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).

In vivo photophysiology was characterized for all red algal species at the end of the experiment by measuring the rate of oxygen evolution produced by algal thalli at seven irradiance levels. Following the final buoyant mass measurement, a small piece of thallus (mean ± SE: B. orbigniana: 0.17 ±0.02g; Crusticorallina spp.: 0.53 ± 0.03g; C. ruprechtiana: 0.07 ± 0.003g) was taken from haphazardly selected individuals (n=3 treatment-1 species-1) and placed in a 69mL incubation chamber filled with seawater from the associated aquaria and equipped with a stir bar and an oxygen sensor spot (PreSens SP-PSt4-SA). Sensor spots were calibrated daily using a two-point correction of 100% (air-saturated water) and 0% (1% Na2SO3 and 0.05% Co(NO3)2 standard solution) saturation. Incubation chambers were sealed airtight using clear plexiglass lids affixed with vacuum grease and submerged onto a magnetic stir plate in a temperature-controlled water bath. Full-spectrum aquarium lights (AI Hydra HD) were used to expose thalli in chambers to seven consecutively increasing irradiance levels (~PPFD 0, 20, 70, 140, 320, 425, 720μmol m-2 s-1). A fiber optic O2 sensor (Fibox IV, Presens) was used to record the dissolved oxygen concentration in each chamber at 30, 45 and 60min after each irradiance level was reached. Dissolved oxygen evolution rate (mg O2 min-1) at each irradiance level was calculated using linear regression, corrected against paired chamber controls (no algae), and normalized to chamber volume and thalli wet mass (mg O2 g-1 min-1 L-1).