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, 41°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 (September-October) in 2023 and 2025. With the help of local collaborators and/or 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. At least 20 spawners per sex and population were used if available. In 2023, the northernmost site of Iquique was sampled twice; we denoted this in the data as fertilization 1 and fertilization 2. 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

In both years, offspring were reared at the Dichato Marine Research Station; in 2023 the experimental setup was located at the INCAR facility (Centro Interdisciplinario para la Investigación Acuícola), whereas in 2025 the setup was housed at the Experimental Marine Biology Laboratory (aka ‘Acuario’) of the Universidad de Concepción. We used four 900-1200L recirculating tanks representing four temperature treatments (2023: 11, 14, 18, 23°C; 2025: 14, 17, 20, 23°C). Each tank housed up to 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 MX2201 temperature logger (Onset) that recorded conditions every 30 minutes in 2023 and every 15 minutes in 2025. 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 (=excess) rations of newly hatched brine shrimp nauplii (Artemia salina, San Francisco strain, Brine Shrimp Direct) produced daily on site. Ad libitum rations were ensured by never allowing rearing containers to become nauplii depleted. 1-3 days post hatch, the number of fish in each rearing container was equalized to 130 to avoid density-dependent growth effects. Another standardization to n = 80 per container occurred 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 peak hatch, when up to 20 hatchlings were preserved in 95% ethanol (0-hatch). The first and second samples were taken at an average TL of approximately 15 mm TL (1-15mm, larvae) and 25 mm TL (2-25mm, early juveniles) and preserved in 95% ethanol as well. All individuals from these intermediate samples were measured for TL using the open-source software ImageJ and digital pictures taken with an iPhone 11 in front of a gridded (7mm grid size) white background. The 95% ethanol was replaced 24h after initial fixation. The final sample was taken when individuals on average had reached approximately 35 mm TL (3-35mm). In 2023, survivors were measured for TL using calipers (nearest 0.1 mm) and blotted wet weight (nearest mg) and then preserved frozen (-20°C) in individual plastic bags. In 2025, final samples were only measured for TL via digital pictures in ImageJ 1.53a and preserved in 95% ethanol (same as intermediate samples).

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 basioccipital and the urostyle. In 2025, additional populations from Chimbote and Ancón in Peru were x-rayed by collaborator Victor Aramaya from the Universidad Nacional Mayor de San Marcos, Facultad de Ciencias Biológicas, Lima, Peru.