AEBSF Official records have shown that A mellifera subspecies

Official records have shown that A. mellifera subspecies, including armeniaca, capensis, carnica, caucasica, cypria, ligustica and mellifera were imported to improve the yield of various bee products during the period of 1911–1997 (Supplementary Information Table 1) (An et al., 2004; Sung et al., 2006). Since then, none has been imported officially. In Taiwan, these Western bees of some apiaries are known to give high yield of royal jelly while bees from other yards are better honey producer. It has been generally assumed that A. mellifera in Taiwan is composed of hybrids of different subspecies. Nevertheless, no study has been carried out up to the present to elucidate the makeup of honey bees managed by the beekeepers. We believe that identification of honey bee subspecies via molecular characterization is essential for more advanced management in AEBSF apiculture in Taiwan.
Twenty-eight subspecies of A. mellifera have been identified morphologically. They are classified, according to their geographic origins, into four branches/lineages, i.e., Africa (A), Western Europe (M), South-Eastern Europe (C) and Middle East (O) (Ruttner, 1988; Engel, 1999; Gupta, 2014). However, phylogeographical studies have shown that variations of morphological characters of these subspecies resulted from adaption to local environments often complicate their proper identification. Therefore, molecular methods using DNA markers, such as mitochondrial DNA (mtDNA), microsatellites and single nucleotide polymorphisms (SNPs), have been developed. Among them, analysis of restriction site polymorphisms of mtDNA fragments, characterized by their maternal inheritance, has been widely used to identify subspecies of honey bees. The length polymorphism of tRNAleu-COII region of mtDNA, due to the absence/presence of P AEBSF and different copy numbers of Q element, which correlates with their evolutionary lineages, has been frequently used to classify A. mellifera lineages (Garnery et al., 1992, 1993).
Among the five mitochondrial lineages identified in A. mellifera (Meixner et al., 2013), A, M and C roughly match with the morphological branches A, M and C (Cornuet and Garnery, 1991; Garnery et al., 1992). The original 4th mitochondrial lineage O from the Near East (Franck et al., 2000; Palmer et al., 2000) was split into mitochondrial C and Z lineages (Meixner et al., 2013), with a sub-lineage of A embedded in the latter (Alburaki et al., 2011, 2013). The 5th mitochondrial lineage Y has been identified in Ethiopia (Franck et al., 2001).
The existence of honey bees has been threatened by multiple factors, such as pesticide usage on crops, fragmentation and loss of habitat as well as pathogens and parasites (Antunez et al., 2009; Rosenkranz et al., 2010; Sanchez-Bayo et al., 2016). Colony Collapse Disorder (CCD), a combination of deadly effects, has widely occurred across Europe and America (Stokstad, 2007; van der Zee et al., 2012; Lee et al., 2015). Besides, the natural biodiversity of honey bees may be declined by the selective management of those subspecies that bring forth profits to the beekeepers. In view of the fact that CCD has not been observed in Taiwan and high yield of honey/royal jelly has been achieved, we intended to establish the matrilineal origins of these precious honey bees by analyzing four fragments of mtDNA, i.e., the non-coding region between tRNAleu and cytochrome c oxidase subunit II (intergenic tRNAleu-COII), cytochrome b (Cyt b), large subunit rRNA (Ls rRNA) and cytochrome c oxidase subunit I (COI) by PCR-RFLP and sequencing the tRNAleu-COII fragments of samples collected throughout the island.

Materials and methods

Results and discussion

Conclusions

Acknowledgements
This work was supported by grant 104-2321-B-005-018 from the Ministry of Science and Technology, Taiwan, ROC. We thank Prof. Wu-Chun Tu for the help on collection of samples from east region of Taiwan and beekeepers of 33 apiaries for providing bee samples. We also thank Dr. Chih-Ning Sun for helpful discussions and critical reading of this manuscript.