A new molecular method for the exploration of hybrid zones between two toad species of conservation interest

Analyzing hybrid zones between previously isolated lineages allows insight into processes determining the fate of re-encounter of these taxa. The distributions of Fire-bellied (Bombina bombina) and Yellow-bellied toads (B. variegata) meet in the Carpathian Basin resulting in a narrow contact zone in the foothill regions, where hybrids often appear. Our aim was to explore a transient zone between B. bombina and B. variegata within the Carpathian Basin along a transect including the Borzsony Hills in Hungary and Krupinska Planina in Slovakia. We visited 28 locations in these areas and collected altogether 230 specimens, photographed and sampled them using buccal swabs. In order to distinguish between the two species and determine hybrid individuals, we used mitochondrial markers and designed a novel technique based on the restriction of the Ncx-1 gene. The geographical distribution pattern of these two species delivered unexpected results, as Borzsony Hills was completely colonised by B. bombina including locations which can be classified as typical habitats of B. variegata. Moreover, in Krupinska Planina many locations were also colonised by B. bombina, including high altitude ponds. The most remote sites still harbour B. variegata populations, but seven of these were found with hybrid individuals. This pattern may indicate the northward and altitudinal range expansion of B. bombina and the colonisation of habitats of its sister species in these areas. Our results warrant enhanced attention to hybrid zones, where introgression and changes in population composition may reflect recent rapid environmental alterations and redirect conservation focus.