Diptera: Tephritidae) Does Not Support Current Taxonomy L

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 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 Abstract biosecurity, fruit fly, multi-gene phylogeny, species delimitation Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predomi- Correspondence nantly occur in south-east Asia and the Pacific. Identifying molecular Laura M. Boykin (corresponding author), Plant diagnostics has proven problematic for these four taxa, a situation that Energy Biology, ARC Centre of Excellence, The cofounds biosecurity and quarantine efforts and which may be the result University of Western Australia, M316 of at least some of these taxa representing the same biological species. We Crawley, WA 6009, Australia. E-mail: [email protected] therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide Received: November 12, 2012; accepted: geographic range; sequencing six loci (cox1, nad4-3′, CAD, period, ITS1, February 18, 2013. ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic doi: 10.1111/jen.12047 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, Rosenberg’s reciprocal monophyly measure, P(AB), Rodrigo’s (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.

frugivorous tephritids oviposit into fleshy fruits and Introduction vegetables, where resultant larvae emerge and feed The Tephritidae (true fruit flies) is one of the most on the fruit pulp. Production losses and costs of field species-rich families within the order Diptera. While control are the direct impacts of fruit fly attack, while non-fruit feeding tephritids are rarely pestiferous indirect losses result from the implementation of reg- (Headrick and Goeden 1998), the frugivorous tephrit- ulatory controls and lost market opportunities (Clarke ids contain many genera of major economic impor- et al. 2011). Bactrocera Macquart contains over 500 tance, including Ceratitis, Rhagoletis and Anastrepha described species and is the dominant genus of fruit (White and Elson-Harris 1992). Mature female flies in the Asia/Pacific region (Drew 1989, 2004).

Within this genus, the Bactrocera dorsalis species com- Attempts to identify DNA markers for these four plex contains 75 species and includes some of the species of the B. dorsalis complex have met with most pestiferous species of the genus, especially the mixed success. An early study of the 18S rDNA, Cu/ Oriental fruit fly, B. dorsalis s.s. (Hendel), and the Zn superoxide dismutase enzyme and 12S rDNA cod- Asian papaya fruit fly, B. papayae Drew and Hancock ing genes found these loci could not differentiate (1994); Clarke et al. 2005). The B. dorsalis complex is B. dorsalis s.s., B. carambolae and B. papayae (White, a monophyletic group of species of relatively recent 1996). Similarly, while within the larger B. dorsalis evolutionary origin, with an estimated age of 6.2 mil- complex, the species B. occipitalis (Bezzi) and B. kandi- lion years to their most recent common ancestor ensis Drew & Hancock could be resolved as separate (Krosch et al. 2012a). species using the 16S gene, B. dorsalis s.s., B. papayae, Bactrocera dorsalis s.s., B. papayae, B. philippinensis B. carambolae and B. philippinensis could not be sepa- Drew & Hancock and B. carambolae Drew & rated (Muraji and Nakahara 2002). In contrast, the Hancock are found predominately in south-east nDNA regions 18S + ITS1, and ITS1 and ITS2 were Asia and the Pacific, and are the members of the found to reliably distinguish B. carambolae from B. dorsalis complex which are of most concern to B. dorsalis s.s. (Armstrong et al. 1997; Armstrong and pest managers and plant biosecurity officials in the Cameron 2000). A series of papers by Nakahara and region. These four species form a true sibling spe- colleagues (Nakahara et al. 2000, 2001, 2002; Muraji cies complex for which both morphological and and Nakahara 2002) targeting the mitochondrial DNA molecular diagnostics have proven problematic D-loop + 12S and 16S suggested the four species (Clarke et al. 2005). The initial taxonomic work could be distinguished from each other, although the that separated these taxa relied on very subtle char- different target sites did not distinguish all species acter state differences (Drew and Hancock 1994), equally (e.g. B. papayae and B. carambolae were poorly but many of these character states have since been or not separated using 16S). Other tightly focused shown to be variable and continuous between the procedures, for example, a microarray test developed taxa (Krosch et al. 2012b; Schutze et al. 2012a). All from EPIC (exon primed intron crossing)-RFLP of four species are polyphagous pests (Allwood et al. muscle actin can distinguish B. dorsalis s.s., B. papayae 1999; Clarke et al. 2001) that have invaded regions and B. carambolae (Naeole and Haymer 2003). beyond their natural ranges (Smith 2000; Cantrell One common feature – and weakness – for nearly et al. 2001; Duyck et al. 2004), hence accurate all of the above studies is a failure to separate what diagnosis for quarantine and field management is may be variation at the intra- vs. inter-specific level. critical. Taxa are often represented by very small sample sizes, Diagnostic development for these species has been sometimes as few as one individual, rarely more than confounded by their close genetic, morphological, five or six (e.g. Muraji and Nakahara 2002); or in behavioural and physiological similarities (Clarke cases where sample sizes are greater they are gener- et al. 2005; Schutze et al. 2012b). While some ally drawn from only one geographic population (e.g. researchers have identified morphological and molec- Nakahara et al. 2001). As a result, it remains impossi- ular markers considered to be diagnostic of different ble to determine whether such diagnostic markers are species (Drew and Hancock 1994; Iwahashi 1999; resolving species or population level differences, as Muraji and Nakahara 2002; Naeole and Haymer 2003; already recognized: for example, ‘In order to confirm Drew et al. 2008), others have found no such mark- the genetic interrelationship among the B. dorsalis ers, or markers which separate some but not all of the complex species, analyses of field populations using four species (Medina et al. 1998; Tan 2000, 2003; many other genetic markers are needed’ (Muraji and Wee and Tan 2000a,b, 2005). Consequently, the Nakahara 2002). We specifically address this issue in debate continues as to whether these four taxa repre- this study. sent good biological species for which species-specific As part of a larger project investigating the species diagnostic markers exist but which are yet to be iden- limits of the target taxa within the B. dorsalis species tified and universally agreed upon; or whether they complex (i.e. B. dorsalis s.s., B. papayae, B. philippinen- may in fact represent a group where one biological sis and B. carambolae = ingroup taxa) (Krosch et al. species has been incorrectly taxonomically split, in 2012b; Schutze et al. 2012a,b), we undertook new which case species-level diagnostic markers simply do field collections of specimens from multiple sites not exist and any observed variation reflects popula- across the geographic ranges of the four taxa. We also tion level differences (Harrison 1998; Sites and included outgroup taxa from within the complex Marshall 2004). [B. cacuminata (Hering), B. opiliae (Drew & Hardy),

B. occipitalis (Bezzi)] and outside the complex [B. mu- known distributions. As discrimination amongst in- sae (Tryon), B. tryoni (Froggatt)]. We sequenced six group species is difficult due to high morphological loci (cox1, nad4-3′, CAD, period, ITS1, ITS2) for approx- similarity, we made collections of in-group species imately 20 individuals from each of 16 sample sites, from locations where each is regarded as allopatric to including two or more sites for each of the ingroup the other three based on the descriptions provided in taxa. Data were analysed within maximum likelihood Drew and Hancock (1994). For collection sites where and Bayesian phylogenetic frameworks for both the more than one of the in-group taxa occur sympatri- individual loci and concatenated data sets for which cally (primarily B. papayae and B. carambolae), we we applied multiple monophyly and species delimita- identified species based on published descriptions tion tests. Using this data set of multiple individuals (Drew and Hancock 1994) and host use data (Clarke sampled from a number of populations, we specifi- et al. 2001). cally tested whether there was phylogenetic support Samples of male flies were collected from 2009 to for the four described pest species: B. dorsalis s.s., 2010 from 13 locations across seven countries B. papayae, B. philippinensis and B. carambolae. (Table 1). The principle method of collection consisted of luring male flies into methyl eugenol (ME) insecticide-baited hanging traps containing propylene Materials and Methods glycol as a preserving agent (Vink et al. 2005; Thomas 2008). These traps were either distributed as part of Target species and outgroup selection ‘collection parcels’ to collaborators throughout south- The aim of this study was to use phylogenetic meth- east Asia who placed the traps in the field, or deployed ods to resolve species limits among the following four during collection trips undertaken by MKS in Decem- target species of the B. dorsalis species complex: B. dor- ber 2010. salis s.s., B. papayae, B. philippinensis and B. carambolae Exceptions to above collection methods are as fol- (Sites and Marshall 2004). For the purposes of this lows. Bactrocera tryoni were collected using the same study, we refer to these four taxa as the ‘ingroup spe- technique as above, but using Cue-lure instead of cies’. We also selected a number of species to repre- ME as the male attractant. Bactrocera musae were sent ‘outgroups’, which were chosen because: (i) they sourced from a culture maintained by the Queens- are related to varying degrees to the ingroup species land Government Department of Agriculture, Fisher- (they are either in the B. dorsalis species complex or ies and Forestry (DAFF) in Cairns, Queensland otherwise closely related) but are unambiguously (Australia). Flies from Serdang (Malaysia) were regarded as different species and (ii) they are taxa that reared from Musa acuminata x balbisiana hybrids, vars. are morphologically similar and may be confused with Mas, Berangan and Lemak bananas (which yielded the target species for quarantine purposes (and hence B. papayae) and Averrhoa carambola fruit (which further resolving their molecular relationships with yielded B. carambolae) collected from the field in the ingroup taxa is of wider benefit). The outgroup November 2010. Samples from Lampung (Indonesia) species consisted of three B. dorsalis complex flies: two were collected into dry ME lure traps placed in the Australian species B. cacuminata and B. opiliae, and the field, and flies were promptly preserved in 70% etha- Philippine species B. occipitalis (which occurs sympat- nol. Bactrocera carambolae from Paramaribo (Suri- rically with B. philippinensis); and B. musae which, name) were reared from A. carambola fruit placed in while not belonging to the B. dorsalis complex per se,is the field. closely related to the complex as demonstrated by pre- All samples were returned to the Queensland Uni- vious molecular studies (Armstrong and Cameron versity of Technology (QUT), Brisbane (Australia), for 2000; Krosch et al. 2012a). Finally, we included transfer into absolute ethanol, preliminary morpho- B. tryoni as an outgroup species for tree rooting, as logical identification and preparation for DNA extrac- while it is of the same genus it unambiguously tion. Three legs of each fly (fore, mid and hind) were belongs to a different species complex, the B. tryoni removed and stored in absolute ethanol in new species complex (Krosch et al. 2012b). Eppendorf â tubes for shipment to the Elizabeth Mac- Arthur Agricultural Institute (New South Wales Department of Primary Industries) for genomic DNA Study sites and specimen collection extraction. When numbers allowed, 30 samples per To obtain as many representative samples from across collection site were sent for extraction (Table 1). The as broad a geographic area as possible, we collected remainder of all flies are stored as vouchers in abso- in-group species from multiple locations across their lute ethanol at QUT.

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

Collection Location Country Latitude Longitude Date Species method

Brisbane, Queensland Australia 27°27′29″S 152°58′56″E 10 July 2009 Bactrocera tryoni Cue-lure September– Bactrocera cacuminata ME Lure November 2009 Cairns, Queensland Australia DEEDI culture DEEDI culture 5 June 2009 Bactrocera musae Culture Noonamah, Northern Australia 12°38′33″S 131°5′58″E 24 December 2009 Bactrocera opiliae ME Lure Territory 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″N98°53′42″E 12 March 2010 Bactrocera dorsalis s.s. ME Lure Chatuchuk, Bangkok Thailand 13°50′32″N 100°34′23″E14–21 December Bactrocera dorsalis s.s. ME Lure 2009 Muang District, Nakhon Thailand 8°25′12″N99°53′48″E 25 October–15 Bactrocera papayae ME Lure Si Thammarat November 2009 25 October–15 Bactrocera carambolae ME Lure November 2009 Tikus Pulau, Penang Malaysia 5°25′50″N 100°18′38″E17–26 November Bactrocera papayae ME Lure 2009 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″E15–17 May 2009 Bactrocera papayae ME Lure 15–17 May 2009 Bactrocera carambolae ME Lure Paramaribo Suriname 5°49′20″N55°10′05″W August 2009 Bactrocera carambolae ex Averrhoa carambola

from Porter and Collins (1991). CAD primers are rede- DNA extraction, PCR and sequencing signed after Moulton and Wiegmann (2004, 2007) Tubes containing fly legs were pulse spun, the ethanol after comparison with GenBank tephritid sequences. removed and air-dried. Samples were transferred Primers for period are from Barr et al. (2005) and Vir- to À80°C for 15 min, after which each tube was gilio et al. (2009). Primer sequences for all loci are dipped in liquid nitrogen while fly legs were crushed given in Table 2. with a sterile micropestle. DNA was extracted using The PCR conditions for ITS1, ITS2 and ND4-2 con- the Qiagen DNeasyâ (QIAGEN Inc., Valencia, CA) sisted of 2 ll of template DNA being added to a final Blood and Tissue kit as per the manufacturer’s volume of 30 ll of reaction mix containing 200 lM of instructions. Two mitochondrial (mt) protein-coding dNTPs, 200 nM of each forward and reverse primer, 1 genes (cox1 and nad4-3′), two nuclear protein-coding 9 Accutaq PCR buffer (Sigma Australia), and 0.02U of genes (CAD, period) and two nuclear ribosomal RNA AccuTaq polymerase. The cycling conditions consisted regions (ITS1, ITS2) were analysed. Primers for cox1 of an initial denaturation at 94°C for 2 min, followed are after Folmer et al. (1994). Those for nad4-3′ were by 35 cycles of denaturation at 94°C for 15 s, anneal- newly designed by comparison of tephritid mt ing at 60°C, 55°C and 60.5°C for ITS1, ITS2 and ND4- genomes on GenBank (Spanos et al. 2000; Nardi et al. 2 respectively, followed by an extension time of 2003; Yu et al. 2007), targeting regions that appeared 1 min at 68°C and final extension of 5 min at 68°C. more variable than cox1 but for which PCR amplifica- All PCR products were visualized on 1.5% agarose tion was still reliable across taxa. The forward ITS1 gels run at 90V for 45 min and post-stained with ethi- primer, ITS7, was designed de novo by KFA; reverse dium bromide. All PCR products were sent to AGRF primer ITS6 was taken from Armstrong and Cameron (Australian Genome Research Facility Ltd) in 96-well (2000), and ITS2 primers FFA and FFB were modified plates for purification and sequencing. AGRF is

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) accredited by NATA to the ISO/IEC17025:2005 Qual- as missing. For ease of comparison, these data sets are ity Standard. Australian Genome Research Facility Ltd limited to specimens for which all six loci have been operates the AB3730xl 96-capillary sequencer for low successfully sequenced (235 specimens, 1219, 1002, to high throughput DNA sequencing. 528 and 686 bp respectively). Polymerase chain reaction conditions for cox1, CAD Dataset #2 A concatenated data set including only and period consisted of 1 ll DNA template in a final specimens for which all six loci were successfully volume of 20 ll containing 100 nM each forward and sequenced (235 specimens, 3435 bp alignment). reverse primer 10 ll Go Taq Green enzyme master Dataset #3 Dataset #2 with heterozygous sites mix (ProMega, Sydney, Australia) and 7 ll of steril- removed from CAD and period alignments (235 speci- ized water. PCR cycling conditions consisted of an ini- mens, 3094 bp) tial denaturation step at 94°C for 2 min, followed by Dataset #4 Dataset #2 with CAD and period removed 40 cycles of denaturation at 95°C for 30 s, annealing from alignment altogether (235 specimens, 2221 bp) at 50°C (for cox1 and period)or54°C (for CAD) for Dataset #5 Specimens for which at least two of the 30 s. and extension at 72°C for 1 min.; there was a four loci (i.e. excluding CAD and period) were success- final run-out extension step at 72°C for 7 min. All fully sequenced (313 specimens, 2221 bp) PCR products were visualized on 1% agarose gels con- Dataset #1 was designed to allow testing of the varia- taining 10X dilution of SYBER Safe (Life Technolo- tion between loci and to apply a species-tree recons- gies, Victoria, Australia) and run at 80V for 30 min. truction approach (Edwards 2008); however, due to Sequencing was performed using ABI BigDyeâ ver. 3 the poor resolution in Datasets #1.2–1.4, the additi- dye terminator chemistry and run on an ABI 3130xl onal, concatenation-based data sets were produced capillary sequencer. Chromatograms were checked (after Gatesy et al. 1999; Gatesy and Baker 2005). and sequence contigs assembled with SEQUENCHER ver Dataset #2 includes a large number of heterozygous 4.2 (Gene Codes Corporation 2004) to produce com- sites in the CAD and period gene partitions, which may pleted sequences. 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 Analytical strategy basis (#3) or by removing the CAD and period gene par- The following series of five data sets were analysed to titions entirely (#4). Dataset #5 tests how significant test the phylogenetic signal of different loci and to missing partitions were for the inferred phylogeny. account for the failure to sequence all loci for all specimens: Alignment and analysis Datasets #1.1–1.4 Each linked inheritance groups as a separate alignment; 1.1: mitochondrial genes Sequences for each locus were aligned by eye (pro- (cox1 + nad4-3′), 1.2: ribosomal RNA genes (ITS1 tein-coding genes) or using ClustalX (rRNA regions) + ITS2), 1.3: CAD; 1.4: period. The two mitochondrial (Thompson et al. 1997). For the ITS 1 and ITS2 data and two ribosomal loci are concatenated as they are set, indels were treated as missing due to the coinherited. For the ITS data sets, indels were treated constraints of Bayesian and RAxML analyses. Hetero-

© 2013 Blackwell Verlag, GmbH 5 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. zygous sites in the CAD and period loci, observed tion plugin (Masters et al. 2010) for Geneious clearly as two bases in the forward and reverse (Drummond et al. 2010) was used to calculate sequences, were labelled according to the IUPAC Rosenberg’s reciprocal monophyly, P (AB) (Rosen- code. Models of molecular evolution for each loci, and berg 2007) and Rodrigo’s P (RD) (Rodrigo et al. each codon position within each protein-coding gene, 2008) measures. The (Cummings et al. 2008) statis- were determined using MODELTEST ver. 3.6 (Posada tic was calculated in R based on the estimated tree and Crandall 1998). Concatenations for multilocus and the assignment file that contains user specified data sets were done in MACCLADE ver. 4.06 (Maddison groups (see http://www.genealogicalsorting.org/). and Maddison 2003). For each data set, phylogenetic Two different assignment files were generated for trees were inferred in parallel by both Maximum the gsi for each data set: one based on previously Likelihood and Bayesian analyses. Likelihood analy- defined taxonomic groups and the other containing ses were conducted using RAxML ver 7.2.8 imple- groups within those as determined using the tip to mented on the RAxML BlackBox webserver (http:// root approach of species delimitation (Boykin et al. phylobench.vital-it.ch/raxml-bb/index.php) (Stamat- 2012). Each of the assignment files was run with akis et al. 2008). Data were analysed with a Gamma the known phylogeny and an R script that specifies model of rate heterogeneity, the proportion of invari- the number of permutations (100 000 permutations able sites was estimated, and for concatenated, mul- across four processors). All of the gsi analyses were tilocus data sets, the alignment was partitioned and run using R on the BeSTGRID computer cluster branch lengths optimized on a per locus basis. Bayes- (Jones et al. 2011). To assess the significance of the ian analyses were conducted using MRBAYES ver 3.2 gsi P-values, the Bonferroni correction was used. (Ronquist et al. 2012) using parallel implementation on the BeSTGRID computer cluster (Jones et al. Results 2011), or using direct implementation on local desk- top computers. Analyses were run for 10 (Datasets #1, Sequence data collection 3, 4, 5) or 50 million generations (Dataset #2, due to a longer time for independent runs to converge) with The six loci (cox1, nad4-3′, ITS1, ITS2, CAD and per- sampling every 1000 generations, partitioned data sets iod) were successfully amplified for the majority of and parameter estimation for each partition unlinked. specimens examined across all species. Success/fail- Each analysis consisted of two independent runs, each ure of sequencing individual loci for each specimen, utilizing four chains, three cold and one hot. Conver- along with their GenBank accession numbers, are gence between runs was monitored by finding a pla- shown in Table S1. Of these, nad4-3′ was the only teau in the likelihood score (standard deviation of one of five additional mt genes (data not shown) tri- split frequencies <0.0015) and the potential scale alled in this study that was successfully amplified reduction factor (PSRF) approaching one. Conver- across the range of species here. Due to the low lev- gence of other parameters within the runs was also els of molecular variation previously found within checked using TRACER v1.5.4 (Rambaut and Drum- the dorsalis complex for cox1 (Armstrong and Ball mond 2010), with ESS values above 200 for each run. 2005), the additional mitochondrial genes trialled The first 12.5% of each run was discarded as burnin were chosen in an effort to maximize variability for the estimation of consensus topology and the pos- based on the previous analyses of dipteran mt ge- terior probability of each node. Bayesian & RAxML nomes (Cameron et al. 2007; Nelson et al. 2012). run files are available from the authors upon request. The trade-off for gene variability is primer reliability, Phylogenetic trees generated from Datasets #2 and whereby sequence variability at the priming sites #5 were used as input in the species monophyly causes mismatches and loss of efficacy. Thus, finding and delimitation analyses. Species delimitation was only one more variable mt gene, which could be addressed using the standard Kimura two-parameter reliably amplified, is not surprising. The nad4-3′ gene (K2P) inter-species distance and Rodrigo’s P (ran- region was confirmed here to be more variable, hav- domly distinct) (Rodrigo et al. 2008) measure. Spe- ing 104 of 577 positions parsimony informative cies monophyly was measured by clade support, compared to 101 of 642 parsimony informative for posterior probability or bootstrap resampling for cox1. Bayesian and likelihood analyses respectively, The ribosomal ITS loci each had significant indels Rosenberg’s reciprocal monophyly measure, P (AB) (33–84 bp in ITS1, 31–40 bp in ITS2), but there were (Rosenberg 2007) and the genealogical sorting index few heterozygous sites, consistent with the concerted (gsi) (Cummings et al. 2008). The species delimita- evolution previously found for these loci (e.g.

Eickbush and Eickbush 2007). In contrast, both (Dataset #1.3), only B. musae (BA & ML) and B. cac- nuclear protein-coding genes had a large proportion uminata (ML only) were monophyletic whereas for of heterozygous sites; 179 of 528 bp in CAD and 162 period (Dataset #1.4), B. musae (BA & ML), B. cacumi- of 686 bp in period. These sites unfortunately made up nata (BA only) and B. opiliae (BA only) were mono- almost all of the variable sites within these two genes, phyletic. The majority of specimens of the remaining with only 10 of the remaining 349 homozygous sites species formed unresolved combs. Due to the poor in CAD and 15 of 524 in period being parsimony infor- resolution across these four data sets, species-tree mative. Intraspecific variation for each gene is shown reconstruction based on individual gene trees was not in Table 3. attempted. Analyses of concatenated data sets were conducted to determine whether larger data sets would be bet- Phylogenetic analyses ter resolved and display higher nodal support than For each data set, Bayesian (BA) and likelihood analy- was achieved analysing each linkage group separately ses (ML) yielded similar topologies; however, nodal (Datasets #1.1–1.4). Further, due to the high propor- support was much greater for the set of Bayesian anal- tion of heterozygous sites within CAD and period, yses. Of the single linkage group analyses (Datasets and the significant number of individuals for which #1.1–1.4), only the mitochondrial gene trees (Dataset one or more genes failed to amplify/sequence (57 #1.1) were well resolved, with each species other than specimens, approximately 25%), a series of different B. dorsalis, B. philippinensis, B. papyae and B. carambo- concatenation data sets were analysed to determine lae monophyletic with significant nodal supported whether either factor resulted in artefactual relation- (BA pp = 0.9–1.0; ML bs >70%) (Fig. S1). For the ships. The same species boundaries were inferred for ribosomal ITS loci (Dataset #1.2), several species were all four concatenated data sets, and the interspecies monophyletic, for example, B. musae, B. occipitalis, relationships were also quite constant. The heterozy- B. opiliae, B. carambolae, whereas B. cacuminata and gous positions within CAD and period had a limited B. dorsalis s.s., formed paraphyletic combs with respect effect on inferred species relationships, as the only to other species (Fig. S2). For example, in the Bayes- difference was in the position of a single specimen, ian analysis of Dataset #1.2, B. cacuminata specimens Bd413 an unidentifiable member of the dorsalis- formed 17 of 19 branches in a polytomy with a mono- group complex. This specimen was sister to all the phyletic B. opiliae (node support not significant in BA dorsalis-group flies with inclusion of these gene or ML) and a single significantly supported clade regions (#2-BA) or the sister-group of B. occipitalis which included all B. dorsalis s.s., B. philippinensis, with their exclusion (#2-ML, #3-#5-BA & ML) B. papaya and B. carambolae specimens. The trees (fig. 1; Figs S5–7). Similarly, the inclusion of speci- inferred for each of the nuclear protein-coding genes mens for which up to half of the loci were missing were almost totally unresolved (Figs S3–4). For CAD (#5) did not result in a different topology from those inferred from specimens where all genes were pres- Table 3 The average intraspecific distances for each gene shown in % ent (#3–#4). calculated using MEGA Below the species level, there was significant vari- Species ITS1 ITS2 ND4 CO1 per CAD ability in topology and nodal support across the different concatenated data sets with few clades larger Bactrocera tryoni 0.000 0.000 1.284 0.809 0.275 0.210 than 2–3 specimens shared between analyses. The Bactrocera musae 0.000 0.000 0.127 0.031 0.033 0.038 only notable exception is the clade containing B. cara- Bactrocera 0.000 0.000 0.101 0.051 0.047 0.000 mbolae specimens from Paramaribo (Suriname, South cacuminata America). This invasive population forms a strongly Bactrocera occipitalis 0.000 0.000 0.972 0.329 0.244 0.682 supported, monophyletic clade to the exclusion of Bactrocera opiliae 0.000 0.000 0.604 0.603 0.291 0.101 Bactrocera 0.158 0.093 0.924 0.611 0.597 2.294 the SE Asian specimens of B. carambolae in Datasets carambolae #1.1, 3–5 (both BA & ML analyses). In Datasets #1.4 Bactrocera dorsalis 0.203 0.081 0.765 0.568 0.505 1.471 and 2, this clade is still recovered however several SE (sensu stricto) Asian B. carambolae specimens were included within Bactrocera 0.216 0.094 0.602 0.641 0.413 1.259 it also. As Datasets #1.1, 3–5 either omit the nuclear philippinensis protein-coding genes altogether (#1.1, 4, 5) or Bactrocera papayae 0.261 0.071 0.595 0.513 0.157 2.471 remove all ambiguous sites (#3), it is likely that the Bactrocera dorsalis 0.224 0.081 0.806 0.632 0.472 1.680 (sensu lato) monophyly of the B. carambolae specimens from Suri- name reflects a genetic bottleneck associated with its

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 (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. establishment in South America and/or that the depending on the combination of data set and infer- source population for this invasion was not present in ence method. this study. The combined phylogeny thus supports the mono- Species delimitation analysis and subclade groupings phyly of the dorsalis-group and sister-group relationships between B. cacuminata and B. opiliae and The use of species delimitation analyses within our between B. dorsalis s.l. and B. carambolae. The mono- phylogenetic framework revealed a number of statisti- phyly of B. musae, B. occipitalis, B. opiliae, B. cacumi- cally well-resolved groupings for (i) each of the out- nata and B. carambolae is each very strongly group species, (ii) B. carambolae and (iii) B. dorsalis s.l. supported (BA pp = 1.0 for each), whereas the (B. dorsalis/papayae/philippinensis) (Tables 3–5; figs 1 monophyly of the remaining B. dorsalis s.l. (B. dor- and 4). Each of the six clades is statistically supported salis/papayae/philippinensis) is slightly weaker by at least four of the five species delimitation (pp = 0.93). Bactrocera papayae and B. philippinensis measures; especially in the case of ‘Dataset #5′ (for were never monophyletic and were essentially indis- which all individuals are represented by at least two tinguishable from B. dorsalis s.s. in the tree. Speci- loci) (fig. 4). Notably, B. carambolae resolves as a taxo- mens morphologically identified as B. papayae and nomically distinct clade, rating 5/5 for all analyses in B. philippinensis occurred in, respectively, 8–17 and 2 both Datasets #2 and #5, while unambiguous species –26 different subclades of the B. dorsalis clade/grade (B. occipitalis, B. cacuminata and B. opiliae) achieve 4/5

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 À235 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

in at least one of the datasets; a result which we Discussion believe lends greater support to the ongoing specific status of B. carambolae. This study represents the most comprehensive Species delimitation statistical analyses undertaken phylogenetic analysis undertaken to-date for four for Dataset #2 revealed considerable support for pestiferous and morphologically cryptic members of each of the six a priori defined groups: B. musae (5/5 the B. dorsalis species complex. The study incorpo- statistically significant), B. occipitalis (4/5), B. cacumi- rates individuals collected from a broad geographic nata (4/5), B. opiliae (4/5), B. carambolae (5/5) and distribution and likely represents a range of intra- B. dorsalis s.l. (i.e. B. dorsalis/papayae/philippinensis) (4/ specific populations for these species. Six indepen- 5) (Table 4). Tip-to-root analysis (examining all dent loci have been targeted and subsequently resolved clades) demonstrated a limited number of examined using a range of analyses, with a clear subclades which were statistically significant for at signal emerging: B. carambolae is a distinct mono- least four of the five statistics applied, with three subc- phyletic clade, whereas B. dorsalis s.s., B. papayae lades resolved within B. carambolae and four in the and B. philippinensis form a single sister clade to B. dorsalis s.l. clade (Table 5; fig. 1). B. carambolae. A priori groups and subclade support increased following analysis of Dataset #5, with all five statistical Phylogenetic analyses and species delimitation analyses significant for four of the a priori defined clades (B. musae, B. occipitalis, B. opiliae and B. cara- The individual gene trees in this study were unre- mbolae) and 4/5 for the remaining two (B. cacuminata solved and therefore prevented the use of the species- and B. dorsalis s.l.). Meanwhile, tip-to-root analysis tree software (e.g.,Ane et al. 2007; Liu 2008; Liu et al. revealed nine subclades to have 4/5 support measures 2009; Kubatko 2009; Kubatko et al. 2009; Heled and statistically significant, with three occurring in the Drummond 2010; Than and Nakhleh 2009; Than B. carambolae clade (one of which consisted exclu- et al. 2008; Huang et al. 2010; Knowles and Kubatko sively of all Suriname individuals), five occurring in 2010). We recognize the caveats of using concate- the B. dorsalis s.l. clade (including one subclade which nated DNA sequence data to generate a species-tree consisted exclusively of B. philippinensis individuals), hypothesis (Degnan and Salter 2005; Kubatko and and one in B. musae (Table 6; fig. 4). Degnan 2007; Kubatko et al. 2011); however, as

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 À235 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

Inter dist – P (Randomly Rosenberg’s Subclades Closest (K2P) distinct) Clade Support 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

there was no conflict among individual gene tree phy- for B. carambolae and B. dorsalis s.l. With respect to logenies, the benefit of using a single multilocus phy- Dataset #5 (fig. 4), subclade ‘b’ within the B. carambo- logeny to confidently delimit species was considered lae clade consists exclusively of every individual col- appropriate (Rokas et al. 2003; Belfiore et al. 2008; lected from Suriname, located in northern South Sanderson et al. 2011). America and constituting part of the invasive range of Species delimitation statistics reveal additional sub- B. carambolae. Bactrocera carambolae was first recorded structure is present within several clades, particularly in South America in 1975 (undescribed at that stage),

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 for flies from the Philippines vs. flies from mainland (Java apple) in Suriname and thought to have been south-east Asia have been demonstrated (Schutze accidentally introduced from south-east Asia (van et al. 2012a). Contrary to the Suriname B. carambolae Sauers-Muller 1991). The emergence of a well-sup- sub-clade, not all individuals from the Philippines ported ‘Suriname subclade’ within the more diverse occur within this group, as six individuals fall outside south-east Asian B. carambolae clade is not unex- subclade ‘d’ (all from Imus; fig. 4) and are unresolved pected given such a recent introduction for which a from other B. dorsalis s.s. and B. papayae; emphasizing ‘genetic bottleneck’ is likely to exist. Similarly, sub- the low resolution within the B. dorsalis s.l. clade as a group ‘d’ in the B. dorsalis s.l. clade consists of all whole. B. philippinensis individuals collected from one of two Four of five measures were used to identify four geographically proximate locations in the Philippines sub-groupings within the B. dorsalis s.l. clade in (Quezon City and Imus) (fig. 4). Philippine flies may Dataset #2 (fig. 1; Clade 6): ‘d’, ‘e’, ‘f’ and ‘g’. For be expected to be genetically divergent from other example, clade ‘e’ consists of four individuals from members of the B. dorsalis s.l. clade considering the each of the three species in the larger B. dorsalis s.l. increased geographic separation between Philippine clade, these being: B. papayae from Penang (Malay- flies relative to those from among mainland south- sia); B. philippinensis (two individuals from Imus, Phil- east Asia and western Indonesian archipelago sites ippines); and B. dorsalis s.s. from San Pa Tong (however, human-mediated movement may limit (northern Thailand). In this case, conspecific repre- this). Indeed, significant isolation-by-distance effects sentatives for each of these species are also repre-

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. of COI and COII genes of Bactrocera species revealed B. clade. The clades identified using the tip-to-root musae to occur within the dorsalis complex clade: sister method provide a basis for further biological research. to B. occipitalis, B. philippinensis, B. dorsalis s.s., In the difficult Bemisia tabaci species complex, for B. papayae and B. carambolae, with B. kandiensis Drew example, the discovery of previously unrecognized & Hancock (a ‘true’ dorsalis complex fly) sister to all of clades through similar analytical approaches has pro- these species (Nakahara and Muraji 2008; Krosch ven a basis for deeper taxonomic and biological et al. 2012a). Furthermore, restriction enzyme analy- research, which is helping to elucidate this equally sis of 25 species of Bactrocera revealed B. musae to difficult group (Boykin et al. 2012). 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 Relationships of outgroup species compared to all other species (B. dorsalis s.s., B. papa- Bactrocera musae and three members of the B. dorsalis yae and B. philippinensis were indistinguishable) (Arm- complex: B. occipitalis, B. opiliae and B. cacuminata strong and Cameron 2000). Indeed it appears the resolve as taxonomically distinct groups and sister to main distinguishing morphological character separat- the ingroup taxa according to all analyses (figs 1–4). ing B. musae from B. dorsalis s.l. is the occasional Bactrocera musae, while taxonomically a member of a absence of the medial longitudinal band on the abdo- different species complex (the B. musae complex), has men for some individuals (Drew 1989); the presence historically demonstrated a very close relationship to of which is typical of dorsalis complex species (Drew dorsalis complex flies. An earlier phylogenetic analysis and Hancock 1994). We therefore recommend further

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 resolv- 1981). Bactrocera opiliae and B. dorsalis s.s. were only sepa- ing its association with the B. dorsalis complex. rable using ecological, physiological and genetic mea- Our results show B. occipitalis (a species occurring in sures, for which colour variation was the only visual sympatry with B. philippinensis in the Philippines) is more difference subsequently observed between the two, with distantly related to the ingroup taxa relative to the Aus- fine-scale differences in ovipositor and egg morphology tralian species B. opiliae and B. cacuminata (figs 1–4). also diagnostic (Drew and Hardy 1981). In contrast, While B. occipitalis has been regarded a closely related B. cacuminata is morphologically distinct, possessing a species of B. dorsalis (Muraji and Nakahara 2002; Naka- characteristic black lanceolate pattern on the mesonotum hara and Muraji 2008; Krosch et al. 2012a), it is morpho- and thereby rendering it easily identifiable from pest logically distinct in having significantly shorter genitalia members of the dorsalis complex (Drew 1989). However, with colour markings distinct as from B. philippinensis as species-level diagnoses are often required for juvenile (Drew and Hancock 1994; Iwahashi 1999). Bactrocera cac- stages (hence adult characters are absent), the genetic uminata and B. opiliae have rarely been directly compared resolution of these non-pest Australian species obtained with pest species of the dorsalis complex as they are here is of practical use for quarantine and plant protec- innocuous and exist in allopatry with respect to the all tion officers. known pests from the complex; however, B. opiliae is at least very similar to B. dorsalis s.s., having been described The unusual case of specimen #413 in 1981 from northern Australian samples and initially regarded as Dacus (Bactrocera) dorsalis due to high mor- We cannot explain the unusual placement of spec- phological similarity with this species (Drew and Hardy imen #413 in any of our phylogenetic reconstruc-

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

Inter Dist – Clade Subclades closest (K2P) P (Randomly Distinct) 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

tions (figs 1–4). For Datasets #2, this specimen identifies as either B. dorsalis s.s. or B. papayae emerges as sister to the entire B. dorsalis s.l. clade, based on existing keys, and examination by Prof. and in Datasets #3, #4 and #5, it is sister to B. oc- R.A.I. Drew confirmed it as one of these two spe- cipitalis. Specimen #413 was collected from Nakhon cies and likely to be B. papayae (pers. comm.). Si Thammarat (southern Thailand) and hence However, our study included only four economi- occurred where B. dorsalis s.s. and B. papayae geo- cally important and three additional out-group spe- graphically abut or overlap according to recorded cies from the B. dorsalis complex, and the inclusion geographic distributions for these species (e.g. of more members from the complex may help to Drew and Hancock 1994). Morphologically, #413 resolve the placement of specimen #413.

and Kitching 1995; Yong 1995; Iwahashi 2000, 2001; Implications and future studies for B. dorsalis taxonomy Muraji and Nakahara 2002; Smith et al. 2003; Tan A number of previous studies have failed to find reso- 2003; Armstrong and Ball 2005; Tan et al. 2011; lution between B. dorsalis s.s, B. papayae and B. philip- Krosch et al. 2012b; Schutze et al. 2012a) that can all pinensis based on molecular (cox1 and microsatellites) be considered to date as supporting, or at least not morphological (wing shape and aedeagus length) or refuting, the possibility that these cryptic species, behavioural (mating and chemical ecology) data namely B. dorsalis s.s., B. papayae and B. philippinensis (Medina et al. 1998; Tan 2000, 2003; Wee and Tan are the same biological species. However, given the 2000a,b, 2005; Krosch et al. 2012b; Schutze et al. risk that severe quarantine and trade implications 2012a). The results of the current study do not contra- could result from changes to the taxonomic delimita- dict this, and in addition, the design here overcomes tion of species relevant to global biosecurity (Boykin the potential weaknesses of earlier studies by sam- et al. 2012), it is critical that there is a high level of pling much larger numbers of individuals across a scientific support for a revision such as that implicated wider geographic range. However, while this body of here for pest species in the B. dorsalis complex. evidence fails to reject the hypothesis, that these three ‘species’ are in fact one, it also fails to distinguish Acknowledgements between this as a result of inappropriate diagnostics or incorrect taxonomy (Drew et al. 2008; Schutze et al. We wish to sincerely thank the following colleagues 2012b). While the former was tested here by use of who assisted us with supplying specimens for this loci that could clearly distinguish other well-recog- study: Mary Finlay-Doney, Richard Bull, Yuvarin nized and closely related biological species within the Boontop, Keng-Hong Tan, Sotero Resilva, Ju-Chun dorsalis complex, that is, B. cacuminata, B. opiliae and Hsu, Alies van Sauers-Muller, Vijay Shanmugam, B. carambolae, as well as B. musae for which a number Hanifah Yahaya, Wigunda Rattanapun and Peter of previous studies have found problematic (White Leach. Vladimir Mencl, Markus Binsteiner and Yuriy 1996; Muraji and Nakahara 2002), there are still some Halytskyy at the New Zealand eScience Infrastructure methodological issues. Given concerted evolution of (NeSi- http://www.nesi.org.nz) were instrumental in the rDNA loci, one might expect these three taxa to the HPC analyses. LMB and KFA were funded by the share a common ITS sequence, but this was not the Tertiary Education Council of New Zealand. The paper case and much of the phylogenetic information in the was produced with research support through CRC CAD and period loci was obscured by the inability to National Plant Biosecurity projects 20115 and 20183. produce true sequence from the many combinations The authors would like to acknowledge the support of of heterozygous alleles. The main source of distinc- the Insect Pest Control Laboratory (Seibersdorf) of the tion, or lack of for B. dorsalis s.s, B. papayae and B. phi- Joint FAO/IAEA Division of Nuclear Techniques in lippinensis, came from two linked mitochondrial loci. Food and Agriculture and the Australian Govern- However, mitochondrial DNA is characterized by ment’s Cooperative Research Centres Program. complex evolutionary dynamics. For example, selec- Acknowledgement also goes to an anonymous tive sweeps that help to differentiate taxa can in the reviewer who helped to significantly improve the case of recently diverged taxa be offset by the homog- manuscript. enizing effect of hybrid introgression (Galtier et al. 2009). Certainly, this has been found in wild popula- References tions of very closely related dipteran species (e.g. Bachtrog et al. 2006), such that any correlation with Allwood AJ, Chinajariyawong A, Drew RAI, Hamacek EL, other taxonomic distinctions are lost. Of course there Hancock DL, Hengsawad C, Jinapin JC, Jirasurat M, may be other nuclear genes that might support the Kong Krong C, Kritsaneepaiboon S, Leong CTS, current taxonomy, and this may become more feasi- Vijaysegaran S, 1999. Host plant records for fruit flies ble to test as genomic data continues to accumulate. (Diptera: Tephritidae) in South-East Asia. Raffles Bullet. Nonetheless, we stress that this work should be exam- Zool. 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Wee SL, Tan KH, 2000a. Interspecific mating of two sibling Figure S2. Dataset 1.2. Phylogenetic reconstruction species of the Bactrocera dorsalis complex in a field cage. based on sequence data for specimens for which ribo- In: Area-wide control of fruit flies and other insect pests. somal DNA (ITS1 and ITS2) were sequenced for Bac- Ed. by Tan KH, Penerbit Universiti Sains Malaysia, trocera spp. in the current study. Penang Malaysia. Figure S3. Dataset 1.3. Phylogenetic reconstruction Wee SL, Tan KH, 2000b. Sexual maturity and intraspecific based on sequence data for specimens for which mating success of two sibling species of the Bactrocera dor- nuclear DNA (CAD gene) was sequenced for Bactrocera – salis complex. Entomol. Exp. Appl. 94, 133 139. spp. in the current study. Wee SL, Tan KH, 2005. Evidence of natural hybridization Figure S4. Dataset 1.4. Phylogenetic reconstruction between two sympatric sibling species of Bactrocera dor- based on sequence data for specimens for which salis complex based on pheromone analysis. J. Chem. nuclear DNA (period gene) was sequenced for Bactro- Ecol. 31, 845–858. cera spp. in the current study. White IM, Elson-Harris MM, 1992. Fruit flies of economic Figure S5. Dataset #2. Phylogenetic reconstruction significance: their identification and bionomics. C.A.B International in association with ACIAR, Wallingford, based on sequence data for specimens for which all Oxon. six loci were sequenced for Bactrocera spp. in the cur- White IM, 1996. Fruit fly taxonomy: recent advances and rent study (236 specimens, 3435 bp alignment). new approaches. In: Fruit fly pests. A world assessment Figure S6. Dataset #3. Phylogenetic reconstruction of their biology and management. Ed. by McPheron BA, based on sequence data for specimens for which all Steck GJ. St Lucie Press, Delray Beach, FL, 253–258. six loci (cox1, nad4-3′, ITS1, ITS2, CAD and per) were Yong HS, 1995. Genetic differentiation and relationships sequenced for Bactrocera spp. in the current study. in five taxa of the Bactrocera dorsalis complex (Insecta: Figure S7. Dataset #4. Phylogenetic reconstruction Diptera: Tephritidae). Bull. Entomol. Res. 85, 431–435. based on sequence data for specimens for which four Yu DJ, Xu L, Nardi F, Li JG, Zhang RJ, 2007. The complete loci were sequenced (cox1, nad4-3′, ITS1 and ITS2) for nucleotide sequence of the mitochondrial genome of the Bactrocera spp. in the current study (236 specimens, oriental fruit fly, Bactrocera dorsalis (Diptera: Tephriti- 2221 bp). dae). Gene 396, 66–74. Figure S8. Dataset #5. Phylogenetic reconstruction based on sequence data for specimens for which at ′ Supporting Information least two of four loci (cox1, nad4-3 , ITS1 and ITS2) were sequenced for Bactrocera spp. in the current Additional Supporting Information may be found in study (315 specimens, 2221 bp). the online version of this article: Table S1. Collection and GenBank accession infor- Figure S1. Dataset #1.1. Bayesian phylogenetic mation for the samples included in this study. reconstruction based on sequence data for specimens for which mtDNA (cox1and nad4-3′) were sequenced for Bactrocera spp. in the current study.