead during 6 week experiment, low confidence in this data collection * Tray_Number: Tray ID 1-6, 10 buckets placed in each tray. * Bucket_Number: Mesocosm ID 1-60 * Plate_Number: Number assigned to each oyster plate - not used as an ID variable in analyses, redundant of Bucket ID * Plate_Side: Oysters attached to both sides of plate, A or B * Oyster_Position: Oyster replication (1-6) ID on plate * Initial_Area_1:  Initial oyster area in cm2 measure in ImageJ replicate 1 * Initial_Area_2: Initial oyster area in cm2 measure in ImageJ replicate 2 * Initial_Area_3: Initial oyster area in cm2 measure in ImageJ replicate 3 * Avg_Initial_Area: Average of three initial area measurements for each oyster in cm2 * Final_Area_1: Final oyster area in cm2 measure in ImageJ replicate 1 * Final_Area_2: Final oyster area in cm2 measure in ImageJ replicate 2 * Final_Area_3: Final oyster area in cm2 measure in ImageJ replicate 3 * Avg_Final_Area: Average of three final area measurements for each oyster in cm2 * Growth*Rate:* Average Initial Area subtracted from Average Final Area to get areal expansion of oysters in cm2 * Header: Header sump tank that fed seawater to buckets in different treatments * Treatment: Temperature and pH treatments combined into one ID column * Species_Condition: Eelgrass present or oysters grown in monoculture.  Missing data codes: NA and N/A ## Code/software Data analyses were conducted in R.  See R scripts archived with these data sets. 1) JEcol0837*AllEelgrassOysterAnalyses.R, and 2) JEcol0837*WaterChem.R.   ## Access information Other publicly accessible locations of the data: * Dryad Data was derived from the following sources: * Collected by researchers at the Schiller Coastal Studies Center, Bowdoin College, ME, USA, Summer 2021