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1 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. Boykin 1,2, M. K. Schutze 1,3, M. N. Krosch 1,3, A. Chomic 1,2, T. A. Chapman 1,4, A. Englezou 1,4, K. F. Armstrong 1,2, A. R. Clarke 1,3, D. Hailstones 1,4 & S. L. Cameron 1,3 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. lboykin@mac.com Received: November 12, 2012; accepted: February 18, doi: /jen Abstract Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south-east Asia and the Pacific. Identifying molecular diagnostics has proven problematic for these four taxa, a situation that cofounds biosecurity and quarantine efforts and which may be the result of at least some of these taxa representing the same biological species. We therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide geographic range; sequencing six loci (cox1, nad4-3, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic frameworks for individual loci and concatenated data sets for which we applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, 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. Introduction The Tephritidae (true fruit flies) is one of the most species-rich families within the order Diptera. While non-fruit feeding tephritids are rarely pestiferous (Headrick and Goeden 1998), the frugivorous tephritids contain many genera of major economic importance, including Ceratitis, Rhagoletis and Anastrepha (White and Elson-Harris 1992). Mature female frugivorous tephritids oviposit into fleshy fruits and vegetables, where resultant larvae emerge and feed on the fruit pulp. Production losses and costs of field control are the direct impacts of fruit fly attack, while indirect losses result from the implementation of regulatory controls and lost market opportunities (Clarke et al. 2011). Bactrocera Macquart contains over 500 described species and is the dominant genus of fruit flies in the Asia/Pacific region (Drew 1989, 2004) Blackwell Verlag, GmbH 1

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

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

4 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. Table 1 Collection details of Bactrocera specimens used in the current study Location Country Latitude Longitude Date Species Collection method Brisbane, Queensland Australia S 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 S E 24 December 2009 Bactrocera opiliae ME Lure Territory Quezon City, Diliman Philippines N E 17 December 2009 Bactrocera occipitalis ME Lure 17 December 2009 Bactrocera philippinensis ME Lure Imus, Cavite Philippines N E 20 December 2009 Bactrocera philippinensis ME Lure Taipei City, Tawian China N E 16 March 2010 Bactrocera dorsalis s.s. ME Lure San Pa Tong, Chiang Mai Thailand N E 12 March 2010 Bactrocera dorsalis s.s. ME Lure Chatuchuk, Bangkok Thailand N E December Bactrocera dorsalis s.s. ME Lure 2009 Muang District, Nakhon Thailand N E 25 October 15 Bactrocera papayae ME Lure Si Thammarat November October 15 Bactrocera carambolae ME Lure November 2009 Tikus Pulau, Penang Malaysia N E November Bactrocera papayae ME Lure 2009 Serdang Malaysia N E November 2010 Bactrocera papayae ex Musa acuminata x balbisiana November 2010 Bactrocera carambolae ex Averrhoa carambola Lampung, South Sumatra Indonesia S E May 2009 Bactrocera papayae ME Lure May 2009 Bactrocera carambolae ME Lure Paramaribo Suriname N W August 2009 Bactrocera carambolae ex Averrhoa carambola 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 â (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 amplification was still reliable across taxa. The forward ITS1 primer, ITS7, was designed de novo by KFA; reverse primer ITS6 was taken from Armstrong and Cameron (2000), and ITS2 primers FFA and FFB were modified from Porter and Collins (1991). CAD primers are redesigned after Moulton and Wiegmann (2004, 2007) after comparison with GenBank tephritid sequences. Primers for period are from Barr et al. (2005) and Virgilio et al. (2009). Primer sequences for all loci are given in Table 2. The PCR conditions for ITS1, ITS2 and ND4-2 consisted of 2 ll of template DNA being added to a final volume of 30 ll of reaction mix containing 200 lm of dntps, 200 nm of each forward and reverse primer, 1 9 Accutaq PCR buffer (Sigma Australia), and 0.02U of AccuTaq polymerase. The cycling conditions consisted of an initial denaturation at 94 C for 2 min, followed by 35 cycles of denaturation at 94 C for 15 s, annealing 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 ethidium bromide. All PCR products were sent to AGRF (Australian Genome Research Facility Ltd) in 96-well plates for purification and sequencing. AGRF is Blackwell Verlag, GmbH

5 L. M. Boykin et al. Phylogeny of B. dorsalis pest flies 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 Quality Standard. Australian Genome Research Facility Ltd operates the AB3730xl 96-capillary sequencer for low to high throughput DNA sequencing. Polymerase chain reaction conditions for cox1, CAD and period consisted of 1 ll DNA template in a final volume of 20 ll containing 100 nm each forward and reverse primer 10 ll Go Taq Green enzyme master mix (ProMega, Sydney, Australia) and 7 ll of sterilized water. PCR cycling conditions consisted of an initial denaturation step at 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) or54 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 containing 10X dilution of SYBER Safe (Life Technologies, Victoria, Australia) and run at 80V for 30 min. Sequencing was performed using ABI BigDye â ver. 3 dye terminator chemistry and run on an ABI 3130xl capillary sequencer. Chromatograms were checked and sequence contigs assembled with SEQUENCHER ver 4.2 (Gene Codes Corporation 2004) to produce completed sequences. Analytical strategy The following series of five data sets were analysed to test the phylogenetic signal of different loci and to account for the failure to sequence all loci for all specimens: Datasets # 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 specimens, 3094 bp) Dataset #4 Dataset #2 with CAD and period removed from alignment altogether (235 specimens, 2221 bp) Dataset #5 Specimens for which at least two of the four loci (i.e. excluding CAD and period) were successfully sequenced (313 specimens, 2221 bp) Dataset #1 was designed to allow testing of the variation between loci and to apply a species-tree reconstruction approach (Edwards 2008); however, due to the poor resolution in Datasets # , the additional, concatenation-based data sets were produced (after Gatesy et al. 1999; Gatesy and Baker 2005). Dataset #2 includes a large number of heterozygous sites in the CAD and period gene partitions, which may have resulted in artefactual results. Datasets #3 and #4 are attempts to correct for this potential problem by removing the heterozygous sites either on a site by site basis (#3) or by removing the CAD and period gene partitions entirely (#4). Dataset #5 tests how significant missing partitions were for the inferred phylogeny. Alignment and analysis Sequences for each locus were aligned by eye (protein-coding genes) or using ClustalX (rrna regions) (Thompson et al. 1997). For the ITS 1 and ITS2 data set, indels were treated as missing due to the constraints of Bayesian and RAxML analyses. Hetero Blackwell Verlag, GmbH 5

6 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. 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 (Maddison and Maddison 2003). For each data set, phylogenetic trees were inferred in parallel by both Maximum Likelihood and Bayesian analyses. Likelihood analyses were conducted using RAxML ver implemented on the RAxML BlackBox webserver ( phylobench.vital-it.ch/raxml-bb/index.php) (Stamatakis et al. 2008). Data were analysed with a Gamma model of rate heterogeneity, the proportion of invariable sites was estimated, and for concatenated, multilocus data sets, the alignment was partitioned and branch lengths optimized on a per locus basis. Bayesian analyses were conducted using MRBAYES ver 3.2 (Ronquist et al. 2012) using parallel implementation on the BeSTGRID computer cluster (Jones et al. 2011), or using direct implementation on local desktop computers. Analyses were run for 10 (Datasets #1, 3, 4, 5) or 50 million generations (Dataset #2, due to a longer time for independent runs to converge) with sampling every 1000 generations, partitioned data sets and parameter estimation for each partition unlinked. Each analysis consisted of two independent runs, each utilizing four chains, three cold and one hot. Convergence between runs was monitored by finding a plateau in the likelihood score (standard deviation of split frequencies <0.0015) and the potential scale reduction factor (PSRF) approaching one. Convergence of other parameters within the runs was also checked using TRACER v1.5.4 (Rambaut and Drummond 2010), with ESS values above 200 for each run. The first 12.5% of each run was discarded as burnin for the estimation of consensus topology and the posterior probability of each node. Bayesian & RAxML run files are available from the authors upon request. Phylogenetic trees generated from Datasets #2 and #5 were used as input in the species monophyly and delimitation analyses. Species delimitation was addressed using the standard Kimura two-parameter (K2P) inter-species distance and Rodrigo s P (randomly distinct) (Rodrigo et al. 2008) measure. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenberg s reciprocal monophyly measure, P (AB) (Rosenberg 2007) and the genealogical sorting index (gsi) (Cummings et al. 2008). The species delimitation plugin (Masters et al. 2010) for Geneious (Drummond et al. 2010) was used to calculate Rosenberg s reciprocal monophyly, P (AB) (Rosenberg 2007) and Rodrigo s P (RD) (Rodrigo et al. 2008) measures. The (Cummings et al. 2008) statistic was calculated in R based on the estimated tree and the assignment file that contains user specified groups (see 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 ( permutations across four processors). All of the gsi analyses were run using R on the BeSTGRID computer cluster (Jones et al. 2011). To assess the significance of the gsi P-values, the Bonferroni correction was used. Results Sequence data collection The six loci (cox1, nad4-3, ITS1, ITS2, CAD and period) were successfully amplified for the majority of specimens examined across all species. Success/failure of sequencing individual loci for each specimen, along with their GenBank accession numbers, are shown in Table S1. Of these, nad4-3 was the only one of five additional mt genes (data not shown) trialled in this study that was successfully amplified across the range of species here. Due to the low levels of molecular variation previously found within the dorsalis complex for cox1 (Armstrong and Ball 2005), the additional mitochondrial genes trialled were chosen in an effort to maximize variability based on the previous analyses of dipteran mt genomes (Cameron et al. 2007; Nelson et al. 2012). The trade-off for gene variability is primer reliability, whereby sequence variability at the priming sites causes mismatches and loss of efficacy. Thus, finding only one more variable mt gene, which could be reliably amplified, is not surprising. The nad4-3 gene region was confirmed here to be more variable, having 104 of 577 positions parsimony informative compared to 101 of 642 parsimony informative for cox1. The ribosomal ITS loci each had significant indels (33 84 bp in ITS1, bp in ITS2), but there were few heterozygous sites, consistent with the concerted evolution previously found for these loci (e.g Blackwell Verlag, GmbH

7 L. M. Boykin et al. Phylogeny of B. dorsalis pest flies Eickbush and Eickbush 2007). In contrast, both nuclear protein-coding genes had a large proportion of heterozygous sites; 179 of 528 bp in CAD and 162 of 686 bp in period. These sites unfortunately made up almost all of the variable sites within these two genes, with only 10 of the remaining 349 homozygous sites in CAD and 15 of 524 in period being parsimony informative. Intraspecific variation for each gene is shown in Table 3. Phylogenetic analyses For each data set, Bayesian (BA) and likelihood analyses (ML) yielded similar topologies; however, nodal support was much greater for the set of Bayesian analyses. Of the single linkage group analyses (Datasets # ), only the mitochondrial gene trees (Dataset #1.1) were well resolved, with each species other than B. dorsalis, B. philippinensis, B. papyae and B. carambolae monophyletic with significant nodal supported (BA pp = ; ML bs >70%) (Fig. S1). For the ribosomal ITS loci (Dataset #1.2), several species were monophyletic, for example, B. musae, B. occipitalis, B. opiliae, B. carambolae, whereas B. cacuminata and B. dorsalis s.s., formed paraphyletic combs with respect to other species (Fig. S2). For example, in the Bayesian analysis of Dataset #1.2, B. cacuminata specimens formed 17 of 19 branches in a polytomy with a monophyletic B. opiliae (node support not significant in BA or ML) and a single significantly supported clade which included all B. dorsalis s.s., B. philippinensis, B. papaya and B. carambolae specimens. The trees inferred for each of the nuclear protein-coding genes were almost totally unresolved (Figs S3 4). For CAD Table 3 The average intraspecific distances for each gene shown in % calculated using MEGA Species ITS1 ITS2 ND4 CO1 per CAD Bactrocera tryoni Bactrocera musae Bactrocera cacuminata Bactrocera occipitalis Bactrocera opiliae Bactrocera carambolae Bactrocera dorsalis (sensu stricto) Bactrocera philippinensis Bactrocera papayae Bactrocera dorsalis (sensu lato) (Dataset #1.3), only B. musae (BA & ML) and B. cacuminata (ML only) were monophyletic whereas for period (Dataset #1.4), B. musae (BA & ML), B. cacuminata (BA only) and B. opiliae (BA only) were monophyletic. The majority of specimens of the remaining species formed unresolved combs. Due to the poor resolution across these four data sets, species-tree reconstruction based on individual gene trees was not attempted. Analyses of concatenated data sets were conducted to determine whether larger data sets would be better resolved and display higher nodal support than was achieved analysing each linkage group separately (Datasets # ). Further, due to the high proportion of heterozygous sites within CAD and period, and the significant number of individuals for which one or more genes failed to amplify/sequence (57 specimens, approximately 25%), a series of different concatenation data sets were analysed to determine whether either factor resulted in artefactual relationships. The same species boundaries were inferred for all four concatenated data sets, and the interspecies relationships were also quite constant. The heterozygous positions within CAD and period had a limited effect on inferred species relationships, as the only difference was in the position of a single specimen, Bd413 an unidentifiable member of the dorsalisgroup complex. This specimen was sister to all the dorsalis-group flies with inclusion of these gene regions (#2-BA) or the sister-group of B. occipitalis with their exclusion (#2-ML, #3-#5-BA & ML) (fig. 1; Figs S5 7). Similarly, the inclusion of specimens for which up to half of the loci were missing (#5) did not result in a different topology from those inferred from specimens where all genes were present (#3 #4). Below the species level, there was significant variability in topology and nodal support across the different concatenated data sets with few clades larger than 2 3 specimens shared between analyses. The only notable exception is the clade containing B. carambolae specimens from Paramaribo (Suriname, South America). This invasive population forms a strongly supported, monophyletic clade to the exclusion of the SE Asian specimens of B. carambolae in Datasets #1.1, 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 Suriname reflects a genetic bottleneck associated with its 2013 Blackwell Verlag, GmbH 7

8 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. Figure 1 Dataset #2. Phylogenetic reconstruction based on sequence data for specimens for which all six loci were sequenced for Bactrocera spp. in the current study (236 specimens, 3435 bp alignment). Bayesian posterior probabilities are listed above each branch, maximum likelihood bootstrap values below. For clarity only supports for backbone nodes are shown; in cases where actual nodal support is absent, posterior probability support values are >0.5 except for those marked with an asterisk (>0.95). All nodes <0.5 are collapsed. Results of clade monophyly statistics are shown as boxes (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 identifications of individual specimens within the dorsalis complex ingroup have been colour coded [i.e. B. dorsalis s.s. (purple), B. papayae (dark blue), B. philippinensis (light blue) and B. carambolae (green)]. See supplementary files for all nodal supports and all individual specimen data. establishment in South America and/or that the source population for this invasion was not present in this study. The combined phylogeny thus supports the monophyly of the dorsalis-group and sister-group relationships between B. cacuminata and B. opiliae and between B. dorsalis s.l. and B. carambolae. The monophyly of B. musae, B. occipitalis, B. opiliae, B. cacuminata and B. carambolae is each very strongly supported (BA pp = 1.0 for each), whereas the monophyly of the remaining B. dorsalis s.l. (B. dorsalis/papayae/philippinensis) is slightly weaker (pp = 0.93). Bactrocera papayae and B. philippinensis were never monophyletic and were essentially indistinguishable from B. dorsalis s.s. in the tree. Specimens morphologically identified as B. papayae and B. philippinensis occurred in, respectively, 8 17 and 2 26 different subclades of the B. dorsalis clade/grade depending on the combination of data set and inference method. Species delimitation analysis and subclade groupings The use of species delimitation analyses within our phylogenetic framework revealed a number of statistically well-resolved groupings for (i) each of the outgroup species, (ii) B. carambolae and (iii) B. dorsalis s.l. (B. dorsalis/papayae/philippinensis) (Tables 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 (for which all individuals are represented by at least two loci) (fig. 4). Notably, B. carambolae resolves as a taxonomically distinct clade, rating 5/5 for all analyses in both Datasets #2 and #5, while unambiguous species (B. occipitalis, B. cacuminata and B. opiliae) achieve 4/ Blackwell Verlag, GmbH

9 L. M. Boykin et al. Phylogeny of B. dorsalis pest flies Table 4 The hypothesized species of the Bactrocera dorsalis species complex were tested for species distinctiveness as measured by the Geneious species delimitation plugin (Masters et al. 2010) and the genealogical sorting index (gsi) (Cummings et al.2008). The species delimitation plugin generates: average pairwise tree distance between members of the group of interest and its sister taxa (K2Pdistance), P (Randomly Distinct), Clade Support: Bayesian posterior probability (PP), and Rosenberg s P AB : Reciprocal monophyly and lastly, the gsi statistic and associated P-value are included. Bold Values indicates significance, and this was determined by: >1% difference (K2P)/>0.05 [P (Randomly Distinct)]/>0.80 (PP)/>0.008 (gsi). Dataset 2 contained a concatenation of all specimens for which all six loci were successfully sequenced 235 specimens, 3435 bp alignment. Dataset 5 consisted of specimens for which at least two of the four loci (i.e. excluding CAD and period) were successfully sequenced (313 specimens, 2221 bp) Inter dist - Closest (K2P) P (randomly distinct) Clade support Rosenberg s P (AB) gsi P-value Dataset 2 Clade 1: musae Bd51/ E E-04 Clade 2: occipitalis 739/ E E-04 Clade 3: cacuminata 231/ E E-04 Clade 4: opiliae 1080/ E E-04 Clade 5: carambolae 1111/ E E-04 Clade 6: dorsalis 818/ E E-04 Dataset 5 Clade 1: musae Bd51/ E E-04 Clade 2: occipitalis 739/ E E-04 Clade 3: cacuminata 231/ E E-04 Clade 4: opiliae 1080/ E E-04 Clade 5: carambolae 1111/ E E-04 Clade 6: dorsalis 818/ E E-04 in at least one of the datasets; a result which we believe lends greater support to the ongoing specific status of B. carambolae. Species delimitation statistical analyses undertaken for Dataset #2 revealed considerable support for each of the six a priori defined groups: B. musae (5/5 statistically significant), B. occipitalis (4/5), B. cacuminata (4/5), B. opiliae (4/5), B. carambolae (5/5) and B. dorsalis s.l. (i.e. B. dorsalis/papayae/philippinensis) (4/ 5) (Table 4). Tip-to-root analysis (examining all resolved clades) demonstrated a limited number of subclades which were statistically significant for at least four of the five statistics applied, with three subclades resolved within B. carambolae and four in the B. dorsalis s.l. clade (Table 5; fig. 1). A priori groups and subclade support increased following analysis of Dataset #5, with all five statistical analyses significant for four of the a priori defined clades (B. musae, B. occipitalis, B. opiliae and B. carambolae) and 4/5 for the remaining two (B. cacuminata and B. dorsalis s.l.). Meanwhile, tip-to-root analysis revealed nine subclades to have 4/5 support measures statistically significant, with three occurring in the B. carambolae clade (one of which consisted exclusively of all Suriname individuals), five occurring in the B. dorsalis s.l. clade (including one subclade which consisted exclusively of B. philippinensis individuals), and one in B. musae (Table 6; fig. 4). Discussion This study represents the most comprehensive phylogenetic analysis undertaken to-date for four pestiferous and morphologically cryptic members of the B. dorsalis species complex. The study incorporates individuals collected from a broad geographic distribution and likely represents a range of intraspecific populations for these species. Six independent loci have been targeted and subsequently examined using a range of analyses, with a clear signal emerging: B. carambolae is a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis form a single sister clade to B. carambolae. Phylogenetic analyses and species delimitation The individual gene trees in this study were unresolved and therefore prevented the use of the speciestree software (e.g.,ane et al. 2007; Liu 2008; Liu et al. 2009; Kubatko 2009; Kubatko et al. 2009; Heled and Drummond 2010; Than and Nakhleh 2009; Than et al. 2008; Huang et al. 2010; Knowles and Kubatko 2010). We recognize the caveats of using concatenated DNA sequence data to generate a species-tree hypothesis (Degnan and Salter 2005; Kubatko and Degnan 2007; Kubatko et al. 2011); however, as 2013 Blackwell Verlag, GmbH 9

10 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. Table 5 Tip to root approach (Boykin et al. 2012) for species delimitation of the Bactrocera dorsalis species complex utilizing Dataset 2 (Concatenation of all specimens for which all six loci were successfully sequenced 235 specimens, 3435 bp alignment). See Table 3 for full description of the species delimitation statistics and fig. 1 for a visual representation of these results Subclades Inter dist Closest (K2P) P (Randomly distinct) Clade Support Rosenberg s P (AB) gsi P-value Clade 1: musae Bd51/67 Bd E Bd E Bd E E-04 Clade 2: occipitalis 739/800 Bd E E-04 Bd E E-04 Bd E E-04 Clade 3: cacuminata 231/244 Bd E E-04 Bd E Bd E Clade 4: opiliae 1080/1082 No additional subclades to test Clade 5: carambolae 1111/189 Bd E E-04 Bd E E-04 Bd E E-04 Bd E Bd Bd E-04 Bd E Bd E Clade 6: dorsalis 818/399 Bd E E-04 Bd E Bd E Bd E Bd E E-04 Bd E-04 Bd E-04 Bd E E-04 Bd E E-04 Bd E E-04 Bd E E-04 Bd E E-04 Bd E E-04 Bd E Bd E Bd E Bd E Bd E Bd E Bd E Bd E Bd E E-04 there was no conflict among individual gene tree phylogenies, the benefit of using a single multilocus phylogeny to confidently delimit species was considered appropriate (Rokas et al. 2003; Belfiore et al. 2008; Sanderson et al. 2011). Species delimitation statistics reveal additional substructure is present within several clades, particularly for B. carambolae and B. dorsalis s.l. With respect to Dataset #5 (fig. 4), subclade b within the B. carambolae clade consists exclusively of every individual collected from Suriname, located in northern South America and constituting part of the invasive range of B. carambolae. Bactrocera carambolae was first recorded in South America in 1975 (undescribed at that stage), Blackwell Verlag, GmbH

11 L. M. Boykin et al. Phylogeny of B. dorsalis pest flies Figure 2 Dataset #3. Phylogenetic reconstruction based on sequence data for specimens for which all six loci (cox1, nad4-3, ITS1, ITS2, CAD and per) were sequenced for Bactrocera spp. in the current study. Ambiguous sites removed from CAD and per alignments (236 specimens, 3094 bp). Node supports and tree annotation as per fig. 1. where it was first reared from Syzygium samarangense (Java apple) in Suriname and thought to have been accidentally introduced from south-east Asia (van Sauers-Muller 1991). The emergence of a well-supported Suriname subclade within the more diverse south-east Asian B. carambolae clade is not unexpected given such a recent introduction for which a genetic bottleneck is likely to exist. Similarly, subgroup d in the B. dorsalis s.l. clade consists of all B. philippinensis individuals collected from one of two geographically proximate locations in the Philippines (Quezon City and Imus) (fig. 4). Philippine flies may be expected to be genetically divergent from other members of the B. dorsalis s.l. clade considering the increased geographic separation between Philippine flies relative to those from among mainland southeast Asia and western Indonesian archipelago sites (however, human-mediated movement may limit this). Indeed, significant isolation-by-distance effects for flies from the Philippines vs. flies from mainland south-east Asia have been demonstrated (Schutze et al. 2012a). Contrary to the Suriname B. carambolae sub-clade, not all individuals from the Philippines occur within this group, as six individuals fall outside subclade d (all from Imus; fig. 4) and are unresolved from other B. dorsalis s.s. and B. papayae; emphasizing the low resolution within the B. dorsalis s.l. clade as a whole. Four of five measures were used to identify four sub-groupings within the B. dorsalis s.l. clade in Dataset #2 (fig. 1; Clade 6): d, e, f and g. For example, clade e consists of four individuals from each of the three species in the larger B. dorsalis s.l. clade, these being: B. papayae from Penang (Malaysia); B. philippinensis (two individuals from Imus, Philippines); and B. dorsalis s.s. from San Pa Tong (northern Thailand). In this case, conspecific representatives for each of these species are also repre Blackwell Verlag, GmbH 11

12 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. Figure 3 Dataset #4. Phylogenetic reconstruction based on sequence data for specimens for which four loci were sequenced (cox1, nad4-3, ITS1 and ITS2) for Bactrocera spp. in the current study (236 specimens, 2221 bp). Node supports and tree annotation as per fig. 1. sented throughout the remainder of the B. dorsalis s.l. clade. The clades identified using the tip-to-root method provide a basis for further biological research. In the difficult Bemisia tabaci species complex, for example, the discovery of previously unrecognized clades through similar analytical approaches has proven a basis for deeper taxonomic and biological research, which is helping to elucidate this equally difficult group (Boykin et al. 2012). Relationships of outgroup species Bactrocera musae and three members of the B. dorsalis complex: B. occipitalis, B. opiliae and B. cacuminata resolve as taxonomically distinct groups and sister to the ingroup taxa according to all analyses (figs 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 analysis of 25 species of Bactrocera revealed B. musae to exhibit the least degree of differentiation between it and B. dorsalis s.s., B. papayae and B. philippinensis (and a non-dorsalis fly, B. curvipennis (Froggatt)) as compared to all other species (B. dorsalis s.s., B. papayae and B. philippinensis were indistinguishable) (Armstrong and Cameron 2000). Indeed it appears the main distinguishing morphological character separating B. musae from B. dorsalis s.l. is the occasional absence of the medial longitudinal band on the abdomen for some individuals (Drew 1989); the presence of which is typical of dorsalis complex species (Drew and Hancock 1994). We therefore recommend further Blackwell Verlag, GmbH

13 L. M. Boykin et al. Phylogeny of B. dorsalis pest flies Figure 4 Dataset #5. Phylogenetic reconstruction based on sequence data for specimens for which at least two of four loci (cox1, nad4-3, ITS1 and ITS2) were sequenced for Bactrocera spp. in the current study (315 specimens, 2221 bp). Node supports and tree annotation as per fig. 1. work on B. musae be undertaken towards fully resolving its association with the B. dorsalis complex. Our results show B. occipitalis (a species occurring in sympatry with B. philippinensis in the Philippines) is more distantly related to the ingroup taxa relative to the Australian species B. opiliae and B. cacuminata (figs 1 4). While B. occipitalis has been regarded a closely related species of B. dorsalis (Muraji and Nakahara 2002; Nakahara and Muraji 2008; Krosch et al. 2012a), it is morphologically distinct in having significantly shorter genitalia with colour markings distinct as from B. philippinensis (Drew and Hancock 1994; Iwahashi 1999). Bactrocera cacuminata and B. opiliae have rarely been directly compared with pest species of the dorsalis complex as they are innocuous and exist in allopatry with respect to the all known pests from the complex; however, B. opiliae is at least very similar to B. dorsalis s.s., having been described in 1981 from northern Australian samples and initially regarded as Dacus (Bactrocera) dorsalis due to high morphological similarity with this species (Drew and Hardy 1981). Bactrocera opiliae and B. dorsalis s.s. were only separable using ecological, physiological and genetic measures, for which colour variation was the only visual difference subsequently observed between the two, with fine-scale differences in ovipositor and egg morphology also diagnostic (Drew and Hardy 1981). In contrast, B. cacuminata is morphologically distinct, possessing a characteristic black lanceolate pattern on the mesonotum and thereby rendering it easily identifiable from pest members of the dorsalis complex (Drew 1989). However, as species-level diagnoses are often required for juvenile stages (hence adult characters are absent), the genetic resolution of these non-pest Australian species obtained here is of practical use for quarantine and plant protection officers. The unusual case of specimen #413 We cannot explain the unusual placement of specimen #413 in any of our phylogenetic reconstruc Blackwell Verlag, GmbH 13