Substygophily in Dinaric Karst: a model case of locally endemic minnows Phoxinellus (Leuciscinae)

The Dinaric Karst stretches along the Adriatic coast of the Western Balkan Peninsula and is home to a group of paraphyletic "karst minnows" of the genera Delminichthys, Phoxinellus and Telestes, which have adapted to the highly variable water conditions in karst, spending up to several months underground but requiring surface habitats for spawning. The three species of the genus Phoxinellus, P. alepidotus, P. pseudalepidotus and P. dalmaticus, are defined by restricted ranges, which make them vulnerable to pollution and introduced/ invasive fish species. In this study, complete mitochondrial genomes of the three Phoxinellus species were assembled to revise their phylogenetic relationships within the genus and their phylogenetic position within the subfamily Leuciscinae (Cypriniformes: Leuciscidae). In addition, historical specimens, including locally extinct populations and type material, were analyzed for a short fragment of cytochrome oxidase I to assess genetic diversity within populations. In addition, selected DNA samples with good DNA quality were sent for low-coverage whole-genome sequencing and analyzed to infer interspecific and intrapopulation structure. This study also includes some field observations on Phoxinellus populations during the last twenty years and morphological analysis of characters possibly related to their partially hypogean lifestyle. The results confirmed the conservation status of the three species, all of which are endangered. Molecular and morphological analyses confirmed the delimitation of the three Phoxinellus species and their placement within the subfamily Leuciscinae. The intrapopulation structure was less clear, either due to gene flow between populations or lack of signal due to insufficient data. Based on morphological analysis and field observations, P. dalmaticus was classified as an advanced substygophile, P. alepidotus as a basic substygophile, and P. pseudalepidotus as a possible basic substygophile, as there is no direct evidence that this species occurs in hypogean habitats.