An extensive multi-approaches distribution map of Apis mellifera mitochondrial DNA lineages in Italy

The conservation of the genetic integrity of honey bee subspecies is a matter of concern in Europe. Specific mitochondrial DNA (mtDNA) lineages characterize several Apis mellifera subspecies. In this study we produced a distribution map of the main A. mellifera mtDNA lineages in Italy by combining two approaches: (1) mtDNA analyses of individual honey bees; (2) mtDNA analyses from honey environmental DNA. This second approach was also applied to obtain a distribution map of mtDNA lineages over four years in the North of Italy. DNA was extracted from ~800 worker bees collected in 2020 and 730 pupae collected in 2021 from different colonies. DNA was also extracted from about 1,600 honey samples produced in the North of Italy over four years (2018-2021) and additional 400 honey samples produced in 2018 in the Central and South of Italy and in the two main Italian islands (Sardinia and Sicily). PCR amplified fragments were sequenced or separated using a size-based assay. Both approaches showed that the C lineages were the most frequent mitotypes all over Italy except in Sicily, where the A lineage was highly represented. Individual bee analyses showed that the C1 mitotype, which mainly characterizes the A. m. ligustica subspecies, was the most frequent mtDNA haplotype in the investigated cohorts. Nevertheless, mtDNA haplotypes associated with non-endemic subspecies (A and M lineages) were present in almost all Italian regions and constantly represented over years. The obtained information can be useful to design conservation strategies of A. mellifera genetic resources in Italy.