The COI-COII Intergenic Spacer for Discriminating Honeybee Subspecies: Capabilities and Constraints

Accurate identification of honeybee (Apis mellifera) subspecies is essential for conservation and breeding programs. The mitochondrial COI-COII intergenic spacer has been proposed as a genetic marker for subspecies discrimination, but its reliability remains debated. We analyzed 41 COI-COII sequences from diverse subspecies and geographic origins using phylogenetic reconstruction, network analysis, and population genetic statistics. Our results show that although this marker successfully distinguishes some regional populations (e.g., Russian, French, and Maltese specimens), it exhibits limited resolution for closely related subspecies. We detected only 27 polymorphic sites and found identical sequences shared among geographically distant and taxonomically distinct groups. Designed primers showed variable specificity, with subspecies-specific amplification achieved for A. m. ruttneri but not for A. m. lamarckii or A. m. carnica. These findings confirm that the COI-COII region alone is insufficient for comprehensive subspecies identification and should be combined with nuclear markers or morphological data in taxonomic and conservation applications.