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Charophytes are amongst the most endangered primary producers in freshwater and coastal ecosystems. In spite of the extensive research on the group and its ecological and conservational relevance, scarce information is available on Mediterranean environments, especially rivers and small water reservoirs, where charophytes face challenging summer droughts and changes in hydrological regimes, as well as pervasive anthropogenic pressures. This research aimed, through repeated field observations, detailed analyses of population traits, and extensive characterization of the colonized environments, to foster an understanding of the distribution, biodiversity, and ecology of charophytes in an area of exceptional environmental value and that is still uninvestigated in relation to its charophyte flora, the southern Campania region (Italy). Overall, 17 populations were discovered, belonging to 4 taxa of the Chara genus: C. globularis, C. gymnophylla, C. vulgaris, and C. vulgaris var. papillata, reduced to 12 populations and to the first 3 taxa by the end of the study. The species occupied different ecological niches and colonized environments such as rivers and small ponds, with environment-dependent morphotypes. The occurrence of few taxa with a wide distribution, often forming ephemeral populations, suggests ongoing constraints on charophyte biodiversity in the area, favoring opportunistic species that are able to benefit from temporary refugia. [ABSTRACT FROM AUTHOR]