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Global change effects, such as warming and increases in nitrogen loading, alter vulnerable Mediterranean aquatic systems, and charophytes can be one of the most affected groups. We addressed the possible interaction between these factors on two populations of the cosmopolitan charophytes Chara hispida and Chara vulgaris. Populations were taken from two different environments, a nitrate-poor mountain lake and a nitrate-rich Mediterranean coastal spring. The laboratory experiment had a 2 x 2 factorial design based on two nitrate levels (similar to and double the local conditions) and two temperatures. Increased temperatures favoured the growth of the four populations, but an increase in nitrate did not have any effect on their growth or architecture. Both species took up and stored more nitrogen (measured as %N in plant tissue) when more nitrate was supplied, and warming favoured this increase in %N and, consequently, in N:P ratio. The effects of both factors depended on the local conditions where the populations originated and on the species. Chara vulgaris, a pioneer species, exhibited more phenotypic plasticity than C. hispida, and its ecotype from the coastal spring was better adapted to changes in temperature and nitrate level. These differential responses to warming conditions and nitrate pollution may modify charophyte diversity, which might be reflected in ecosystem performance, a matter of concern in vulnerable Mediterranean water bodies where these species co-occur.