Background: Post-Pleistocene diversification of threespine stickleback in fresh water offers a valuable opportunity to study how changes in environmental salinity shape physiological evolution in fish. In Alaska, the presence of both ancestral oceanic populations and derived landlocked populations, including recent lake introductions, allows us to examine rates and direction of evolution of osmoregulation following halohabitat transition.

Hypotheses: Strong selection for enhanced freshwater tolerance will improve survival of recently lake-introduced stickleback in ion-poor conditions compared with their oceanic ancestors. Trade-offs between osmoregulation in fresh water and seawater will allow members of the ancestral population to survive better in response to seawater challenge, as mediated by upregulating salt-secreting transporters in the gill. Poorer hypo-osmoregulatory performance of derived fish will be marked by higher levels of taurine and other organic osmolytes.

Methods: We reared clutches at a common salinity from an anadromous and a descendant population, Scout Lake, which has been landlocked for only two generations. We challenged 6-week-old juveniles with extreme low and high salinity treatments and sampled fish over 10 days to investigate putative molecular mechanisms underlying differences in halotolerance. We measured whole-body organic osmolyte content as well as gill Na⁺/K⁺-ATPase (NKA) activity and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) protein abundance. Other juveniles from these populations and also from Cheney Lake, a fourth-generation landlocked descendant, were gradually salt-acclimated to determine maximum halotolerance limits.

Results: Scout Lake stickleback exhibited 67% higher survival in fresh water than the ancestral anadromous population, but individuals from both groups exhibited similar seawater tolerance. Likewise, the gradual salinity threshold for each population was equivalent (71 ppt). Gill NKA activity and NKCC abundance were both higher in seawater-challenged fish, but did not differ between populations. Sticklebacks from both populations responded to acute salinity stress by transiently increasing osmolyte levels in seawater and decreasing them in fresh water.

Conclusion: Enhanced freshwater tolerance has evolved rapidly in recently landlocked stickleback compared with their anadromous ancestors (0.569 haldanes), but the former have retained ancestral seawater-osmoregulatory function.