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In groundwater-fed springs, habitat characteristics are primarily determined by a complex combination of geomorphic features and physico-chemical parameters, while species assemblages are even more intricate. Springs host species either inhabiting the spring mouth, or colonizing spring habitats from the surface or from the aquifers which feed the springs. Groundwater species living in springs have been claimed as good candidates for identifying dual aquifer flowpaths or changes in groundwater pathways before reaching the spring outlets. However, the reliability of spring species as hydrological biotracers has not been widely investigated so far. Our study was aimed at analysing a large karstic spring system at three nested spatial scales in order: i) to assess, at whole spring system scale, the presence of a groundwater divide separating two aquifers feeding two spring units within a single spring system, by combining isotope analyses, physico-chemistry, and copepod distribution patterns; ii) to test, at vertical spring system scale, the effectiveness of copepods in discriminating surface and subsurface habitat patches within the complex mosaic spring environment; iii) to explore, at local spring unit level, the relative role of hydrochemistry and sediment texture as describers of copepod distribution among microhabitats. The results obtained demonstrated the presence of a hierarchical spatial structure, interestingly reflected in significant differences in assemblage compositions. Copepod assemblages differed between the two contiguous spring units, which were clearly characterized by their hydrochemistry and by significant differences in the groundwater flowpaths and recharge areas, as derived by the isotope analyses. The biological results suggested that stygobiotic species seem to be related to the origin of groundwater, suggesting their potential role as hydrological biotracers. At vertical scale, assemblage composition in surface and subsurface habitats was significantly different, both between spring units and among microhabitats, supporting strong habitat preferences of copepod species. At the smaller local scale, the response to habitat patchiness of subsurface copepod assemblages resulted in distribution patterns primarily defined by sediment texture, while the sensitivity to differences in hydrochemistry was negligible.