DNA Barcodes for Biosecurity: Invasive Species Identification Author(s): K. F. Armstrong and S. L. Ball Source: Philosophical Transactions: Biological Sciences, Vol. 360, No. 1462, DNA Barcoding of Life (Oct. 29, 2005), pp. 1813-1823 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/30040928 Accessed: 06/05/2009 00:44

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http://www.jstor.org PHILOSOPHICAL TRANSACTIONS -oF Phil. Trans.R. Soc. B (2005) 360, 1813-1823 THE ROYAL doi:10.1098/rstb.2005.1713 SOCIETY B Publishedonline 15 September2005

DNA barcodes for biosecurity: invasive species identification K. F. Armstrong* and S. L. Ball National Centrefor Advanced Bio-ProtectionTechnologies, PO Box 84, Lincoln University,Canterbury, New Zealand Biosecurity encompasses protecting against any risk through 'biological harm', not least being the economic impact from the spread of pest . Molecular diagnostic tools provide valuable support for the rapid and accurate identification of morphologically indistinct alien species. However, these tools currently lack standardization. They are not conducive to adaptation by multiple sectors or countries, or to coping with changing pest priorities. The data presented here identifies DNA barcodes as a very promising opportunity to address this. DNA of tussock and fruit fly specimens intercepted at the New Zealand border over the last decade were reanalysed using the coxi sequence barcode approach. Species identifications were compared with the historical dataset obtained by PCR-RFLP of nuclear rDNA. There was 90 and 96% agreement between the methods for these species, respectively. Improvements included previous tussock moth 'unknowns' being placed to family, genera or species and further resolution within fruit fly species complexes. The analyses highlight several advantages of DNA barcodes, especially their adaptability and predictive value. This approach is a realistic platform on which to build a much more flexible system, with the potential to be adopted globally for the rapid and accurate identification of invasive alien species. Keywords: mitochondrial DNA; cytochrome oxidase subunit I; COI; molecular diagnostics; quarantine; insects

1. INTRODUCTION New Zealand is very sensitive to the potential Biosecurity is emerging as one of the most important impact that such pests could have on the primary issues facing the international community. Tradition- industries and natural ecosystems that underpin its ally it has been associated with risks from infectious economy. This is apparent by internationally having diseases, living modified organisms and biological the most comprehensive biosecurity approach based weapons, but in the very broadest sense it encompasses on its Biosecurity Act of 1993 (Meyerson & Reaser minimizing risk through 'biological harm' (Meyerson 2002). Nevertheless, one of the main weaknesses et al. 2002). Not least is the economic risk from invasive recognized with this is the difficulty to predict new alien species (IAS) that threaten ecosystem stability, IAS which limits the implementation of appropriate producer livelihoods and consumer confidence (Cock risk management strategies (Parliamentary Commis- et al. 2003). That risk is facilitated by the movement of sioner for the Environment 2000). A critical aspect of exotic species around the world through increasing prediction, and also monitoring, is the ability to international tourism and trade, and is influenced by accurately identify any intercepted specimen to the changes in climate and land use. Of those species species-level. This is essential for support of early introduced to novel environments an estimated one detection systems. It is also a means of collecting percent is anticipated to become invasive and with complete and accurate data about which species are serious economic impacts (Williamson 1996). An actually entering for the assessment of risk. However, example relevant to the following discussion is provided development of a comprehensive identification capa- by Japan, where on average four exotic species bility is hindered by the growing imbalance worldwide have become established each year for the last 50 years. between diagnostic needs and the availability of trained Of these 74% were economic pests, but just two, the taxonomic experts. Long-term research strategies are Oriental fruit fly, Bactroceradorsalis, and the melon fly, also required to address the deficiencies in existing Bactroceracucurbitae, have cost equivalent to more than taxonomic keys to deal with morphologically indistinct EUR200 million to eradicate (Kiritani 1998). Also in immature life stages, cryptic species and damaged the USA, the potential cumulative economic losses specimens. For a few of the most economically from Asian gypsy moth (Lymantria dispar) and nun significant and global pests morphotaxonomic keys moth (Lymantria monacha) establishment between are now supported by molecular diagnostic technology, 1990 and 2004 were estimated in the range equivalent e.g. fruit flies (Tephritidae; Armstrong et al. 1997a), to EUR28-46 billion (Cock et al. 2003). tussock (Lymantriidae; Armstrong et al. 2003), leafroller moths (Tortricidae; Dugdale et al. 2002) and Toda & * thrips (Thripidae; Komazaki 2002). However, Author for correspondence ([email protected]). such methods are developed on an ad hoc and often One contribution of 18 to a Theme Issue 'DNA barcoding of life'. reactive basis with immediate local needs in mind and

1813 c 2005The RoyalSociety 1814 K. F. Armstrong & S. L. Ball DNA barcodesfor biosecurity little or no coordination between institutes, regions or Methods designed out of necessity for a predeter- taxa. The following discussion illustrates how DNA mined range of taxa also undermines the potential to barcodes can provide a very realistic, practical and cope with the unpredicted arrivals. The entry of flexible framework for species identification in the species found on inanimate objects, such as vehicles context of biosecurity. Specific examples are given (Armstrong et al. 2003) or solid wood packaging from a New Zealand perspective for two global (Wittenburg & Cock 2001), are more difficult to economically significant agricultural pest insect predict compared to those that are closely associated groups, the fruit flies and tussock moths. However with their host material such as fruit flies (Armstrong the principles could equally apply to other taxa, sectors et al. 1997a). Others may not be predicted because they and countries. We propose that adoption of the method are innocuous or minor pests in their native range. would enable the international IAS diagnostic com- However, they can become a significant pest in a new munity to better cope with changing and localized environment with no specific natural enemies or species priorities, to capitalize on the efforts of others competition. This has been a significant issue in, for and to address the international standardization of example, North America where of the six most technologies that has been recommended for a more devastating forestry pests introduced only the Euro- effective and coherent diagnostic effort (e.g. Klijn pean strain of the gypsy moth was known as a pest in its 2004). indigenous range (Cock et al. 2003). Similarly in New Zealand the unanticipated arrival in 1999 of Teia anartoides (: Lymantrii- 2. LIMITSOF PREVIOUS MOLECULAR dae) from Australia, where it is a minor localized pest, DIAGNOSTICS FOR BIOSECURITY was predicted to have a significant impact and so an eradication was initiated. The cost to New A variety of stand-alone molecular methods exist for programme Zealand if it is not eradicated is to be identification of regulated pest species. Immunological anticipated to EUR33-205 million over the next 20 (e.g. Symondson et al. 1999; Trowell et al. 2000) or equivalent 3 A more flexible protein-based (e.g. Miles 1979; Soares et al. 2000) years (Case study 2002). significantly methods are not widely used being highly taxon- and anticipatory diagnostic system is required to specific, difficult to adapt, vulnerable to environmental provide timely support for management of these events. factors and reliant on good quality, fresh tissue. The majority of molecular diagnostic methods are instead (b) Diverse methodologies polymerase chain reaction (PCR) based DNA analyses There has been little or no consistency in the PCR- which are not limited in these ways. However, a decade based technologies used. A number of different or more into the application of molecular diagnostics methods have been designed, such as species-specific for biosecurity and other identification purposes, two PCR (e.g. Kohlmayr et al. 2002; Lu et al. 2002; Liu major hurdles exist that preclude the building of a 2004), PCR restriction fragment length polymorphism smarter, more co-ordinated and anticipatory IAS (PCR-RFLP; e.g. Armstrong et al. 1997a,b; Brunner identification system. et al. 2002), multiplex PCR (Kumar et al. 1999; Kengne et al. 2001), DNA sequencing (e.g. Brown (a) Finite range of taxa et al. 2002; Dugdale et al. 2002) and oligonucleotide The number of taxa that can be accommodated by any array analyses (Naeole & Haymer 2003). Even the one method is predetermined and limited to various idiosyncrasies of similar methods means that they are degrees from one to around fifty species. Due initially rarely directly transferable between laboratories or for to the relative expense of method development, and for use with different taxa and data cannot be shared. other pragmatic reasons, the approach has been to There is also no consistency in the gene or parts of develop tests for those taxa predicted to be the most genes used to identify species. For insect identification likely invaders, i.e. for species known to be invasive and examples of mitochondrial DNA (mtDNA) used are spreading elsewhere (Cock et al. 2003). Examples are cytochrome oxidase subunit I (coxl; e.g. Brunner et al. for species within the fruit flies (Haymer et al. 1994; 2002; Kohlmayr et al. 2002), non-transcribed region Armstrong et al. 1997a), tussock moths (Armstrong between coxl and tRNAleu (Stauffer 1997), 16S rDNA et al. 2003), leafroller moths (Dugdale et al. 2002) and (Brown et al. 2002) and cytochrome B (Khemakhem thrips (Toda & Komazaki 2002). Taxa within these and et al. 2001, 2002). For nuclear gene regions the rDNA others are prioritized differently amongst countries internal transcribed spacer regions ITS1 plus ITS2 according to matching host and climate, access to (Armstrong et al. 1997a,b), ITS1 only (Chiu et al. existing pathways of entry and anticipated economic 2001) and ITS2 only (Pfeifer et al. 1995) have been impact. Modification of protocols to accommodate used, as well as an actin gene intron (He & Haymer additional species as the need arises, or to adapt for 1997) and randomly amplified polymorphic DNA different sectors or countries with differing taxa (RAPD; Kengne et al. 2001). To a certain extent priorities may not be practical. This can be especially choice is dependent on the taxonomic range involved difficult when diagnosis is reliant on a few single and appropriate evolutionary rate of the gene, but there nucleotide polymorphisms that form the basis of may also be elements of convenience regarding primers primers required for specific PCR or for polymorphic available and in-house experience. The consequences restriction sites or in the design of oligonucleotide of this disparity have been recognized to be an issue of arrays. Finding additional informative polymorphisms much broader dimensions across the field of phyloge- within such methods can be problematic. netics (Caterino et al. 2000).

Phil. Trans.R. Soc. B (2005) DNA barcodesfor biosecurity K. F. Armstrong & S. L. Ball 1815

In essence therefore, molecular diagnostic tests, previously designed PCR-RFLP and specific-PCR which are more and more being accepted as an methods. To test for improvement on the previous inevitable and essential component of the biosecurity methods, all specimens that were previously unidentifi- toolbox (Martin et al. 2000), remain very limited. They able by those methods were also included. The latter are not flexible enough to accommodate the growing were either because offailure to PCR amplify the 1.5 kb number of IAS, to identify unanticipated arrivals or to ITS rDNA region for subsequent RFLP analysis, capitalize on the efforts of others that collectively work ambiguous RFLP patterns, RFLP patterns that were across a very diverse taxonomic range. not recognized amongst those established for the target list of species or failure to amplify a nested species- PCR 3. IDENTIFICATIONUSING DNA BARCODES specific product. DNA previously obtained for border specimens and The emergence of DNA barcoding as a means of specimens of the morphologically identified species species identification (Hebert et al. 2003a) has the contributing to the 'profile' data (see Electronic potential to address the shortcomings outlined above. Appendix, table 3), was PCR amplified and sequenced In contrast to the molecular diagnostic methods was for the coxl Folmer region according to established available to date, DNA barcoding proposes to use procedure (Hebert et al. 2003a,b). The only variation information within a single gene region common across was use of the Expand High Fidelity (Roche Diag- all taxa and to access that information by DNA nostics) polymerase system instead of Taqin the PCR. sequencing under universal conditions. These features For use with the tussock moth profile data set, lend it well to standardization across species and sequence data for other Lepidopteran species was also laboratories, thus providing a platform for global included from the Barcode of Life Database (BOLD) exchange of homologous data and capitalizing on the and GenBank (see Electronic Appendix, table 2). efforts of others to build a more flexible system. Sequences were aligned and truncated to a ca 650 bp There is a growing literature demonstrating that coxl homologous region using SEQUENCHER(Gene Codes will reliably discriminate a diverse range of taxa at the Corp.). A profile neighbour-joining (NJ) tree of species level (e.g. Hebert et al. 2003a,b; Hogg & Hebert Kimura-2-parameter (K2P) distances was constructed 2004; Whiteman et al. 2004; Ball et al. 2005; Shander & from the sequence data using MEGAv.2.1. The K2P Willassen 2005). This gene, along with 16S, 18S, and model provides a suitable metric model when genetic elongation factor- la genes, has also been encouraged distances are low (Nei & Kumar 2000) as anticipated as a standard target for insect phylogenetics (Caterino with many of the species here. The simple NJ algorithm et al. 2000). Of enormous benefit to the international was considered at this juncture to be an appropriate diagnostics community is the very large amount of coxi starting point for the analyses, given that specimen sequence information that already exists in the identification is based entirely on sequence similarity, literature for a diverse range of insect taxa. However rather than on strictly phylogenetic relationships, and from a biosecurity perspective, where accuracy is the speed of analysis that is necessary for biosecurity critical, the robustness of identifications and genetic diagnostic purposes. limits of this gene need to be established. Potential complications arising from discordance with morpho- (a) Case study 1: tussock moths logically established species limits, species sequence Background: around 30 species of tussock moths have overlap or divergence across intra-specific geographic been determined to be unwanted under the ranges also need to be examined. Even so, if the organisms Biosecurity Act (MAF Biosecurity, Unwanted concept can be verified to operational agencies as Organ- isms Based on their sound, it needs to be demonstrated that DNA Register). pest status, polyphagous nature and invasive potential, seven northern hemi- sequencing is a practical and rapid alternative to the sphere species are considered to present the greatest current technologically more accessible methods. risk to New Zealand forestry. These are Asian and European gypsy moth (L. dispar), nun moth 4. TESTING COX1 DNA BARCODES FOR EXOTIC (L. monacha), pink or rose gypsy moth (L. mathura), INSECT IDENTIFICATION vapourer or rusty tussock moth ( antiqua), white To examine the suitability of coxl sequence as a marked tussock moth (Orgyia leucostigma),Douglas fir diagnostic tool for biosecurity, two datasets that exist tussock moth (Orgyia pseudotsugata)and white spotted for molecular identifications of specimens intercepted tussock moth (Orgyia thyellinax; Armstrong et al. at the New Zealand border over the past decade have 2003). Specific life history strategies, such as long been revisited. The datasets are for the tussock moths overwintering phases in the egg stage and indiscrimi- (Lepidoptera: Lymantriidae) and fruit flies (Diptera: nate oviposition on inanimate surfaces, such as Tephritidae). Several species within these groups are containers, ship superstructures, forestry equipment considered internationally to be significant economic and used vehicles could enable them to arrive in New pests. They are not established in New Zealand, but are Zealand. considered high risk to New Zealand's forestry and Of these, the Asian gypsy moth is particularly well horticultural industries, respectively. DNA from a equipped to invade as the females are capable of random selection of specimens that have been inter- sustained flight (Keena et al. 2001) and are attracted to cepted at the border was used. To test accuracy of the lights of vehicles and ports (Wallner et al. 1995). The barcode method, identifications so determined were species also has variable occlusion cues enabling compared to those that, to all intents and purposes, had hatching to coincide with favourable environmental been successfully identified to species using the conditions (Walsh 1993) and has a proven invasive

Phil. Trans.R. Soc. B (2005) 1816 K. F. Armstrong & S. L. Ball DNA barcodesfor biosecurity

Table 1. Comparisonsof previousmolecular species identifications with DNA barcodeidentifications for New Zealandborder specimens. taxon total n previouslyidentified by RFLP or specificPCR previouslyunidentifiable by RFLP n (% of total) disagreement(% of identified) n (% of total) identifiedby barcoding(% of unidentified) barcodeversus barcodeversus RFLP sspb tussockmoth 57 49 (86.0%) 1 (2.0%) 4 (8.2%) 8 (7.1%) 5 (62.5%) fruitfly 81 79a (97.5%) 2 (2.5%) 1 (1.2%) 2 (<0.1%) 2 (100%) a Includes10 caseswhere common RFLP patterns indicated 2-3 possiblespecies; final identifications determined by speciesand geographic originof thehost fruit. b Species-specificprimers. ability (Savotikov et al. 1995). Consequently the their prior morphological identification, i.e. in all cases tussock moth egg masses commonly intercepted on test sequences grouped more closely with their imported used vehicles had previously been assumed conspecifics than with any other species. This result is by quarantine officers to be Asian gypsy moth. consistent with previous DNA barcoding studies of Unfortunately, while this species can be readily Lepidoptera (Hebert et al. 2003a). Testing this further distinguished from the others based on adult as a biosecurity tool, new data are presented here for morphology the early life stages cannot. This is specimens intercepted at the border that had previously compounded by their arrival on inanimate objects been identified to species, or otherwise, by PCR-RFLP with unknown origin, providing limited host or or specific PCR. geographic information to indicate their likely identity. Results: of the 57 border interception specimens Consequently there has been no accurate record of analysed here, 49 had previously been identified to which species actually arrive in New Zealand. This has species by RFLP or by L. dispar-specific primers. Eight serious implications for the suitability of the post- others had been unidentifiable (table 1). Of the latter, border quarantine systems that are in place. five could be placed with confidence (80-99% boot- To improve interception records, a molecular strap support) by their coxl sequence to a genus or diagnostic method was developed based on PCR- species within the profile tree (table 1). This improved RFLP of ca 1.5 kb nuclear ribosomal DNA (rDNA) the previous RFLP identification rate from 86% incorporating partial 18S plus complete ITS1, 5.8S to 93%. and ITS2 regions. This has since been used to routinely Interestingly four of the five additional identifi- identify the egg masses intercepted on imported used cations were not tussock moths. Specimen MAF812 vehicles (Armstrong et al. 2003). Samples also arrive in associated closely with two species of Spodopteraand very poor condition with potentially degraded DNA. MAF773 with two species of Clostera (Electronic Although not a risk in themselves, their accurate Appendix, figure 1). While these appear to be the identification is necessary for a comprehensive risk most likely congeners, the interspecifc divergences analysis. Consequently a species-specific PCR method suggest that the actual species are not represented in was designed to supplement the main RFLP diagnoses the profile dataset, e.g. 7.3% between Clostera albos- for specimens failing to amplify the 1.5 kb nuclear tigma and Clostera apicalis is of the same order as rDNA region (unpublished). Specific PCR primers between MAF773 and each of those species (6.4% and were designed to amplify a 150-300 bp nested region of 8.4%, respectively). A third specimen, MAF775, the ITS1 for the Asian species, L. dispar, L. mathura, previously unamplifiable for subsequent RFLP or by L. monacha and O. thyellina. Used together with a L. dispar-specific primers, was amplified with the control amplicon of 350 bp of the 18S rDNA, positive universal species coxl primers. The sequence identified amplification indicated a positive identification. Incor- it as possibly a species of Dasychira, although there was porating these original methods into operational only one species in the profile dataset (Dasychira procedures the large majority of specimens were dorsipennata) representing this genus (Electronic confirmed to be gypsy moth, plus two other high risk Appendix, figure 1). A fourth specimen, MAF913 species, L. monacha and O. thyellina. Of concern produced an ambiguous RFLP haplotype, but was however were the specimens that could not be identified here as Hyphantria cunea (Electronic Appen- identified. Some failed to PCR amplify. Others dix, figure 1) with a mean sequence divergence of 1.4% produced novel RFLP patterns for which no species from three H. cunea profile sequences. A fifth speci- or even genus could be inferred. men, MAF891, appears to be a divergent form of Recently, coxl barcodes have been demonstrated to L. disparwith 2.4% sequence divergence from all other hold great potential for tussock moth species identifi- non-Hokkaidoensis L. dispar specimens. The remain- cation (Ball & Armstrong 2005). In that study 81 'test' ing three of the eight previous unidentifiables, specimens were used to interrogate a coxl sequence MAF816, MAF851 and MAF912, could not be placed profile composed of 18 lymantriid species across four with confidence in the profile dataset. They grouped genera. 100% of the coxl identifications agreed with most closely with Hypnea humuli, Leuhdorfiajaponica

Phil. Trans.R. Soc. B (2005) DNA barcodesfor biosecurity K. F. Armstrong & S. L. Ball 1817

and Calophasia lunula, respectively (Electronic Appen- for an increasing number of species (Carroll et al. dix, figure 1), but the bootstrap supports were weak 2004). Unfortunately it is usually the early instar larvae (12, 34 and 33%, respectively). The coxl sequences or eggs that are intercepted at the border in fruit of suggest that these specimens belong to taxa not commercial consignments or accompanying overseas represented in this dataset, ranging from other species travellers cannot be identified morphologically beyond to other families. It is no surprise therefore that they the family level. were outside the diagnostic scope of the original Since 1994 a molecular diagnostic technique based 'tussock moth specific' RFLP method and explains on PCR-RFLP of the ITS1, 5.8S plus ITS2 rDNA why they were difficult to analyse. It also suggests very regions (Armstrong et al. 1997b), has been used positively for barcoding that, with greater represen- routinely to rapidly identify fruit fly intercepted at the tation of lepidopteran taxa in the profile dataset, this New Zealand border to the species level. Also, in a method could achieve a 100% identification rate which similar manner to the tussock moth method, a species- is not possible by other current molecular methods. specific PCR amplifying a 200 bp nested region has Of the 49 previously identified specimens, the been used for the identification of degraded DNA barcode identification disagreed with five of them associated with eggs found in cooked breadfruit (table 1). Each had been diagnosed as L. dispar. Four (unpublished). Again while these do not present a of them were identified using the L. dispar specific threat themselves, accurate identification is necessary PCR, as they had been difficult to analyse by the PCR- for comprehensive risk assessment. These methods RFLP. Three of those were indicated by barcoding to effectively replaced the need to rear immature stages belong to species not represented in the profile dataset. through to adults for identification, which was often MAF915 and MAF729 associated with Spilosoma sp. unsuccessful or at best too slow for making timely (99% bootstrap support) and Hyphantria sp. (identical biosecurity management decisions (Armstrong et al. sequence) respectively, belonging to another lepidop- 1997a). However, this approach has evolved from use teran family, the Arctiidae, and MAF839 came out on a with 19 original species to 49 (unpublished) and relies long branch within the Lymantriidae, between L. xylina heavily on the host and geographic origin information and L. dispar (80% bootstrap support). The fourth, to limit the list of likely species. Continuing to add MAF914, associated with high bootstrap support to more species has increasingly compromised the diag- the Korean haplotype of L. mathurawhich is genetically nostic sensitivity of the method as overlapping RFLP divergent from those in Japan (Electronic Appendix, patterns become more common. Using DNA barcodes figure 1). This implies the potential use of the barcode was therefore considered here as a method that might data to provide useful geographic origin information enable accurate identifications amongst a large number that was not possible previously. Data for the four of species. The first data towards a fruit fly coxl species specimens also indicate that the original species- profile, including the only two tephritid barcode region specific primer test (unpublished) was not as broad as sequences available in Genbank at the time (see it had needed to be and again demonstrates the Electronic Appendix, table 3), is presented here and limitation of previous methods to deal with species used to re-identify specimens intercepted at the border. outside the anticipated taxonomic range. The fifth Results: one hundred and ninety three sequences, specimen amongst those that disagreed, MAF795, was representing 60 species were used to create a tephritid previously identified as L. dispar by RFLP, but 100% coxl reference profile (Electronic Appendix, figure 2). coxi homology to H. cunea suggests otherwise. This Forty one species were represented by 2-14 specimens specimen has been flagged as one that needs further taken from across their geographic range where analysis to determine its true identity. possible. Nineteen species were represented by only one specimen. The profile NJ analysis generally (b) Case study 2: fruit flies resolved the taxa according to their morphologically Background: Of the some 4000 species of fruit fly, derived as genera, subgenera, species and around 250 are considered economic pests (White & species complexes (White & Elson-Harris 1992). Elson-Harris 1992). New Zealand remains the only Bootstrap support was high at the nodes (greater than major fruit producing country in the world that is free 80%) for species that were not part of a species from them and significant investment has been placed complex (discussed below). There were two excep- in monitoring and surveillance systems to ensure their tions. Bactrocerapsidii 'clustered' weakly with Bactro- early detection and to minimize pathway risk (Cowley cera trilineola;sequence divergence between them was & Frampton 1989; Frampton 2000; Stephenson et al. 2.9%, compared to intra-specific 1.8% and 2.0%, 2003). The species, however, present different degrees respectively. Also, Bactrocera curvipennis was placed of risk to New Zealand based on their host and climatic within the Bactrocera tryoni complex, but with weak preferences and differing quarantine actions can result, bootstrap support. Interestingly, the latter are also i.e. to treat, re-ship or destroy the imported produce. difficult to distinguish by the PCR-RFLP method Distinction of regulated and non-regulated species (Armstrong & Cameron 2000) although the adults are groups is the minimum diagnostic requirement, but morphologically distinct. Others that came out on identification to species is essential for accurate long branches, Bactrocera minuta, Bactrocera arecae, interception data and assessment of pathway risk. As Bactroceradistincta and Dacus demmerezi,suggests that for the tussock moths the majority of these species can there is insufficient taxonomic representation around be readily distinguished based on their adult mor- these species within the current dataset. There were phology (White & Elson-Harris 1992), and late instar also two discrepancies that warrant further investi- larval morphological keys are also becoming available gation. One is the Bactrocera cognata specimen 1009

Phil. Trans.R. Soc. B (2005) 1818 K. F. Armstrong & S. L. Ball DNA barcodesfor biosecurity which did not cluster with its conspecifics (specimens well as the groupings within the species complexes is 975, 976 and 1011) that were correctly located within underway. Finally, of the 81 border intercepted speci- the B. dorsalisspecies complex (Drew & Hancock 1994; mens analysed, two were previously classified as figure 2 in the Electronic Appendix, here considered to 'unknown', due to novel RFLP patterns. The coxl include the Asian and Australian species within the analysis clearly placed them within the B. (B.) dorsalis branch that includes B. dorsalis(specimen 726) through complex, but interestingly not within the clusters to B. endiandrae (specimen 789)). The second was containing the only four species (B. dorsalis,Bactrocera B. arecae which is part of the B. dorsalis complex but philippinensis, Bactrocera papayae and Bactrocera placed distantly from it. These specimens are flagged carambolae) for which RFLP profiles had been for morphological and/or molecular re-examination. determined. Importantly, besides some exceptions within the Conclusion:as the analysis stands the fruit fly coxl species complexes, there were no sequences that were sequences provide slightly better resolution, and also shared by different species. This is in contrast to the quantitative support in terms of bootstraps and current nuclear rDNA method where some RFLP divergence values, for species-level identification than haplotypes are common to several species. was previously possible. The dataset however high- The coxl sequences appear to be limited in their lights the limit of coxl to provide confident identifi- ability to distinguish taxa within the species complexes cations within species complexes. For those outside the of B. dorsalis,B. tryoni (Morrow et al. 2000; figure 2 in complexes, the coxl data sort the species well in terms the Electronic Appendix, species B. tryoni, B. neohu- of their morphologically-based taxonomy, from genus meralis and B. aquilonis) and A. fraterculus (Norrbom to species. Ambiguity within the complexes may be a et al. 1999; figure 2 in the Electronic Appendix, all consequence of insufficient variation in coxl to species within the branch starting from A. ludens accurately reconstruct such recent divergences, but through to A. fraterculus). This is interesting given that reasoning does not appear to hold for other sub- that there were cases here, as for the tussock moth data species separations, such as that within B. cucurbitae. set, where known subspecies could be distinguished. The disparity may be a function of the status of the For example, the B. cucurbitaestrains A and B (3.5% alpha taxonomy of this genus which is still under divergence between them here, versus 0.0% and 0.8% scrutiny (Smith et al. 2003) and also the amount of respectively within each) are anecdotally separated by systematic interest that certain taxa have received. In host range (unpublished), and Bactroceraxanthodes and this case, in contrast to B. cucurbitae, the B. dorsalis Bactrocera paraxanthodes (7.2% divergence between complex has been extensively studied taxonomically. them here, versus 0.0% within each, respectively) are Until recently B. dorsaliswas considered the single most also separated by host (Drew et al. 1997). Each of these significant fruit fly pest species in Asia. A recent and other aspects of the reference profile data, such as revision by Drew & Hancock (1994) now recognizes the placement of B. xanthodesand B. paraxanthodesetc, it as part of a complex of 52 sibling species of which are the subject of a more in-depth barcoding treatment eight are economically important, and more species in a separate publication in preparation. continue to be described (see Clarke et al. 2005). In Eighty one border intercepted specimens were addition, with the highly specialized taxonomic identified by appending their coxl sequences to the expertise required to distinguish these species with profile dataset (Electronic Appendix, figure 2). Based any confidence, their apparent 'poor resolution' in the on the closest species which they associated with in the current analysis may in part be a consequence of NJ tree, 73 (94%) of these identifications were in mistaken specimen identification by the suppliers. agreement with the previous RFLP method (table 1) Does this limitation at the level of species complexes with high bootstrap support (84-100%). The mean invalidate the ability of this method to correctly identify sequence divergence between the intercepted speci- unknowns? As the method stands, it appears to be no mens and the profile sequences they grouped with was less accurate than the existing PCR-RFLP method. 0.9% (range =0.1%-5.3%). This is consistent with For the B. dorsalis complex, interspersion of species intraspecific coxl divergences observed for a variety of such as B. dorsalis sensu stricto, B. papayae and insect taxa (Hebert et al. 2003a,b). Four identifications B. philippinensis is no less informative than before, were in disagreement. Three of these were supported and from a biosecurity point this is not an issue as they by high bootstrap support (Electronic Appendix, are all regulated species. In fact there have been no figure 2). Specifically, MAF274 was identified within molecular studies that have been able to satisfactorily the B. tyroni species complex by PCR-RFLP but as distinguish these three species to date (Clarke et al. B. facialis here, MAF665 was B. passiflorae but 2005). The method does however promise to be more B. facialis here and MAF940 was within the B. tyroni informative for other species within the complex such complex but Dirioxa pornia here. The fourth, MAF 144, as Bactrocera kandiensis and Bactrocera caryeae which was weakly supported as being within the B. dorsalis form a distinct group; interestingly this identified two species complex. The previous PCR-RFLP method border intercepted specimens that previously could had identified it either as Bactrocera kirki, Bactrocera only be resolved as far as the species complex. trilineolaor Bactrocerafrauenfeldi which share common B. carambolaealso forms a discrete group although it restriction profiles. However, bootstrap support for the is not well supported. This is consistent with the PCR- entire B. dorsaliscomplex node was generally low in the RFLP method although it was very confidently profile dataset, indicating little confidence in the ability distinguished from the others in the complex with of these sequences to identify 'unknowns' within the that method. Additional notable improvements on complex. Further evaluation of these aberrant results as the previous method are the separation of the species

Phil. Trans.R. Soc. B (2005) DNA barcodesfor biosecurity K. F. Armstrong & S. L. Ball 1819

B. kirki, B. trilineolaand B. frauenfeldi,which previously (b) Test cases shared a common haplotype. Also, specimens that The two groups of taxa analysed here presented previously gave novel (unknown) PCR-RFLP haplo- different challenges to using coxl barcodes for species types are now identifiable, associating with reasonable identification. For the tussock moths there had been bootstrap support to particular profile taxa and difficulty in placing previous RFLP 'unknowns' to providing clues as to the species gaps that need filling. species. This obstacle was overcome here to a certain extent with subsequent inclusion of a much broader taxonomic range of lepidopteran coxl sequence data, available through BOLD and to a lesser extent in 5. DISCUSSION Genbank. That enabled several unknowns to be barcodes (a) Comparative utility ofDNA assigned to likely genus and species within the In contrast to the other molecular methods diagnostic Noctuidae and Arctiidae. In retrospect it is not referred to DNA has some here, barcoding significant unrealistic to expect that other, non-lymantriid moth a more accurate and robust advantages: (i) it provides species were being intercepted. Females of other to all of the approach diagnosis by using targeted lepidoptera, besides gypsy moth (Leonard 1981), data. PCR and PCR-RFLP genetic Species-specific oviposit indiscriminately, including on inanimate on the other utilize small windows of diagnoses, hand, objects during population outbreaks or when attracted the data at priming or restriction sites, ignoring most to lights of human settlements. As arrival of these of the information. genetic (ii) incorporating the range species can not be easily predicted the design of a more of intra-specific polymorphism by adding as many comprehensive PCR-RFLP method was not possible. reference sequences as possible clearly enhances the In contrast, placing sequences of the unknowns within robustness of any key and assignment of species a broader taxonomic context was made possible by identification. In contrast, our PCR-RFLP procedures publically available lepidopteran coxl data. This now actively avoided using restriction enzymes that detected provides a guide as to how best to target further sub-specific polymorphism because of the ambiguity in collections and fill the reference species gaps. interpreting it as species- or population-level variation. Of the previously unknown 'tussock moth' species, (iii) using a tree-based approach enables all the data to four were identified as the fall web worm, Hyphantria be observed at a glance; this is very cumbersome to cunea. This evidence of multiple entry events could manage with PCR-RFLP. (iv) the NJ analysis also elevate this species in terms of risk. In fact, around the provides quantitative data with sequence divergences same time in 2003, this species was found to have and bootstrap values that give a measure of confidence established a localized population in New Zealand. in the identifications. In general terms, our conclusions Fortunately, this was eradicated. An active surveillance about the relative benefits of sequence 'tree'-based campaign has operated since that time and interest- species identification methods, concur with those of ingly, two more finds have been made recently. Fall other similar molecular identification keys for thrips webworm had never been found in New Zealand or (Brunner et al. 2002), nematodes (Floyd et al. 2002) Australia prior to 2003. It is native to North America and whales (Ross et al. 2003). and Mexico, but since establishing in Europe and parts From a practical perspective, identifications can be of Asia in the 1940s and 1950s, it has become a achieved on a par with RFLP analyses, within a 24 h significant pest of trees in these continents. The period from extraction to NJ analysis and for around identification here of immature life stages on imported the same cost, if not less. This is given in-house used vehicles is evidence that a pathway of entry exists. sequencing facilities and an appropriate reference It also highlights that species other than gypsy moth dataset. However, even if the latter is not available, it enter New Zealand via the imported used vehicle is much easier to build this up over a relatively short pathway more frequently than was originally thought. period of time compared to the same for a diagnostic In contrast to the tussock moths, the species of suite of restriction patterns. Assuming that the same immature fruit flies entering and the pathways involved care is taken over sequence quality and interpretation are more predictable due to their close host association. as it would be for quality and interpretation of Consequently it has been easier to target species that electrophoretic gels, DNA barcodes provide a robust should be included in an appropriately comprehensive alternative to PCR-RFLP. With the exception of speed, coxl reference dataset. This was reflected here with this is also the case with species-specific primer considerable confidence in the dataset for making methods. However, that appeared here to be the least identifications within the Tephritidae. The challenge reliable method which is not unexpected given the for barcoding instead was recent evolutionary diver- potential ambiguity associated with presence/absence gences, at the level of species complexes where blurry of a PCR product and assumed 'specificity' of PCR species boundaries exist and undermine confidence in primers. Brunner et al. (2002) decided not to dispose of identifications. In spite of this, for some cases it was an the PCR-RFLP profiles for the benefit of other improvement over the PCR-RFLP method. For laboratories that do not have convenient DNA example, it was possible to place specimens previously sequencing facilities. However, this is unlikely to identified as 'B. dorsalis complex' with reasonable present a barrier in the future. Even now, DNA bootstrap support (75% see figure 2 in the Electronic sequencing technology is becoming more accessible Appendix) to a likely species, B. caryeae. through a number of dedicated local and offshore Distinguishing recently diverged taxa is no less an commercial sequencing facilities structured for com- issue for morphologically-based identifications. The petitive pricing and rapid turnarounds. reasons for this are varied, but not least is the status of

Phil. Trans.R. Soc. B (2005) 1820 K. F. Armstrong & S. L. Ball DNA barcodesfor biosecurity the alpha taxonomy. Confusing species limits may exist within the B. dorsaliscomplex, for which an additional through species being 'oversplit' or 'overlumped' and gene region may be appropriate. However it clearly the phenotypic boundaries not adequately reflecting separates races within other species. This implies issues the speciation process (Funk & Omland 2003). Never- with the alpha taxonomy. It also highlights the theless precise species identification remains very fundamental need for continued participation of important for biosecurity. Firstly, some species within taxonomists to provide accurately identified reference these complexes are more aggressive in their ability to material and to describe new species potentially invade and reach pest status than others. B. dorsalis discovered during the process. sensu stricto and B. carambolae are very successful Employing DNA sequences from known adult invaders, but they are difficult to distinguish from, specimens to identify their morphologically indistinct and have overlapping host and geographic ranges with immature life stages, as has been considered here, B. verbascifoliae,which is not a recognized pest. In illustrates the power of molecular data to complement reality many fruit fly species, especially tropical ones, (and enhance) the morphological approach to insect are unlikely to be serious pests per se in New Zealand, diagnoses. Using barcode data in this context, i.e. but accurate diagnosis of regulated pests is still matching sequences within taxa, should not suffer from necessary to avoid trade restrictions of significant the same potential for misidentification as in other impact. Secondly, some morphologically indistinct diagnostic situations where incongruence with a priori regulated species, such as B. philippinensis and predictions based on morphology requires a more B. papayae, have different host and geographic ranges, comprehensive taxonomic approach (Paquin & Hedin which is important information for assessing the 2004). However as with other molecular diagnostic specific risk and pathway involved. In that situation, methods DNA barcode-based identifications will extension of the barcode system to include a second (or clearly depend heavily on the availability of appropriate more) gene region for resolving what is not possible reference taxa to avoid problems associated with with the coxl Folmer region is justified. This might inadequate taxon sampling (Moritz & Cicero 2004). involve a nested approach to include regions such as 3' Despite this, given the largely unique nature of coxi coxl or the hypervariable 16S rDNA that may evolve at sequences at the species level demonstrated here a slightly faster rate. It also warrants considerable effort (outside of species complexes) and in other studies to include specimens from across the host and (e.g. Hebert et al. 2003a,b; Hogg & Hebert 2004; geographic range. Whiteman et al. 2004; Ball et al. 2005; Janzen et al. 2005; Shander & Willassen 2005), missing taxa are (c) Barcodes for biosecurity in the future likely to lead to non-identification, not misidentifica- Access to 'historical' datasets and the same specimens tion. Non-identification will be recognized by the has provided a valuable opportunity to comparatively 'unknown' appearing on a relatively long branch with test the appropriateness and power of the barcoding low bootstrap support. Misidentification on the other approach for identifying unknown organisms. The test hand could result from close proximity to a congener cases here support the view that coxl barcodes offer the and absence of a conspecific reference sequence. This best opportunity to date to form the foundation of a is most likely to occur for recently diverged taxa, and is flexible and accurate identification system for invasive no less an issue for any other diagnostic method. insect species. Given the potential universality of the Robust diagnostic procedures should however be able application, this approach promises to address the to pre-empt this in part or completely by making every standardization and efficiency needs that are severely effort to understand reference taxa from a 'whole lacking at the moment for the international biosecurity organism' point of view, i.e. using common sense to community. The approach is even more appealing with incorporate relevant biological aspects into identifi- the ease of technical transfer between laboratories, cation of unknowns. enabling consistency of the process from PCR to the Accuracy of identifications is also dependent on alignment of homologous sequence data and sharing of reliability of the simple sequence-similarity approach. sequence data from diverse and unrelated sources. It In this case using the NJ model the analysis is not lends itself well to automation and as a basis for constrained by potential rate variation and different development of the next generation of diagnostic tools base composition amongst taxa as are phylogenetic such as micro-array technology (Kochzius et al. 2004). treatments of the data such as the use of maximum DNA barcoding may also contribute to digitized likelihood. In fact the need for rapid identifications in collections more easily than that proposed for mor- biosecurity would preclude the use of a phylogenetic phologically based identifications (Gaston & O'Neil analysis with the computation of such large datasets 2004). taking several days. Conversely, occasional phyloge- Nevertheless there are a number of issues that will netic treatment of the data could provide a useful need to be taken into account. There continues to be strategy for determining the addition of species when debate as to the reliability of a cox1 barcode species taxon gaps are inferred by anomalous placings and long identifier given issues of diversity related to mtDNA branches. Indeed, the most important improvement of phylogeography and the rules determining 'species' the barcode method over all other diagnostic methods limits (Moritz & Cicero 2004). Suggesting that just one is the ability to continually add in more species. This gene can supply the diagnostic needs for all would be introduces a highly desirable, more anticipatory rather naive. Even with our data set there are diagnostic approach that will enable the international inconsistencies. For example with the fruit flies cox1 IAS diagnostic community to better cope with does not confidently discriminate some of the species changing and local species priorities, to capitalize on

Phil. Trans.R. Soc. B (2005) DNA barcodesfor biosecurity K. F. Armstrong & S. L. Ball 1821

the efforts of others and to standardize technologies. Ball, S. L., Hebert, P. D. N., Burian, S. K. & Webb,J. M. There is, nevertheless, unlikely to be a 'one size fits all' 2005 Biological identificationsof mayflies (Ephemerop- molecular diagnostic approach to biosecurity which tera) using DNA barcodes.JNABS 24, in press. benefits from the use of tools on a case-by-case basis. Brown,S., McLaughlin,W., Jerez, I. T. & Brown,J. K. 2002 Identification For example DNA barcoding would not replace and distribution of Bemisia tabaci (Genna- dius; Homoptera: Aleyrodidae) haplotypes in Jamaica. routine species-specific portable tests such as the Trop.Agric. 79, 140-149. Lateral Flow Devices (LFDs) used in the serological Brunner, P. C., Fleming, C. & Frey, J. E. 2002 A molecular field for confirming plant pathogen identification identification key for economically important thrips (Hughes et al. 2005). However inclusion of DNA species (Thysanoptera: Thripidae) using direct sequencing barcoding in the molecular diagnostics 'toolbox' does and a PCR-RFLP-based approach. Agric. For. Entomol. 4, offer a level of transparency for species identification 127-136. (10.1046/j.1461-9563.2002.00132.x.) across countries, and sectors within countries, that is Carroll, L. E., Norrbom, A. L., Dallwitz, M. J., Thompson, not possible with the current uncoordinated adoption F. C. 2004 (onwards) Pest fruit flies of the world-larvae. of numerous different diagnostic methods. Version:13th April 2005. http://delta-intkey.com. Aspects that need to be seriously considered towards Case study 3 2002 Case study 3-response to the incursionof the adopting this on a global scale will be the ability to painted apple moth. Ministry of agriculture and forestry: obtain validated specimens for building robust refer- managementof biosecurityrisks. ISBN 0-477-02898-5. M. S. & F. A. 2000 The current ence profiles, an agreed framework for the sharing and Caterino, S., Chol, Sperling, state of insect molecular systematics: a thriving tower of quality control of sequence data, confidence in the Babel. Annu. Rev. Entomol. 45, 1-54. current bioinformatic and rules user-friendly analyses Chiu, Y-C., Wu, W-J. & Shih, C-J. 2001 Identification of or standardized criteria for interpreting barcode results. three Aulacaspis species (Homoptera: Diaspididae) by Importantly, the rigorous assessment of these will now PCR-RFLP analysis for quarantine application. Formosan be possible through the Barcode of Life initiative Entomol. 21, 365-375. (http://www.barcodinglife.org/), assisted by the Con- Clarke, A. R., Armstrong, K. F., Carmichael, A. E., Milne, sortium for the Barcode of Life (http://www.barcoding. J. R., Raghu, S., Roderick, G. K. & Yeates, D. K. 2005 si.edu/). The significant international momentum Invasive phytophagous pests arising through a recent gathering for this programme will facilitate the tropical evolutionary radiation: The Bactrocera dorsalis emergence of a globally collaborative and less frag- complex of fruit flies. Annu. Rev. Entomol. 50, 293-319. mented approach to molecular diagnostics and is Cock, M. J. W., Kenis, M., Wittenburg, R. 2003 Biosecurity entirely appropriate for international biosecurity. and forests: an introduction-with particular emphasis on forest pests. FAO Forest Health and Biosecurity Working We thank the organizersof the inauguralmeeting of the Paper FBS/2E. Consortium for the Barcodingof Life (London 2005) for Cowley, J. M. & Frampton, E. R. 1989 Significance of and invitingpresentation of this work. We thank Robyn Cowan, monitoring for alien fruit flies (Tephritidae). Proc. 42nd Ruth Frampton and Barney Stephenson for constructive N.Z. 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