Sporophytic specimens of Pterosiphonia bipinnata and Corallina vancouveriensis were collected from the mid to low intertidal zone on San Juan Island, Washington (Deadman Bay: 48 30' 48.93"N, 123 8'56.14"W), immediately transported to Friday Harbor Laboratories (FHL), and maintained in an outdoor seawater table for up to one week before testing. Reproductive parent fronds were held at ambient levels of seawater pH/pCO2 and were only exposed to experimental conditions while releasing spores. Spore release and settlement was performed in the Ocean Acidification Environmental Laboratory (OAEL) at FHL, allowing precise control of pH and temperature in a flow-through system. Two pH treatments were established by bubbling CO2 (7.75, 7.30 total scale) at 11C and confirmed with carbonate water chemistry analyses. Specifically, spectrophotometric pH was determined as per SOP6 and total alkalinity was measured using an open cell titrator as outlined in SOP3b (Dickson et al. 2007). Ambient pH values for the Salish Sea are approximately 7.8, and pH as low as 7.3 has been documented in nearshore environments in Washington. 

 The spore settlement apparatus consisted of a carriage system that slowly (1-2.5 cm/hr.) drove reproductive algal thalli across a glass settlement plate (0.6 x 7.6 x 60 cm), while releasing spores. The working section (14 x 1.5 cm) was defined at one end of the plate. Spores landing in the working section had a decreasing gradient of attachment time, determined by the rate parent thalli were driven across the plate and the time the plate was allowed to set. Error in attachment time was estimated to be 6-12 minutes given sinking rates of spores of comparable spore size (50-100 um) and release height (5-10 cm).

The shear flume was designed to release a tall column of water that flushes quickly across the working section of the spore settlement plate. Water column height was varied to create a range of shear stresses.  Shear stresses generated by each water column height were calculated according to Schultz et al. 2000, using the height of the channel (4.3 mm), the pressure gradient across the working section (measured with a manometer), and the length of the working section.  Most spores were removed in the first 10 seconds of continuous exposure to shear and longer exposure times result in little additional detachment, so trials were limited to 15 seconds. 

Prior to each test, the shear flume was fitted on the settlement plate with clamps and the released spores were photographed using a microscope (Steindorff SXC, New York Microscopes) connected to a camera (Nikon Coolpix S3300), using the lines drawn in the working section for reference. For the attachment time assay, a low shear stress (1 Pa) was applied and the remaining spores were photographed and counted. 

For the attachment strength assay, spores were allowed to set in stationary water for 35-48 hours in P. bipinnata and for 5-10 hours in C. vancouveriensis, since maximum attachment of each species was found in these time frames using the logistic regression analyses of attachment time. For P. bipinnata, replication was achieved by repeating the experiment in time since one frond moved across the entire settlement plate in each run. Due to limitations with C. vancouveriensis spore release, several fronds were used to provide sufficient spores for this assay. In this case, each frond that released spores was considered a replicate, and this was repeated across several days. All spore releases were natural, as attempts to artificially induce spore release (osmotic, temperature, or light) were unsuccessful. 

Attached spores were exposed to increasing shear stresses (1, 4, 7, 17 and 20 Pa). Shear stresses in the flume represented boundary layer velocities of 0.2 – 4 ms-1, similar to intertidal field conditions. At each position and at each shear stress applied, spores were counted through photo-analysis in ImageJ (version 1.48; U.S. National Institutes of Health, Bethesda, MD). With each successive application of shear stress, we expected an increasing percentage of spores to detach from the settlement plate, up to a maximum value assuming some spores would be stronger than our assay. The effect of shear stress on spore detachment in each species was tested using an exponential rise-to-maximum non-linear regression (SigmaPlot 11.0, R2 = 0.84 - 0.99), which provided estimates of two parameters: the maximum percentage of spores detached and the initial dependence of detachment on shear stress. The first derivatives of fitted, non-linear regressions were plotted to estimate frequency distributions of spore attachment strength.