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Multi-genephylogeneticanalysisofsouth-eastAsianpestmembersoftheBactroceradorsalisspeciescomplex(Diptera:Tephritidae)doesnotsupportcurrenttaxonomy

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ORIGINAL CONTRIBUTION

Multi-gene phylogenetic analysis of south-east Asian pestmembers of the Bactrocera dorsalis species complex (Diptera:Tephritidae) does not support current taxonomyL. M. Boykin1,2, M. K. Schutze1,3, M. N. Krosch1,3, A. Chomi�c1,2, T. A. Chapman1,4, A. Englezou1,4,K. F. Armstrong1,2, A. R. Clarke1,3, D. Hailstones1,4 & S. L. Cameron1,3

1 CRC for National Plant Biosecurity, Bruce, ACT, Australia

2 Bio-Protection Research Centre, Lincoln University, Lincoln, Christchurch, New Zealand

3 School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, Qld, Australia

4 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. carambo-

lae are serious pest fruit fly species of the B. dorsalis complex that predomi-

nantly 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 clo-

sely 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 mono-

phyly was measured by clade support, posterior probability or bootstrap

resampling for Bayesian and likelihood analyses respectively, Rosenberg’s

reciprocal monophyly measure, P(AB), Rodrigo’s (P(RD)) and the genea-

logical sorting index, gsi. We specifically tested whether there was phylo-

genetic 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 monophy-

letic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are

unresolved. These data add to the growing body of evidence that B. dor-

salis 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 tephrit-

ids contain many genera of major economic impor-

tance, including Ceratitis, Rhagoletis and Anastrepha

(White and Elson-Harris 1992). Mature female

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 reg-

ulatory 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).

© 2013 Blackwell Verlag, GmbH 1

J. Appl. Entomol.

Within this genus, the Bactrocera dorsalis species com-

plex 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 mil-

lion 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 spe-

cies 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 char-

acter 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 molec-

ular 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 mark-

ers, 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 repre-

sent good biological species for which species-specific

diagnostic markers exist but which are yet to be iden-

tified 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 popula-

tion level differences (Harrison 1998; Sites and

Marshall 2004).

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 cod-

ing 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. kandi-

ensis 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 sepa-

rated (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 gener-

ally drawn from only one geographic population (e.g.

Nakahara et al. 2001). As a result, it remains impossi-

ble 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. philippinen-

sis 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, GmbH2

Phylogeny of B. dorsalis pest flies L. M. Boykin et al.

B. occipitalis (Bezzi)] and outside the complex [B. mu-

sae (Tryon), B. tryoni (Froggatt)]. We sequenced six

loci (cox1, nad4-3′, CAD, period, ITS1, ITS2) for approx-imately 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 delimita-

tion tests. Using this data set of multiple individuals

sampled from a number of populations, we specifi-

cally 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 meth-

ods to resolve species limits among the following four

target species of the B. dorsalis species complex: B. dor-

salis 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 spe-

cies’. We also selected a number of species to repre-

sent ‘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 sympat-

rically 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 pre-

vious 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

known distributions. As discrimination amongst in-

group 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 sympatri-

cally (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 con-

sisted 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 south-

east Asia who placed the traps in the field, or deployed

during collection trips undertaken by MKS in Decem-

ber 2010.

Exceptions to above collection methods are as fol-

lows. 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 Queens-

land Government Department of Agriculture, Fisher-

ies 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% etha-

nol. Bactrocera carambolae from Paramaribo (Suri-

name) were reared from A. carambola fruit placed in

the field.

All samples were returned to the Queensland Uni-

versity of Technology (QUT), Brisbane (Australia), for

transfer into absolute ethanol, preliminary morpho-

logical identification and preparation for DNA extrac-

tion. 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 Mac-

Arthur 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 abso-

lute ethanol at QUT.

© 2013 Blackwell Verlag, GmbH 3

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies

DNA extraction, PCR and sequencing

Tubes containing fly legs were pulse spun, the ethanol

removed and air-dried. Samples were transferred

to �80°C 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 manufacturer’s

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 amplifica-

tion 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

from Porter and Collins (1991). CAD primers are rede-

signed after Moulton and Wiegmann (2004, 2007)

after comparison with GenBank tephritid sequences.

Primers for period are from Barr et al. (2005) and Vir-

gilio et al. (2009). Primer sequences for all loci are

given in Table 2.

The PCR conditions for ITS1, ITS2 and ND4-2 con-

sisted 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 94°C for 2 min, followed

by 35 cycles of denaturation at 94°C for 15 s, anneal-

ing at 60°C, 55°C and 60.5°C for ITS1, ITS2 and ND4-

2 respectively, followed by an extension time of

1 min at 68°C and final extension of 5 min at 68°C.All PCR products were visualized on 1.5% agarose

gels run at 90V for 45 min and post-stained with ethi-

dium bromide. All PCR products were sent to AGRF

(Australian Genome Research Facility Ltd) in 96-well

plates for purification and sequencing. AGRF is

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

Location Country Latitude Longitude Date Species

Collection

method

Brisbane, Queensland Australia 27°27′29″S 152°58′56″E 10 July 2009 Bactrocera tryoni Cue-lure

September–

November 2009

Bactrocera cacuminata ME Lure

Cairns, Queensland Australia DEEDI culture DEEDI culture 5 June 2009 Bactrocera musae Culture

Noonamah, Northern

Territory

Australia 12°38′33″S 131°5′58″E 24 December 2009 Bactrocera opiliae ME Lure

Quezon City, Diliman Philippines 14°38′00″N 121°01′00″E 17 December 2009 Bactrocera occipitalis ME Lure

17 December 2009 Bactrocera philippinensis ME Lure

Imus, Cavite Philippines 14°07′18″N 120°58′00″E 20 December 2009 Bactrocera philippinensis ME Lure

Taipei City, Tawian China 25°00′53″N 121°32′18″E 16 March 2010 Bactrocera dorsalis s.s. ME Lure

San Pa Tong, Chiang Mai Thailand 18°37′37″N 98°53′42″E 12 March 2010 Bactrocera dorsalis s.s. ME Lure

Chatuchuk, Bangkok Thailand 13°50′32″N 100°34′23″E 14–21 December

2009

Bactrocera dorsalis s.s. ME Lure

Muang District, Nakhon

Si Thammarat

Thailand 8°25′12″N 99°53′48″E 25 October–15

November 2009

Bactrocera papayae ME Lure

25 October–15

November 2009

Bactrocera carambolae ME Lure

Tikus Pulau, Penang Malaysia 5°25′50″N 100°18′38″E 17–26 November

2009

Bactrocera papayae ME Lure

Serdang Malaysia 3°00′20″N 101°42′00″E November 2010 Bactrocera papayae ex Musa

acuminata

x balbisiana

November 2010 Bactrocera carambolae ex Averrhoa

carambola

Lampung, South Sumatra Indonesia 5°40′43″S 105°36′38″E 15–17 May 2009 Bactrocera papayae ME Lure

15–17 May 2009 Bactrocera carambolae ME Lure

Paramaribo Suriname 5°49′20″N 55°10′05″W August 2009 Bactrocera carambolae ex Averrhoa

carambola

© 2013 Blackwell Verlag, GmbH4

Phylogeny of B. dorsalis pest flies L. M. Boykin et al.

accredited by NATA to the ISO/IEC17025:2005 Qual-

ity 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 steril-ized water. PCR cycling conditions consisted of an ini-

tial denaturation step at 94°C for 2 min, followed by

40 cycles of denaturation at 95°C for 30 s, annealing

at 50°C (for cox1 and period) or 54°C (for CAD) for

30 s. and extension at 72°C for 1 min.; there was a

final run-out extension step at 72°C for 7 min. All

PCR products were visualized on 1% agarose gels con-

taining 10X dilution of SYBER Safe (Life Technolo-

gies, 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 com-

pleted 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 spec-

imens:

Datasets #1.1–1.4 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

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 speci-

mens, 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 success-

fully sequenced (313 specimens, 2221 bp)

Dataset #1 was designed to allow testing of the varia-

tion between loci and to apply a species-tree recons-

truction approach (Edwards 2008); however, due to

the poor resolution in Datasets #1.2–1.4, the additi-

onal, 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 par-

titions 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 (pro-

tein-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. Hetero-

Table 2 Primer sequences used in the current study

Gene Name Direction Sequence Reference

cox1 LCO1490 F GGT CAA CAA ATC ATA AAG ATA TTG G Folmer et al. (1994)

HCO2198 R TAA ACT TCA GGG TGA CCA AAA AAT CA Folmer et al. (1994)

nad4-3′ Teph_ND4F1 F TAG AGT WTG TGA AGG TGC TTT RGG Herein

Teph_ND4R1 R AGC WAC WGA WGA ATA AGC AAT TAA WGC C Herein

ITS1 ITS7 F GAA TTT CGC ATA CAT TGT AT Herein

ITS6 R AGC CGA GTG ATC CAC CGC T

ITS2 FFA F TGT GAA CTG CAGG ACA CAT

Shortened FFB R TCG CTA TTT TAA AGA AAC AT Herein

CAD CAD-Bd-F F CCG GTA AAT TTT GAA TGG TTC Moulton and Wiegmann (2004, 2007)

CAD-Bd-R R GCR GTK GCG AGC ARY TGA TG Moulton and Wiegmann (2004, 2007)

period F2508 F CAA CGA CGA AAT GGA GAA ATT C Barr et al. (2005)

R3270 R AGG TGT GAT CGA GTG GAA GG Virgilio et al. (2009)

© 2013 Blackwell Verlag, GmbH 5

L. M. Boykin et al. 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 analy-

ses were conducted using RAxML ver 7.2.8 imple-

mented on the RAxML BlackBox webserver (http://

phylobench.vital-it.ch/raxml-bb/index.php) (Stamat-

akis et al. 2008). Data were analysed with a Gamma

model of rate heterogeneity, the proportion of invari-

able sites was estimated, and for concatenated, mul-

tilocus data sets, the alignment was partitioned and

branch lengths optimized on a per locus basis. Bayes-

ian 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 desk-

top 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. Conver-

gence between runs was monitored by finding a pla-

teau in the likelihood score (standard deviation of

split frequencies <0.0015) and the potential scale

reduction factor (PSRF) approaching one. Conver-

gence of other parameters within the runs was also

checked using TRACER v1.5.4 (Rambaut and Drum-

mond 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 pos-

terior 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 Rodrigo’s P (ran-

domly distinct) (Rodrigo et al. 2008) measure. Spe-

cies monophyly was measured by clade support,

posterior probability or bootstrap resampling for

Bayesian and likelihood analyses respectively,

Rosenberg’s reciprocal monophyly measure, P (AB)

(Rosenberg 2007) and the genealogical sorting index

(gsi) (Cummings et al. 2008). The species delimita-

tion plugin (Masters et al. 2010) for Geneious

(Drummond et al. 2010) was used to calculate

Rosenberg’s reciprocal monophyly, P (AB) (Rosen-

berg 2007) and Rodrigo’s P (RD) (Rodrigo et al.

2008) measures. The (Cummings et al. 2008) statis-

tic 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 per-

iod) were successfully amplified for the majority of

specimens examined across all species. Success/fail-

ure 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) tri-

alled in this study that was successfully amplified

across the range of species here. Due to the low lev-

els 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 ge-

nomes (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′ generegion was confirmed here to be more variable, hav-

ing 104 of 577 positions parsimony informative

compared to 101 of 642 parsimony informative for

cox1.

The ribosomal ITS loci each had significant indels

(33–84 bp in ITS1, 31–40 bp in ITS2), but there were

few heterozygous sites, consistent with the concerted

evolution previously found for these loci (e.g.

© 2013 Blackwell Verlag, GmbH6

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 infor-

mative. Intraspecific variation for each gene is shown

in Table 3.

Phylogenetic analyses

For each data set, Bayesian (BA) and likelihood analy-

ses (ML) yielded similar topologies; however, nodal

support was much greater for the set of Bayesian anal-

yses. Of the single linkage group analyses (Datasets

#1.1–1.4), only the mitochondrial gene trees (Dataset

#1.1) were well resolved, with each species other than

B. dorsalis, B. philippinensis, B. papyae and B. carambo-

lae monophyletic with significant nodal supported

(BA pp = 0.9–1.0; 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 Bayes-

ian analysis of Dataset #1.2, B. cacuminata specimens

formed 17 of 19 branches in a polytomy with a mono-

phyletic 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 S3–4). For CAD

(Dataset #1.3), only B. musae (BA & ML) and B. cac-

uminata (ML only) were monophyletic whereas for

period (Dataset #1.4), B. musae (BA & ML), B. cacumi-

nata (BA only) and B. opiliae (BA only) were mono-

phyletic. 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 bet-

ter resolved and display higher nodal support than

was achieved analysing each linkage group separately

(Datasets #1.1–1.4). Further, due to the high propor-

tion 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 relation-

ships. The same species boundaries were inferred for

all four concatenated data sets, and the interspecies

relationships were also quite constant. The heterozy-

gous 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 dorsalis-

group 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 S5–7). Similarly, the inclusion of speci-

mens 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 pres-

ent (#3–#4).Below the species level, there was significant vari-

ability in topology and nodal support across the dif-

ferent concatenated data sets with few clades larger

than 2–3 specimens shared between analyses. The

only notable exception is the clade containing B. cara-

mbolae 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, 3–5 (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, 3–5 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 Suri-

name reflects a genetic bottleneck associated with its

Table 3 The average intraspecific distances for each gene shown in %

calculated using MEGA

Species ITS1 ITS2 ND4 CO1 per CAD

Bactrocera tryoni 0.000 0.000 1.284 0.809 0.275 0.210

Bactrocera musae 0.000 0.000 0.127 0.031 0.033 0.038

Bactrocera

cacuminata

0.000 0.000 0.101 0.051 0.047 0.000

Bactrocera occipitalis 0.000 0.000 0.972 0.329 0.244 0.682

Bactrocera opiliae 0.000 0.000 0.604 0.603 0.291 0.101

Bactrocera

carambolae

0.158 0.093 0.924 0.611 0.597 2.294

Bactrocera dorsalis

(sensu stricto)

0.203 0.081 0.765 0.568 0.505 1.471

Bactrocera

philippinensis

0.216 0.094 0.602 0.641 0.413 1.259

Bactrocera papayae 0.261 0.071 0.595 0.513 0.157 2.471

Bactrocera dorsalis

(sensu lato)

0.224 0.081 0.806 0.632 0.472 1.680

© 2013 Blackwell Verlag, GmbH 7

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies

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 mono-

phyly of the dorsalis-group and sister-group relation-

ships between B. cacuminata and B. opiliae and

between B. dorsalis s.l. and B. carambolae. The mono-

phyly of B. musae, B. occipitalis, B. opiliae, B. cacumi-

nata 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. dor-

salis/papayae/philippinensis) is slightly weaker

(pp = 0.93). Bactrocera papayae and B. philippinensis

were never monophyletic and were essentially indis-

tinguishable from B. dorsalis s.s. in the tree. Speci-

mens morphologically identified as B. papayae and

B. philippinensis occurred in, respectively, 8–17 and 2

–26 different subclades of the B. dorsalis clade/grade

depending on the combination of data set and infer-

ence method.

Species delimitation analysis and subclade groupings

The use of species delimitation analyses within our

phylogenetic framework revealed a number of statisti-

cally well-resolved groupings for (i) each of the out-

group species, (ii) B. carambolae and (iii) B. dorsalis s.l.

(B. dorsalis/papayae/philippinensis) (Tables 3–5; 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′ (forwhich all individuals are represented by at least two

loci) (fig. 4). Notably, B. carambolae resolves as a taxo-

nomically 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

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 boot-

strap values below. For clarity only supports for backbone nodes are shown; in cases where actual nodal support is absent, posterior probability sup-

port 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 (1–5 = a priori group analysis; a–g = root-to tip analysis), with only those achieving 4/5 (orange) or 5/5 (red) shown. A priori taxonomic iden-

tifications 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.

© 2013 Blackwell Verlag, GmbH8

Phylogeny of B. dorsalis pest flies L. M. Boykin et al.

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. cacumi-

nata (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 subc-

lades resolved within B. carambolae and four in the

B. dorsalis s.l. clade (Table 5; fig. 1).

A priori groups and subclade support increased fol-

lowing 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. cara-

mbolae) 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 exclu-

sively 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).

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 incorpo-

rates individuals collected from a broad geographic

distribution and likely represents a range of intra-

specific populations for these species. Six indepen-

dent loci have been targeted and subsequently

examined using a range of analyses, with a clear

signal emerging: B. carambolae is a distinct mono-

phyletic 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 unre-

solved and therefore prevented the use of the species-

tree 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 concate-

nated DNA sequence data to generate a species-tree

hypothesis (Degnan and Salter 2005; Kubatko and

Degnan 2007; Kubatko et al. 2011); however, as

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 gener-

ates: 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 Rosenberg’s 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 con-

tained 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 Rosenberg’s P (AB) gsi P-value

Dataset 2

Clade 1: musae Bd51/67 2.263 0.05 1 1.40E-11 1 1.00E-04

Clade 2: occipitalis 739/800 1.653 0.77 1 1.40E-11 0.952 1.00E-04

Clade 3: cacuminata 231/244 0.858 0.05 1 1.40E-11 1 1.00E-04

Clade 4: opiliae 1080/1082 0.858 0.05 1 1.40E-11 1 1.00E-04

Clade 5: carambolae 1111/189 1.575 0.05 1 3.00E-42 1 1.00E-04

Clade 6: dorsalis 818/399 1.368 0.9 85 3.00E-42 1 1.00E-04

Dataset 5

Clade 1: musae Bd51/67 2.914 0.05 1 1.50E-12 0.886 1.00E-04

Clade 2: occipitalis 739/800 2.531 0.05 87 3.95E-03 0.944 1.00E-04

Clade 3: cacuminata 231/244 1.117 0.16 100 1.50E-12 1 1.00E-04

Clade 4: opiliae 1080/1082 1.117 0.05 100 1.50E-12 1 1.00E-04

Clade 5: carambolae 1111/189 1.542 0.05 100 1.50E-12 1 1.00E-04

Clade 6: dorsalis 818/399 1.542 0.39 93 1.50E-12 1 1.00E-04

© 2013 Blackwell Verlag, GmbH 9

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies

there was no conflict among individual gene tree phy-

logenies, the benefit of using a single multilocus phy-

logeny to confidently delimit species was considered

appropriate (Rokas et al. 2003; Belfiore et al. 2008;

Sanderson et al. 2011).

Species delimitation statistics reveal additional sub-

structure is present within several clades, particularly

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

Dataset #5 (fig. 4), subclade ‘b’ within the B. carambo-

lae clade consists exclusively of every individual col-

lected 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),

Table 5 Tip to root approach (Boykin et al. 2012) for species delimitation of the Bactrocera dorsalis species complex utilizing Dataset 2 (Concatena-

tion 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

Rosenberg’s

P (AB) gsi P-value

Clade 1: musae Bd51/67 Bd61-62 0.13 0.05 71 5.50E-04 1 0.00139986

Bd57-71 0.156 0.09 88 9.20E-05 1 0.00019998

Bd54-70 0.13 0.05 77 1.30E-06 1 1.00E-04

Clade 2: occipitalis 739/800 Bd791-795 0.43 0.75 60 3.00E-05 1 1.00E-04

Bd784-796 0.43 0.05 86 2.30E-04 1 1.00E-04

Bd788-786 0.581 0.07 67 1.30E-05 1 1.00E-04

Clade 3: cacuminata 231/244 Bd1088-1099 0.197 0.05 59 3.10E-04 1 1.00E-04

Bd1086-1095 0.185 0.05 100 1.36E-03 1 0.00089991

Bd1083-1085 0.185 0.05 100 1.36E-03 1 0.00209979

Clade 4: opiliae 1080/1082 No additional subclades to test

Clade 5: carambolae 1111/189 Bd204-1242 0.357 0.05 51 1.80E-07 1 1.00E-04

Bd201-225 0.377 0.05 63 1.80E-07 0.8533 1.00E-04

Bd191-226 0.357 0.05 100 5.10E-05 1 1.00E-04

Bd189-227 0.396 0.05 100 5.10E-05 1 0.00119988

Bd1237-1236 0.58 0.05 50 0.01 0.664 0.00019998

Bd1224-1232 0.58 0.05 100 0.01 1 1.00E-04

Bd419-415 0.889 0.05 61 6.30E-07 0.664 0.00019998

Bd1119-1126 0.889 0.05 98 3.00E-07 0.664 0.00019998

Clade 6: dorsalis 818/399 Bd1122-1197 0.421 0.05 94 6.00E-04 1 1.00E-04

Bd1127-1129 0.263 0.05 99 3.64E-03 1 0.00069993

Bd1114-1117 0.263 0.05 97 3.64E-03 1 0.00179982

Bd399-589 0.306 0.05 100 1.69E-03 1 0.00149985

Bd740-758 0.23 0.05 51 5.80E-15 1 1.00E-04

Bd819-1181 0.397 0.05 85 0.01 1 1.00E-04

Bd1176-1164 0.372 0.05 77 0.01 1 1.00E-04

Bd403-1123 0.403 0.05 99 7.80E-13 1 1.00E-04

Bd781-1200 0.298 1 60 1.10E-11 1 1.00E-04

Bd1175-774 0.422 0.05 80 3.80E-09 1 1.00E-04

Bd580-1215 0.245 0.61 84 3.80E-09 1 1.00E-04

Bd1136-1145 0.389 1 89 9.80E-08 1 1.00E-04

Bd1209-1203 0.384 0.05 71 9.80E-08 1 1.00E-04

Bd400-817 0.297 1 71 9.80E-08 1 0.00029997

Bd1194-1205 0.291 0.05 100 3.40E-06 1 0.00139986

Bd821-829 0.384 0.05 69 3.40E-06 1 0.00089991

Bd816-1148 0.367 1 100 3.40E-06 1 0.00159984

Bd775-780 0.198 0.61 96 3.40E-06 1 0.00149985

Bd769-773 0.247 0.05 82 3.40E-06 1 0.00169983

Bd757-759 0.14 0.05 85 3.40E-06 1 0.00079992

Bd751-768 0.14 0.05 62 3.40E-06 1 0.00179982

Bd744-752 0.159 0.46 57 3.40E-06 1 1.00E-04

© 2013 Blackwell Verlag, GmbH10

Phylogeny of B. dorsalis pest flies L. M. Boykin et al.

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-sup-

ported ‘Suriname subclade’ within the more diverse

south-east Asian B. carambolae clade is not unex-

pected given such a recent introduction for which a

‘genetic bottleneck’ is likely to exist. Similarly, sub-

group ‘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 south-

east Asia and western Indonesian archipelago sites

(however, human-mediated movement may limit

this). Indeed, significant isolation-by-distance effects

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 (Malay-

sia); B. philippinensis (two individuals from Imus, Phil-

ippines); and B. dorsalis s.s. from San Pa Tong

(northern Thailand). In this case, conspecific repre-

sentatives for each of these species are also repre-

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.

© 2013 Blackwell Verlag, GmbH 11

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies

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 pro-

ven 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 1–4).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

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 analy-

sis 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. papa-

yae and B. philippinensiswere indistinguishable) (Arm-

strong and Cameron 2000). Indeed it appears the

main distinguishing morphological character separat-

ing B. musae from B. dorsalis s.l. is the occasional

absence of the medial longitudinal band on the abdo-

men for some individuals (Drew 1989); the presence

of which is typical of dorsalis complex species (Drew

and Hancock 1994). We therefore recommend further

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.

© 2013 Blackwell Verlag, GmbH12

Phylogeny of B. dorsalis pest flies L. M. Boykin et al.

work on B. musae be undertaken towards fully resolv-

ing 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 Aus-

tralian species B. opiliae and B. cacuminata (figs 1–4).While B. occipitalis has been regarded a closely related

species of B. dorsalis (Muraji and Nakahara 2002; Naka-

hara andMuraji 2008; Krosch et al. 2012a), it is morpho-

logically distinct in having significantly shorter genitalia

with colour markings distinct as from B. philippinensis

(Drew and Hancock 1994; Iwahashi 1999). Bactrocera cac-

uminata 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 mor-

phological similarity with this species (Drew and Hardy

1981). Bactrocera opiliae and B. dorsalis s.s.were only sepa-

rable using ecological, physiological and genetic mea-

sures, 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 protec-

tion officers.

The unusual case of specimen #413

We cannot explain the unusual placement of spec-

imen #413 in any of our phylogenetic reconstruc-

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.

© 2013 Blackwell Verlag, GmbH 13

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies

tions (figs 1–4). 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. oc-

cipitalis. Specimen #413 was collected from Nakhon

Si Thammarat (southern Thailand) and hence

occurred where B. dorsalis s.s. and B. papayae geo-

graphically abut or overlap according to recorded

geographic distributions for these species (e.g.

Drew and Hancock 1994). Morphologically, #413

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 spe-

cies and likely to be B. papayae (pers. comm.).

However, our study included only four economi-

cally important and three additional out-group spe-

cies from the B. dorsalis complex, and the inclusion

of more members from the complex may help to

resolve the placement of specimen #413.

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 Rosenberg’s P (AB) gsi P-value

Clade 1: musae Bd51/67 Bd61-62 0.234 0.05 59 5.50E-04 1 0.00059994

Bd56-71 0.309 0.42 51 2.10E-05 1 1.00E-04

Bd54-70 0.234 0.05 85 1.30E-06 1 1.00E-04

Clade 2: occipitalis 739/800 Bd783&786 0.331 0.05 61 0.05 1 0.00089991

Bd794&799 0.331 0.05 91 0.05 1 0.00059994

Clade 3: cacuminata 231/244 No additional subclades to test

Clade 4: opiliae 1080/1082 Bd1081&88 0.279 0.05 88 6.40E-04 1 0.00069993

Bd1083&85 0.258 0.05 90 6.40E-04 1 0.00119988

Bd1086&95 0.345 0.05 100 6.40E-04 1 0.00029997

Bd1089&90 0.258 0.05 86 6.40E-04 1 0.00069993

Clade 5: carambolae 1111/189 Bd405&1241 0.364 0.05 68 1.90E-05 1 0.00069993

Bd1255-1262 0.345 0.05 100 1.90E-05 1 1.00E-04

Bd1238&58 0.256 0.05 77 6.90E-08 1 0.00069993

Bd1234-1121 0.55 0.05 73 6.20E-09 1 1.00E-04

Bd419-1263 0.256 0.05 70 6.70E-10 1 1.00E-04

Bd1225-1239 0.543 0.05 99 1.90E-15 1 1.00E-04

Bd191-216 0.343 0.05 100 8.00E-18 1 1.00E-04

Clade 6: dorsalis 818/399 Bd818&1168 0.596 0.05 92 9.50E-07 1 0.00129987

Bd1195-1200 0.344 0.06 100 1.90E-08 0.498 0.0009999

Bd418&827 0.345 0.05 87 1.00E-06 1 0.0009999

Bd579&1179 0.303 1 64 1.00E-06 1 0.00139986

Bd580&1164 0.284 0.09 50 1.00E-06 1 0.00109989

Bd583&1142 0.292 0.71 57 1.00E-06 1 0.00039996

Bd772&775 0.254 0.13 82 1.00E-06 1 0.00079992

Bd816&1148 0.333 0.05 100 1.00E-06 1 0.00039996

Bd823&1181 0.377 0.05 100 1.00E-06 1 0.00079992

Bd1143&1145 0.4 0.05 100 1.00E-06 1 0.00049995

Bd1206&1210 0.224 0.05 72 1.00E-06 1 0.00069993

Bd1211&1253 0.404 0.05 55 1.00E-06 1 0.00079992

Bd1244&1250 0.231 0.41 64 1.00E-06 1 0.00069993

Bd1202-1215 0.269 1 92 2.00E-08 1 1.00E-04

Bd1246-1249 0.224 0.16 54 2.00E-08 1 1.00E-04

Bd825-1209 0.401 1 63 5.40E-10 1 1.00E-04

Bd1194-1205 0.263 1 99 5.40E-10 1 1.00E-04

Bd585-1183 0.426 0.94 80 1.70E-11 1 1.00E-04

Bd744-781 0.361 0.05 100 1.10E-30 0.953 1.00E-04

Bd593-1123 0.37 1 55 6.80E-36 1 1.00E-04

© 2013 Blackwell Verlag, GmbH14

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 reso-

lution between B. dorsalis s.s, B. papayae and B. philip-

pinensis 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 contra-

dict this, and in addition, the design here overcomes

the potential weaknesses of earlier studies by sam-

pling 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-recog-

nized 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 distinc-

tion, or lack of for B. dorsalis s.s, B. papayae and B. phi-

lippinensis, came from two linked mitochondrial loci.

However, mitochondrial DNA is characterized by

complex evolutionary dynamics. For example, selec-

tive sweeps that help to differentiate taxa can in the

case of recently diverged taxa be offset by the homog-

enizing effect of hybrid introgression (Galtier et al.

2009). Certainly, this has been found in wild popula-

tions 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 feasi-

ble to test as genomic data continues to accumulate.

Nonetheless, we stress that this work should be exam-

ined 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

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 delimita-

tion 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 Govern-

ment’s 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.

Figure S2. Dataset 1.2. Phylogenetic reconstruction

based on sequence data for specimens for which ribo-

somal DNA (ITS1 and ITS2) were sequenced for Bac-

trocera 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 Bactro-

cera 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 cur-

rent 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 infor-

mation for the samples included in this study.

© 2013 Blackwell Verlag, GmbH 19

L. M. Boykin et al. Phylogeny of B. dorsalis pest flies