Exploration of a hybrid zone between two toad species in Central Europe's Carpathian region with a new molecular marker

Analyzing hybrid zones allows insight into interactions between previously isolated lineages. The distributions of firebellied toads (Bombina bombina) and yellow-bellied toads (B. variegata) meet in the Carpathian Basin. We aimed to explore the genetic composition of a transient zone between the two sister species along a transect in the Carpathian foothill region. We collected 230 specimens from 21 locations and sampled them using buccal swabs. We used mitochondrial markers and designed a novel test based on the restriction of the nuclear Ncx-1 gene to distinguish between the two species and determine hybrid individuals. According to our results, the complete Borzsony Hills and many locations in Krupinska Planina were colonized by B. bombina, including typical B. variegata habitats at higher elevations. However, in Krupinska Planina, the most remote sites still harbor B. variegata populations, often mixed with hybrid individuals. This pattern may indicate the northward and altitudinal range expansion of B. bombina. Our results warrant enhanced attention to hybrid zones, where introgression and other changes in the populations' genetic composition may reflect recent rapid environmental alterations.