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 hypothesised 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.

Academia Copernicana Department of Animal Physiology and Neurobiology Faculty of Biological and Veterinary Sciences Nicolaus Copernicus University in Torun Torun Poland

BIOPOLIS Program in Genomics Biodiversity and Land Planning CIBIO Vairão Portugal

CIBIO Centro de Investigação em Biodiversidade e Recursos Genéticos InBIO Laboratório Associado Universidade Do Porto Vairão Portugal

CIMO LA SusTEC Instituto Politécnico de Bragança Campus de Santa Apolónia 5300 253 Bragança Portugal

CITAB Center for Research and Technology of Agro Environment and Biological Sciences University of Trás os Montes and Alto Douro Vila Real Portugal

Department of Animal Physiology and Neurobiology Faculty of Biological and Veterinary Sciences Nicolaus Copernicus University Toruń Poland

Department of Ecology and Vertebrate Zoology Faculty of Biology and Environmental Protection University of Lodz Banacha 12 16 90 237 Lodz Poland

Institute of Nature Conservation Polish Academy of Sciences al Adama Mickiewicza 33 31 120 Kraków Poland

Institute of Vertebrate Biology Czech Academy of Sciences Brno Czech Republic

MORE Laboratório Colaborativo Montanhas de Investigação Associação Bragança Portugal

Norwegian Institute for Nature Research Sognsveien 68 0855 Oslo Norway

River Ecology and Management Research Group RivEM Department of Environmental and Life Sciences Karlstad University 65188 Karlstad Sweden

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