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A molecular phylogeny of the genus Apis suggests that the Giant Honey Bee of
the Philippines, A-breviligula Maa, and the Plains Honey Bee of southern
India, A-indica Fabricius, are...

Article  in  Systematic Entomology · December 2009

DOI: 10.1111/j.1365-3113.2009.00504.x

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Systematic Entomology (2010), 35, 226–233 DOI: 10.1111/j.1365-3113.2009.00504.x

A molecular phylogeny of the genus Apis suggests that

the Giant Honey Bee of the Philippines, A. breviligula
Maa, and the Plains Honey Bee of southern India,
A. indica Fabricius, are valid species
1 1 2
N A T H A N L O , R O S A L Y N S . G L O A G , D E N I S L . A N D E R S O N and
1
BENJ AMIN P. OLDROYD
1 Behaviour and Genetics of Social Insects Laboratory, School of Biological Sciences, University of Sydney, Sydney, Australia

and 2 CSIRO Division of Entomology, Black Mountain Laboratory, Canberra, Australia

Abstract. Two new taxa have been added to the existing molecular phylogenies of
the genus Apis. The new phylogeny supports the accepted phylogenetic relationships of
{dwarf honey bees [giant honey bees (cavity-nesting honey bees)]}. Based on Bayesian
and maximum parsimony trees, our analysis supports recognition of Apis indica, the
Plains Honey Bee of south India, as a separate species from A. cerana. Our analysis
also supports recognition of the Giant Philippines Honey Bee, A. breviligula, as a
separate species from A. dorsata.

Introduction
The taxonomy of the honey bees, genus Apis Linnaeus has
been confused for 100 years. At the extremes, Maa (1953)
recognized 24 species and four subgenera, whereas Buttel-
Reepen (1906) argued for a single genus and just three
species. In a heroic effort, Engel (1999) reviewed the vast
literature (there are over 175 names) and settled on three
extant subgenera and seven species. In their review of the
biology of the Asian honey bees, Oldroyd & Wongsiri (2006)
adopted Engel’s classification, but followed Otis (1996),
who recognized two additional species. The Giant Mountain
Honey Bee from mountainous regions of Asia, A. laboriosa
(Smith, 1871), has distinct colour and morphology (Sakagami
et al., 1980), and probably reproductive isolation (Underwood,
1990) from the more broadly distributed lowland A. dorsata
(Fabricius, 1793), and is associated with a unique species of
parasitic mite (Anderson & Morgan, 2007). The Mountain
Honey Bee from Borneo, A. nuluensis (Tingek et al., 1996),
also has distinct coloration (Tingek et al., 1996) and mating
Correspondence: Benjamin P. Oldroyd, Behaviour and Genetics
of Social Insects Laboratory, School of Biological Sciences A12,
University of Sydney, NSW 2600, Australia. E-mail: boldroyd@
usyd.edu.au
time and place (Koeniger et al., 1996) from the more broadly
distributed A. cerana (Tanaka et al., 2001).
Two recent phylogenies based on multiple mitochondrial
and nuclear genes (Arias & Sheppard, 2005; Raffiudin &
Crozier, 2007) have produced the most comprehensive and
well-supported phylogenies of the honey bees to date. These
analyses included the nine species recognized by Oldroyd and
Wongsiri, and a tenth taxon, A. dorsata binghami (Cockerell,
1906), from Sulawesi. Raffiudian and Crozier’s Bayesian
consensus trees gave 100% support for grouping A. d. binghami
and A. dorsata as a group distinct from A. laboriosa, supporting
recognition of A. laboriosa as a valid species. Arias &
Sheppard (2005) found that A. dorsata was paraphyletic with
respect to A. d. binghami : A. dorsata from Thailand was more
closely related to A. d. binghami from Indonesia than it was to
a sample of A. dorsata from Sri Lanka. The genetic distance
between the Sri Lankan sample and those from Thailand and
Indonesia was similar to that found between well-accepted
species, suggesting that A. dorsata may represent at least two
species.
The two recent molecular phylogenies (Arias & Sheppard,
2005; Raffiudin & Crozier, 2007) did not include two taxa
that are of considerable interest. First, Maa (1953) described
a species A. (Megapis) breviligula from Los Banõs on the
Philippine island of Luzon. Giant bees from this area are
strikingly divergent from most other A. dorsata owing to

© 2009 The Authors

226 Journal compilation © 2009 The Royal Entomological Society

black rather than yellow coloration of the abdomen (personal
observations of BPO; Maa, 1953). This ecotype is also
behaviourally divergent from mainland species in that it never
forms colony aggregations as do A. laboriosa and A. dorsata
(Morse & Laigo, 1969). Except for Palawan, the islands of
the Philippines were never connected to Borneo or mainland
Asia (George, 1981). Thus the giant bees of most of the
Philippines probably arose by means of a single chance
introduction by sea, providing ideal conditions for speciation
through a combination of genetic drift and selection (Oldroyd
& Wongsiri, 2006).
The second taxon of interest not included in the two recent
molecular phylogenies is the yellow ‘Plains’ morph of cavity-
nesting honey bees from southern India. Based on a region
that spans both coding and non-coding regions between the
tRNAleu and COII genes of the mitochondria, Oldroyd et al.
(2006) showed that the mitochondrial haplotype carried by
this morphotype is dramatically diverged from the ubiquitous
‘mainland Asia’ family of haplotypes that are carried by other
A. cerana (Fabricius, 1793) across its range (Smith & Hagen,
1996). In addition, drones of the ‘Plains’ morph have an
earlier mating time than drones of the black ‘Hill’ morph,
which is sympatric in south India. The ‘Hill’ morph carries the
‘mainland Asia’ (Smith & Hagen, 1996) haplotype. Combined
molecular and behavioural evidence suggests reproductive
isolation of the ‘Plains’ and ‘Hill’ morphs (Oldroyd et al.,
2006). Thus the Plains morphotype may be a separate species
from A. cerana.
Here we re-analyse the data of Raffiudin & Crozier (2007)
[which included considerable data from Arias & Sheppard
(2005)], but include new sequence data from the two additional
taxa mentioned above, based on the same genetic partitions.
Our phylogeny therefore includes representatives of all known
candidate honey bee species in a single analysis.
Materials and methods
The methods used in this study closely followed those used
by Raffiudin & Crozier (2007). We produced new sequences
from the Plains morphotype of the cavity-nesting bee from
South India (from individuals of four different colonies), and
from samples of giant honey bees collected by Denis Anderson
from the major islands of the Philippine archipelago (one
individual from each collection site) (Table 1, Fig. 1). DNA
was obtained by grinding the hind leg of individual bees in 5%
Chelex solution (1 mm Tris HCl pH 7.6, 0.1 mm EDTA pH
8) and incubation at 100◦ C for 15 min (Walsh et al., 1991).
Supernatant containing DNA was diluted 1:1 in 250 μL of
distilled water. Target DNA from two mitochondrial genes
(rrnL, cox2 ) and one nuclear gene (itpr) was amplified using
the primers and protocols described by Raffiudin & Crozier
(2007) (polymerase chain reaction was attempted on the gene
nd2 (Arias & Sheppard 2005), but it was not successfully
amplified). Products were sequenced in both directions in
a commercial sequencing facility (Macrogen Inc., Korea).
GenBank accession numbers are given in Table 1.
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, Systematic Entomology, 35, 226–233
Honey bee phylogenetics 227
Alignment, data partitioning, and model selection
Sequences obtained in this study were aligned with those
reported in Raffiudin & Crozier (2007) by eye. The rrnL
alignment was 501 bp in length. One region corresponding
to positions 236–274 could not be aligned unambiguously,
and was removed from analyses. Of the remaining characters,
167 were variable and 114 were parsimony-informative. The
cox2 alignment was 675 bp in length; 284 bp were variable,
197 bp were parsimony-informative. The itpr alignment
was 538 bp in length; 131 bp were variable, 40 bp
were parsimony-informative. The cox2 and itpr sequences
were partitioned into first, second and third codon positions
(following partitioning of itpr into coding and non-coding
(a single intron of 93 bp) sequences) using paup*4.0. Base
compositional bias was examined for each partition using
TreePuzzle 5.2. In the cases where non-stationarity (Jermiin
et al., 2004) was suggested, sequences were recoded into
purines and pyrimidines and checked again for stationarity. The
appropriate substitution model for each partition retaining the
original coding and those recoded was then determined using
MrModeltest (Nylander, 2004) and TreePuzzle (Schmidt
et al., 2002) respectively (in the latter case, manual likelihood
ratio tests were performed using nested models). The partitions
used and the models selected are shown in Table 2.
Phylogenetic analyses
MrBayes 3.1 (Schmidt et al., 2002) was used to estimate
tree topologies from the combined dataset. The appropriate
model (Table 2) was used for each partition. Two independent
analyses were run for 1 million generations, each starting
with different random trees with four chains, with sampling
every 100th generation. The program awty (Nylander et al.,
2008) was used to plot likelihood scores for each sampled
generation. Stationarity was confirmed to have occurred after
3000 sampled generations by inspection of the cumulative
split frequencies. The program Tracer 1.3 (A. Rambaut and
A. J. Drummond, http://evolve.zoo.ox.ac.uk) was also used to
confirm convergence and stability [with effective sample size
of all parameters >100; Abecasis et al. (2007)]. The first 3000
sampled generations were considered burn-in and discarded.
Posterior probabilities were determined by constructing a 50%
majority-rule tree of the 7000 sampled post-burn-in trees.
A maximum-parsimony bootstrap tree (1000 replicates; 10
random addition replicates per bootstrap replicate) was also
generated using paup*.
Results
The Bayesian consensus tree obtained in this study (Fig. 2)
was identical to that obtained using maximum parsimony (not
shown), with the exception that some nodes generated in
the latter analysis were not supported in >50% of bootstrap
replicates. The relationships found here were similar to those
in fig. 3 of Raffiudin & Crozier (2007) and in fig. 5 of
228 N. Lo et al.

Table 1. Collection sites and collectors of honey bee specimens used in this study.

GenBank accession number

Group Species Location Collector/s itpr rrnL cox2

Bumble bee (OG)

Bombus terrestris Switzerland RSa DQ468668 AY588427 AY588419
Stingless bee (OG)
Trigona fimbriata Chiang Mai, Thailand RRa DQ468667 AY588426 AY588418
Melipona bicolor Sao Pãulo SD and MCAa na AF466146 AF466146
Dwarf honey bees
A. andreniformis Sumatra, Indonesia RRa DQ468666 AY588425 AY588417
A. florea Chiang Mai, Thailand RRa DQ468665 L22894 AY587548
Giant honey bees
A. dorsata Sabah, Malaysia RRa DQ468664 L22984 AY588415
Palawan, Philippines DLAa FJ932643 FJ932651 FJ932635
A. laboriosa Kathmandu, Nepal JPa DQ468662 AY588424 AY587547
A. d. binghami Sulawesi, Indonesia GWOa DQ468663 AY588423 AY587548
A. breviligula Luzon Philippines DLAb FJ932639 FJ932647 FJ932631
Mindoro, Philippines DLAb FJ932640 FJ932648 FJ932632
Bohol, Philippines DLAb FJ932638 FJ932646 FJ932630
Cebu, Philippines DLAb FJ932637 FJ932645 FJ932629
Luzon, Philippines DLAb FJ932641 FJ932649 FJ932633
Mindanao, Phillipines DLAb FJ932642 FJ932650 FJ932634
Cavity-nesting honey bees
A. mellifera Sydney, Australia BPOa DQ468657 NC001566 AY587542
NC001566
A. koschevnikovi Sabah, Malaysia RRa DQ468661 L22895 AY587546
A. cerana Sabah, Malaysia RRa DQ468659 L22892 AY587544
A. nigrocincta Sulawesi, Indonesia SHa DQ468660 NC001421 AY587545
A. nuluensis Sabah, Malaysia RRa DQ468658 AY588420 AY587543
‘A. indica’ Kanataka, India BPOb FJ932636 FJ932644 FJ932628
a Sequences obtained from Raffiudin & Crozier (2007).
b New sequences.
Collector abbreviations: BPO, Ben P. Oldroyd; DLA, Denis L. Anderson; GWO, Gard W. Otis; SH, Soesilowati Hadisoesilo; RS, Regula Schmid-
Hempel; RR, Rika Raffiudin; SD and MCA, D. Silvestre and M.C. Arias. OG, outgroup.

Arias & Sheppard (2005), and reflect the broadly accepted
evolutionary relationships of the honey bees (Oldroyd &
Wongsiri, 2006). The topology shows three clear groupings
corresponding to the dwarf, giant and cavity-nesting honey
bees. The accepted species (Engel, 1999) generally group with
posterior probabilities that exceed 0.95, which is regarded as
significant (Wilcox et al., 2002).
With the exception of A. mellifera, the cavity-nesting bees
show robust groupings that reflect our current understanding
of the biogeography of the honey bees. The Plains cavity-
nesting bee from Kanataka groups with mainland A. cerana,
although the two are genetically quite divergent: the value of
2.1% is similar to the distance (2.2%) between the examined
representatives of A. cerana and A. nigrocincta (we note
that four ‘Plain’ individuals from four different colonies were
sequenced; in each case the sequences were identical for all
three genes). This result is consistent with a previous study
(Oldroyd et al., 2006) and supports the idea that the Plain
Honey Bee represents a new species.
Apis mellifera was found to group with other cavity-
nesting bees (A. koschevnikovi, A. nuluensis, A. cerana and
A. nigrocincta) with relatively low support [0.52 posterior
probability compared with 1.0 in fig. 3 of Raffiudin & Crozier
(2007) and <50% bootstrap support]. This may be because
our analysis was based on three genes rather than four, and
because the fragment of itpr that we examined was shorter.
Within the giant bees, the specimen from Palawan groups
with A. dorsata in Sabah. In contrast, all the giant bees from
the islands of Mindanao, Luzon and Bohol group together
and show little divergence from each other, perhaps with
the exception of the specimen from Cebu (Fig. 2). This
suggests that the giant bees of the eastern islands of the
Philippine archipelago are a separate species from all others.
Apis laboriosa and A. d. binghami are separate from the Sabah
and Palawan samples, although support values for the clades
containing the first two species are not high.
The dwarf bees A. florea and A. andrenformis are strongly
diverged from all others and from each other.
Discussion
Our analysis contains representatives of all known honey bee
taxa. The phylogeny (Fig. 3) is similar to that presented by
Oldroyd & Wongsiri (2006) but shows the likely relationships
of the cavity-nesting honey bees and the giant honey bees in
greater detail.

© 2009 The Authors

Journal compilation © 2009 The Royal Entomological Society, Systematic Entomology, 35, 226–233
 Honey bee phylogenetics 229

Fig. 1. (A) Collection sites across Asia; (B) collection sites within the Philippines.

Based on recent findings (Oldroyd et al., 2006; Raffiudin
& Crozier, 2007) we propose that three honey bee taxa
(A. breviligula, A. laboriosa, A. indica) be elevated to species
status, and suggest that further work on the Giant Sulawesi
Honey Bee Apis dorsata binghami Cockerell is required to
determine its status. Below we follow Engel (1999) wherever
possible.
Apis breviligula Maa (1953): The Giant Philippine Honey Bee
Our analysis shows that the giant bees of the Philippine
islands of Luzon, Mindanao, Bohol and Cebu cluster together,
and as a group are strongly differentiated from our single
specimen from Palawan and from A. dorsata from Sabah.
We suggest that this, combined with the strongly divergent

© 2009 The Authors

Journal compilation © 2009 The Royal Entomological Society, Systematic Entomology, 35, 226–233
230 N. Lo et al.

Table 2. Substitution models and coding schemes for partitions (1, 2 Philippine samples, and why the samples from the eastern
and 3 refer to codon positions for cox2 and itpr ). Philippine islands (A. breviligula) show little divergence from
each other. Apis breviligula may have arisen from a Pleistocene
Gene Partition
colonization event in which a founder population crossed the
All 1 2 3 Intron
strait from Palawan/Borneo to what are now the eastern islands
rrnL F81+G of the Philippine group.
cox2 GTR+G F81+G RY, F81+G
itpr JC F81 RY, F81 RY, F81
Apis dorsata binghami Cockerell (1906): The Giant Sulawesi
Honey Bee
coloration and nesting behaviour, is sufficient evidence to
support Maa’s classification of species status for A. breviligula In our analysis the placement of A. d. binghami is
separate from A. dorsata. Apis breviligula has also been shown ambiguous, and is not clearly distinguished from either A.
to host its own unique species of parasitic mite (Anderson & dorsata sensu stricto or A. laboriosa. The consensus trees of
Morgan, 2007). Raffiudin & Crozier (2007) and Arias & Sheppard (2005) did
About 18 000 years ago, during the Pleistocene cooling, the give strong support to grouping A. d. binghami with A. dorsata
Philippine island of Palawan was joined to Borneo. To the east, and separate from A. laboriosa. This topological difference
the other Philippine islands, including Mindanau, Luzon, Cebu, may be a result of the addition of the taxa from the Philippines
Laguna and Bohol, were joined to each other, but separated in our study. Further studies that include the same genes from
from Palawan and Borneo by the Cotabato-Sagihe deep ocean all the taxa in our study and that of Arias & Sheppard (2005)
trench that delineates the famous Wallace line which broadly are required to clear this up (the latter used nd2 and EF1-alpha,
separates the Eurasian and Australian biotas (George, 1981). neither of which was included in our study).
This ancient geography probably explains why our Palawan An earlier name, Apis zonata (Smith, 1865), exists for a
sample groups with our Borneo sample rather than with other species of giant honey bee collected in Celebes (= Sulawesi).

Fig. 2. Phylogenetic relationships among Apis spp., inferred from a combined dataset of rrnL, cox2 and itpr. The Bayesian inference (BI) topology
was inferred using the program MrBayes; the models of sequence evolution used for each partition are shown in Table 2. The tree was rooted with
Bombus terrestris, Trigona fimbriata and Melipona bicolor. Identical topologies and highly similar support values were found in two additional BI
replicates. A maximum parsimony (MP) 50% majority-rule bootstrap analysis (1000 replicates) produced a tree that was very similar to that shown,
although slightly less well resolved. Support values above and below branches are posterior probabilities (PP) and bootstrap percentage values
(BV) respectively. In the case where only a PP value is shown, the bootstrap value for the grouping was <50%. For A. brevigula and A. dorsata,
collection locations are shown. The scale bar represents substitutions/site (note that some sites were recoded as purines and pyrimidines).

© 2009 The Authors

Journal compilation © 2009 The Royal Entomological Society, Systematic Entomology, 35, 226–233

Fig. 3. Phylogenetic relationships between the extant honey bee taxa.
Maa (1953) rejected this name because it had also been used
for the Philippine species, which he (correctly in our opinion)
regarded as being distinct from the Sulawesi species. The Giant
Sulawesi Honey Bee is black and similar in appearance to
A. breviligula and A. laboriosa (Maa, 1953).
Giant honey bees are known to have colonized the volcanic
island of Krakatoa, at least 50 km distant from Sumatra, within
26 years of a catastrophic eruption that denuded the island
of all plant life and probably all invertebrates (Thornton &
New, 1988). This demonstrates the ability of giant honey
bees to colonize across significant stretches of ocean. During
the Pleistocene cooling, the island of Sulawesi either was
connected via Sundaland to mainland Asia or was separated
by a stretch of ocean of less than 100 km (George, 1981). This
relatively recent connection probably provided the opportunity
for honey bee populations from what is now the island
of Sulawesi to exchange genetic material with honey bee
populations on what is now the island of Borneo for a
protracted period.
Apis laboriosa Smith (Moore et al., 1871): The Giant
Mountain Honey Bee
The clear differentiation of A. laboriosa from A. dorsata
(Arias & Sheppard, 2005; Raffiudin & Crozier, 2007), its
distinct coloration (Sakagami et al., 1980), preference for
habitats above 1200 m, probable reproductive isolation from A.
dorsata owing to a differing mating time (Underwood, 1990)
and its own parasitic mite species (Anderson & Morgan, 2007)
compel us to recognize A. laboriosa as a valid species. Engel
recommended the common name Giant Himalayan Honey Bee,
but this now seems inappropriate given reports of this species
in mountainous regions of Vietnam (Trung et al., 1996). We
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, Systematic Entomology, 35, 226–233
Honey bee phylogenetics 231
therefore suggest Giant Mountain Honey Bee. Our study
found that A. laboriosa was closer to A. dorsata than to A.
dorsata binghami, although support values were low. A more
detailed study of A. dorsata and relatives is required to clear
this up.
Apis indica Fabricius (1798 p. 370)
Oldroyd et al. (2006) demonstrated that the yellow Plain
morph of cavity-nesting bee from southern India carries a
mitochondrial haplotype that is distinct from those of the
black Hill morph and all other A. cerana. Our current analysis
includes additional genes, and the branch lengths of our new
phylogeny also suggest considerable divergence of the Plain
morph from A. cerana.
The name ‘A. indica’ is based on two specimens of worker
honey bees collected from India and lodged in the Copenhagen
Museum (Fabricius, 1798). The name is listed by Smith (1865)
without description. Smith synonymized A. indica with A.
peronii from Timor, and four other Indian specimens. Smith
notes that A. indica is also present in Java, Sumatra, Flores,
Malacca and Borneo, suggesting confusion with A. cerana.
The subspecies name ‘Apis cerana indica’ is often used by
Indian researchers to refer to any cavity-nesting honey bee
from India. Ruttner (1988) used A. c. indica as one of four
subspecies of A. cerana. Based on principal component anal-
ysis of morphometric characters, Ruttner classified specimens
from Thailand and Indonesia in A. c. indica. As Hepburn et al.
(2001) have put it: ‘the meaningfulness of the designation “A.
cerana indica”. . . is put under considerable pressure when it
is remembered that the “cerana indica” of Thailand, Borneo
and Malaysia are certainly not the same bees which occur in
India and Sri Lanka’.
232 N. Lo et al.
We suggest that the name A. cerana indica or A. indica
be restricted to the yellow plains morph of cavity-nesting
bees from southern India. Further molecular studies on
representatives from Sri Lanka are required to confirm whether
it is closely related to the southern Indian populations.
Diagnosis is by means of coloration of the first three anterior
tergites of the workers. Workers in which the first three tergites
are completely yellow are classified as A. indica; workers in
which one or more of the first three tergites show partial or
complete black coloration are the A. cerana of the ‘Hill’ variety
(Kapil, 1956; Oldroyd et al., 2006).
We note that Arias & Sheppard (2005) found that a cavity-
nesting bee from Sri Lanka was strongly diverged from
A. cerana specimens from Sulawesi and Sabah, supporting
the existence of a different species (presumably A. indica) in
Sri Lanka.
Acknowledgements
We thank members of the Behaviour and Genetics of Social
Insects Laboratory for comments on this manuscript.
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Accepted 14 August 2009
First published online 8 December 2009