Analysis of Apis mellifera mitotypes in Emilia‑Romagna: eight years of investigation using Honey eDNA

Honey contains DNA from all organisms involved in its production, including honey bees (Apis mellifera), making it a valuable, non-invasive source of genetic information for analysing population structure and authenticating its entomological origin. In this study, we applied our previously developed molecular assays, which distinguish three mitochondrial DNA (mtDNA) lineages (A, C and M) of A. mellifera and differentiate two mitotypes within the mtDNA C lineage: the C1 mitotype, predominantly associated with A. m. ligustica, and the C2 mitotype, specific to A. m. carnica, to characterize the genetic origin of honey samples from Emilia-Romagna region (Italy). A total of 1,200 honey samples produced in Emilia-Romagna over the last eight years (20182025) were analysed. The C1 mitotype was confirmed as the most frequent in Emilia-Romagna, reflecting the predominance of A. m. ligustica in local beekeeping. A smaller fraction of samples carried the C2 mitotype, while some samples showed both C1 and C2 mitotypes, representing the presence of admixed populations in Emilia Romagna region. A small number of honey samples also contained A and/or M lineages, other than C lineage. Geographical information associated with the honey samples allowed us to map the distribution of mitotypes across the Emilia-Romagna territory, providing insights into spatial patterns and potential local variation in honey bee populations. This approach demonstrates the reliability of using honey as a DNA source for population genetic studies and for tracing the A. mellifera genetic origin of honey. This study can be helpful to develop conservation strategies of honey bee genetic resources, and improve the authentication of honey at both regional and international levels.