We quantified thermal tolerance and developmental acclimation across populations using LT50 methodology with an aluminium heat bar (Kuo and Sanford, 2009; Cheng et al., 2017). The heat bar was drilled to accommodate 5-ml centrifuge tubes that can house individual snails that are then exposed to a gradient of temperatures along the length of the heat bar. This heat bar was constructed with a solid aluminium block similar to Kuo and Sanford (2009), but heat was applied with a silicone heating element (Omega SRFGA-406/2-P 60 watt, Omega Engineering, Norwalk, CT, USA) and adjusted with a proportional integral derivative (PID) controller (ITC-100, Inkbird, Shenzhen, PRC). Cooling was maintained by circulating 3°C–5°C water through the opposing end of the heat bar. Although Urosalpinx experiences aerial and aquatic thermal stress, this species is commonly found in both subtidal and low-intertidal habitats with limited aerial exposure (Carriker, 1955; Cheng and Grosholz, 2016; Cheng et al., 2017). Thus, we chose to quantify thermal tolerance in water to avoid the confounding effect of aerial desiccation (Stillman and Somero, 2000).

In heat bar trials, individual snails were placed in 5-ml centrifuge tubes filled with 5 ml of aerated seawater at the same acclimation temperature the snail experienced during development. We inserted a 2 × 2-cm, 200-μm nitex mesh square into the tube using a plastic collar so that ~0.5 ml of the tube’s water was above the mesh. This prevented the snail from crawling out of the water, ensured free exchange of oxygen with the water in the tube and enabled us to record water temperature without disturbing the snail. We randomly assigned one of the three possible row positions along the heat bar, so that each population was represented in a column but in a random row. Thus, we tested up to three different population-acclimation treatments (each of which was defined as a ‘trial’) at a time on the heat bar array (Fig. S1). Each heat bar ‘run’ was defined as a ramping of 3 trials in the heat bar with 18–30 snails from 3 populations and a single acclimation temperature. We quantified wet weight of each live snail (Ohaus Pioneer PX Scale, Ohaus Corporation, Parsippany, NJ, USA) prior to the run to account for age and size effects, as age and age-linked size can affect thermal tolerance (Nyamukondiwa and Terblanche, 2009; Truebano et al., 2018). However, there was little evidence that age (as measured by centered and scaled body mass) predicted survivorship (Table S2). Therefore, we removed body mass as a predictor from our models. The shell length of these juvenile snails ranged from 1–2 mm.

We used the PID controller to control the temperature ramp along the heat bar, increasing the controller setpoint by 5°C every 30 minutes in steps from 25°C to 60°C for a total period of 4 hours. In the final hour, we held the heat bar at 60°C, so each snail was exposed to a heat ramp lasting 5 hours (Table S3, Fig. S2). We measured the temperature in each column every hour using a thermocouple. After the heat ramp, we removed the centrifuge tubes from the heat bar and allowed them to recover in aerated seawater at the appropriate acclimation temperature (20°C or 24°C) overnight. After the recovery period, we evaluated snails for mortality using a stereomicroscope and a probe classifying snails that did not retract their foot upon stimulus as dead and those that reacted as alive (Cheng et al., 2017). In total, we conducted 22 independent heat bar trials (20°C, n = 14; 24°C, n = 8) for 7 populations using a total of 652 juvenile snails (Table S4). Individual snail sample sizes between acclimations were uneven, with 418 at 20°C and 234 at 24°C, due to egg case availability.

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