An over-time distribution map of Apis mellifera mitotypes in Italy constructed using an environmental DNA approach.

For the role that Apis mellifera plays in the agroecological systems, a growing interest across Europe is emerging to preserve the genetic integrity of honey bee populations and subspecies. Honey is an incredible source of environmental DNA that contains traces of all organisms that directly or indirectly were involved in its production, including that of the honey bees that produced it. Specific mitochondrial DNA (mtDNA) lineages that characterize several Apis mellifera subspecies can be detected from the same honey sample, providing approximate population genetic information useful to estimate the diffusion and frequency of honey bee mitotypes. In this study, more than 2400 honey samples produced over the last five years (2018–2022) in several Italian regions were collected from different beekeepers. Honey samples were produced in nine regions of the North of Italy, in Sardinia and Sicily. DNA was isolated from all these samples and PCR amplified fragments were separated using a sizebased assay to detect A, M and C mtDNA lineages. A second assay based on Sanger sequencing was used to discriminate C1 and C2 mitotypes, which are associated with the A. m. ligustica and A. m. carnica subspecies, respectively. The results showed that the C lineages were the most frequent mitotypes all over Italy and the C1 mitotype was the most prevalent, as expected. Nevertheless, a similar distribution of honey with different mitotypes was obtained in all other regions of the North of Italy and mtDNA haplotypes associated with non-endemic subspecies (A and M lineages) were present and continuously represented over years. The A lineage was highly represented in Sicily. Metadata about the geographical origin, the altitude of the production site and beekeeper information were collected and merged with the results of mtDNA analysis to produce a distribution map of the main honey bee mitotypes. The obtained results will be useful to design conservation strategies of A. mellifera genetic resources in Italy. Moreover, we demonstrated that eDNA from honey can be exploited to design cost-effective non-invasive and simple methods to obtain information on the genetic distribution of honey bee mitotypes in a large geographic area. Acknowledgements This study was supported by Regione Emilia-Romagna BEE-RER3 project. KJ is supported by the Fulbright Program. We thank Osservatorio Nazionale Miele e Lucia Piana srl for providing samples and many beekeepers for the collaboration