Cold tolerance strategies of freshwater mussels across latitudes

Freshwater mussels across Europe exhibit physiological and behavioural adaptations to survive winter conditions. Climate change projections, including more frequent extreme weather events, are expected to intensify pressures on these ecosystems. In this study, we tested the temperature-size hypothesis, which posits that larger body size in ectothermic organisms is an adaptation to colder climates. We predicted that Anodonta anatina populations in northern regions would have larger shells than those in central and southern regions. Additionally, we hypothesized that harsher winters in northern regions require mussels to maintain higher glycogen levels as an energy reserve. We also explored whether shell size varies between lowland and upland populations, following the temperature-size rule, and whether supercooling (SCP) occurs primarily in northern populations as a complementary survival strategy. Northern populations had the highest glycogen levels, reflecting adaptations to colder conditions. SCP was rare (2.5%) and observed predominantly in northern mussels, suggesting limited reliance on freeze avoidance. Instead, it is likely that mussels employ mixed strategies, such as metabolic reduction and burrowing, to withstand winter. These findings link shell size, glycogen levels, and SCP to specific survival strategies, providing new insights into the cold tolerance mechanisms of freshwater mussels and their potential vulnerability to climate change.