Mitogenomic analysis of Slovak Carniolan honey bees reveals C-lineage affiliation and regional mitochondrial variation

Abstract The Carniolan honey bee ( Apis mellifera carnica ) represents a key subspecies in Central European apiculture and is the only officially recognized honey bee subspecies in Slovakia. In this study, we performed a complete mitogenomic analysis of eight honey bee colonies, all originating from officially registered Carniolan breeding lines with one exception originating from a non-selected colony from a relatively isolated apiary (“wild”). Phylogenetic analysis based on whole mitogenome alignment placed all Slovak samples within the C evolutionary lineage, clustering closely with A. m. carnica reference sequences. Analysis of the tRNA-Leu–COX2 intergenic region and in silico DraI restriction confirmed their affiliation with the C1/C1a haplotype group. Sequence comparison and BLASTn analysis revealed three distinct haplotypes among the eight Slovak samples: one predominant variant shared by five lines (wild, Sitňanka, Košičanka, Júlia, and Carnica Sokol), a second shared by two lines (Vojničanka and Tatranka), and a third represented by a single unique (singleton) haplotype (Šahanka). These results are consistent with the placement of Slovak Carniolan honey bees within the Southeast European lineage C and their affiliation with the C1/C1a haplotype group, while also revealing region-specific mitochondrial variants that may reflect natural diversification within managed breeding lines.