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. carambo- lae 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 clo- sely 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 mono- phyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenberg’s reciprocal monophyly measure, P(AB), Rodrigo’s (P(RD)) and the genea- logical sorting index, gsi. We specifically tested whether there was phylo- genetic support for the four ‘ingroup’ pest species using a data set of multiple individuals sampled from a number of populations. Based on our combined data set, Bactrocera carambolae emerges as a distinct monophy- letic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are unresolved. These data add to the growing body of evidence that B. dor- salis s.s., B. papayae and B. philippinensis are the same biological species, which poses consequences for quarantine, trade and pest management. 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). © 2013 Blackwell Verlag, GmbH 1 Phylogeny of B. dorsalis pest flies L. M. Boykin et al. 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), 2 © 2013 Blackwell Verlag, GmbH L. M. Boykin et al. Phylogeny of B. dorsalis pest flies 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.