Field sampling of spawners, offspring production, transport

At each of four locations along the Chilean coast (north to south: Iquique [IQ, 20°S], Caleta Sierra [CS, 31°S], Dichato [DI, 37°S], Puerto Montt [PM, 42°S]), flowing-ripe adults of the Chilean silverside (Odontesthes regia) (urn:lsid:marinespecies.org:taxname:281830) were obtained during the species’ spawning season in austral spring 2023 (September-October). With the help of local artisanal fishermen, adults were caught during night or early morning hours with gill nets of various lengths (2 cm stretched mesh). Fish were separated by sex and kept alive for up to one hour, before they were strip-spawned into plastic dishes filled with clean seawater. Our target was to use at least 20 spawners per sex and population, but the realized number of spawners varied. The northernmost site of Iquique was sampled twice; we denoted this in the data as IQ1 and IQ2. Fertilized embryos were transported in cooler boxes or thermos containers to the rearing facility in Dichato via car (PM, DI, CS) or airplane (IQ). Upon arrival at the rearing facility no more than 24h post-fertilization, they were randomly distributed among temperature treatments and replicate rearing containers.

Common garden experiment

Offspring were reared at the INCAR (Centro Interdisciplinario para la Investigación Acuícola) facility of the Dichato Marine Research Station of the Universidad de Concepción. We used four 1200L recirculating tanks representing the four temperature treatments (11, 14, 18, 23°C). Each tank housed 12 rearing containers that represented the 3 replicates for each of the 4 populations (4 temperatures × 4 populations × 3 replicates = 48 containers). The container design was identical to previous silverside experiments (e.g., Baumann & Conover 2011, Murray et al. 2014), i.e., we used round, white 20L containers equipped with individual airlines and mesh-screened (150 µm) holes, which guaranteed ~100% oxygenation and water exchange with the surrounding tank while retaining food. Tank water of ~ 30 psu was drawn from Coliumo Bay, filtered/UV-sterilized, and controlled for temperature via thermostats connected to commercial aquarium heaters or chillers. Each tank was equipped with a HOBO® Pendant MX temperature logger (Onset) that recorded conditions every 30 minutes. For biofiltration we used 4 large FX4-250 (Fluval) canister filters. The photoperiod was 15h light: 9h dark throughout the entire experiment.

Starting at hatch, silverside larvae were fed ad libitum rations of newly hatched brine shrimp nauplii (Artemia salina, San Francisco strain, from Brine Shrimp Direct) produced daily on site. Ad libitum rations were ensured by never allowing rearing containers to become nauplii depleted. A few days after successful first feeding, the number of fish in each rearing container was equalized to 130 avoid density-dependent growth effects. Another standardization to n = 80 per container occurred during when larvae reached approximately 15 mm total length (TL), and a final standardization to n = 40 occurred when larvae reach approximately 25 mm TL. When larvae reached approximately 35 mm TL, the rearing concluded and all survivors were sampled.

Samples

All collected adults – both spawners and non-spawners - were measured for TL and then preserved frozen (-20°C) for later vertebral number analysis. Experimental samples were first taken on the temperature- and population-specific day of hatch, when up to 200 hatchlings were preserved in 95% ethanol (0-hatch). The first and second larval samples were taken at approximately 15 mm TL (1-15mm) and 25 mm TL (2-25mm) and preserved in 95% ethanol as well. All larvae from these intermediate samples were measured for TL using the open-source software ImageJ (version 1.53a) and digital pictures taken with an I-Phone 11 in front of a gridded (5mm grid size) white background. The 95% ethanol was replaced once, at 24h after initial fixation. The final sample was taken at approximately 35 mm TL (3-35mm). All survivors were measured for TL using calipers (nearest 0.1 mm) and blotted wet weight (Mettler-Toledo; nearest mg) and then preserved frozen (-20°C) in individual plastic bags.

Vertebral number

The number of vertebrae was determined for all adult fish, regardless of whether they were used to produce offspring or not. Fish were X-rayed at the Oceanside Animal Hospital in Sandwich, MA, using a Vet Ray veterinarian X-ray system (SEDECAL, model A6504-25) and settings that were slightly modified from those recommended for small exotic pets (lizards). Specifically, we used a voltage of 60 kVp, power of 320 mA, and a shutter speed of 16 milliseconds. Digital x-ray pictures were analyzed using the multipoint tool in ImageJ to mark each vertebra between but excluding the basiocciptal and the urostyle.