Molecular Ecology (2004) 13, 2317–2331 doi: 10.1111/j.1365-294X.2004.02236.

Nest architecture and genetic differentiation in a species

Blackwell Publishing, Ltd.

complex of Australian stingless bees

P . F R A N C K ,*†‡ E . C A M E R O N ,† G . G O O D ,† J . - Y . R A S P L U S * and B . P . O L D R O Y D †
*Centre de Biologie et de Gestion des Populations, CS30016, Campus International de Baillarguet, 34988 Montferrier Cedex, France,
†School of Biological Sciences, A12, University of Sydney, NSW 2006, Australia

Abstract
We investigated the taxonomic significance of nest shape and its putative role in speciation
in Trigona (Heterotrigona) carbonaria and T. (H. ) hockingsi, two sibling species of stingless
bee species from eastern Australia. These species are primarily distinguished by their nest
architecture, as in all other respects they are nearly identical. We genotyped 130 colonies
from six locations in Queensland at 13 microsatellite loci together with 106 additional colon-
ies from six other Indo-Pacific Trigona species. Whether they were present in allopatry
or in sympatry, colonies that displayed the T. carbonaria or the T. hockingsi nest architecture
could be unambiguously differentiated at the genetic level. However, T. hockingsi colonies
were classifiable into two highly differentiated paraphyletic and geographically separate
populations, one in northern and one in southern Queensland. These two populations
probably belong to two distinct species, T. hockingsi and T. davenporti nov. sp. Our results
suggest that nest architecture characters are relevant but not sufficient criteria to identify
species in this group. Consequently, modifications of nest architecture are probably not of
prime importance in the speciation process of Australian stingless bees, although nest
architecture differences probably result from relatively simple mechanisms. The rare inter-
specific hybrid colonies detected did not display a nest with an intermediate form between
T. hockingsi and T. carbonaria.
Keywords: Australia, microsatellite, phylogeography, taxonomy, Thailand, Trigona (Heterotrigona)
davenporti nov. sp.

Received 10 February 2004; revision received 5 April 2004; accepted 5 April 2004

demonstrate that small changes in the rules followed dur-

Introduction
In social bees, combs are often remarkably complex struc-
tures that are seemingly perfectly adapted to the fabric of
the society. Not only does the comb provide the substrate
on which the workers live, food is stored and brood is
reared, but the wax itself plays a critical role in the dis-
persal of pheromones and the generation of the nest-
specific odour (Breed et al. 1988; Hepburn 1998). Despite
their high complexity, the construction of combs can be
realistically simulated using simple mathematical models
based on self-organization and self-assembly theories (e.g.
Belic et al. 1986; Bonabeau et al. 1997). These simulations
Correspondence: P. Franck. ‡Present address: UMR Ecologie des
Invertébrés, INRA-UAPV, Agro Parc Domaine St-Paul, 84914
AVIGNON Cedex 9, France. Fax: +33 (0)4 3272 2602; E-mail:
pfranck@avignon.inra.fr
ing comb construction can lead to very different patterns of
comb formation (Camazine 1991; Karsai & Penzes 1998).
This suggests that a fundamental shift in nest architecture
could arise from just a small change in the rules followed
by comb-building workers. Consequently, the taxonomic
significance of nest shape and its importance to the process
of speciation is of great interest.
The eusocial stingless bees (Apidae, Apinae, Meliponini)
comprise some 374 species, which are broadly distributed
over tropical regions (Kerr & Maule 1964; Michener 2000).
Two genera occur in Australia: the endemic Austroplebia
Moure, and the more cosmopolitan Trigona Jurine. The five
Australian Trigona species belong to the subgenus Tetra-
gonula Moure, which is sometimes considered to be part of
the subgenus Heterotrigona Schwarz (Michener 1990). Tetra-
gonula is the largest subgenus of Indo-Pacific stingless bees
and includes around 20 species, some of which are very

© 2004 Blackwell Publishing Ltd

2318 P . F R A N C K E T A L .

common and widespread. Tetragonula has been subdivided

into five species groups: geissleri, clypearis, laeviceps, fuscobalteata
and carbonaria (Sakagami 1978; Dollin et al. 1997). Each species
group encompasses a number of very similar species that
are currently recognized based on morphometric studies
(Sakagami 1978; Sakagami & Inoue 1985; Dollin et al. 1997).
However, identification is often made difficult because of
within-species size variation along latitudinal clines (Sakagami
1978; Dollin et al. 1997) and the probable existence of cryptic
species (Sakagami 1978; Sakagami & Inoue 1985). Further-
more, many Tetragonula species occur in sympatry, often aggre-
gating their nests at the same place (Starr & Sakagami 1987).
Very few studies have analysed the genetic variability
within and among Tetragonula species. Allozyme poly-
morphism was observed at three loci in T. fuscobalteata
populations from peninsular Malaysia (Yong 1986; Yong
et al. 1987). However, no variability was observed at 12 Fig. 1 Nest architectures in the carbonaria species group. (a)
Entrance hole of Trigona carbonaria nests. (b) Projecting entrance
allozyme loci among T. carbonaria populations from eastern
tubes of T. mellipes nests. (c) Top view of the spiral comb of brood
Australia (Wagner & Briscoe 1983). Furthermore, pre- cells in T. carbonaria nests. (d) Top view of the comb of brood cells
liminary studies did not detect any variation among in T. hockingsi and T. mellipes nests.
species of the carbonaria group in partial sequence of the
cytochrome b gene (Tsang 1996; Allsop 1997).
Nest architecture is often the key feature that has been overlapping distribution in eastern Queensland, and some
used to accord species status to morphometrically and of their close relatives from Australia and southeastern
genetically similar taxa of Tetragonula (Michener 1961; Sak- Asia. We use the variability at 13 microsatellite loci and at
agami et al. 1983; Starr & Sakagami 1987; Dollin et al. 1997). a more conserved anonymous DNA sequence to test inde-
This is particularly the case with the three species of the pendently the taxonomic significance of nest architecture
Australian carbonaria group. These species (T. carbonaria within the carbonaria species complex and to shed light on
Smith, T. hockingsi Cockerell, and T. mellipes Friese) are the speciation process among the Australian Trigona.
most readily distinguished by the structural characteristics
of their nests (Michener 1961; Dollin et al. 1997). A dark
Materials and methods
mixture of resin and wax usually surrounds the entrance
holes of T. carbonaria nests (Fig. 1a), whereas T. hockingsi
Sampling and identification
has little or no lining at its nest entrance. The majority of T.
mellipes nests have projecting entrance tunnels (Fig. 1b) Figure 2 shows the distribution of the collection sites.
whereas this structure is almost always absent in the nests We collected Trigona carbonaria and T. hockingsi samples
of T. carbonaria and T. hockingsi. In T. carbonaria, larval cells (130 colonies) at six sites in eastern Queensland, Australia
are continuously constructed along a spiral horizontal (numbered 1–6 from south to north). Collection sites are
comb (Fig. 1c). In contrast, cells of the brood comb are clustered in two different climatic regions. Collection sites
irregularly arranged in T. mellipes and T. hockingsi (Fig. 1d). from southern Queensland (sites 1 and 2) are within the
Although nest architecture has long been used to classify temperate zone. Collection sites from northern Queens-
genera within the Meliponini tribe (Michener 2000), tax- land (sites 3–6) are within the tropical zone. At each site,
onomists caution that nest morphology should be used 14 – 30 colonies were sampled within a radius of 15 km. Five
with due care for species identification because it can to 10 worker bees were collected per colony within the nest
be strongly affected by environmental variables (Schwarz or at its entrance. Worker bees were brought back to the
1948; Michener 1961). For example, the open comb archi- laboratory in 70% ethanol. The taxonomic status of each
tecture in T. hockingsi (Fig. 1d) may arise in response to colony was principally determined based on nest arch-
warm temperatures, whereas the more compact form of T. itecture features (entrance hole and brood comb, Fig. 1).
carbonaria (Fig. 1c) may arise in response to cooler temper- However, we also measured the head width (HW) on two
atures or because of restricted cavity size. Similarly, the to four worker bees per colony, notably to discriminate the
form of the nest entrance may be strongly influenced by the 17 colonies from site 6 for which we did not have access to
level of exposure to sun or to wind. the internal nest architecture. HW are usually 0.1 mm
In this study we investigate the genetic variability among smaller in T. carbonaria than in T. hockingsi, but vary consider-
T. carbonaria and T. hockingsi populations, which have an ably along latitudinal clines (Dollin et al. 1997). Consequently,

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331

 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2319

Fig. 2 Natural distribution of the Trigona (Heterotrigona) subgenus sensu Michener (1990). Trigona collina Smith have formerly been
recognized as belonging to the subgenus Tetragonilla Moure. All other species studied have formerly been classified within the subgenus
Tetragonula Moure, which is subdivided into five morphologically distinctive groups (Sakagami 1978; Sakagami & Inoue 1985; Dollin et al.
1997). Trigona clypearis Friese and T. pagdeni Schwartz belong to the iridipennis group, T. carbonaria Smith, T. mellipes Friese and T. hockingsi
Cockerell belong to the carbonaria group and T. sapiens Cockerell and T. laeviceps Smith belong to the laeviceps group. Each group is defined
with a different symbol. Complete distributions of each species in the Indo-Pacific area are provided in Dollin et al. (1997) and Sakagami
(1978). (a) Geographic origins of the identified colonies based on morphological measurements. Abbreviations ChM, ChB, Da, Ku, Ka, Co
and Ca refer to the collection sites Chiang Mai, Chantabury, Darwin, Kununurra, Katherine, Cook Town and Cairns, respectively. (b)
Geographic origin of the T. carbonaria and T. hockingsi colonies determined using the shape of their nests. Collection sites (Elanora, Gatton,
Gladstone, Rockhampton, Kunwarara and Herberton) are numbered 1–6 from South to North. Collection sites from southern Queensland
(sites 1, 2) and from northern Queensland (sites 3 – 6) are outside and inside the tropical zone, respectively. Trigona carbonaria and T. hockingsi
were only observed in sympatry at sites 1 and 6. N indicates the number of colonies analysed per collection site and species.

we preferred features based on nest architecture for the

identification of T. carbonaria and T. hockingsi colonies when
such criteria were available. One worker bee per colony
was genotyped.
Additional Trigona (Heterotrigona) samples (106 colonies)
collected in Thailand and Australia were used for com-
parison. We identified these colonies based on worker wing
length/head width ratios (WL2/HW), which were com-
pared with previously published values (Sakagami 1978;
Dollin et al. 1997). Samples from Australia were identified
as T. sapiens (four colonies), T. clypearis (two colonies), and
T. mellipes (13 colonies). Samples from Thailand were iden-
tified as T. collina (35 colonies), T. laeviceps (41 colonies) and
T. pagdeni (11 colonies).
DNA protocols
We extracted total DNA from the two anterior legs
of individual bees using 500 µL 5% of Chelex 100 (Biorad)
resin solution (Walsh et al. 1991). These extracts were used
as DNA templates for polymerase chain reaction (PCR)
amplifications. An alternative DNA extraction based on a
high-salt method (Green et al. 2001) was used to optimize
PCR protocols.
A total of 13 microsatellite loci were scored for 236
worker bees. Primers for 10 of these loci (Tc4.287, Tc4.63,
Tc3.155, Tc.4.214, Tc1.20, Tc7.13, Tc3.302, Tc3.56, Tc4.326
and Tc4.349) had been identified from a partial genomic
library of T. carbonaria (Green et al. 2001). Primers used for

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331

2320 P . F R A N C K E T A L .
the three additional microsatellite loci (B124, Mbi278AAG
and T4-171) had been identified from three other apid spe-
cies (Estoup et al. 1993; Peters et al. 1998; Paxton et al. 1999).
Touchdown PCR and electrophoresis were conducted as in
Green et al. (2001). Amplifications were carried out in 10 µL
reaction volumes containing 10 mm Tris–HCl, pH 8.3,
50 mm KCl, 40 µm each dNTP, 0.4 µm each primer, 1.5–
2.0 mm MgCl2, 0.5 units Taq DNA polymerase, 0.2 mg/mL
bovine serum albumin with 2 µL of DNA template. The
reverse primer for each pair was fluorescently labelled
with HEX (Gibco BRL). PCR products were visualized on
6% polyacrylamide denaturing gels using a GS2000 auto-
mated DNA fragment analyser (Corbett Research).
We selected one to three T. carbonaria and T. hockingsi
individuals from each of the six collection sites and one
individual each of other six Trigona species for sequencing.
A 432 base pair (bp) fragment was PCR-amplified with
primers CB1 and CB2 (Jermiin & Crozier 1994) located
within the mitochondrial gene coding for Cytochrome b
(Cytb). PCR amplifications were carried out in 50 µL reac-
tion volumes containing 10 mm Tris–HCl, pH 8.3, 50 mm
KCl, 0.2 mm each dNTP, 10 pm each primer, 3 mm MgCl2,
1 unit Taq DNA polymerase and 3 µL DNA template.
Thirty cycles of amplification were performed (92 °C for
1 min, 48 °C for 1 min and 72 °C for 1 min) followed by a
final extension step at 72 °C for 6 min. PCR products were
subsequently gel-purified by electrophoresis on 1% agar-
ose gel stained with ethidium bromide. DNA fragments at
432 bp were excised, then extracted from agarose with a
QIAquick gel extraction kit (QIAgen). The purified PCR
products were directly sequenced with CB1 and CB2 as
sequencing primers on an ABI 373 automated sequencer
using TaqFS and dye-labelled terminators (Perkin-Elmer).
Standard analyses of microsatellite patterns
In our first analysis of microsatellite variability, we pre-
defined population based on collection sites. Each of the six
collection sites from Queensland was considered as a popu-
lation sample regardless of the phenotypes of the colonies
collected. Four additional population samples (one T. collina,
one T. pagdeni and two T. laeviceps), for which a sufficient
number of colonies were available from the same sites
(N > 11), were used for comparison.
Proportions of polymorphic microsatellite loci were
estimated using 95% and 99% criteria (Estoup et al. 1995).
Unbiased estimates and standard deviations of gene
diversity at each locus were calculated according to Nei
(1978). Estimates of allele diversity were computed using
a rarefaction method that adjusted the observed number
of alleles to a common sample size for each population
(Hurlbert 1971). The variability of a microsatellite locus is
usually proportional to its size (repeat count), with larger
loci being generally more polymorphic than smaller ones
(Estoup & Cornuet 1999). Size can be differentially con-
strained among species and subject to directional selection
(Schlötterer 2000). To quantify this effect, the mean repeat
counts of microsatellite alleles were calculated per locus
and per population sample. At each locus, the size and the
sequence of a cloned allele was used as reference to define
the repeat count of each allele. The mean repeat count for
a locus in a population was then calculated by weighting
the repeat count of each allele by its proportion within
the population. Differences among population samples in
average repeat count and in average gene and allele diver-
sities were assessed by Mann–Whitney’s U-test (Sokal &
Rohlf 1995).
Exact tests for Hardy–Weinberg equilibrium, genotypic
linkage equilibrium and genetic structure (genotypic
differentiation) were computed using the genepop package
version 3.2 (Raymond & Rousset 1995). Multilocus FST
estimates (Weir & Cockerham 1984) between population
samples and isolation by distance among and within
population samples were computed and tested using the
genepop package. The correlations between geographical
distance and FST/(1 − FST) values between population sam-
ples (Rousset 1997) or â estimates (Rousset 2000) between
individuals within population samples were tested using
the permutation procedure of Mantel (1967).
Genetic assignment of individuals
In our second analysis, individuals were assigned to popu-
lations based only on genotypic microsatellite data. In other
words, individuals were assigned to populations (panmictic
groups) irrespective of their geographical origin, their
morphology or the nest architecture of their colony, but
requiring that the estimated population structure resulted
in Hardy–Weinberg equilibrium and linkage equilibrium
within the estimated populations. For this purpose, we
used a Bayesian clustering method implemented in the
software structure (Pritchard et al. 2000). The number of
groups, K, into which individuals could be assigned was
assayed by constructing six alternative models, each with
a different K-value (we assumed that K is lower than the
number of collection sites). In each of the six models,
individuals could have a mixed genome of any of the K
groups. The number of groups, K, present in our data set
was inferred from the most likely of the six models. Each
individual was then a posteriori assigned according to its
level of admixture, Q, to one of the K groups determined
from the most likely model. Individuals with Q-values
higher than 90% were considered as correctly assigned.
Individuals with Q-values less than 90% were considered
as potential hybrids. For these putative hybrid individuals,
we determined a more accurate estimate of Q-values by
again running the most likely model but including the
groups of those individuals assigned in the previous
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2321
analysis. Individuals that still had Q-values of less than
90% in this new analysis were considered to be hybrids. A
final model that incorporated group information for all the
individuals was used to assess whether these hybrids had
recent ancestors in one of the K groups. For all models,
we performed runs of 500 000 iterations after a ‘burn-in’
period of 100 000 iterations.
Sequence analyses and tree construction
We manually edited and aligned DNA sequences using the
software bioedit version 5.0.6 (Hall 1999) using the Cytb
sequence from Apis mellifera (Crozier & Crozier 1993) as a
reference. The amino acid translation of each sequence was
computed online with the Deambulatum of infobiogen using
the insect mitochondrial code. We then built a neighbour-
joining (Saitou & Nei 1987) tree based on the p-distance
between DNA sequences and using the A. mellifera sequence
as outgroup. Bootstraps values were computed over 2000
replications, resampling polymorphic sites in the DNA
sequences.
A neighbour-joining tree based on the shared allele dis-
tance (Chakraborty & Jin 1993) was built as described
in Bowcock et al. (1994) to assess relationships among the
236 individual samples genotyped with microsatellite loci.
Those loci that did not amplify for a particular specimen
were coded as missing data. A classification index (Ic) was
computed as in Estoup et al. (1995) to test the robustness of
each putative group within the tree.
Results
Species identification
Figure 2 presents the geographical distribution of the eight
putative Trigona species identified based on worker morpho-
logy and nest architecture. Our worker HW measures were
consistently smaller than those reported in Dollin et al.
(1997). Worker HW measures in colonies with T. carbonaria
nest architecture were 1.73 ± 0.03 mm. Worker HW meas-
ures in colonies with T. hockingsi nest architecture were
1.84 ± 0.07 mm. Colonies identified as T. carbonaria and
T. hockingsi were not randomly distributed among the six
collection sites. Trigona carbonaria colonies were mainly
found in southern Queensland (48/52 colonies) whereas
T. hockingsi colonies were mainly found in northern
Queensland (62/78 colonies). We found both T. carbonaria
and T. hockingsi colonies at the same collection site only
at either end of the transect (sites 1 and 6). The taxonomic
status of the colonies from these two sites was more
difficult to determine. At site 1, four colonies out of 30 were
designated as T. hockingsi on the basis of nest architecture.
However, worker specimens from these T. hockingsi colonies
had similar HW (1.72 ± 0.03 mm) to T. carbonaria workers.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
At site 6, two colonies out of 17 were defined as T. hockingsi
on the base of worker morphology. Worker HW measures
in these two colonies (1.89 ± 0.01 mm) were slightly higher
than those usually observed in T. hockingsi. Worker HW
measures of the 15 other colonies (1.73 ± 0.03 mm) were
similar to those usually observed in T. carbonaria.
Patterns of microsatellite variation
A total of 236 worker bees were genotyped (130 T. carbon-
aria and T. hockingsi individuals from collection sites 1– 6
and 106 additional Trigona individuals from the various taxa
that we used for comparison). Three loci did not amplify in
a few taxa: Tc4.326 in T. collina, Tc1.20 in T. laeviceps and
Tc4.63 in T. laeviceps, T. pagdeni and T. clypearis. Further-
more, 22% of T. hockingsi specimens collected in northern
Queensland did not amplify at the locus Tc4.349, sug-
gesting a high proportion of null alleles at this locus in this
region (estimates of null allele frequencies are 27, 40 and
57% in samples from sites 3, 4 and 5, respectively).
The 13 loci are polymorphic across the eight taxa.
The average variabilities for the 13 microsatellite loci are
detailed for 10 Trigona population samples in Table 1 (allele
frequencies at each locus and genotypic data are available
as supplementary material at the journal’s web page). With
respect to the 10 Trigona population samples, the average
repeat count was positively correlated with the allele
diversity at each microsatellite locus (r = 0.485, d.f. = 124,
P < 0.01). However, the average repeat count at these loci
was not significantly different between samples (45 pair-
wise comparisons; 84.5 > U > 63.5, 0.999 > P > 0.430).
The proportion of polymorphic loci was particularly low
(54%) in the samples from sites 3–5 that comprised only T.
hockingsi colonies (Table 1). The allele and gene diversities
were also significantly lower in these three samples than in
the three other Australian collection sites (U = 939.5 and
U = 979, respectively, P < 0.005). In contrast, the allele and
gene diversities were significantly higher in those sites where
we found both T. carbonaria and T. hockingsi colonies (sites
1 and 6) than in the four other Australian collection sites
(U = 1156 and U = 1099, respectively, P < 0.001). Multi-
locus tests per collection site revealed significant departures
from Hardy–Weinberg equilibrium for the samples from
collection sites 1 and 6 (Fisher’s tests, P = 10 −5 and P = 0.013,
respectively). Tests for linkage equilibrium revealed four
instances of significant (after Bonferroni’s correction) dis-
equilibria out of 258 comparisons (P < 0.00019). The four
disequilibria were detected in the sample from collection
site 1. Three out of the four disequilibria involved the locus
Tc4.326.
Pairwise multilocus FST values between Trigona popula-
tion samples ranged from 0.007 to 0.688 (Table 2). The larg-
est FST values were between the samples of T. hockingsi and
T. collina. The smallest FST value was between the samples
 2322 P . F R A N C K E T A L .

Table 1 Genetic polymorphisms in Trigona samples at 13 microsatellite loci: proportion of polymorphic loci at the 95% and 99% criteria and mean and standard deviation (SD) of repeats from sites 3 and 5. Multilocus tests of genotypic differenti-

(0.20)
(0.30)
(0.26)
(0.28)
(0.27)
(0.33)
(0.25)
(0.32)
(0.23)
(0.23)

Polymorphism is given per collection site. Abbreviations of collection sites follow Fig. 2. 2N indicates the mean number of genes sampled per collection site. The allele diversities were
(SD)
ation were not significant at the 5% level only for the com-

Gene diversities
parison between the samples from site 3 and 5 (Fisher’s
test, P = 0.296, Table 2). No significant correlation between
genetic differentiation and geographical distance was
Mean

0.53
0.43
0.25
0.23
0.27
0.43
0.42
0.36
0.65
0.30
observed among the six T. carbonaria and T. hockingsi
collection site samples (Mantel’s test, P = 0.058, r = 0.638),
or among T. carbonaria colonies and among T. hockingsi
colonies within sites 2 and 4, respectively (Mantel’s tests,
(0.26)
(0.31)
(0.27)
(0.26)
(0.29)
(0.32)
(0.23)
(0.33)
(0.23)
(0.21)
(SD)

P > 0.251, −0.124 < r < 0.073).

Heterozygote
proportions

Genetic assignment of individuals

Mean

0.47
0.42
0.27
0.22
0.29
0.38
0.40
0.37
0.61
0.29
Based on their microsatellite genotype, the number of
groups, K, to which the 130 T. carbonaria and T. hockingsi
individuals could most parsimoniously be assigned was
estimated assuming a uniform prior K between one and
(2.27)
(2.55)
(1.07)
(1.48)
(1.21)
(2.49)
(2.97)
(2.01)
(2.66)
(1.21)
(SD)

six. The model that best described the genetic data was for
Allele diversities

K = 4 (P = 0.992). This model is consistent with the assign-

ment of individuals according to a combination of their
taxonomic status and their collection sites (Table 3). The 67
Mean

specimens with T. hockingsi phenotype were assigned to

4.12
3.66
1.94
2.21
2.02
3.88
3.58
2.79
5.63
2.62

two groups, one in northern and one in southern Queens-

land (A and B, respectively). The 61 specimens from sites
3–5 (Q > 0.97) and the two specimens from site 6 (Q = 0.59
(3.50)
(3.38)
(3.20)
(3.31)
(3.53)
(3.20)
(6.66)
(5.89)
(4.91)
(4.33)
(SD)

and 0.87, respectively) were assigned to group A. The

four specimens from site 1 (Q = 0.99, 0.99, 0.99 and 0.63,
Repeat counts

respectively) were assigned to group B. The 63 specimens

displaying T. carbonaria phenotypes were also assigned
Mean

11.48
11.30
11.29
11.50
11.66
10.78
11.08
12.30
11.02
11.18

to two geographically distinct groups in northern and

southern Queensland (C and D, respectively). The 15
specimens collected at site 6 were assigned to group C
(Q > 0.92), while the 48 specimens sampled at sites 1 and 2
polymorphic loci

99%

1.00
0.85
0.54
0.54
0.62
0.85
0.92
0.67
1.00
0.91

were assigned to group D (Q > 0.78). Six specimens out of

Proportions of

130 were assigned to a group with Q-values lower than

90%. By again running the model K = 4, considering group
count, allele and gene diversities, and heterozygote proportions

information for all those individuals previously assigned

95%

1.00
0.85
0.54
0.54
0.62
0.77
0.83
0.58
1.00
0.81

to a group with Q higher than 90%, only two specimens

computed for the smallest sample (2N = 22) in T. pagdeni.

continued to have Q-values significantly lower than 90%.

Those specimens were from sites 1 and 6. The specimen
from site 1 had a high proportion of alleles from group B
2N

60
44
28
38
56
34
22
70
32
50

(Q = 0.55), but one of its parents seems very likely to be

from group D (P = 0.915). The specimen from site 6 was
assigned to group A (P = 0.616, Q = 0.54), but shared
[ChM]
[ChB]
[ChB]
[ChB]

numerous alleles with group B (Q = 0.34).

Site

[1]
[2]
[3]
[4]
[5]
[6]

The dendrogram based on microsatellite data (Fig. 3)

confirms a high level of differentiation between the eight
Trigona species. Almost all individuals were grouped with
specimens from their morphologically recognized species
carbonaria/hockingsi

carbonaria/hockingsi

(Ic = 100%). The only exception concerns the T. hockingsi

individuals that were grouped in two clearly paraphyletic
carbonaria

groups (Ic = 91%); a cluster formed by the samples from

hockingsi
hockingsi
hockingsi

laeviceps
laeviceps
Species

pagdeni
collina

sites 3 – 6 (hockingsi A, Ic = 100%) and a cluster formed by the

samples from site 1 (hockingsi B, Ic = 100%). This classification

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331

 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2323

Table 2 Pairwise multilocus FST estimates (below diagonal) and P-values of the genotypic differentiation (above diagonal)

Species carbonaria and hockingsi pagdeni collina laeviceps

[1] [2] [3] [4] [5] [6] [ChB] [ChB] [ChB] [ChM]

carbonaria [1] *** *** *** *** *** *** *** *** ***
and [2] 0.035 *** *** *** *** *** *** *** ***
hockingsi [3] 0.449 0.388 ** NS *** *** *** *** ***
[4] 0.485 0.421 0.020 * *** *** *** *** ***
[5] 0.468 0.414 0.010 0.007 *** *** *** *** ***
[6] 0.235 0.215 0.383 0.416 0.398 *** *** *** ***
pagdeni [ChB] 0.528 0.447 0.665 0.671 0.655 0.536 *** *** ***
collina [ChB] 0.551 0.483 0.658 0.672 0.656 0.546 0.590 *** ***
laeviceps [ChB] 0.448 0.386 0.515 0.535 0.531 0.442 0.393 0.484 ***
[ChM] 0.604 0.526 0.680 0.688 0.675 0.621 0.553 0.608 0.318

NS indicates non significant differences, *P < 0.05, **P < 0.01, ***P < 0.001. Trigona samples are abbreviated per collection sites (in
bracket). Abbreviations of collection sites follow Fig. 2.

Table 3 Assignment proportions of Trigona carbonaria and T. hockingsi specimens according to their taxonomic identification and their
collection sites

Phenotype hockingsi Phenotype carbonaria

Group A Group B Group C Group D

Site N Q > 0.9 0.5 < Q < 0.9 Q > 0.9 0.5 < Q < 0.9 Q > 0.9 0.5 < Q < 0.9 Q > 0.9 0.5 < Q < 0.9

[1] 30 3 1* 26
[2] 22 22
[3] 14 14
[4] 19 19
[5] 28 28
[6] 17 1 1 15

*Specimen having a parent in population C (see text).

Assignment in the four panmictic groups A, B, C and D was performed using the software structure solely based on the genetic data. N
indicates the total number of specimens analysed per collection site. Q indicates the genome proportion of the specimens assigned in the
group.

is exactly the same as the A and B groupings established by
structure. The two T. hockingsi individuals from site 6
branch at the base of the hockingsi A group. The T. carbonaria
specimens cluster in two subgroups. The first encompasses
individuals from site 6 (Ic = 100%) and the second, individuals
from sites 1 and 2 (Ic = 100%). Again, this grouping is exactly
the same as groups C and D established by structure.
Similar geographically based groupings were found within
T. laeviceps individuals from northern and southern Thai-
land (Ic = 98% and 94% in sites ChM and ChB, respectively).
The groupings described above remain unaltered if we
arbitrarily code the suspected null alleles at loci Tc4.326,
Tc1.20, Tc4.63 as identical alleles in the T. collina, T. laevi-
ceps, T. pagdeni and T. clypearis individuals. The topology
of the dendrogram is then slightly modified. Individuals
recognized as belonging to the carbonaria species group
(T. mellipes, T. hockingsi and T. carbonaria) form a mono-
phyletic group in this case; the hockingsi B specimens
branch at the base of this group (data not shown).
Sequence divergence among taxa
Only four of the amplified DNA sequences out of 15
(genbank accession numbers AY575080 to AY575094)
apparently coded for cytochrome b. Although they dis-
play c. 77% similarity with Apis and Trigona mitochondrial
Cytb sequences, the 11 other sequences were noncoding.
We assumed that these 11 sequences are nuclear mitochon-
drial pseudogenes and we have designated them ψ Cytb.
The four coding Cytb sequences were obtained from one
specimen of each T. collina, T. clypearis, T. laeviceps and
T. hockingsi species. Nucleotide divergences among these

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331

2324 P . F R A N C K E T A L .

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331

 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2325
Fig. 4 Neighbour-joining tree based on p-distance between DNA
sequences of the mitochondrial cytochrome b gene (Cytb) and its
nuclear pseudogenes (ψ Cytb). Specimens sequenced are referenced
according to their taxonomic identification and the abbreviation
of their collection site in brackets. The tree is rooted with Cytb
sequence of Apis mellifera (L06178). Bootstraps have been computed
over 2000 iterations by resampling the informative sites. Only
bootstraps values greater than 80% are reported.
four sequences (18 – 24%) were slightly smaller than their
divergences from the Cytb sequence of A. mellifera (25–
28%). The 11 ψ Cytb sequences (AY575084 to AY575094)
were obtained from T. pagdeni, T. sapiens and T. mellipes and
two individuals from each of the A, B, C, D groupings.
Nucleotide divergences among the 11 ψ Cytb sequences
were particularly low (< 2.4%).
The ψ Cytb sequences suggest that species of the carbon-
aria group are monophyletic (Fig. 4). ψ Cytb sequences of T.
carbonaria, T. hockingsi and T. mellipes differ from T. pagdeni
and T. sapiens sequences by two transitions (positions 127 and
161) and one transversion (position 18). Within the carbonaria
group, the same ψ Cytb sequences were observed in four T.
carbonaria and T. hockingsi individuals (Fig. 4). Only T. hock-
ingsi B individuals were clearly differentiated within the
carbonaria group (Fig. 4). ψ Cytb sequences of those specimens
differ to all other sequences by two transitions (positions
275 and 341), and one base pair deletion (position 363).
Discussion
Our main conclusions from the analysis of microsatellite
variation of Australian stingless bees can be summarized
as follows: (i) Trigona carbonaria and T. hockingsi colonies
show unambiguous genetic differences whether or not
they exist in allopatry or in sympatry (Table 3 and Fig. 3);
(ii) individuals from northern and southern Queensland
Fig. 3 Neighbour-joining tree based on the shared allele distance between individual bees. The tree was computed coding nonamplified
loci for an individual bee as missing data. Individuals are labelled according to their collection site (see Fig. 2). Individuals recognized as
Trigona hockingsi belong to two paraphyletic groups: hockingsi A and hockingsi B (Ic = 91%). Individual bees from the seven other recognized
species belong to monophyletic groups (Ic = 100%). Symbols defined in Fig. 2 indicate the roots of each recognized species. Individual bees
identified as hybrid are indicated using an asterisk (see Table 3).
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
are genetically differentiated within both of the currently
recognized taxa, T. hockingsi and T. carbonaria (Table 3 and
Fig. 3); (iii) T. hockingsi samples from northern and southern
Queensland are paraphyletic; (iv) T. hockingsi samples
from northern Queensland display low genetic variability
compared with other T. hockingsi and T. carbonaria samples
(Table 1); (v) collection sites where both T. hockingsi and
T. carbonaria were observed show comparatively high gen-
etic variation (Table 1), significant departures to Hardy–
Weinberg equilibrium and a few colonies that appeared to
be hybrids (Table 3).
DNA sequences (ψ Cytb) confirm that T. hockingsi
samples from southern Queensland show high genetic
divergence from T. hockingsi samples from northern Queens-
land and from all other taxa of the carbonaria group (Fig. 4).
Genetic differentiation at microsatellite loci and taxonomy
Based on morphology and nest architecture the 236 colonies
analysed here belonged to eight different species (Fig. 2).
Variation at the 13 microsatellite loci studied generally
confirm these identifications (Fig. 3). Colonies displaying the
same phenotypes are genetically similar, even when different
phenotypes were sampled at the same collection site (e.g. sites
1 and 6). The only exception concerns colonies displaying
the T. hockingsi nest architecture whose individuals cluster
in two highly differentiated paraphyletic groups, one in
northern and one in southern Queensland (Fig. 3, Table 3).
This marked differentiation is confirmed by significant dif-
ferences in their DNA sequences (Fig. 4) and head width
measures. A posteriori, we found several morphological
characters that discriminate specimens from these two
regions (see Appendix). The specimens from the few colonies
collected in southern Queensland consistently differ in size,
coloration and pilosity from those from northern Queensland
(Table 4). Consequently, we postulated that those colonies
from southern Queensland should be classified as a fourth
species (T. davenporti nov. sp., see Appendix for description)
within the Australian carbonaria species group.
Microsatellite loci also revealed significant differenti-
ation among geographically distant specimens classified
as the same species. Such differentiation was observed
between samples of T. carbonaria from northern and south-
ern Queensland as well as between samples of T. laeviceps
from northern and southern Thailand (Table 2, multilocus
FST = 0.215 and 0.318, respectively). In T. carbonaria, no
morphological diagnostic character was detected between
the two geographical samples. However, in T. laeviceps,
2326 P . F R A N C K E T A L .

Table 4 Coloration, pilosity and measured differences among species of the carbonaria group

Trigona Trigona Trigona Trigona

Species mellipes carbonaria hockingsi davenporti

Coloration CI II 50%
III 20% 38%
IV 28% 45% 12%
V 10% 50% 35%
VI 90% 22%
Pilosity PI I 30% 63%
II 50% 11% 10% 37%
III 20% 89% 90%
Morphometry HL Range 1.34–1.46 1.32–1.42 1.44–1.56 1.38 –1.42
Mean 1.40 1.36 1.50 1.41
(SD) (0.04) (0.04) (0.04) (0.01)
HW Range 1.66–1.80 1.66–1.78 1.78–1.96 1.68 –1.76
Mean 1.72 1.73 1.86 1.72
(SD) (0.05) (0.03) (0.05) (0.03)
HTL Range 1.50–1.68 1.48–1.56 1.66–1.78 1.52–1.64
Mean 1.57 1.52 1.71 1.57
(SD) (0.07) (0.03) (0.03) (0.05)
HTW Range 0.57–0.65 0.48–0.58 0.56–0.64 0.48 – 0.58
Mean 0.60 0.53 0.59 0.50
(SD) (0.02) (0.03) (0.03) (0.04)
WL2 Range 1.00–1.05 1.05–1.20 1.04–1.28 1.14 –1.18
Mean 1.02 1.13 1.17 1.17
(SD) (0.02) (0.04) (0.06) (0.02)

Trigona carbonaria (two colonies from Elanora [site 1], one colony from Mudgeeraba [1], two colonies from Laidley [2], one colony from Esk
[2], three colonies from Herberton [6]); T. mellipes (three colonies from Darwin [Da], one colony from Kathrine [Ka], one colony from
Kununurra [Ku]); T. hockingsi (two colonies from Gladstone [3], three colonies from Rockhampton [4], two colonies from Kunwarara [5],
two colonies from Herberton [6], one colony from Hockings [Cockerell’s holotype* and paratype]), T. davenporti (four colonies from
Mudgeeraba [1]). Two workers per colony were analysed. Coloration index (CI) has been established on the flagellum (see Dollin et al. 1997
for a definition of the six levels of colour). Pilosity index (PI) on the malar space is defined as follows: glabrous (I), discontinuous line of
bristles (II), continuous line of bristles (III). Structural dimensions (HL, HW, HTL, HTW and WL2) are referred in mm following Sakagami
(1978).
Morphometric measures on T. hockingsi QMBA holotype are as follow: HL 1.50, HW 1.96, HTL 1.78, HTW 0.62; WL2 1.06.

pilosity on the vertex clearly differentiates the specimens
from northern and southern Thailand (see supplementary
material at the journal’s web page). These results probably
indicate that T. laeviceps encompasses at least two taxa. This
demonstrates the difficulty of making taxonomic infer-
ences based solely on genetic differentiation, particularly
when the differentiation is linked with the geography.
Population genetic structure revealed by microsatellite loci
depends on both current and ancestral gene flows among
populations or taxa and their demographic histories. In the
case of demographic differences among taxa, the level of
differentiation is not proportional to the level of genetic
exchange and divergence time. This explains why such
highly polymorphic genetic markers are not well suited for
making phylogenetic inferences, particularly when limited
numbers of loci are used (Schlötterer 2001). For example,
the genetic analyses presented here fail to prove the mono-
phyly of the carbonaria group (T. carbonaria, T. mellipes, T.
hockingsi and T. davenporti) whereas such monophyly is
supported by DNA sequences (Fig. 4) and morphological
characters such as the pilosity of the meso- and metathorax
(see Appendix).
Nevertheless, our results clearly show how genetic ana-
lyses using microsatellite loci can be helpful to identify
closely related species. Internal nest architecture currently
appears as an efficient but not sufficient character to dis-
criminate Australian species in the carbonaria species group.
Colonies displaying different nest architectures unequi-
vocally belong to different species (e.g. T. carbonaria and T.
hockingsi), but different species may also have similar internal
nest architectures (i.e. T. hockingsi and T. davenporti nov. sp.).
Demographic patterns and genetic differentiation
The differences in the degree of heterozygosity observed
among collection sites and species (Table 1) suggest that

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 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2327
the taxa analysed have experienced different demographic
histories. Note, however, that heterozygosity differences
among taxa can also result from biases inherent in the
genetic markers themselves (e.g. presence of null alleles
and directional evolution of microsatellite sizes).
Null alleles reduce genetic diversity and the proportion
of heterozygotes estimated for a population and may cause
artificial departures from Hardy–Weinberg equilibrium.
Individuals that did not amplify at some microsatellite loci
usually belonged to the same morphological taxon, which
is an indirect proof of the presence of null alleles in our
data set. However, such alleles would only marginally
affect variability within the populations studied, because
the two collection sites (sites 1 and 6) that showed signi-
ficant departures from Hardy–Weinberg equilibrium also
showed higher genetic diversity than other sites (Table 1).
Furthermore, the samples from these two collection sites
displayed significant linkage disequilibria, phenotypically
distinct colonies (Fig. 2) and a few colonies that appeared
to be hybrids (Table 3). Furthermore, we cannot exclude
the possibility that the high levels of differentiation
observed between T. carbonaria samples from northern
and southern Queensland arise partially from gene flow
between T. carbonaria and T. hockingsi in northern Queens-
land. In fact, the proportion of T. hockingsi alleles (2.6%) is
only slightly smaller than the proportion of private alleles
(3.3%) in T. carbonaria samples from northern Queensland.
A more complete sampling of T. hockingsi bees from this
region will be necessary to clarify this issue.
Size constraints on microsatellite loci may differ among
closely related species and consequently affect the level
of variability detectable in each species (Schlötterer 2000).
However, the average repeat count of microsatellite loci
was not statistically different among the Trigona samples,
suggesting that differences in heterozygosity observed
among taxa are not the consequence of directional evolu-
tion of microsatellite size. The T. hockingsi samples (sites 3–
5) display the lowest level of polymorphism (Table 1),
which may reflect either small population sizes or past
reduction of population sizes. As we were unable to detect
evidence of demographic variation through time in these
samples (test performed with bottleneck, Piry et al. 1999;
data not shown), the hypothesis that T. hockingsi popula-
tions have a low number of colonies compared with other
species seems the more plausible. Nonetheless, genetic
drift should have a stronger effect in T. hockingsi than in
other Trigona species, which may explain larger levels of
differentiation in pairwise comparisons with T. hockingsi
than between other Trigona species.
Speciation in Australian Trigona
Our evidence that hybridizations occasionally occur in the
carbonaria group demonstrates that disruption of gene flow
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
is incomplete among taxa. This raises the problem of the
definition of species within this group according to the
biological concept (Mayr 1963). However, hybridizations
seem to have only minor evolutionary consequences. Gene
flow between some of these species is probably limited to
the first generation because of break down of second-
generation hybrids. In agreement with this hypothesis, the
only davenporti × carbonaria colony observed at site 1 is very
likely to be a first-generation hybrid (Table 3). Further-
more, taxonomic criteria based on nest architecture cur-
rently used for species identification in the carbonaria species
group are strongly correlated with the observed genetic
patterns. Consequently, we argue that the rank of species
should be retained for the four taxa currently described in
the carbonaria group.
Estimates of mutation rates of nuclear mitochondrial
pseudogenes remain too imprecise (Bensasson et al. 2001)
to accurately estimate the divergence times among species
of the carbonaria species complex. However, nucleotide
divergences among these species are large enough (mean
divergence = 0.6%) to suggest that the four species of the
carbonaria diverged several hundreds of thousands years
ago. As with other Australian Trigona species, the ances-
tors of the carbonaria group probably came from southeast-
ern Asia (Kerr & Maule 1964). The absence of similar forms
among the Asian Trigona and the broad distribution of the
carbonaria group over diverse Australian biotopes suggests
that the group diverged within Australia. Although our
sampling was restricted, meaning that we do not have an
accurate picture of species distributions over the entire
range of the carbonaria complex, our data suggest that spe-
ciation with the carbonaria has occurred in allopatry. The
migration of T. sapiens and T. clypearis to Australia prob-
ably occurred independently and more recently.
How did nest architecture evolve in the carbonaria species
group? A comb with irregularly arranged cells (Fig. 1d) is
likely to be the ancestral character of the carbonaria group,
because this pattern is observed in three out of four species
including T. davenporti, and because combs with irregu-
lar cells are found in related taxa of southeastern Asia
(Michener 1961; Sakagami et al. 1983; Starr & Sakagami
1987). Consequently, modification of the internal nest arch-
itecture toward spiral comb probably occurred just once in
the T. carbonaria lineage. Modification of nest architecture
does not seem the primary force for speciation in this group.
Note, however, that modification of nest architecture prob-
ably results from apparently trivial mechanisms. Indeed,
comb architecture need not necessarily be genetically deter-
mined. Reproductive swarming in stingless bees often
involves exchange of food and workers between mother
and daughter colonies for several weeks or months after
swarming (Inoue et al. 1984). Such behaviour may suggest
that some aspects of nest construction could be culturally
transmitted from the mother colony (Cavalli-Sforza et al.
2328 P . F R A N C K E T A L .
1982). Interestingly, the davenporti × carbonaria hybrid col-
ony observed at site 1 displays exactly the same internal
nest architecture as other T. davenporti colonies (i.e. comb
with irregularly arranged cells, not comb with an inter-
mediate form between T. davenporti and T. carbonaria). This
suggests that internal nest architecture is either inherited
from only one parent, culturally transmitted from the
mother colony, or that the spiral-comb pattern is a reces-
sive phenotype (the irregularly arranged cell patterns
being the dominant phenotype). A more complete genetic
investigation of such hybrid colonies would undoubtedly
provide a better understanding of the speciation process in
the carbonaria group.
Acknowledgements
We are grateful to the Australian Native Bee Research Centre,
David Low, Peter Davenport, Les Felhaber, Morrie Sweeney,
Brendan Dodd, Tom Carter, Tadd Bartereau and Russel Zabel for
their help in collecting samples. We thank Jenny Bower for her
drawings of nest architectures, Bryan Danforth for his comments
on the manuscript, Emanuel Barrau and Laurent Soldati for the
preparation of insects for electronic microscopy and particularly
Anne Dollin for her help in obtaining samples and identifying
Trigona species. The project was funded in part by an Australian
Research Council International Fellowship to Pierre Franck.
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The present study was mainly carried out in 2001 during Pierre Franck’s
postdoctoral fellowship at the Behaviour and Genetic of Social
Insects lab managed by Ben Oldroyd (http://www.bio.usyd.edu.au/
Social_InsectsLab/Social_InsectsLab.htm). Emilie Cameron com-
pleted her honour thesis and Greg Good was the technician at the
bee lab during this period. Jean-Yves Rasplus is senior scientific
working on systematic, evolution and conservation genetic of insects.
Pierre Franck currently works on integrated pest management in
apple trees, notably population genectics of Lepidoptera species.
2330 P . F R A N C K E T A L .
Appendix
Trigona (Heterotrigona) davenporti Franck, nov. sp.
Description
Coloration. Thorax and abdomen cuticle chestnut brown,
yellow to orange-brown on the legs.
Pilosity. Vertex densely and uniformly covered with black
ramified bristles (25 µm) and few black and tick simple
hairs (30 µm in the middle, 80 µm at the lateral peri-
phery). Frons and genae uniformly covered with brownish
and faintly plumose bristles. Surpraclypeus and clypeus
densely covered with whitish and strongly plumose
bristles. Malar space (Fig. 4a) glabrous or subglabrous
(bearing one or two fine and simple setae on the side).
Mesopleuron and metapleuron densely covered with short
plumose hairs. Mesoscutum sparsely covered with yellow-
brown and ramified bristles with admixed black hairs
about twice as long as the ramified bristles. Mesoscutum
uniformly pilose, without longitudinal glabrous band.
Sixth tergite (Fig. 5a) bearing mostly simple hairs (100 µm)
and sparse ramified hairs (35 µm).
Morphometry. Worker structural dimensions (range, mean
± SD) are referred in mm (following Sakagami 1978):
maximum head length (HL) 1.38 –1.42, 1.41 ± 0.01; maximum
head width (HW) 1.68 –1.76, 1.72 ± 0.03; maximum hind
tibia length (HTL) 1.52 –1.64, 1.57 ± 0.05; maximum hind tibia
width (HTW) 0.48 – 0.58 0.50 ± 0.04; wing diagonal (WL2)
1.14–1.18, 1.17 ± 0.02.
Nest. Entrance tunnel is lacking, but spaced dots of orange-
brown resin are usually observed around the entrance
hole. Cells of the brood comb are yellow and irregularly
arranged in horizontal layers. Involucrum layers and storage
pots with dark red-brown cerumen surround brood cells
(see Michener 1961 for terminology).
Material examined
Workers specimens examined are from four colonies
(Mud.24, Mud.25, Mud.26, Mud.27).
Holotype. Worker Mud.26.3, AUSTRALIA, QLD, Mudgeer-
aba, Austinville Road, V. 1990, P. Davenport leg. [deposited
in Queensland Museum BrisbAne]
Paratypes. Four workers (Mud.24), four workers (Mud.25),
four workers (Mud.26) and four workers (Mud.27), same
locality [deposited in QMBA (2); Australian National Insect
Collection, Canberra (two); Natural History Museum,
London (two); Musée d’Histoire Naturelle, Paris (two);
INRA collection, Montpellier (eight)].
Notes
Trigona (Heterotrigona) davenporti workers display densely
covered mesopleuron and metapleuron characteristic of
the carbonaria group. Characters of coloration, pilosity and
morphometry only marginally differ from those of T.
carbonaria Smith, T. hockingsi Cockerell and T. mellipes Friese
(Table 4). External and internal architectures of T. davenporti
nests is similar with those of T. hockingsi nests. Only few
morphological differences were observed between these
two species: (i) short bristles on vertex are white in T. hockingsi
vs. black in T. davenporti; (ii) malar space bears a continued
line of hairs in T. hockingsi whereas is glabrous or subgla-
brous in T. davenporti (Fig. 5); (iii) sixth tergus covered with
numerous small hairs in T. hockingsi, but bearing a few
scattered small hairs in T. davenporti (Fig. 6); (iv) general
coloration of cuticle is consistently darker in T. hockingsi
than in T. davenporti (T. hockingsi types examined, QMBA,
Hy3722). Additional pictures comparing morphology of Aus-
tralian Trigona species are available at the journal’s web page.
Etymology. I dedicate this species to P. Davenport who
collected the four colonies analysed in this work.
Fig. 5 Worker pilosity on malar space. (a)
Trigona davenporti nov. sp., Mudgeeraba
(site 1); (b) T. hockingsi, Herberton (site 6).
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
 G E N E T I C S T R U C T U R E O F A U S T R A L I A N T R I G O N A 2331
Remarks
Among the variation observed in T. davenporti, specimens
from the colony Mud.25 display characters of coloration,
pilosity and morphometry marginally distributed. Mud.25
bees are darker and they have larger tibia (HTL and HTW)
and wider head (HW) than other T. davenporti specimens.
© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 2317–2331
Fig. 6 Worker pilosity on the sixth tergite.
(a) Trigona davenporti nov. sp., Mudgeeraba
(site 1); (b) T. hockingsi, Kunwarara (site 5).
Their malar space is partially haired and their short bristles
on vertex are brown as in T. carbonaria. These results are in
agreement with molecular data that recognized the colony
Mud.25 as a davenporti × carbonaria hybrid.
Some specimens collected by P. Davenport and deter-
mined by A. Dollin as T. hockingsi (Dollin et al. 1997; p. 883)
may belong to the current T. davenporti species.