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Leuciscine fishes represent an important component of freshwater ichthyofauna endemic to northern Mediterranean areas. This lineage shows high intra-specific morphological variability and exhibits high levels of hybridization, two characteristics that contribute to systematic uncertainties, misclassification of taxa and, potentially, the mismanagement of biodiversity. This study focused on brook chub,  Squalius lucumonis , an endemic taxon of Central Italy. The taxonomic status of this species has long been questioned, and a hybrid origin from sympatric leusciscines ( S. squalus  x  Rutilus rubilio , or  S. squalus  x  Telestes muticellus ) has been hypothesised. A phenotypic (evaluating shape and meristic counts) and genetic (using mitochondrial and nuclear markers) investigation of these four taxa was conducted to test species delimitation in sympatric areas and to evaluate the taxonomic status of  S. lucumonis . One hundred and forty-five individuals of all four taxa were collected within streams of the lowest portion of the Tiber River basin and analysed; this region encompasses a large portion of the  S. lucumonis  distribution. The different morphological and genetic approaches were individually examined, compared, and then combined in a quantitative model to both investigate the limits of each approach and to identify cases of misclassification. The results obtained confirm the cladogenetic non-hybrid origin of  S. lucumonis , highlight the need for immediate conservation actions and emphasise the value of an integrated approach in the study of leuciscines evolution.

Testing Species Delimitations in Four Italian Sympatric Leuciscine Fishes in the Tiber River: A Combined Morphological and Molecular Approach