Biogeographic study of the honey bee (Apis mellifera L.) from Serbia, Bosnia and Herzegovina and Republic of Macedonia based on mitochondrial DNA analyses.

In this work, Apis mellifera carnica and A. m. macedonica honey bees from Serbia, Bosnia and Herzegovina and Republic of Macedonia were analysed using molecular techniques in order to improve our knowledge about biogeography of A. mellifera on the Balkan peninsula. This is the first time that the indigenous honey bees from Bosnia and Herzegovina and Republic of Macedonia have been analyzed using a molecular approach. Sampling was carried out from 560 stationary apiaries where bees were kept in traditional hives (woven skeps). The COI-COII regions of 1680 samples were PCR-amplified and sequenced. To reveal the haplotype of studied bees, the obtained sequences were aligned with published sequence data of haplotypes that belong to A. mellifera C phylogenetic lineage. The C2D mtDNA haplotype was found in all honey bees sampled from Serbia, Bosnia and Herzegovina and Republic of Macedonia. These results show that A. m. carnica and A. m. macedonica share the same C2D mtDNA haplotype. COI gene segments of 1680 samples were PCR-amplified and digested with restriction enzymes NcoI and StyI in order to discriminate A. m. macedonica from A. m. carnica. Amplified fragment patterns produced by both restriction enzymes matched with diagnostic pattern characteristic for A. m. macedonica in case of samples from east, south and south-west parts of Serbia, and Republic of Macedonia, fragments of samples from northern part of Serbia and Bosnia and Herzegovina did not include NcoI and StyI restriction sites. These results indicate that honey bees from east, south and south-west parts of Serbia, and Republic of Macedonia belong to the A. m. macedonica, and honey bees from northern part of Serbia and Bosnia and Herzegovina belong to another subspecies, probably to the A. m. carnica. Therefore A. m. macedonica has much wider area of distribution than it was previously considered.