Chemical mutagenesis and thermal selection of coral photosymbionts induce adaptation to heat stress with trait trade-offs

Despite the relevance of heat-evolved microalgal endosymbionts to coral reef res-toration, to date, few Symbiodiniaceae strains have been thermally enhanced via experimental evolution. Here, we investigated whether the thermal tolerance of Symbiodiniaceae can be increased through chemical mutagenesis followed by ther-mal selection. Strains of Durusdinium trenchii, Fugacium kawagutii and Symbiodinium pilosum were exposed to ethyl methanesulfonate to induce random mutagenesis, and then underwent thermal selection at high temperature (31/33 degrees C). After 4.6- 5 years of experimental evolution, the in vitro thermal tolerance of these strains was assessed via reciprocal transplant experiments to ambient (27 degrees C) and elevated (31/35 degrees C) tem-peratures. Growth, photosynthetic efficiency, oxidative stress and nutrient use were measured to compare thermal tolerance between strains. Heat-evolved D. trenchii, F. kawagutii and S. pilosum strains all exhibited increased photosynthetic efficiency under thermal stress. However, trade- offs in growth rates were observed for the heat-evolved D. trenchii lineage at both ambient and elevated temperatures. Reduced phosphate and nitrate uptake rates in F. kawagutii and S. pilosum heat-evolved lin-eages, respectively, suggest alterations in nutrition resource usage and allocation processes may have occurred. Increased phosphate uptake rates of the heat-evolved D. trenchii strain indicate that experimental evolution resulted in further trade- offs in this species. These findings deepen our understanding of the physiological responses of Symbiodiniaceae cultures to thermal selection and their capacity to adapt to el-evated temperatures. The new heat-evolved Symbiodiniaceae developed here may be beneficial for coral reef restoration efforts if their enhanced thermal tolerance can be conferred in hospite.