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Karst ecosystems are considered as priority environments for the protection of biodiversity on a global scale. This study provides a first comparative analysis of epilithic diatom flora from karst springs in two Mediterranean geographic areas (Spain and Italy) with contrasting characteristics (islands vs mainland). We investigated twenty-three springs with different anthropogenic impact levels once in the winter season between 2007 and 2017 (N = 23). A total of 176 diatom taxa (56 genera) were found of which 101 (44 genera) were observed in single sites. A general good biotic integrity was revealed by structural indices (species richness, diversity and evenness). However, crenophilous species were generally present and abundant in less impacted springs. Comparing islands and mainland, significant differences were found in species composition and diversity (H’) based on multivariate analyses (global R = 0.610; p = 0.001) and t-test (t = 2.304; p = 0.031). Discharge and Cl− were the most significant variables in determining diatom assemblages. Our results confirm the role of springs as multiple ecotones and refuges for rare species and suggest that the geographic insularity may be an important factor in maintaining diatom biodiversity.

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