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Our aims were to test the hypothesis that in deep lakes the co-occurrence patterns of macrophytes are not random, and to compare the relative contribution of the main environmental determinants (light, water and sediment parameters, phytoplankton) in structuring aquatic vegetation. We collected data from five deep Chara-dominated lakes in Central Italy along gradients of depth (33 to 165 m), dimension (1.7 to 114.5 km2) and water trophic conditions (12.4 to 41.3 μg L-1 of total phosphorous). Twenty-five sampling plots per lake were randomly selected at five predetermined depths (1.5, 3.0, 6.0, 12.0 and 20.0 m; n=5) within homogenous littoral sectors. Data were explored by a null model analysis using the checkerboard score (C-score) index, and Canonical Correspondence Analysis. Our data verify the not random co-occurrence patterns of macrophyte’ communities in deep lakes. However, present data suggested that C-scores are strictly dependent on lake’ trophic status: low nutrient loads, in both water and sediments, seemed to be reflected in a not random co-occurrence zonation of macrophytes. Summarizing, it is fundamental evaluate the local effects of lake trophy on the macrophyte community dynamics both in time and space before inquiring about mutual links. If it fails to assess macrophyte co-occurrence patterns, it may be not possible to identify the determinants of the spatial arrangement of macrophytes and, in turn, the conservation status or the ongoing dynamics of lakes.