359
Australian Journal of Entomology, 1997, 36: 359-364
Mitochondria1 Sequence Characterisation of Australian Commercial
and Feral Honeybee Strains, Apis mellifera L. (Hymenoptera:
Apidae), in the Context of the Species Worldwide
S. KOULIANOS* and R. H. CROZIER
School of Genetics and Human Variation, La Trobe University, Bundoora, Vic. 3083.
*Present address: ETH Zurich, Experimental Ecology, ETH Zentrum NW, 8092 Zurich, Switzerland.
ABSTRACT
The history of honeybee (Apisrneliifera) importations and management in Australia
is largely anecdotal and therefore a survey and characterisation of this agriculturally important
insect is of value. We give information on the genetic composition of 42 feral and commercial strains
by sequencing sections of the ATPase 6, cytochrome oxidase 111, cytochrome b and ND2
mitochondrial genes to determine the relationship of the strains to each other. O u r phylogenetic
analysis shows novel associations between A . 0 7 . inellifera. A . in. scutellaia, A . m. ligusiica and
A . m. caucasica.
Introduction
The "English black bee", Apis mellifera mellijiera
L., was first introduced to Sydney in 1822. In
1831, swarms were sent from there to Tasmania
by Dr T. B. Wilson. By this time they were well
established in the Sydney area. During a journey
by the explorer James Stuart Laurie to rescue
Burke and his party, it was noticed that honeybees
were also common in the bush ". . . pasturage is
abundant and the bees find a convenient domicile
in hollow trees" (Laurie 1853). The bees had
spread throughout the state of Western Australia
by 1881 (Coleman 1956). With the advent of
migratory beekeeping in the 1896 (Smith 1964),
bees became well established as feral colonies
throughout Australia. In addition to A . m.
mellijiera, A . m. ligustica bees are now extensively
used by apiarists and A . m. caucasica and A. m.
carnica have been used in bee breeding programs.
T h e history of bee importations a n d
management in Australia is largely anecdotal;
occurrences are determined by human activities
and classifications are usually based on the
judgment of apiarists. Therefore, a survey and
characterisation of both feral and commercial
strains of this agriculturally important insect are
of value. Here we present a characterisation of
Australian feral and commercial honeybees based
on DNA sequences from the ATPase 6,
cytochrome oxidase 111, cytochrome b and ND2
mitochondria1 genes. All reference material (see
Table 1) was amplified for the size polymorphic
COI/COII region, and restriction digests of the
NDI /ND4 region were checked against published
restriction maps because of the difficulty in
locating reliable reference populations of certain
subspecies status in Australia. The DNA was
sequenced from single bees, allowing the
development of rapid diagnostic tests for
particular lineages. Once the information was
available in terms of the sequence of nucleotides
of mtDNA, new lineages were identified as such
to the extent allowed by the variation actually
uncovered, and their relationship along the
maternal line t o other lineages was determined.
Materials and methods
Bees were supplied by the University of Western
Sydney, the Department of Agriculture (Western
Australia), The Department of Agriculture
(Tasmania), Dr M. Schwarz, Dr D. Paton,
Crestline Apiaries, T. Brown, G. Wheen, B. White
and R. Stephens Apiaries. A . m. mellifera
specimens were collected by Dr B. Oldroyd and
SK. The areas which were sampled are indicated
in Fig. 1. A . m. scutellata specimens were supplied
in alcohol by Dr H. G . Hall (University of
Florida).
A . m. mellifera and A . m. ligusfica reference
specimens were sent to the US Department of
Agriculture in Baton Rouge for identification (for
methods see Oldroyd el al. 1995).
Total DNA was extracted from single bees by
modification of standard procedures as reported
by Crozier et al. (1991). The ATPase 6/COIII,
cytochrome b, ND2 and COI/COII regions were
amplified using primers in Koulianos and Crozier
(1996) designed from the known complete
sequence (Crozier and Crozier 1993) and Taq
polymerase (United States Biochemical
Corporation) in a Perkin Elmer Cetus thermal
cycler for 35 cycles under the following conditions:
denaturation at 92 "C for 1 min, annealing at 53 "C
for 1 min, extension at 70°C for 1 min. The
NDI/ND4 region was amplified for 30 cycles
under the following conditions: denaturation at
92 "C for 1.5 min, annealing at 50°C for 1 min,
extension at 70°C for 5 mins. All products were
purified with Millipore Ultrafree units. The
C O I / C O I I P C R product was sized by
electrophoresis on a 2% agarose gel against
a x 1 7 4 cut with HaeIII. The bands were visualised
by ethidium bromide staining. The NDI/ND4
region was digested with PvuII, SpeI and Ndel as
360
S. KOULIANOS and R. H. CROZIER
recommended by the supplier (Promega). The
digested fragments were separated on a 2%
agarose gel and visualised by ethidium bromide
staining. Direct sequencing of the ATPase
6/COIII, cytochrome b and ND2 gene regions
from single-stranded PCR product was achieved
by standard procedures (Kessing et al. 1989;
Koulianos and Crozier 1991).
Divergence between haplotypes (d) was
estimated by the method of Galtier and Gouy
(1995). Phylogenetic analyses were carried out
using the maximum likelihood method
implemented using the program DNAML in
PHYLIP version 3.52 (Felsenstein 1993) and
maximum parsimony using PAUP version 3.1.1
(Swofford 1993). Maximum likelihood trees were
obtained by searching for the best tree with global
rearrangements. Parsimony trees were obtained by
the branch and bound method. Support for the
nodes was estimated using a minimum of 1,000
bootstrap replications. The transition/transversion
ratio used for both methods obtained by
maximising the likelihood was 5.4 for the whole
data set and 4.0 for the reduced data set containing
haplotypes of known heritage. Further, we used
the LogDet transformation (Lockhart et al. 1994)
with the inclusion of all sites implemented using
the program SplitsTree 1.0 (Huson and Wetzel
1994) to avoid sequences being grouped on the
basis of their nucleotide composition alone. The
Kishino and Hasegawa (1989) test was used to
determine whether the parsimony and maximum
likelihood trees were significantly different from
each other.
Results
The 13 haplotypes found, voucher specimen
identity codes (IC) and the locality of the colonies
are shown in Table 1. The distribution of the
Australian haplotypes is shown in Fig. 1. The
haplotypes are named by a three-letter code which
is dependent on the variation found in the three
mitochondria1 regions sequenced. For example, in
AAA, the first A represents allele A of the ATPase
6/cytochrome oxidase 111 region, the second A
represents allele A of the cytochrome b region and
the third A represents allele A of the ND2 region.
This coding sequence is maintained for all
haplotypes. The actual nucleotide sequences are
accessible from GenBank (accession numbers
U72266-U72289). 12 haplotypes in total were
I
/
Fig. 1. Regions sampled and corresponding haplotypes in
Western Australia, South Australia (including Kangaroo
Island), New South Wales, Victoria and Tasmania. Note that
AAA and AFI are A. m. ligustica, ACE and ACF are A . m.
caucasica and BEB is A . m. mellifera.
Table 1. The 12 Australian haplotypes found. The 13th haplotype (CGK) represents the Africanised bee. Identity codes (IC)
for voucher specimens kept at La Trobe University are also given. Numbers in brackets represent the number of colonies sampled
which had that haulotyue.
Haplotype
Origin
Commercial and feral colonies from NSW, WA and
AAA (13) IC: ABYG-I; ABYU-I; ABZB-I, 2, 3, 4, 6, 9,
Victoria of unknown or ligustican heritage (our
10, 12, 14, IS; ABZV
ligustica reference).
Feral colonies from Tasmania.
AAJ (2) 1C: T-8, 1 1
Commercial colonies from NSW of unknown heritage.
ACC (2) IC: ABYG-2; ABYU-2
Colonies from Krasnopoliansky in Russia of Caucasian
ACE (2) IC: ABZN-3, 4
heritage (our caucasica reference).
Colony from Krasnopoliansky in Russia of Caucasian
ACF (1) IC: ABZN-2
heritage (our caucasica reference).
Commercial and feral colonies from NSW of unknown
ACG (4) IC: ABYZ-2; ABZA-I, 2; SYDNEY
heritage.
Commercial colonies from Tasmania of ligustican heritage
AFI (2) IC: T-49, 52
(our ligustica reference).
Feral colony from WA.
BAB (1) IC: ABZB-I 1
Feral colony from WA.
BBB (1) IC: ABZB-19
Commercial and feral colonies from Kangaroo Island of
BDD (8) IC: ABZO-I, 2, 3, 4, 6 , 7, 10; ACAO
ligustican or melliferan heritage, and a feral colony
from SA.
Commercial colony from NSW of unknown heritage.
BDH (1) IC: ABYZ-I
Colonies from Tarraleah in Tasmania of melliferan
BEB (4) IC: Tas-81, 86; W-22, 23
heritage and feral colonies from Victoria (our mellifera
reference).
A. m. scutellata
CGK (1) IC: ABZR-4
CHARACTERISATION OF AUSTRALlAN HONEYBEE STRAINS
361
too have been found to be morphologically A. m.
mellifera (for results see Oldroyd et al. 1995).
The number of nucleotide substitutions between
haplotypes varies from 0.07% to as much as
2.17% (between BBB and CGK). The nucleotide
diversity (Nei 1987) for haplotypes (weighted by
their relative frequencies) was equal to 0.004. The
mean divergence (Galtier and Gouy 1995) between
the mtDNA lineages is shown in Table 3.
Divergence between mtDNA haplotypes was
always higher between than within lineages (only
distances between haplotypes of known origin are
shown).
Phylogenetic trees were inferred from the
sequence data using maximum IikeIihood,
parsimony and LogDet transformation methods.
Fig. 2 contains trees obtained for the whole data
set. The best tree obtained by maximum likelihood
(a) differs slightly from that obtained by
maximum parsimony a n d the LogDet
transformation (b); tree (b) was not significantly
worse than tree (a) by the Kishino and Hasegawa
(1989) test. The major difference between the two
trees is the position of the Africanised haplotype
(CGK); DNAML clustered CGK within A . m.
ligustica whereas parsimony and the LogDet
transformation recognised it as a distinct lineage.
Maximum likelihood, parsimony and the LogDet
transformation returned the same tree (Fig. 3) for
the reduced data set containing haplotypes of
known heritage only, except for minor differences
in the length of branches.
found in the Australian honeybees. Haplotype
AAA was identical in sequence to A . m. ligustica
in Crozier and Crozier (1993). The 13th haplotype
(CGK) is from A . m. scutellata. All reference
populations show size classes that are consistent
with that subspecies (Table 2). These size classes
result from different combinations of t w o
sequence fragments named P and Q in Cornuet
et al. (1991). The Q sequence is observed in our
A . m. ligustica reference populations (AAA and
AFI). It is also found in our A . m. caucasica
reference populations (ACE and ACF) and all
other northern Mediterranean races (Garnery et
al. 1992). The other sizes (PQ, PQQ and PQQQ)
are known in A . m. mellifera and several African
races (Garnery et al. 1992). Therefore, we can
distinguish A . m. mellgera and African haplotypes
from those of the Northern Mediterranean species.
The PQQQ sequence is observed in our A . m.
melliferu reference population (BEB) and the P Q
sequence is observed in A . m. scutelluta (CGK).
The presence or absence of BglII, HindIII,
PvuII, SpeI and NdeI restriction sites are also
shown in Table 2. All reference populations show
restriction patterns typical of their subspecies,
except the population from Tarraleah, Tasmania
which differs from published A . m. mellifera maps
(Garnery et ul. 1992) by 1 NdeI site. Tarraleah bees
were described by Ruttner (1976) as true A . m.
meflifera on morphological grounds; our samples
have been sent to the US Department of Agriculture in Baton Rouge for identification and they
Table 2. Restriction site and length polymorphisms for the Australian haplotypes and A . m. scutellata. Presence of a restriction
site is indicated by + and the absence by - . Presence/absence of PvuII, SpeI and Ndel sites was obtained from the restriction
digests of the NDl/ND4 region. Presencelabsence of BglIl and Hind111 sites was obtained from the cytochrome b and ND2
sequences, respectively. Corresponding restriction sites of Garnery et a/. (1992) are shown in brackets. The length polyrnorphisms
result from different combinations of 2 sequence fragments named P and Q in Cornuet et al. (1991).
Bglli
(b)
Sample
Hindlll
(0
Pvull
(a)
Spel
(C)
SpeI
(t)
Ndel
(u)
Ndel
(v)
Length
Poly.
+
i
+
AAA
+
+
Q
+
AAJ
+
+
++
+
Q
ACC
+
+
+
Q
ACE
+
t
+
Q
ACF
+
++
Q
++
ACG
+
+
++
Q
AFI
+
++
+
+
Q
+
BAB
+
++Q
BBB
+
+
++
PQQ
+
BDD
+
+
t
PQQ
+
BDH
+
+
+
PQ
BEB
++
+
+PQQQ
CGK
+
+
t
PQ
Note: AAA and AFI is A . m. ligustica, ACE and ACF is A . m. caucasica, BEB is A . m. mellifera and CGK is A . m. scutellata.
-
-
Table 3. Distances (Galtier and Gouy 1995) and standard deviations within and between the lineages A ( A . m. scutellata),
C ( A . tn. ligustica and A . tn. caucasica) and M ( A . in. ineNifera). Only distances from haplotypes of known subspecies status
are shown.
C
A . m. ligusrico
A . m. caucasit,a
m. ligustica
C
A.
M
A
A. m. caucasica
A. m. mellifera
A. m. scutellata
SD
0.001 I
= 0.0013
M
..
A
A . m. tiiellifera
A . tn. scutella
0.01 18
SD
= 0.0011
-
0.0072
SD
= 0.001 1
0.01 80
-
362
S. KOULIANOS and R. H. CROZIER
(A. m. scutellata)
CGK
(A. m. melliferu)
BEB
BDD
BDH
BBB
(A.
111.
ligustica) AA
BAB
AFI (A. m. ligustica)
(A. m. caucasica) ACE
ACF
(A. m. caiicasica)
U
0.00 I
(A. m. mellijera)
B EB
BDD
100
BAB
(A. m. ligustica) AAA 5 1
AFI (A. m. ligiistica)
59
(A. m. caucasica) ACE
1
ACF
(A. nz. caucasica)
U
0.001
Fig. 2. Relationship of the 13 haplotypes to each other. (a) was obtained using maximum likelihood. (b) was obtained using
parsimony and the LogDet transformation. Length of branches were obtained by the usertree option of maximum likelihood.
For (b) numbers at nodes are bootstrap percentages (1,000 replicates).
CHARACTERISATION OF AUSTRALIAN HONEYBEE STRAINS
363
When considering length polymorphism of the region. Although different from BEB, the
region between the cytochrome oxidase I and I1 restriction sites are compatible with A. m.
genes, restriction data and phylogenetic analysis mellifera except for the HindIII site which is
of the sequence data, then it seems likely that: usually absent.
I , A A J is A. rn. ligustica because it has the same
length polymorphism and restriction sites, and
Discussion
differs only by one base from known A . rn.
Ruttner et al. (1978) grouped the honeybee
ligust ica;
2. ACC and ACG are A. m. caucasica because subspecies into three lineages based o n
they have the same length polymorphism and morphometry and phylogeography: lineage A
restriction sites, and differ only by one base from included subspecies from Africa; lineage M
both ACE and ACF. Further, a high bootstrap included subspecies from northern Europe, Spain,
value supports their inclusion with A. rn. Portugal and northern Africa; and lineage C
included subspecies from eastern Europe, northern
caucasica;
Mediterranean
and the Middle East. Distances
3 . BAB is A. rn. rnellifera by the ATPase 6,
COIII and ND2 fragments, but A . rn. ligustica by between the lineages obtained from A. m.
the cytochrome b fragment. It has the sequence ligustica, A. m. caucasica, A. rn. mellifera and A.
fragment Q which is compatible with A . rn. rn. scutellata reference populations in this study
ligustica and all other northern Mediterranean are similar to those obtained by Garnery et al.
races, but not with A. rn. rnellifera. It differs by (1992) and Arias and Sheppard (1996). Again the
one restriction site from our data and published divergence between A and M is greatest, followed
maps (Garnery et al. 1992) of A. rn. ligustica and by C and M, and A and C, respectively. Also,
by two from published maps of A. rn. rnellifera mtDNA variability found is much higher between
lineages than within, which is consistent with the
(Garnery et al. 1992);
4. BBB is A . rn. rnelliferabecause it has the same existence of long term barriers to gene flow during
restriction sites as BEB and differs by only 4 of the Pleistocene (Smith 1991).
The phylogenies presented here do not agree
21 base changes from known A . rn. rnellifera. It
also has the sequence fragment PQQ which is with those from previous molecular studies. The
main difference is the terminal position of A . rn.
compatible with A. rn. rnelliyera;
5 . BDD is A. rn. mellifera from the phylogenetic caucasica on lineage C, which is clear in Fig. 2.
analysis and the length of the size polymorphic Lineage C is largely unresolved in Arias and
region. Therefore there are at least two restriction Sheppard (1996) by parsimony and neighbour
site differences (HindIII and Spel) between it and joining methods, and from our reanalysis using
published maps of A. rn. rnellifera (Garnery et al. the LogDet transformation method. However, A .
1992); and
rn. caucasica consistently joins the base of lineage
6 . BDH is A. m. rnelliferafrom the phylogenetic C under both parsimony and neighbour joining
analysis and the length of the size polymorphic (using distances calculated according to Kimura
(A. tn. ligustica) AAA
78
(A. m. ligusticu) AFI
U
0.00 1
Fig. 3. Relationship of the six haplotypes of known origin. The tree shown is that obtained using maximum likelihood, parsimony
and the LogDet transformation. Length of branches were obtained by the usertree option of maximum likelihood. Numbers
at nodes are bootstrap percentages (1,000 replicates).
S. KOULIANOS and R. H. CROZIER
364
(1980)) methods in the analyses of Cornuet and
Garnery (1991) and Garnery et al. (1992). This
alternative is significantly worse from our data by
the Kishino and Hasegawa (1989) test.
Reanalysing the sequences in Garnery et al. ( 1 992)
using the LogDet transformation method results
in a tree more congruent with ours; A. m. ligustica
becomes basal to both A. m. caucasica and A. m.
carnica. If this is the case, then there is no clear
correlation between the geographic distribution of
lineage C haplotypes and their phylogeny because
the more derived haplotypes are from the
Caucasus and Austria (rather than Italy and
Austria).
High AT content has been reported for all
honeybee mitochondrial genes (Crozier and
Crozier 1993). Differences in nucleotide
frequencies may result in some methods of
phylogenetic inference/estimation returning
erroneous topologies (Lockhart et a f . 1994). The
LogDet transformation can compensate for the
effects of biased nucleotide compositions
(Lockhart et al. 1994). For example, using
mitochondrial sequences from Apis species
Lockhart el a f . (1994) were able to show that
parsimony and neighbour joining methods
grouped the sequences according to their A, G,
C, T contents. However, after transforming the
data using the LogDet method, the resulting tree
was congruent with those from other biological
data. Similarly, we find that honeybee
intraspecific phylogenies are affected by
differences in base composition bias. These results
emphasise the warning of Lockhart et al. (1994)
which expresses a need for caution when
reconstructing trees from taxa with differing
nucleotide frequencies, and that many published
studies should be reconsidered.
Acknowledgments
We thank the University of Western Sydney, the Department
of Agriculture (Western Australia), the Department of
Agriculture (Tasmania), Dr H. G . Hall, Dr M. Schwarz, Dr
D. Paton, Crestline Apiaries, T. Brown, G. Wheen, B. White
and R. Stephens Apiaries for supplying the bees, Jean-Marie
Cornuet for critical comments on an earlier version of the
manuscript, and the Australian Research Council and the
Honey Research and Development Council for grant support
to RHC.
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