Biosecurity encompasses protecting against any risk through ‘biological harm’, not least being the economic impact from the spread of pest insects. 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 moth and fruit fly specimens intercepted at the New Zealand border over the last decade were reanalysed using the cox1 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.

▪ Abstract The Bactrocera dorsalis complex of tropical fruit flies (Diptera: Tephritidae: Dacinae) contains 75 described species, largely endemic to Southeast Asia. Within the complex are a small number of polyphagous pests of international significance, including B. dorsalis sensu stricto, B. papayae, B. carambolae, and B. philippinensis. Most species within the complex were described in 1994 and since then substantial research has been undertaken in developing morphological and molecular diagnostic techniques for their recognition. Such techniques can now resolve most taxa adequately. Genetic evidence suggests that the complex has evolved in only the last few million years, and development of a phylogeny of the group is considered a high priority to provide a framework for future evolutionary and ecological studies. As model systems, mating studies on B. dorsalis s.s. and B. cacuminata have substantially advanced our understanding of insect use of plant-derived chemicals for mating, but such studies have not been applied to help resolve the limits of biological species within the complex. Although they are commonly regarded as major pests, there is little published evidence documenting economic losses caused by flies of the B. dorsalis complex. Quantification of economic losses caused by B. dorsalis complex species is urgently needed to prioritize research for quarantine and management. Although they have been documented as invaders, relatively little work has been done on the invasion biology of the complex and this is an area warranting further work.

Summary: 1. DNA barcoding studies use Kimura's two‐parameter substitution model (K2P) as thede factostandard for constructing genetic distance matrices. Distances generated under this model then provide the basis for most downstream analyses, but uncertainty in model choice is rarely explored and could potentially affect how reliably DNA barcodes discriminate species.2. Using information‐theoretic approaches for a data set comprising 14 472 DNA barcodes from 14 published studies, we tested whether the K2P model was a good fit at the species level and whether applying a better fitting model biased error rates or changed overall identification success.3. We report that the K2P was a poorly fitting model at the species level; it was never selected as the best model and very rarely selected as a credible alternative model. Despite the lack of support for the K2P model, differences in distance between best model and K2P model estimates were usually minimal, and importantly, identification success rates were largely unaffected by model choice even when interspecific threshold values were reassessed.4. Although these conclusions may justify using the K2P model for specimen identification purposes, we found simpler metrics such aspdistance performed equally well, perhaps obviating the requirement for model correction in DNA barcoding. Conversely, when incorporating genetic distance data into taxonomic studies, we advocate a more thorough examination of model uncertainty.

Species delimitation directly impacts on global biosecurity. It is a critical element in the decisions made by national governments in regard to the flow of trade and to the biosecurity measures imposed to protect countries from the threat of invasive species. Here we outline a novel approach to species delimitation, “tip to root”, for two highly invasive insect pests, Bemisia tabaci (sweetpotato whitefly) and Lymantria dispar (Asian gypsy moth). Both species are of concern to biosecurity, but illustrate the extremes of phylogenetic resolution that present the most complex delimitation issues for biosecurity; B. tabaci having extremely high intra-specific genetic variability and L. dispar composed of relatively indistinct subspecies. This study tests a series of analytical options to determine their applicability as tools to provide more rigorous species delimitation measures and consequently more defensible species assignments and identification of unknowns for biosecurity. Data from established DNA barcode datasets (COI), which are becoming increasingly considered for adoption in biosecurity, were used here as an example. The analytical approaches included the commonly used Kimura two-parameter (K2P) inter-species distance plus four more stringent measures of taxon distinctiveness, (1) Rosenberg's reciprocal monophyly, (P(AB)), ¹ (2) Rodrigo's (P(randomly distinct)), ² (3) genealogical sorting index, ( gsi), ³ and (4) General mixed Yule- coalescent (GMYC). ^{4 , 5} For both insect datasets, a comparative analysis of the methods revealed that the K2P distance method does not capture the same level of species distinctiveness revealed by the other three measures; in B. tabaci there are more distinct groups than previously identified using the K2P distances and for L. dipsar far less variation is apparent within the predefined subspecies. A consensus for the results from P(AB), P(randomly distinct) and gsi offers greater statistical confidence as to where genetic limits might be drawn. In the species cases here, the results clearly indicate that there is a need for more gene sampling to substantiate either the new cohort of species indicated for B. tabaci or to detect the established subspecies taxonomy of L. dispar. Given the ease of use through the Geneious species delimitation plugins, similar analysis of such multi-gene datasets would be easily accommodated. Overall, the tip to root approach described here is recommended where careful consideration of species delimitation is required to support crucial biosecurity decisions based on accurate species identification.

Reliable and rapid identification of exotic pest species is critical to biosecurity. However, identification of morphologically indistinct specimens, such as immature life stages, that are frequently intercepted at borders is often impossible. Several DNA-based methods have been used for species identification; however, a more universal and anticipatory identification system is needed. Consequently, we tested the ability of DNA "barcodes" to identify species of tussock moths (Lymantriidae), a family containing several important pest species. We sequenced a 617 base pair fragment of the mitochondrial gene cytochrome c oxidase 1 for 20 lymantriid species. We used these, together with other Noctuoidea species sequences from GenBank and the Barcoding of Life Database to create a "profile" or reference sequence data set. We then tested the ability of this profile to provide correct species identifications for 93 additional lymantriid specimens from a data set of mock unknowns. Of the unknowns, 100% were correctly identified by the cytochrome c oxidase 1 profile. Mean interspecific sequence (Kimura 2-parameter) divergence was an order of magnitude greater (14%) than mean intraspecific divergence (<1%). Four species showed deeper genetic divergences among populations. We conclude that DNA barcodes provide a highly accurate means of identifying lymantriid species and show considerable promise as a universal approach to DNA-based identification of pest insects.