Molecular Identification of Honey Bee Species

To confirm the morphospecies identification, molecular analyses were performed using highly conserved regions of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which is referred to as the DNA barcoding region. Genomic DNA was isolated from the whole bodies of the A. cerana samples (Figure 1 and Supplementary Table S1) using a DNA purification kit (PureLink Genomic DNA Mini Kit, Invitrogen, Carlsband, CA, USA) according to the manufacturer’s instructions. The primer pair was used to amplify a partial fragment DNA of the COI gene (listed in Table S2). The PCR amplification was performed in 25 µL reactions containing 1X PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.5 µM forward primer, 0.5 µM reverse primer, 1U Taq DNA polymerase (Invitrogen, Carlsband, CA, USA), and 50 ng of DNA template. The PCR cycling conditions were 94 °C for 5 min, and 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, followed by a final step at 72 °C for 7 min. The resulting PCR products were separated by size on 1.5% agarose gel electrophoresis, and the nucleotide sequences were analyzed to distinguish the honey bee species. The DNA sequences were deposited in GenBank with accession numbers (see Supplementary Figure S1 and Table S3).