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Southern Europe hosts a large number of critical catchments for freshwater biodiversity, including endemic fish species. Unfortunately, these areas are severely threatened due to direct and indirect anthropogenic effects. In this context, with the aim to improve the effectiveness of threatened fish protection, the Life project BARBIE (LIFE13 NAT/IT/001129) started in 2014 and focused on three congeneric species of the genus Barbus: two of “priority interest” sensu Habitats Directive [Barbus caninus (Bonaparte, 1839), and B. plebejus (Bonaparte, 1839)], and one alien [Barbus barbus (Linnaeus, 1758)]. Our main objective was to assess the contribution of a complex of protected areas included in the Natura 2000 network – located in the provinces of Parma, Piacenza and Reggio Emilia (Norther Italy) – to support the presence of the three barbel species in analysis. Additionally, we explored the role of a set of environmental variables (i.e., physical, chemical, biological, and land-use descriptors) to drive the current conditions of the study sites and the responses of Barbus species. As a general rule, the present study confirmed a clear decline of the local native barbel populations, and confirmed the existence of a zonation pattern of the barbel taxa. Hence, we observed a strong altitude segregation between native vs. alien species, with the exotic B. barbus currently limited to plain and only sporadically present in the Apennine areas as genetic introgression. These evidences mirrored the altitudinal gradients of anthropogenic disturbance. The main causes were the progressive disappearance of well-structured riparian stripes, and the intense land use change, ranging from semi-natural patches (mountain and hill sectors) to land clearing for intensive agriculture (lowland sectors). This highlights the need to take into account the spatial dynamics of alien invasive species in programming recovery actions that could have unexpected impacts to the native fish populations, for example the reduction of the hydraulic fragmentation along rivers.