Study sites, fish collection and maintenance conditions
Fish used for this experiment were also part of a candidate gene study, and more methodological details can be found in Taugbøl et al. (2014). Adult sticklebacks were captured at two locations near Oslo, Norway (Figure 1 ), during May and June 2010. The marine site, Sandspollen, has a salinity varying between 22-29 ‰, while the freshwater pond, Glitredammen, is stable at 0 ‰. Coastal stickleback populations in Norway are considered to be purely marine, potentially with some gene flow from nearby freshwater populations (Klepaker 1996). Fish from Sandspollen breed locally (are not anadromous). The two locations are geographically isolated by approximately 35 km (shortest distance through water) (Figure 1b ), where about 8.5 km is through the river Sandvikselva that contains several steep waterfalls and dams. This makes downstream movement of fish from Glitredammen towards the marine sampling site possible, but upstream movement from the fjord impossible. The stickleback in Glitredammen has probably been separated from marine ancestors for at least 7000 years, as the age of the lake has been estimated to 7800 years before present using the program Sealevel32 (Møller 2003). The lake Glitredammen is located at 82.2 m above sea level.
After capture, the fish were acclimated to holding conditions in their native salinity (30 or 0 ‰) for minimum three weeks prior to the experiment. To reduce potential male nesting behaviour, the tanks were not equipped with any environmental enrichment, leaving the tanks free of sand and vegetation. The temperature in the tanks was maintained at room temperature (about 20°C), and the light regime was set at a 12:12 light: dark-cycle. The fish were fed two times a day with frozen red bloodworms throughout the acclimation period. More details on fish maintenance can be found in Taugbøl et al. (2014a).