Comparing Morphometric and Mitochondrial DNA Data from Honeybees and Honey Samples for Identifying <i>Apis mellifera ligustica</i> Subspecies at the Colony Level

The ability to differentiate between different <i>Apis mellifera</i> subspecies can have significant implications for their conservation. In this study, we explored the possibility of obtaining multi-level information that could be useful in assigning a colony to the <i>A. m. ligustica</i> subspecies. This was accomplished by combining (1) standard morphometric characteristics of the honeybees with mitochondrial DNA (mtDNA) sequence data obtained from both the (2) honeybees and (3) honey present in a honeycomb sampled from the same colony. Eighty colonies were sampled in the Emilia-Romagna region (Northern Italy). From these colonies, a total of 413 honeybees were analyzed to obtain sequence information of the tRNALeu-COII mtDNA region. When we combined the results obtained from all three methods, 63.75% of the colonies were found to be compatible with being assigned to <i>A. m. ligustica</i>. For 36.25% of the colonies, morphometric analysis indicated a hybrid origin and mtDNA from honeybees or honey samples indicated the presence of mtDNA haplotypes other than those characteristic for this subspecies. Cohen’s Kappa statistic indicated poor agreement in the classification of the colonies between morphometric analysis and the two DNA-based methods, which, in turn, were in substantial agreement with each other. Although not completely effective, mtDNA analysis of honey samples could serve as an initial and practical compromise to begin initiatives aimed at preserving the genetic integrity of <i>A. m. ligustica</i> in the Emilia-Romagna region, as well as in other regions where it is necessary to preserve the genetic integrity of autochthonous honeybee populations.