ISSN 1022-7954, Russian Journal of Genetics, 2016, Vol. 52, No. 10, pp. 1062–1070. © Pleiades Publishing, Inc., 2016.
Original Russian Text © R.A. Ilyasov, A.V. Poskryakov, A.G. Nikolenko, 2016, published in Genetika, 2016, Vol. 52, No. 10, pp. 1176–1184.
Seven Genes of Mitochondrial Genome Enabling Differentiation
of Honeybee Subspecies Apis mellifera
R. A. Ilyasov, A. V. Poskryakov, and A. G. Nikolenko
Institute of Biochemistry and Genetics, Ufa Scientific Center, Russian Academy of Sciences, Ufa, 450054 Russia
Received November 19, 2015
Abstract—On the basis of comparative sequence analysis of 12 honeybee mitochondrial genes, seven genes
enabling us to differentiate honeybees subspecies of the A, M, C, O evolutionary lineages were found. Apply-
ing comparative sequence analysis of ND2 gene mtDNA as an example on a statistically valid sample size, we
showed a high level of differentiating ability of this gene and assumed that each of these seven genes probably
can be used for differentiation of the subspecies within four evolutionary lineages.
Keywords: black honeybee, evolutionary lineages, mitochondrial genome, mtDNA, ND2 gene, subspecies
differentiation, phylogenetic analysis, single nucleotide polymorphisms
The originally phylogenetic structure of honeybee
Apis mellifera was based on morphological studies .
Honeybee Apis mellifera under different environmen-
tal factors was divided into 30 subspecies spread over
all of the Old World [2–8]. The high level of honeybee
genetic diversity observed became a consequence of
continuous prolonged natural selection in different
climatic and ecological conditions [9, 10].
In the first investigations, honeybee subspecies were
divided into three groups: A lineage, uniting African sub-
species (A. m. lamarckii, A. m. yemenitica, A. m. scutel-
lata, A. m. litorea, A. m. adasonii, A. m. capensis);
M lineage, uniting subspecies of North Africa and West-
ern Europe (A. m. mellifera, A. m. iberica, A. m. inter-
missa); and C lineage, uniting honeybee subspecies of
Eastern Europe, Northern Mediterranean, and the
Later, the C lineage was divided into two: C lin-
eage, including subspecies of Eastern Europe and
Northern Mediterranean (A. m. carnica, A. m. ligustica,
A. m. macedonica, A. m. cecropia, A. m. caucasica,
A. m. sicula), and O lineage, including subspecies of
the Near and Middle East (A. m. armeniaca, A. m. meda,
A. m. anatoliaca, A. m. syriaca, A. m. cypria, A. m. adami)
[2, 11–17]. As a whole, molecular-genetic studies [17–
19] confirmed the earlier obtained morphometric data
concerning the A, M, and C lineages ; the O lineage
was isolated according to DNA polymorphism [16,
20–22]. Recently, the Y lineage, including subspecies
A. m. yemenitica in Ethiopia , and the Z lineage,
including subspecies A. m. syriaca in Syria [24, 25]
were isolated, but they have not been generally recog-
nized as yet.
One is constantly faced with informative and non-
informative genetic markers in genetic geography and
phylogenetic studies of honeybees [26, 27]. We
intended to seek and evaluate genetic markers of the
mitochondrial genome making it possible to correctly
differentiate honeybee subspecies belonging to the A,
M, C, and O lineages.
MATERIALS AND METHODS
For comparative analysis of 12 genes of mitochon-
drial DNA nucleotide sequences (ND2, COII, COI,
ATP8, ATP6, COIII, ND3, ND5, ND4, ND4L, ND6,
ND1) to be carried out, we used sequences of the
whole mitochondrial genome of members of the A,
M, C, and O lineages: NC_001566 A. m. ligustica (С)
, KJ396189 A. m. mellifera (M) , KJ396188 A.
m. mellifera (M) , KP163643 A. m. syriaca (O)
, KJ601784 A. m. scutellata (A) , KM458618
A. m. intermissa (A) .
Determination of the nucleotide sequence of
amplified fragments of gene ND2  was performed
at Syntol (Moscow) using the Applied Biosystems
sequencer. For ND2 gene amplification, F 5′-TGATA-
AAAGAAATATTTTGA-3′ and R 5′-GAATCTAAT-
TAATAAAAAA-3′ primers were used .
For purposes of comparative analysis, the nucleo-
tide sequences of the ND2 gene mtDNA of 31 honey-
bee samples belonging to the A evolutionary lineage,
34 samples belonging to the C evolutionary lineage, 3
samples belonging to the M evolutionary lineage, and