Methodology:

Sampling and analytical procedures:

To test the response of the coralline algae Crusticorallina spp. and Bossiella orbigniana to future OA scenarios, we used an 18-aquaria indoor experimental system with flow-through seawater at the Sitka Sound Science Center to simulate three static pHT levels (current summer = 8.0, future summer/current winter = 7.7, future winter = 7.4) under two seasonal light regimes simulated with full-spectrum aquarium lights (AI Prime HD) (summer = PPFD 55μmol m-2 s-1, 13h d-1, winter = PPFD 40μmol m-2 s-1, 6h d-1). We had a total of 3 aquaria for each of the 6 treatment combinations. A full description of the pH control for this system can be found in Kroeker et al. 2021, but in short: pH was regulated using a relay system that controlled mixing of pre-equilibrated low-pH seawater (formed by bubbling pure CO2 gas into seawater: pH6.0) and ambient pH seawater into 9 header buckets (n=3 headers per pH treatment) that then flowed into the experimental aquaria. Each header bucket was equipped with a pH sensor (DuraFET, Honeywell) communicating with a controller (UDA 2152, Honeywell) to regulate flow of the low pH water through solenoid valves to maintain pre-programmed pH setpoints. Experimental pH levels were chosen to reflect current seasonal minimums of coastal pH measured at Harris Is. (57.032N, 135.277W) from 2016-2017, as well as end-of-century projections for Gulf of Alaska pH levels based on RCP 8.5 (-0.3 pHT from current levels). Experimental light regimes were defined using seasonal averages for day length and measured irradiance level at 10m depth at Harris Is.

Within each pH level and light treatment combination, half of the individual Crusticorallina spp. and B. orbigniana were randomly assigned to be paired in close proximity with the fleshy red alga Cryptopleura ruprechtiana (n=6 species treatment-1). All algal individuals were collected on Aug 5, 2017 at Harris Is. Total experimental duration was 45d (Aug 7-Sept 21, 2017).

Total alkalinity (TA) incubations were run in the last week of the experiment on a subset of coralline algae from each treatment (n=3 individuals treatment-1 species-1) by isolating individuals in 245mL glass chambers filled with seawater from their associated aquaria and sealed airtight. Paired Cryptopleura ruprechtiana were not included in incubation chambers in order to isolate the responses of the coralline algae. Chambers were placed on a magnetic stir plate in a water bath at consistent temperature (13℃), with stir bars able to spin freely underneath coralline algae separated by a mesh screen. All incubations were run under a mean PPFD of 80μmol m-2 s-1 for 3h. At the end of the incubation period, seawater from each chamber was collected to measure endpoint TA. Seawater for TA incubation chamber controls was collected from corresponding aquaria at the beginning of each incubation round and used to measure any background TA variation in empty chambers during the incubation period. All discrete water samples for TA were poisoned and processed as outlined in section 2.4.

TA measurements from coralline algal incubations were used to calculate short-term net calcification (Gnet; μmol g-1 DW h-1) using the equation (Smith et al. 1975, Martin et al. 2006): Gnet(CaCO3) = (ΔTA*ν)/(2*DW*Δt ), where ΔTA (μmol kgSW-1) is the change in total alkalinity from the beginning to end of the incubation period corrected to chamber controls, ν (L) is the chamber volume, DW (g) is the dry weight of the alga, and Δt (h) is the total incubation time. Dry weights (DW; g) for the living coralline algae used in TA incubations were estimated from buoyant weight (BW; g) measurements using the equation: DW = BW /(1-(ρSW/ρcalcite)),where we used a seawater density (ρSW) of 1.02g cm-3 (from average T and S data at the time of BW) and a calcite density (ρcalcite) of 2.71g cm-3.

Each coralline algae’s buoyant weight (Jokiel et al. 1978) was measured to the nearest 0.0001g on a balanced platform suspended below a microbalance in a temperature-monitored seawater bath. To ensure precision, buoyant weights were repeated for each individual until measurements differed by less than ±0.005g, and then an average was taken of the measurements falling in this range of precision.

Discrete water samples for laboratory measurements of TA were transported to UCSC for analysis within 8 months of collection. TA measurements were performed using open cell titration (Metrohm, 905 Titrandro) and corrected against certified reference materials of CO2 in seawater (Dickson laboratory, Scripps Institution of Oceanography), with an average standard error of ±0.933μmol kg-1 SW-1 among sample triplicates.