Fish dissection
A subsample of brown trout (Table 1) at each study site was transported alive to the department of Biology and Environmental Sciences at the University of Gothenburg for dissection. Before the dissection, fish were housed in holding tanks (30 L, 300 × 320 × 340 mm), which provided shelter structures (rocks and plastic plants) and fresh 12 °C water from a flow-through filtration system (flow rate: 2 L min-1). Fish were not fed during this period. All fish were dissected within 20 hours after capture and to randomize the time effect, individuals from allopatric and sympatric sites were dissected in alternating order (with even and odd individuals from allopatry and sympatry respectively). Before dissection, fish were euthanized by an overdose of 2-phenoxyethanol (0.5 mL L-1) and body mass and fork length were measured to the nearest 0.1 g and 1.0 mm respectively. Sex of individuals was determined by inspection of the gonads. Heads of fish were removed and fixed in 4% buffered (pH 6.9) paraformaldehyde solution. Brains were then dissected out as described in Gonda et al. (2009) by opening the scull along the anteroposterior axis and removing muscle tissue, nerves and bones until the brain could be lifted up from the skull and stored in 4% buffered paraformaldehyde solution until further procedure. Brains were imaged with a digital camera (EOS 40D with MP-E 65mm lens; Canon Inc., Tokyo, Japan). Images of brains were taken using the dorsal, left lateral and ventral views to calculate the total volume and the volume of cerebellum, optic tectum, telencephalon, olfactory bulb, and hypothalamus. Measurements were completed using ImageJ 1.48 (Schneider et al. 2021) and used to calculate volume with the formulas outlined by Pollen et al. (2007). Brain morphology was assessed by principal component analysis (Supporting Information S2) yielding two dominant principal components. PC1 was positively related to the volume of olfactory bulb, and hypothalamus and negatively related to cerebellum and optic tectum, while PC2 was positively related to the volume of telencephalon.
Fixing fish brains in paraformaldehyde solution in order to do morphological measurements prevented us from performing fatty acid analysis of the brain tissue. Therefore, we have used the muscle tissue as a proxy of n-3 LC-PUFA content in fish body. Muscles provide a good proxy for this purpose, because they represent the majority of fish biomass, and thus muscle metabolic activity and fatty acids content reflects individuals as a whole (Norin & Malte 2012; Gladyshev et al. 2018). In addition, biochemical composition of muscle tissue of salmonids has been shown to respond in similar direction to dietary deprivation of n-3 LC-PUFA as brain tissue, but with higher magnitude (Závorka et al. 2021). Thus, samples (~1 g of wet mass) of dorsal and ventral muscle tissue samples were taken from the left side below the dorsal fin, above and below the lateral line respectively. Bones and skin residuals were mechanically removed from the tissue samples, before they were stored on dry ice and subsequently frozen at -80 °C.