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Multi-gene phylogenetic analysis of south-east

Asian pest members of the Bactrocera dorsalis
species complex (Diptera: Tephritidae) does not
support current taxonomy
ARTICLE in JOURNAL OF APPLIED ENTOMOLOGY MARCH 2013
Impact Factor: 1.65 DOI: 10.1111/jen.12047

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J. Appl. Entomol.

ORIGINAL CONTRIBUTION

Multi-gene phylogenetic analysis of south-east Asian pest

members of the Bactrocera dorsalis species complex (Diptera:
Tephritidae) does not support current taxonomy
L. M. Boykin1,2, M. K. Schutze1,3, M. N. Krosch1,3, A. Chomic1,2, T. A. Chapman1,4, A. Englezou1,4,
K. F. Armstrong1,2, A. R. Clarke1,3, D. Hailstones1,4 & S. L. Cameron1,3
1
2
3
4

CRC for National Plant Biosecurity, Bruce, ACT, Australia

Bio-Protection Research Centre, Lincoln University, Lincoln, Christchurch, New Zealand
School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, Qld, Australia
NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, Australia

Keywords
biosecurity, fruit fly, multi-gene phylogeny,
species delimitation
Correspondence
Laura M. Boykin (corresponding author), Plant
Energy Biology, ARC Centre of Excellence, The
University of Western Australia, M316
Crawley, WA 6009, Australia.
E-mail: lboykin@mac.com
Received: November 12, 2012; accepted:
February 18, 2013.
doi: 10.1111/jen.12047

Abstract
Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south-east Asia and the Pacific. Identifying molecular
diagnostics has proven problematic for these four taxa, a situation that
cofounds biosecurity and quarantine efforts and which may be the result
of at least some of these taxa representing the same biological species. We
therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide
geographic range; sequencing six loci (cox1, nad4-3, CAD, period, ITS1,
ITS2) for approximately 20 individuals from each of 16 sample sites. Data
were analysed within maximum likelihood and Bayesian phylogenetic
frameworks for individual loci and concatenated data sets for which we
applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap
resampling for Bayesian and likelihood analyses respectively, Rosenbergs
reciprocal monophyly measure, P(AB), Rodrigos (P(RD)) and the genealogical sorting index, gsi. We specifically tested whether there was phylogenetic support for the four ingroup pest species using a data set of
multiple individuals sampled from a number of populations. Based on our
combined data set, Bactrocera carambolae emerges as a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are
unresolved. These data add to the growing body of evidence that B. dorsalis s.s., B. papayae and B. philippinensis are the same biological species,
which poses consequences for quarantine, trade and pest management.

Introduction
The Tephritidae (true fruit flies) is one of the most
species-rich families within the order Diptera. While
non-fruit feeding tephritids are rarely pestiferous
(Headrick and Goeden 1998), the frugivorous tephritids contain many genera of major economic importance, including Ceratitis, Rhagoletis and Anastrepha
(White and Elson-Harris 1992). Mature female
2013 Blackwell Verlag, GmbH

frugivorous tephritids oviposit into fleshy fruits and

vegetables, where resultant larvae emerge and feed
on the fruit pulp. Production losses and costs of field
control are the direct impacts of fruit fly attack, while
indirect losses result from the implementation of regulatory controls and lost market opportunities (Clarke
et al. 2011). Bactrocera Macquart contains over 500
described species and is the dominant genus of fruit
flies in the Asia/Pacific region (Drew 1989, 2004).
1

Phylogeny of B. dorsalis pest flies

Within this genus, the Bactrocera dorsalis species complex contains 75 species and includes some of the
most pestiferous species of the genus, especially the
Oriental fruit fly, B. dorsalis s.s. (Hendel), and the
Asian papaya fruit fly, B. papayae Drew and Hancock
(1994); Clarke et al. 2005). The B. dorsalis complex is
a monophyletic group of species of relatively recent
evolutionary origin, with an estimated age of 6.2 million years to their most recent common ancestor
(Krosch et al. 2012a).
Bactrocera dorsalis s.s., B. papayae, B. philippinensis
Drew & Hancock and B. carambolae Drew &
Hancock are found predominately in south-east
Asia and the Pacific, and are the members of the
B. dorsalis complex which are of most concern to
pest managers and plant biosecurity officials in the
region. These four species form a true sibling species complex for which both morphological and
molecular diagnostics have proven problematic
(Clarke et al. 2005). The initial taxonomic work
that separated these taxa relied on very subtle character state differences (Drew and Hancock 1994),
but many of these character states have since been
shown to be variable and continuous between the
taxa (Krosch et al. 2012b; Schutze et al. 2012a). All
four species are polyphagous pests (Allwood et al.
1999; Clarke et al. 2001) that have invaded regions
beyond their natural ranges (Smith 2000; Cantrell
et al. 2001; Duyck et al. 2004), hence accurate
diagnosis for quarantine and field management is
critical.
Diagnostic development for these species has been
confounded by their close genetic, morphological,
behavioural and physiological similarities (Clarke
et al. 2005; Schutze et al. 2012b). While some
researchers have identified morphological and molecular markers considered to be diagnostic of different
species (Drew and Hancock 1994; Iwahashi 1999;
Muraji and Nakahara 2002; Naeole and Haymer 2003;
Drew et al. 2008), others have found no such markers, or markers which separate some but not all of the
four species (Medina et al. 1998; Tan 2000, 2003;
Wee and Tan 2000a,b, 2005). Consequently, the
debate continues as to whether these four taxa represent good biological species for which species-specific
diagnostic markers exist but which are yet to be identified and universally agreed upon; or whether they
may in fact represent a group where one biological
species has been incorrectly taxonomically split, in
which case species-level diagnostic markers simply do
not exist and any observed variation reflects population level differences (Harrison 1998; Sites and
Marshall 2004).
2

L. M. Boykin et al.

Attempts to identify DNA markers for these four

species of the B. dorsalis complex have met with
mixed success. An early study of the 18S rDNA, Cu/
Zn superoxide dismutase enzyme and 12S rDNA coding genes found these loci could not differentiate
B. dorsalis s.s., B. carambolae and B. papayae (White,
1996). Similarly, while within the larger B. dorsalis
complex, the species B. occipitalis (Bezzi) and B. kandiensis Drew & Hancock could be resolved as separate
species using the 16S gene, B. dorsalis s.s., B. papayae,
B. carambolae and B. philippinensis could not be separated (Muraji and Nakahara 2002). In contrast, the
nDNA regions 18S + ITS1, and ITS1 and ITS2 were
found to reliably distinguish B. carambolae from
B. dorsalis s.s. (Armstrong et al. 1997; Armstrong and
Cameron 2000). A series of papers by Nakahara and
colleagues (Nakahara et al. 2000, 2001, 2002; Muraji
and Nakahara 2002) targeting the mitochondrial DNA
D-loop + 12S and 16S suggested the four species
could be distinguished from each other, although the
different target sites did not distinguish all species
equally (e.g. B. papayae and B. carambolae were poorly
or not separated using 16S). Other tightly focused
procedures, for example, a microarray test developed
from EPIC (exon primed intron crossing)-RFLP of
muscle actin can distinguish B. dorsalis s.s., B. papayae
and B. carambolae (Naeole and Haymer 2003).
One common feature and weakness for nearly
all of the above studies is a failure to separate what
may be variation at the intra- vs. inter-specific level.
Taxa are often represented by very small sample sizes,
sometimes as few as one individual, rarely more than
five or six (e.g. Muraji and Nakahara 2002); or in
cases where sample sizes are greater they are generally drawn from only one geographic population (e.g.
Nakahara et al. 2001). As a result, it remains impossible to determine whether such diagnostic markers are
resolving species or population level differences, as
already recognized: for example, In order to confirm
the genetic interrelationship among the B. dorsalis
complex species, analyses of field populations using
many other genetic markers are needed (Muraji and
Nakahara 2002). We specifically address this issue in
this study.
As part of a larger project investigating the species
limits of the target taxa within the B. dorsalis species
complex (i.e. B. dorsalis s.s., B. papayae, B. philippinensis and B. carambolae = ingroup taxa) (Krosch et al.
2012b; Schutze et al. 2012a,b), we undertook new
field collections of specimens from multiple sites
across the geographic ranges of the four taxa. We also
included outgroup taxa from within the complex
[B. cacuminata (Hering), B. opiliae (Drew & Hardy),
2013 Blackwell Verlag, GmbH

L. M. Boykin et al.

B. occipitalis (Bezzi)] and outside the complex [B. musae (Tryon), B. tryoni (Froggatt)]. We sequenced six
loci (cox1, nad4-3, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites,
including two or more sites for each of the ingroup
taxa. Data were analysed within maximum likelihood
and Bayesian phylogenetic frameworks for both the
individual loci and concatenated data sets for which
we applied multiple monophyly and species delimitation tests. Using this data set of multiple individuals
sampled from a number of populations, we specifically tested whether there was phylogenetic support
for the four described pest species: B. dorsalis s.s.,
B. papayae, B. philippinensis and B. carambolae.
Materials and Methods
Target species and outgroup selection

The aim of this study was to use phylogenetic methods to resolve species limits among the following four
target species of the B. dorsalis species complex: B. dorsalis s.s., B. papayae, B. philippinensis and B. carambolae
(Sites and Marshall 2004). For the purposes of this
study, we refer to these four taxa as the ingroup species. We also selected a number of species to represent outgroups, which were chosen because: (i) they
are related to varying degrees to the ingroup species
(they are either in the B. dorsalis species complex or
otherwise closely related) but are unambiguously
regarded as different species and (ii) they are taxa that
are morphologically similar and may be confused with
the target species for quarantine purposes (and hence
further resolving their molecular relationships with
the ingroup taxa is of wider benefit). The outgroup
species consisted of three B. dorsalis complex flies: two
Australian species B. cacuminata and B. opiliae, and the
Philippine species B. occipitalis (which occurs sympatrically with B. philippinensis); and B. musae which,
while not belonging to the B. dorsalis complex per se, is
closely related to the complex as demonstrated by previous molecular studies (Armstrong and Cameron
2000; Krosch et al. 2012a). Finally, we included
B. tryoni as an outgroup species for tree rooting, as
while it is of the same genus it unambiguously
belongs to a different species complex, the B. tryoni
species complex (Krosch et al. 2012b).
Study sites and specimen collection

To obtain as many representative samples from across

as broad a geographic area as possible, we collected
in-group species from multiple locations across their
2013 Blackwell Verlag, GmbH

Phylogeny of B. dorsalis pest flies

known distributions. As discrimination amongst ingroup species is difficult due to high morphological
similarity, we made collections of in-group species
from locations where each is regarded as allopatric to
the other three based on the descriptions provided in
Drew and Hancock (1994). For collection sites where
more than one of the in-group taxa occur sympatrically (primarily B. papayae and B. carambolae), we
identified species based on published descriptions
(Drew and Hancock 1994) and host use data (Clarke
et al. 2001).
Samples of male flies were collected from 2009 to
2010 from 13 locations across seven countries
(Table 1). The principle method of collection consisted of luring male flies into methyl eugenol (ME)
insecticide-baited hanging traps containing propylene
glycol as a preserving agent (Vink et al. 2005; Thomas
2008). These traps were either distributed as part of
collection parcels to collaborators throughout southeast Asia who placed the traps in the field, or deployed
during collection trips undertaken by MKS in December 2010.
Exceptions to above collection methods are as follows. Bactrocera tryoni were collected using the same
technique as above, but using Cue-lure instead of
ME as the male attractant. Bactrocera musae were
sourced from a culture maintained by the Queensland Government Department of Agriculture, Fisheries and Forestry (DAFF) in Cairns, Queensland
(Australia). Flies from Serdang (Malaysia) were
reared from Musa acuminata x balbisiana hybrids, vars.
Mas, Berangan and Lemak bananas (which yielded
B. papayae) and Averrhoa carambola fruit (which
yielded B. carambolae) collected from the field in
November 2010. Samples from Lampung (Indonesia)
were collected into dry ME lure traps placed in the
field, and flies were promptly preserved in 70% ethanol. Bactrocera carambolae from Paramaribo (Suriname) were reared from A. carambola fruit placed in
the field.
All samples were returned to the Queensland University of Technology (QUT), Brisbane (Australia), for
transfer into absolute ethanol, preliminary morphological identification and preparation for DNA extraction. Three legs of each fly (fore, mid and hind) were
removed and stored in absolute ethanol in new
Eppendorf tubes for shipment to the Elizabeth MacArthur Agricultural Institute (New South Wales
Department of Primary Industries) for genomic DNA
extraction. When numbers allowed, 30 samples per
collection site were sent for extraction (Table 1). The
remainder of all flies are stored as vouchers in absolute ethanol at QUT.
3

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Table 1 Collection details of Bactrocera specimens used in the current study

Location

Country

Latitude

Longitude

Date

Species

Collection
method

Brisbane, Queensland

Australia

272729S

1525856E

Bactrocera tryoni
Bactrocera cacuminata

Cue-lure
ME Lure

Cairns, Queensland
Noonamah, Northern
Territory
Quezon City, Diliman

Australia
Australia

DEEDI culture
123833S

DEEDI culture
131558E

10 July 2009
September
November 2009
5 June 2009
24 December 2009

Bactrocera musae
Bactrocera opiliae

Culture
ME Lure

Philippines

143800N

1210100E

Imus, Cavite
Taipei City, Tawian
San Pa Tong, Chiang Mai
Chatuchuk, Bangkok

Philippines
China
Thailand
Thailand

140718N
250053N
183737N
135032N

1205800E
1213218E
985342E
1003423E

Bactrocera occipitalis
Bactrocera philippinensis
Bactrocera philippinensis
Bactrocera dorsalis s.s.
Bactrocera dorsalis s.s.
Bactrocera dorsalis s.s.

ME Lure
ME Lure
ME Lure
ME Lure
ME Lure
ME Lure

Muang District, Nakhon

Si Thammarat

Thailand

82512N

995348E

17 December 2009
17 December 2009
20 December 2009
16 March 2010
12 March 2010
1421 December
2009
25 October15
November 2009
25 October15
November 2009
1726 November
2009
November 2010

Bactrocera papayae

ME Lure

Bactrocera carambolae

ME Lure

Bactrocera papayae

ME Lure

Bactrocera papayae

November 2010

Bactrocera carambolae

1517 May 2009

1517 May 2009
August 2009

Bactrocera papayae
Bactrocera carambolae
Bactrocera carambolae

ex Musa
acuminata
x balbisiana
ex Averrhoa
carambola
ME Lure
ME Lure
ex Averrhoa
carambola

Tikus Pulau, Penang

Malaysia

52550N

1001838E

Serdang

Malaysia

30020N

1014200E

Lampung, South Sumatra

Indonesia

54043S

1053638E

Paramaribo

Suriname

54920N

551005W

DNA extraction, PCR and sequencing

Tubes containing fly legs were pulse spun, the ethanol

removed and air-dried. Samples were transferred
to 80C for 15 min, after which each tube was
dipped in liquid nitrogen while fly legs were crushed
with a sterile micropestle. DNA was extracted using
the Qiagen DNeasy (QIAGEN Inc., Valencia, CA)
Blood and Tissue kit as per the manufacturers
instructions. Two mitochondrial (mt) protein-coding
genes (cox1 and nad4-3), two nuclear protein-coding
genes (CAD, period) and two nuclear ribosomal RNA
regions (ITS1, ITS2) were analysed. Primers for cox1
are after Folmer et al. (1994). Those for nad4-3 were
newly designed by comparison of tephritid mt
genomes on GenBank (Spanos et al. 2000; Nardi et al.
2003; Yu et al. 2007), targeting regions that appeared
more variable than cox1 but for which PCR amplification was still reliable across taxa. The forward ITS1
primer, ITS7, was designed de novo by KFA; reverse
primer ITS6 was taken from Armstrong and Cameron
(2000), and ITS2 primers FFA and FFB were modified
4

from Porter and Collins (1991). CAD primers are redesigned after Moulton and Wiegmann (2004, 2007)
after comparison with GenBank tephritid sequences.
Primers for period are from Barr et al. (2005) and Virgilio et al. (2009). Primer sequences for all loci are
given in Table 2.
The PCR conditions for ITS1, ITS2 and ND4-2 consisted of 2 ll of template DNA being added to a final
volume of 30 ll of reaction mix containing 200 lM of
dNTPs, 200 nM of each forward and reverse primer, 1
9 Accutaq PCR buffer (Sigma Australia), and 0.02U of
AccuTaq polymerase. The cycling conditions consisted
of an initial denaturation at 94C for 2 min, followed
by 35 cycles of denaturation at 94C for 15 s, annealing at 60C, 55C and 60.5C for ITS1, ITS2 and ND42 respectively, followed by an extension time of
1 min at 68C and final extension of 5 min at 68C.
All PCR products were visualized on 1.5% agarose
gels run at 90V for 45 min and post-stained with ethidium bromide. All PCR products were sent to AGRF
(Australian Genome Research Facility Ltd) in 96-well
plates for purification and sequencing. AGRF is
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Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Table 2 Primer sequences used in the current study

Gene

Name

Direction

Sequence

Reference

cox1

LCO1490
HCO2198
Teph_ND4F1
Teph_ND4R1
ITS7
ITS6
FFA
Shortened FFB
CAD-Bd-F
CAD-Bd-R
F2508
R3270

F
R
F
R
F
R
F
R
F
R
F
R

GGT CAA CAA ATC ATA AAG ATA TTG G

TAA ACT TCA GGG TGA CCA AAA AAT CA
TAG AGT WTG TGA AGG TGC TTT RGG
AGC WAC WGA WGA ATA AGC AAT TAA WGC C
GAA TTT CGC ATA CAT TGT AT
AGC CGA GTG ATC CAC CGC T
TGT GAA CTG CAGG ACA CAT
TCG CTA TTT TAA AGA AAC AT
CCG GTA AAT TTT GAA TGG TTC
GCR GTK GCG AGC ARY TGA TG
CAA CGA CGA AAT GGA GAA ATT C
AGG TGT GAT CGA GTG GAA GG

Folmer et al. (1994)

Folmer et al. (1994)
Herein
Herein
Herein

nad4-3
ITS1
ITS2
CAD
period

accredited by NATA to the ISO/IEC17025:2005 Quality Standard. Australian Genome Research Facility Ltd
operates the AB3730xl 96-capillary sequencer for low
to high throughput DNA sequencing.
Polymerase chain reaction conditions for cox1, CAD
and period consisted of 1 ll DNA template in a final
volume of 20 ll containing 100 nM each forward and
reverse primer 10 ll Go Taq Green enzyme master
mix (ProMega, Sydney, Australia) and 7 ll of sterilized water. PCR cycling conditions consisted of an initial denaturation step at 94C for 2 min, followed by
40 cycles of denaturation at 95C for 30 s, annealing
at 50C (for cox1 and period) or 54C (for CAD) for
30 s. and extension at 72C for 1 min.; there was a
final run-out extension step at 72C for 7 min. All
PCR products were visualized on 1% agarose gels containing 10X dilution of SYBER Safe (Life Technologies, Victoria, Australia) and run at 80V for 30 min.
Sequencing was performed using ABI BigDye ver. 3
dye terminator chemistry and run on an ABI 3130xl
capillary sequencer. Chromatograms were checked
and sequence contigs assembled with SEQUENCHER ver
4.2 (Gene Codes Corporation 2004) to produce completed sequences.
Analytical strategy

The following series of five data sets were analysed to

test the phylogenetic signal of different loci and to
account for the failure to sequence all loci for all specimens:
Datasets #1.11.4 Each linked inheritance groups as
a separate alignment; 1.1: mitochondrial genes
(cox1 + nad4-3), 1.2: ribosomal RNA genes (ITS1
+ ITS2), 1.3: CAD; 1.4: period. The two mitochondrial
and two ribosomal loci are concatenated as they are
coinherited. For the ITS data sets, indels were treated
2013 Blackwell Verlag, GmbH

Herein
Moulton and Wiegmann (2004, 2007)
Moulton and Wiegmann (2004, 2007)
Barr et al. (2005)
Virgilio et al. (2009)

as missing. For ease of comparison, these data sets are

limited to specimens for which all six loci have been
successfully sequenced (235 specimens, 1219, 1002,
528 and 686 bp respectively).
Dataset #2 A concatenated data set including only
specimens for which all six loci were successfully
sequenced (235 specimens, 3435 bp alignment).
Dataset #3 Dataset #2 with heterozygous sites
removed from CAD and period alignments (235 specimens, 3094 bp)
Dataset #4 Dataset #2 with CAD and period removed
from alignment altogether (235 specimens, 2221 bp)
Dataset #5 Specimens for which at least two of the
four loci (i.e. excluding CAD and period) were successfully sequenced (313 specimens, 2221 bp)
Dataset #1 was designed to allow testing of the variation between loci and to apply a species-tree reconstruction approach (Edwards 2008); however, due to
the poor resolution in Datasets #1.21.4, the additional, concatenation-based data sets were produced
(after Gatesy et al. 1999; Gatesy and Baker 2005).
Dataset #2 includes a large number of heterozygous
sites in the CAD and period gene partitions, which may
have resulted in artefactual results. Datasets #3 and #4
are attempts to correct for this potential problem by
removing the heterozygous sites either on a site by site
basis (#3) or by removing the CAD and period gene partitions entirely (#4). Dataset #5 tests how significant
missing partitions were for the inferred phylogeny.
Alignment and analysis

Sequences for each locus were aligned by eye (protein-coding genes) or using ClustalX (rRNA regions)
(Thompson et al. 1997). For the ITS 1 and ITS2 data
set, indels were treated as missing due to the
constraints of Bayesian and RAxML analyses. Hetero5

Phylogeny of B. dorsalis pest flies

zygous sites in the CAD and period loci, observed

clearly as two bases in the forward and reverse
sequences, were labelled according to the IUPAC
code. Models of molecular evolution for each loci, and
each codon position within each protein-coding gene,
were determined using MODELTEST ver. 3.6 (Posada
and Crandall 1998). Concatenations for multilocus
data sets were done in MACCLADE ver. 4.06 (Maddison
and Maddison 2003). For each data set, phylogenetic
trees were inferred in parallel by both Maximum
Likelihood and Bayesian analyses. Likelihood analyses were conducted using RAxML ver 7.2.8 implemented on the RAxML BlackBox webserver (http://
phylobench.vital-it.ch/raxml-bb/index.php) (Stamatakis et al. 2008). Data were analysed with a Gamma
model of rate heterogeneity, the proportion of invariable sites was estimated, and for concatenated, multilocus data sets, the alignment was partitioned and
branch lengths optimized on a per locus basis. Bayesian analyses were conducted using MRBAYES ver 3.2
(Ronquist et al. 2012) using parallel implementation
on the BeSTGRID computer cluster (Jones et al.
2011), or using direct implementation on local desktop computers. Analyses were run for 10 (Datasets #1,
3, 4, 5) or 50 million generations (Dataset #2, due to a
longer time for independent runs to converge) with
sampling every 1000 generations, partitioned data sets
and parameter estimation for each partition unlinked.
Each analysis consisted of two independent runs, each
utilizing four chains, three cold and one hot. Convergence between runs was monitored by finding a plateau in the likelihood score (standard deviation of
split frequencies <0.0015) and the potential scale
reduction factor (PSRF) approaching one. Convergence of other parameters within the runs was also
checked using TRACER v1.5.4 (Rambaut and Drummond 2010), with ESS values above 200 for each run.
The first 12.5% of each run was discarded as burnin
for the estimation of consensus topology and the posterior probability of each node. Bayesian & RAxML
run files are available from the authors upon request.
Phylogenetic trees generated from Datasets #2 and
#5 were used as input in the species monophyly
and delimitation analyses. Species delimitation was
addressed using the standard Kimura two-parameter
(K2P) inter-species distance and Rodrigos P (randomly distinct) (Rodrigo et al. 2008) measure. Species monophyly was measured by clade support,
posterior probability or bootstrap resampling for
Bayesian and likelihood analyses respectively,
Rosenbergs reciprocal monophyly measure, P (AB)
(Rosenberg 2007) and the genealogical sorting index
(gsi) (Cummings et al. 2008). The species delimita6

L. M. Boykin et al.

tion plugin (Masters et al. 2010) for Geneious

(Drummond et al. 2010) was used to calculate
Rosenbergs reciprocal monophyly, P (AB) (Rosenberg 2007) and Rodrigos P (RD) (Rodrigo et al.
2008) measures. The (Cummings et al. 2008) statistic was calculated in R based on the estimated tree
and the assignment file that contains user specified
groups (see http://www.genealogicalsorting.org/).
Two different assignment files were generated for
the gsi for each data set: one based on previously
defined taxonomic groups and the other containing
groups within those as determined using the tip to
root approach of species delimitation (Boykin et al.
2012). Each of the assignment files was run with
the known phylogeny and an R script that specifies
the number of permutations (100 000 permutations
across four processors). All of the gsi analyses were
run using R on the BeSTGRID computer cluster
(Jones et al. 2011). To assess the significance of the
gsi P-values, the Bonferroni correction was used.
Results
Sequence data collection

The six loci (cox1, nad4-3, ITS1, ITS2, CAD and period) were successfully amplified for the majority of
specimens examined across all species. Success/failure of sequencing individual loci for each specimen,
along with their GenBank accession numbers, are
shown in Table S1. Of these, nad4-3 was the only
one of five additional mt genes (data not shown) trialled in this study that was successfully amplified
across the range of species here. Due to the low levels of molecular variation previously found within
the dorsalis complex for cox1 (Armstrong and Ball
2005), the additional mitochondrial genes trialled
were chosen in an effort to maximize variability
based on the previous analyses of dipteran mt genomes (Cameron et al. 2007; Nelson et al. 2012).
The trade-off for gene variability is primer reliability,
whereby sequence variability at the priming sites
causes mismatches and loss of efficacy. Thus, finding
only one more variable mt gene, which could be
reliably amplified, is not surprising. The nad4-3 gene
region was confirmed here to be more variable, having 104 of 577 positions parsimony informative
compared to 101 of 642 parsimony informative for
cox1.
The ribosomal ITS loci each had significant indels
(3384 bp in ITS1, 3140 bp in ITS2), but there were
few heterozygous sites, consistent with the concerted
evolution previously found for these loci (e.g.
2013 Blackwell Verlag, GmbH

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Eickbush and Eickbush 2007). In contrast, both

nuclear protein-coding genes had a large proportion
of heterozygous sites; 179 of 528 bp in CAD and 162
of 686 bp in period. These sites unfortunately made up
almost all of the variable sites within these two genes,
with only 10 of the remaining 349 homozygous sites
in CAD and 15 of 524 in period being parsimony informative. Intraspecific variation for each gene is shown
in Table 3.
Phylogenetic analyses

For each data set, Bayesian (BA) and likelihood analyses (ML) yielded similar topologies; however, nodal
support was much greater for the set of Bayesian analyses. Of the single linkage group analyses (Datasets
#1.11.4), only the mitochondrial gene trees (Dataset
#1.1) were well resolved, with each species other than
B. dorsalis, B. philippinensis, B. papyae and B. carambolae monophyletic with significant nodal supported
(BA pp = 0.91.0; ML bs >70%) (Fig. S1). For the
ribosomal ITS loci (Dataset #1.2), several species were
monophyletic, for example, B. musae, B. occipitalis,
B. opiliae, B. carambolae, whereas B. cacuminata and
B. dorsalis s.s., formed paraphyletic combs with respect
to other species (Fig. S2). For example, in the Bayesian analysis of Dataset #1.2, B. cacuminata specimens
formed 17 of 19 branches in a polytomy with a monophyletic B. opiliae (node support not significant in BA
or ML) and a single significantly supported clade
which included all B. dorsalis s.s., B. philippinensis,
B. papaya and B. carambolae specimens. The trees
inferred for each of the nuclear protein-coding genes
were almost totally unresolved (Figs S34). For CAD
Table 3 The average intraspecific distances for each gene shown in %
calculated using MEGA
Species

ITS1

ITS2

ND4

CO1

per

CAD

Bactrocera tryoni
Bactrocera musae
Bactrocera
cacuminata
Bactrocera occipitalis
Bactrocera opiliae
Bactrocera
carambolae
Bactrocera dorsalis
(sensu stricto)
Bactrocera
philippinensis
Bactrocera papayae
Bactrocera dorsalis
(sensu lato)

0.000
0.000
0.000

0.000
0.000
0.000

1.284
0.127
0.101

0.809
0.031
0.051

0.275
0.033
0.047

0.210
0.038
0.000

0.000
0.000
0.158

0.000
0.000
0.093

0.972
0.604
0.924

0.329
0.603
0.611

0.244
0.291
0.597

0.682
0.101
2.294

0.203

0.081

0.765

0.568

0.505

1.471

0.216

0.094

0.602

0.641

0.413

1.259

0.261
0.224

0.071
0.081

0.595
0.806

0.513
0.632

0.157
0.472

2.471
1.680

2013 Blackwell Verlag, GmbH

(Dataset #1.3), only B. musae (BA & ML) and B. cacuminata (ML only) were monophyletic whereas for
period (Dataset #1.4), B. musae (BA & ML), B. cacuminata (BA only) and B. opiliae (BA only) were monophyletic. The majority of specimens of the remaining
species formed unresolved combs. Due to the poor
resolution across these four data sets, species-tree
reconstruction based on individual gene trees was not
attempted.
Analyses of concatenated data sets were conducted
to determine whether larger data sets would be better resolved and display higher nodal support than
was achieved analysing each linkage group separately
(Datasets #1.11.4). Further, due to the high proportion of heterozygous sites within CAD and period,
and the significant number of individuals for which
one or more genes failed to amplify/sequence (57
specimens, approximately 25%), a series of different
concatenation data sets were analysed to determine
whether either factor resulted in artefactual relationships. The same species boundaries were inferred for
all four concatenated data sets, and the interspecies
relationships were also quite constant. The heterozygous positions within CAD and period had a limited
effect on inferred species relationships, as the only
difference was in the position of a single specimen,
Bd413 an unidentifiable member of the dorsalisgroup complex. This specimen was sister to all the
dorsalis-group flies with inclusion of these gene
regions (#2-BA) or the sister-group of B. occipitalis
with their exclusion (#2-ML, #3-#5-BA & ML)
(fig. 1; Figs S57). Similarly, the inclusion of specimens for which up to half of the loci were missing
(#5) did not result in a different topology from those
inferred from specimens where all genes were present (#3#4).
Below the species level, there was significant variability in topology and nodal support across the different concatenated data sets with few clades larger
than 23 specimens shared between analyses. The
only notable exception is the clade containing B. carambolae specimens from Paramaribo (Suriname, South
America). This invasive population forms a strongly
supported, monophyletic clade to the exclusion of
the SE Asian specimens of B. carambolae in Datasets
#1.1, 35 (both BA & ML analyses). In Datasets #1.4
and 2, this clade is still recovered however several SE
Asian B. carambolae specimens were included within
it also. As Datasets #1.1, 35 either omit the nuclear
protein-coding genes altogether (#1.1, 4, 5) or
remove all ambiguous sites (#3), it is likely that the
monophyly of the B. carambolae specimens from Suriname reflects a genetic bottleneck associated with its
7

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Figure 1 Dataset #2. Phylogenetic reconstruction based on sequence data for specimens for which all six loci were sequenced for Bactrocera spp.
in the current study (236 specimens, 3435 bp alignment). Bayesian posterior probabilities are listed above each branch, maximum likelihood bootstrap values below. For clarity only supports for backbone nodes are shown; in cases where actual nodal support is absent, posterior probability support values are >0.5 except for those marked with an asterisk (>0.95). All nodes <0.5 are collapsed. Results of clade monophyly statistics are shown
as boxes (15 = a priori group analysis; ag = root-to tip analysis), with only those achieving 4/5 (orange) or 5/5 (red) shown. A priori taxonomic identifications of individual specimens within the dorsalis complex ingroup have been colour coded [i.e. B. dorsalis s.s. (purple), B. papayae (dark blue),
B. philippinensis (light blue) and B. carambolae (green)]. See supplementary files for all nodal supports and all individual specimen data.

establishment in South America and/or that the

source population for this invasion was not present in
this study.
The combined phylogeny thus supports the monophyly of the dorsalis-group and sister-group relationships between B. cacuminata and B. opiliae and
between B. dorsalis s.l. and B. carambolae. The monophyly of B. musae, B. occipitalis, B. opiliae, B. cacuminata and B. carambolae is each very strongly
supported (BA pp = 1.0 for each), whereas the
monophyly of the remaining B. dorsalis s.l. (B. dorsalis/papayae/philippinensis)
is
slightly
weaker
(pp = 0.93). Bactrocera papayae and B. philippinensis
were never monophyletic and were essentially indistinguishable from B. dorsalis s.s. in the tree. Specimens morphologically identified as B. papayae and
B. philippinensis occurred in, respectively, 817 and 2
26 different subclades of the B. dorsalis clade/grade
8

depending on the combination of data set and inference method.

Species delimitation analysis and subclade groupings

The use of species delimitation analyses within our

phylogenetic framework revealed a number of statistically well-resolved groupings for (i) each of the outgroup species, (ii) B. carambolae and (iii) B. dorsalis s.l.
(B. dorsalis/papayae/philippinensis) (Tables 35; figs 1
and 4). Each of the six clades is statistically supported
by at least four of the five species delimitation
measures; especially in the case of Dataset #5 (for
which all individuals are represented by at least two
loci) (fig. 4). Notably, B. carambolae resolves as a taxonomically distinct clade, rating 5/5 for all analyses in
both Datasets #2 and #5, while unambiguous species
(B. occipitalis, B. cacuminata and B. opiliae) achieve 4/5
2013 Blackwell Verlag, GmbH

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Table 4 The hypothesized species of the Bactrocera dorsalis species complex were tested for species distinctiveness as measured by the Geneious
species delimitation plugin (Masters et al. 2010) and the genealogical sorting index (gsi) (Cummings et al.2008). The species delimitation plugin generates: average pairwise tree distance between members of the group of interest and its sister taxa (K2Pdistance), P (Randomly Distinct), Clade Support:
Bayesian posterior probability (PP), and Rosenbergs PAB: Reciprocal monophyly and lastly, the gsi statistic and associated P-value are included. Bold
Values indicates significance, and this was determined by: >1% difference (K2P)/>0.05 [P (Randomly Distinct)]/>0.80 (PP)/>0.008 (gsi). Dataset 2 contained a concatenation of all specimens for which all six loci were successfully sequenced 235 specimens, 3435 bp alignment. Dataset 5 consisted
of specimens for which at least two of the four loci (i.e. excluding CAD and period) were successfully sequenced (313 specimens, 2221 bp)
Inter dist - Closest (K2P)

P (randomly distinct)

Clade support

Rosenbergs P (AB)

gsi

P-value

Dataset 2
Clade 1: musae Bd51/67
Clade 2: occipitalis 739/800
Clade 3: cacuminata 231/244
Clade 4: opiliae 1080/1082
Clade 5: carambolae 1111/189
Clade 6: dorsalis 818/399

2.263
1.653
0.858
0.858
1.575
1.368

0.05
0.77
0.05
0.05
0.05
0.9

1
1
1
1
1
85

1.40E-11
1.40E-11
1.40E-11
1.40E-11
3.00E-42
3.00E-42

1
0.952
1
1
1
1

1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04

Dataset 5
Clade 1: musae Bd51/67
Clade 2: occipitalis 739/800
Clade 3: cacuminata 231/244
Clade 4: opiliae 1080/1082
Clade 5: carambolae 1111/189
Clade 6: dorsalis 818/399

2.914
2.531
1.117
1.117
1.542
1.542

0.05
0.05
0.16
0.05
0.05
0.39

1
87
100
100
100
93

1.50E-12
3.95E-03
1.50E-12
1.50E-12
1.50E-12
1.50E-12

0.886
0.944
1
1
1
1

1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04

in at least one of the datasets; a result which we

believe lends greater support to the ongoing specific
status of B. carambolae.
Species delimitation statistical analyses undertaken
for Dataset #2 revealed considerable support for
each of the six a priori defined groups: B. musae (5/5
statistically significant), B. occipitalis (4/5), B. cacuminata (4/5), B. opiliae (4/5), B. carambolae (5/5) and
B. dorsalis s.l. (i.e. B. dorsalis/papayae/philippinensis) (4/
5) (Table 4). Tip-to-root analysis (examining all
resolved clades) demonstrated a limited number of
subclades which were statistically significant for at
least four of the five statistics applied, with three subclades resolved within B. carambolae and four in the
B. dorsalis s.l. clade (Table 5; fig. 1).
A priori groups and subclade support increased following analysis of Dataset #5, with all five statistical
analyses significant for four of the a priori defined
clades (B. musae, B. occipitalis, B. opiliae and B. carambolae) and 4/5 for the remaining two (B. cacuminata
and B. dorsalis s.l.). Meanwhile, tip-to-root analysis
revealed nine subclades to have 4/5 support measures
statistically significant, with three occurring in the
B. carambolae clade (one of which consisted exclusively of all Suriname individuals), five occurring in
the B. dorsalis s.l. clade (including one subclade which
consisted exclusively of B. philippinensis individuals),
and one in B. musae (Table 6; fig. 4).

2013 Blackwell Verlag, GmbH

Discussion
This study represents the most comprehensive
phylogenetic analysis undertaken to-date for four
pestiferous and morphologically cryptic members of
the B. dorsalis species complex. The study incorporates individuals collected from a broad geographic
distribution and likely represents a range of intraspecific populations for these species. Six independent loci have been targeted and subsequently
examined using a range of analyses, with a clear
signal emerging: B. carambolae is a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae
and B. philippinensis form a single sister clade to
B. carambolae.
Phylogenetic analyses and species delimitation

The individual gene trees in this study were unresolved and therefore prevented the use of the speciestree software (e.g.,Ane et al. 2007; Liu 2008; Liu et al.
2009; Kubatko 2009; Kubatko et al. 2009; Heled and
Drummond 2010; Than and Nakhleh 2009; Than
et al. 2008; Huang et al. 2010; Knowles and Kubatko
2010). We recognize the caveats of using concatenated DNA sequence data to generate a species-tree
hypothesis (Degnan and Salter 2005; Kubatko and
Degnan 2007; Kubatko et al. 2011); however, as

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Table 5 Tip to root approach (Boykin et al. 2012) for species delimitation of the Bactrocera dorsalis species complex utilizing Dataset 2 (Concatenation of all specimens for which all six loci were successfully sequenced 235 specimens, 3435 bp alignment). See Table 3 for full description of the
species delimitation statistics and fig. 1 for a visual representation of these results

Subclades

Inter dist
Closest (K2P)

P (Randomly
distinct)

Clade Support

Rosenbergs
P (AB)

gsi

P-value

Clade 1: musae Bd51/67

Bd61-62
Bd57-71
Bd54-70

0.13
0.156
0.13

0.05
0.09
0.05

71
88
77

5.50E-04
9.20E-05
1.30E-06

1
1
1

0.00139986
0.00019998
1.00E-04

Clade 2: occipitalis 739/800

Bd791-795
Bd784-796
Bd788-786

0.43
0.43
0.581

0.75
0.05
0.07

60
86
67

3.00E-05
2.30E-04
1.30E-05

1
1
1

1.00E-04
1.00E-04
1.00E-04

Clade 3: cacuminata 231/244

Bd1088-1099
Bd1086-1095
Bd1083-1085

0.197
0.185
0.185

0.05
0.05
0.05

59
100
100

3.10E-04
1.36E-03
1.36E-03

1
1
1

1.00E-04
0.00089991
0.00209979

Clade 4: opiliae 1080/1082

No additional subclades to test

Clade 5: carambolae 1111/189

Bd204-1242
Bd201-225
Bd191-226
Bd189-227
Bd1237-1236
Bd1224-1232
Bd419-415
Bd1119-1126

0.357
0.377
0.357
0.396
0.58
0.58
0.889
0.889

0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

51
63
100
100
50
100
61
98

1.80E-07
1.80E-07
5.10E-05
5.10E-05
0.01
0.01
6.30E-07
3.00E-07

1
0.8533
1
1
0.664
1
0.664
0.664

1.00E-04
1.00E-04
1.00E-04
0.00119988
0.00019998
1.00E-04
0.00019998
0.00019998

Clade 6: dorsalis 818/399

Bd1122-1197
Bd1127-1129
Bd1114-1117
Bd399-589
Bd740-758
Bd819-1181
Bd1176-1164
Bd403-1123
Bd781-1200
Bd1175-774
Bd580-1215
Bd1136-1145
Bd1209-1203
Bd400-817
Bd1194-1205
Bd821-829
Bd816-1148
Bd775-780
Bd769-773
Bd757-759
Bd751-768
Bd744-752

0.421
0.263
0.263
0.306
0.23
0.397
0.372
0.403
0.298
0.422
0.245
0.389
0.384
0.297
0.291
0.384
0.367
0.198
0.247
0.14
0.14
0.159

0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
1
0.05
0.61
1
0.05
1
0.05
0.05
1
0.61
0.05
0.05
0.05
0.46

94
99
97
100
51
85
77
99
60
80
84
89
71
71
100
69
100
96
82
85
62
57

6.00E-04
3.64E-03
3.64E-03
1.69E-03
5.80E-15
0.01
0.01
7.80E-13
1.10E-11
3.80E-09
3.80E-09
9.80E-08
9.80E-08
9.80E-08
3.40E-06
3.40E-06
3.40E-06
3.40E-06
3.40E-06
3.40E-06
3.40E-06
3.40E-06

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

1.00E-04
0.00069993
0.00179982
0.00149985
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
0.00029997
0.00139986
0.00089991
0.00159984
0.00149985
0.00169983
0.00079992
0.00179982
1.00E-04

there was no conflict among individual gene tree phylogenies, the benefit of using a single multilocus phylogeny to confidently delimit species was considered
appropriate (Rokas et al. 2003; Belfiore et al. 2008;
Sanderson et al. 2011).
Species delimitation statistics reveal additional substructure is present within several clades, particularly
10

for B. carambolae and B. dorsalis s.l. With respect to

Dataset #5 (fig. 4), subclade b within the B. carambolae clade consists exclusively of every individual collected from Suriname, located in northern South
America and constituting part of the invasive range of
B. carambolae. Bactrocera carambolae was first recorded
in South America in 1975 (undescribed at that stage),
2013 Blackwell Verlag, GmbH

L. M. Boykin et al.

Phylogeny of B. dorsalis pest flies

Figure 2 Dataset #3. Phylogenetic reconstruction based on sequence data for specimens for which all six loci (cox1, nad4-3, ITS1, ITS2, CAD and
per) were sequenced for Bactrocera spp. in the current study. Ambiguous sites removed from CAD and per alignments (236 specimens, 3094 bp).
Node supports and tree annotation as per fig. 1.

where it was first reared from Syzygium samarangense

(Java apple) in Suriname and thought to have been
accidentally introduced from south-east Asia (van
Sauers-Muller 1991). The emergence of a well-supported Suriname subclade within the more diverse
south-east Asian B. carambolae clade is not unexpected given such a recent introduction for which a
genetic bottleneck is likely to exist. Similarly, subgroup d in the B. dorsalis s.l. clade consists of all
B. philippinensis individuals collected from one of two
geographically proximate locations in the Philippines
(Quezon City and Imus) (fig. 4). Philippine flies may
be expected to be genetically divergent from other
members of the B. dorsalis s.l. clade considering the
increased geographic separation between Philippine
flies relative to those from among mainland southeast Asia and western Indonesian archipelago sites
(however, human-mediated movement may limit
this). Indeed, significant isolation-by-distance effects
2013 Blackwell Verlag, GmbH

for flies from the Philippines vs. flies from mainland

south-east Asia have been demonstrated (Schutze
et al. 2012a). Contrary to the Suriname B. carambolae
sub-clade, not all individuals from the Philippines
occur within this group, as six individuals fall outside
subclade d (all from Imus; fig. 4) and are unresolved
from other B. dorsalis s.s. and B. papayae; emphasizing
the low resolution within the B. dorsalis s.l. clade as a
whole.
Four of five measures were used to identify four
sub-groupings within the B. dorsalis s.l. clade in
Dataset #2 (fig. 1; Clade 6): d, e, f and g. For
example, clade e consists of four individuals from
each of the three species in the larger B. dorsalis s.l.
clade, these being: B. papayae from Penang (Malaysia); B. philippinensis (two individuals from Imus, Philippines); and B. dorsalis s.s. from San Pa Tong
(northern Thailand). In this case, conspecific representatives for each of these species are also repre11

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Figure 3 Dataset #4. Phylogenetic reconstruction based on sequence data for specimens for which four loci were sequenced (cox1, nad4-3, ITS1
and ITS2) for Bactrocera spp. in the current study (236 specimens, 2221 bp). Node supports and tree annotation as per fig. 1.

sented throughout the remainder of the B. dorsalis s.l.

clade. The clades identified using the tip-to-root
method provide a basis for further biological research.
In the difficult Bemisia tabaci species complex, for
example, the discovery of previously unrecognized
clades through similar analytical approaches has proven a basis for deeper taxonomic and biological
research, which is helping to elucidate this equally
difficult group (Boykin et al. 2012).
Relationships of outgroup species

Bactrocera musae and three members of the B. dorsalis

complex: B. occipitalis, B. opiliae and B. cacuminata
resolve as taxonomically distinct groups and sister to
the ingroup taxa according to all analyses (figs 14).
Bactrocera musae, while taxonomically a member of a
different species complex (the B. musae complex), has
historically demonstrated a very close relationship to
dorsalis complex flies. An earlier phylogenetic analysis

12

of COI and COII genes of Bactrocera species revealed B.

musae to occur within the dorsalis complex clade: sister
to B. occipitalis, B. philippinensis, B. dorsalis s.s.,
B. papayae and B. carambolae, with B. kandiensis Drew
& Hancock (a true dorsalis complex fly) sister to all of
these species (Nakahara and Muraji 2008; Krosch
et al. 2012a). Furthermore, restriction enzyme analysis of 25 species of Bactrocera revealed B. musae to
exhibit the least degree of differentiation between it
and B. dorsalis s.s., B. papayae and B. philippinensis
(and a non-dorsalis fly, B. curvipennis (Froggatt)) as
compared to all other species (B. dorsalis s.s., B. papayae and B. philippinensis were indistinguishable) (Armstrong and Cameron 2000). Indeed it appears the
main distinguishing morphological character separating B. musae from B. dorsalis s.l. is the occasional
absence of the medial longitudinal band on the abdomen for some individuals (Drew 1989); the presence
of which is typical of dorsalis complex species (Drew
and Hancock 1994). We therefore recommend further
2013 Blackwell Verlag, GmbH

L. M. Boykin et al.

Phylogeny of B. dorsalis pest flies

Figure 4 Dataset #5. Phylogenetic reconstruction based on sequence data for specimens for which at least two of four loci (cox1, nad4-3, ITS1 and
ITS2) were sequenced for Bactrocera spp. in the current study (315 specimens, 2221 bp). Node supports and tree annotation as per fig. 1.

work on B. musae be undertaken towards fully resolving its association with the B. dorsalis complex.
Our results show B. occipitalis (a species occurring in
sympatry with B. philippinensis in the Philippines) is more
distantly related to the ingroup taxa relative to the Australian species B. opiliae and B. cacuminata (figs 14).
While B. occipitalis has been regarded a closely related
species of B. dorsalis (Muraji and Nakahara 2002; Nakahara and Muraji 2008; Krosch et al. 2012a), it is morphologically distinct in having significantly shorter genitalia
with colour markings distinct as from B. philippinensis
(Drew and Hancock 1994; Iwahashi 1999). Bactrocera cacuminata and B. opiliae have rarely been directly compared
with pest species of the dorsalis complex as they are
innocuous and exist in allopatry with respect to the all
known pests from the complex; however, B. opiliae is at
least very similar to B. dorsalis s.s., having been described
in 1981 from northern Australian samples and initially
regarded as Dacus (Bactrocera) dorsalis due to high morphological similarity with this species (Drew and Hardy
2013 Blackwell Verlag, GmbH

1981). Bactrocera opiliae and B. dorsalis s.s. were only separable using ecological, physiological and genetic measures, for which colour variation was the only visual
difference subsequently observed between the two, with
fine-scale differences in ovipositor and egg morphology
also diagnostic (Drew and Hardy 1981). In contrast,
B. cacuminata is morphologically distinct, possessing a
characteristic black lanceolate pattern on the mesonotum
and thereby rendering it easily identifiable from pest
members of the dorsalis complex (Drew 1989). However,
as species-level diagnoses are often required for juvenile
stages (hence adult characters are absent), the genetic
resolution of these non-pest Australian species obtained
here is of practical use for quarantine and plant protection officers.
The unusual case of specimen #413

We cannot explain the unusual placement of specimen #413 in any of our phylogenetic reconstruc13

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Table 6 Tip to root approach (Boykin et al. 2012) for species delimitation of the Bactrocera dorsalis species complex utilizing Dataset 5 consisted of
specimens for which at least two of the four loci (i.e. excluding CAD and period) were successfully sequenced (313 specimens, 2221 bp). See Table 3
for full description of the species delimitation statistics and fig. 4 for a visual representation of these results

Subclades

Inter Dist
closest (K2P)

P (Randomly Distinct)

Clade
support

Rosenbergs P (AB)

gsi

P-value

Clade 1: musae Bd51/67

Bd61-62
Bd56-71
Bd54-70

0.234
0.309
0.234

0.05
0.42
0.05

59
51
85

5.50E-04
2.10E-05
1.30E-06

1
1
1

0.00059994
1.00E-04
1.00E-04

Clade 2: occipitalis 739/800

Bd783&786
Bd794&799

0.331
0.331

0.05
0.05

61
91

0.05
0.05

1
1

0.00089991
0.00059994

Clade 3: cacuminata 231/244

No additional subclades to test

Clade 4: opiliae 1080/1082

Bd1081&88
Bd1083&85
Bd1086&95
Bd1089&90

0.279
0.258
0.345
0.258

0.05
0.05
0.05
0.05

88
90
100
86

6.40E-04
6.40E-04
6.40E-04
6.40E-04

1
1
1
1

0.00069993
0.00119988
0.00029997
0.00069993

Clade 5: carambolae 1111/189

Bd405&1241
Bd1255-1262
Bd1238&58
Bd1234-1121
Bd419-1263
Bd1225-1239
Bd191-216

0.364
0.345
0.256
0.55
0.256
0.543
0.343

0.05
0.05
0.05
0.05
0.05
0.05
0.05

68
100
77
73
70
99
100

1.90E-05
1.90E-05
6.90E-08
6.20E-09
6.70E-10
1.90E-15
8.00E-18

1
1
1
1
1
1
1

0.00069993
1.00E-04
0.00069993
1.00E-04
1.00E-04
1.00E-04
1.00E-04

Clade 6: dorsalis 818/399

Bd818&1168
Bd1195-1200
Bd418&827
Bd579&1179
Bd580&1164
Bd583&1142
Bd772&775
Bd816&1148
Bd823&1181
Bd1143&1145
Bd1206&1210
Bd1211&1253
Bd1244&1250
Bd1202-1215
Bd1246-1249
Bd825-1209
Bd1194-1205
Bd585-1183
Bd744-781
Bd593-1123

0.596
0.344
0.345
0.303
0.284
0.292
0.254
0.333
0.377
0.4
0.224
0.404
0.231
0.269
0.224
0.401
0.263
0.426
0.361
0.37

0.05
0.06
0.05
1
0.09
0.71
0.13
0.05
0.05
0.05
0.05
0.05
0.41
1
0.16
1
1
0.94
0.05
1

92
100
87
64
50
57
82
100
100
100
72
55
64
92
54
63
99
80
100
55

9.50E-07
1.90E-08
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
1.00E-06
2.00E-08
2.00E-08
5.40E-10
5.40E-10
1.70E-11
1.10E-30
6.80E-36

1
0.498
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.953
1

0.00129987
0.0009999
0.0009999
0.00139986
0.00109989
0.00039996
0.00079992
0.00039996
0.00079992
0.00049995
0.00069993
0.00079992
0.00069993
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04
1.00E-04

tions (figs 14). For Datasets #2, this specimen

emerges as sister to the entire B. dorsalis s.l. clade,
and in Datasets #3, #4 and #5, it is sister to B. occipitalis. Specimen #413 was collected from Nakhon
Si Thammarat (southern Thailand) and hence
occurred where B. dorsalis s.s. and B. papayae geographically abut or overlap according to recorded
geographic distributions for these species (e.g.
Drew and Hancock 1994). Morphologically, #413
14

identifies as either B. dorsalis s.s. or B. papayae

based on existing keys, and examination by Prof.
R.A.I. Drew confirmed it as one of these two species and likely to be B. papayae (pers. comm.).
However, our study included only four economically important and three additional out-group species from the B. dorsalis complex, and the inclusion
of more members from the complex may help to
resolve the placement of specimen #413.
2013 Blackwell Verlag, GmbH

Phylogeny of B. dorsalis pest flies

L. M. Boykin et al.

Implications and future studies for B. dorsalis taxonomy

A number of previous studies have failed to find resolution between B. dorsalis s.s, B. papayae and B. philippinensis based on molecular (cox1 and microsatellites)
morphological (wing shape and aedeagus length) or
behavioural (mating and chemical ecology) data
(Medina et al. 1998; Tan 2000, 2003; Wee and Tan
2000a,b, 2005; Krosch et al. 2012b; Schutze et al.
2012a). The results of the current study do not contradict this, and in addition, the design here overcomes
the potential weaknesses of earlier studies by sampling much larger numbers of individuals across a
wider geographic range. However, while this body of
evidence fails to reject the hypothesis, that these three
species are in fact one, it also fails to distinguish
between this as a result of inappropriate diagnostics or
incorrect taxonomy (Drew et al. 2008; Schutze et al.
2012b). While the former was tested here by use of
loci that could clearly distinguish other well-recognized and closely related biological species within the
dorsalis complex, that is, B. cacuminata, B. opiliae and
B. carambolae, as well as B. musae for which a number
of previous studies have found problematic (White
1996; Muraji and Nakahara 2002), there are still some
methodological issues. Given concerted evolution of
the rDNA loci, one might expect these three taxa to
share a common ITS sequence, but this was not the
case and much of the phylogenetic information in the
CAD and period loci was obscured by the inability to
produce true sequence from the many combinations
of heterozygous alleles. The main source of distinction, or lack of for B. dorsalis s.s, B. papayae and B. philippinensis, came from two linked mitochondrial loci.
However, mitochondrial DNA is characterized by
complex evolutionary dynamics. For example, selective sweeps that help to differentiate taxa can in the
case of recently diverged taxa be offset by the homogenizing effect of hybrid introgression (Galtier et al.
2009). Certainly, this has been found in wild populations of very closely related dipteran species (e.g.
Bachtrog et al. 2006), such that any correlation with
other taxonomic distinctions are lost. Of course there
may be other nuclear genes that might support the
current taxonomy, and this may become more feasible to test as genomic data continues to accumulate.
Nonetheless, we stress that this work should be examined in the broader context of integrative taxonomy,
where final taxonomic conclusions are not based on
one line of evidence but on several integrated lines of
independent evidence (Dayrat 2005; Schlick-Steiner
et al. 2010). In this context, there is a growing body
of international, multidisciplinary literature (Fletcher
2013 Blackwell Verlag, GmbH

and Kitching 1995; Yong 1995; Iwahashi 2000, 2001;

Muraji and Nakahara 2002; Smith et al. 2003; Tan
2003; Armstrong and Ball 2005; Tan et al. 2011;
Krosch et al. 2012b; Schutze et al. 2012a) that can all
be considered to date as supporting, or at least not
refuting, the possibility that these cryptic species,
namely B. dorsalis s.s., B. papayae and B. philippinensis
are the same biological species. However, given the
risk that severe quarantine and trade implications
could result from changes to the taxonomic delimitation of species relevant to global biosecurity (Boykin
et al. 2012), it is critical that there is a high level of
scientific support for a revision such as that implicated
here for pest species in the B. dorsalis complex.
Acknowledgements
We wish to sincerely thank the following colleagues
who assisted us with supplying specimens for this
study: Mary Finlay-Doney, Richard Bull, Yuvarin
Boontop, Keng-Hong Tan, Sotero Resilva, Ju-Chun
Hsu, Alies van Sauers-Muller, Vijay Shanmugam,
Hanifah Yahaya, Wigunda Rattanapun and Peter
Leach. Vladimir Mencl, Markus Binsteiner and Yuriy
Halytskyy at the New Zealand eScience Infrastructure
(NeSi- http://www.nesi.org.nz) were instrumental in
the HPC analyses. LMB and KFA were funded by the
Tertiary Education Council of New Zealand. The paper
was produced with research support through CRC
National Plant Biosecurity projects 20115 and 20183.
The authors would like to acknowledge the support of
the Insect Pest Control Laboratory (Seibersdorf) of the
Joint FAO/IAEA Division of Nuclear Techniques in
Food and Agriculture and the Australian Governments Cooperative Research Centres Program.
Acknowledgement also goes to an anonymous
reviewer who helped to significantly improve the
manuscript.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Dataset #1.1. Bayesian phylogenetic
reconstruction based on sequence data for specimens
for which mtDNA (cox1and nad4-3) were sequenced
for Bactrocera spp. in the current study.

2013 Blackwell Verlag, GmbH

Phylogeny of B. dorsalis pest flies

Figure S2. Dataset 1.2. Phylogenetic reconstruction

based on sequence data for specimens for which ribosomal DNA (ITS1 and ITS2) were sequenced for Bactrocera spp. in the current study.
Figure S3. Dataset 1.3. Phylogenetic reconstruction
based on sequence data for specimens for which
nuclear DNA (CAD gene) was sequenced for Bactrocera
spp. in the current study.
Figure S4. Dataset 1.4. Phylogenetic reconstruction
based on sequence data for specimens for which
nuclear DNA (period gene) was sequenced for Bactrocera spp. in the current study.
Figure S5. Dataset #2. Phylogenetic reconstruction
based on sequence data for specimens for which all
six loci were sequenced for Bactrocera spp. in the current study (236 specimens, 3435 bp alignment).
Figure S6. Dataset #3. Phylogenetic reconstruction
based on sequence data for specimens for which all
six loci (cox1, nad4-3, ITS1, ITS2, CAD and per) were
sequenced for Bactrocera spp. in the current study.
Figure S7. Dataset #4. Phylogenetic reconstruction
based on sequence data for specimens for which four
loci were sequenced (cox1, nad4-3, ITS1 and ITS2) for
Bactrocera spp. in the current study (236 specimens,
2221 bp).
Figure S8. Dataset #5. Phylogenetic reconstruction
based on sequence data for specimens for which at
least two of four loci (cox1, nad4-3, ITS1 and ITS2)
were sequenced for Bactrocera spp. in the current
study (315 specimens, 2221 bp).
Table S1. Collection and GenBank accession information for the samples included in this study.

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