Collection of adults and spawning: Adults of each species were collected from intertidal or shallow subtidal zones from various sites in Los Angeles County and transported to California State University Long Beach, where they were maintained in recirculating seawater tanks at 16 °C until their use in experiments. Experiments were carried out on one species at a time, depending on reproductive seasonality for that species.

Spawning was induced using standard methods (e.g., M. Strathmann, 1987). The echinoids Dendraster excentricus, Lytechnius pictus, Strongylocentrotus purpuratus and S. fragilis were induced to spawn via injection of 0.2-1.0 mL (depending on adult size) 0.53 M KCL into the perivisceral coelom. The asteroids Patiria miniata and Astropecten armatus were induced to spawn by injection of 1-3 mL 100 µM 1-methyladenine. The holothuroid Apostichopus parvimensis was injected with 3 mL of 200 µM NGLWY-amide (Kato et al., 2009). The ophiuroid Ophiothrix spiculata was exposed to 4 °C water in the dark for 15 minutes, then to room temperature water and sunlight for 15 minutes; this treatment was repeated for up to two hours (Selvakumaraswamy & Byrne, 2000). For all species, adults were each induced to spawn in their own isolated containers, allowing us to control subsequent fertilizations. Spawning continued until a minimum of three parents of each sex were obtained. Sperm from each spawning male was combined with eggs from each spawning female. Once the larvae reached the swimming stage (~24 hours), the offspring of all parents were combined to produce a genetically diverse population.

Culturing: Larvae were distributed into seven replicate 2 l beakers, with a total of 500 larvae per beaker (for S. purpuratus, 14 beakers of 500 larvae each were produced since greater numbers were needed for their protein analysis due to their small size). Larvae were fed 6000 cells ml−1 Rhodomonas lens which were isolated from their growth medium via centrifugation, resuspended in FSW, and counted using a BD Accuri C6 flow cytometer. Cultures were maintained in the 16 °C environmental chamber and continuously stirred by a paddle system (Strathmann, 1987). Daily water changes began on the third day post fertilization (dpf), allowing larvae to develop without disturbance for a day while still in or just completing the pre-feeding period. To change the water, cultures were filtered through a 60 µm sieve to capture larvae. The sieve was submerged in shallow water while filtering so larvae were not exposed to air. Larvae were then gently rinsed from the filter with fresh FSW back into their cleaned beakers and fed.

Sampling: For all species except D. excentricus, sampling for images and protein analysis occurred at 5 (“early-development”) and 10 dpf (“mid-development”). Sampling for D. excentricus occurred at 3 and 5 dpf due to their more rapid development and early formation of the rudiment. For imaging, ten individuals were sampled haphazardly from each beaker and placed on a glass slide. The larvae were oriented ventral-side and viewed with an Olympus BX5 compound microscope. The first five correctly oriented larvae encountered were photographed. The microscope was outfitted with both a digital camera and a z-axis drive, both controlled with Micromanager software. This allowed for the creation of an image stack of the entire larva with images taken at 2 µm depth intervals. The ciliated band was measured in three dimensions using maps of pre-determined landmarks. We identified the x, y, and z coordinates of the landmarks with ImageJ and used Excel to calculate the distance between coordinates and sum the total ciliary band length (McEdward, 1985; Rendleman et al., 2018)