The Dinaric Karst extends along the Adriatic coast of the Western Balkan Peninsula and is home to a group of “karst minnows” of the genera Delminichthys, Phoxinellus, and Telestes, which have adapted to the highly variable water conditions in the karst by spending up to several months underground, but require surface habitats for spawning, defining them as substygophiles. The three species of the genus Phoxinellus, P. alepidotus , P. pseudalepidotus, and P. dalmaticus , are defined by restricted ranges, making them vulnerable to pollution and extended draughts caused by the climate change. In this study, the phylogeny of Leusciscinae was reconstructed using 15 Phoxinellus and one Delminichthys adspersus, one Pelasgus epiroticus, and one Telestes polylepis complete mitochondrial genomes and the position of the genus Phoxinellus within the subfamily as sister species to the Chondrostoma clade was confirmed. The inter‐ and intrapopulation structure of the genus Phoxinellus was inferred using molecular (nuclear and mitochondrial data) and morphological analyses. For the molecular analysis, more than 150 historical specimens were analyzed for a short fragment of the cytochrome oxidase I (COI) barcoding region and 15 Phoxinellus specimens were subjected to single nucleotide polymorphism analysis. For morphological analysis, 121 Phoxinellus specimens were analyzed for 51 measurements and 8 counts. All analyses confirmed the clear delimitation of the three Phoxinellus species, but were insufficient to fully resolve the intrapopulation structure within the species. This study also included data from field surveys of Phoxinellus collected over the past 20 years, which showed that abundance is declining and ranges are shrinking. Phoxinellus are also threatened by invasive/introduced species. Based on cave observations/occurrence and morphological analysis, P. dalmaticus was classified as an advanced substygophile and P. alepidotus and P. pseudalepidotus were classified as basic stygophiles.
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