DNA barcoding and forest biosecurity L.M. Humble, J.R. deWaard, R. Hanner and P.D.N. Hebert Introduction Biosurveillance (cont’d) The ability to distinguish non-indigenous Results species (NIS) from native species is critical to The identification engine in the Barcode of Life the success of any surveillance program. database (BOLD-ID) was used to obtain initial Unfortunately there are numerous problems identifications for the 925 specimens (Fig. 3). Figure 4. Argyresthia pruniella (left) and inherent with detection of NIS including: Dichelonia histrionana (right) • ~190 species clusters with a 3% sequence • large samples needed to detect NIS when divergence cut-off (Hebert et al. 2003) they are present at low levels Identification of • immature life stages often intercepted but • 124 clusters assigned to species, 61 to usually cannot be fully identified genus using BOLD-ID (all species immature life stages assignments were also confirmed • often inadequate knowledge of native fauna morphologically) Eggs, larvae or pupae are the most frequently We provide examples from forest bio- intercepted life stages of many quarantine • 66 remaining clusters were identified surveillance in Canada showing how these organisms. They usually cannot be fully morphologically (the only step in the problems can be circumvented by the identified to species’. identification process that required application of DNA barcoding (DNAB) for taxonomic specialists) • DNA barcodes are invariant during a species identification. species development; any life stage can be identified from its DNA sequence What is DNA barcoding? European poplar shoot borer, Gypsonoma aceriana, was first reported from North • DNAB uses the sequence variation in a America in 2001 short, standardized DNA fragment to identify organisms • barcoding of adults in museum collections confirmed its long-term presence in • DNA fragment used for animals is a 658 western Canada (Fig. 5) base pair segment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) • barcoding of larvae from delimitation (Hebert et al. 2003) surveys used to define its range in British Columbia (Humble et al. 2009) • It compares unknown sequences against a reference library of DNA sequences jflandry 1026 G. salicicolana Canada 08-JDWBC-0074 G. fasciolana Canada • It meets or exceeds minimum standards Dun-08-279 Dun-08-279 required for diagnostic protocols under 08-JDWBC-3679 08-JDWBC-3678 ISPM No. 27 (Floyd et al. 2010) 08-JDWBC-3677 08-JDWBC-3676 08-JDWBC-3675 08-JDWBC-3674 PFC-2006-0505 08-JDWBC-3673 08-JDWBC-3672 early instar in 08-JDWBC-3671 leaf mine 08-JDWBC-3667 08-JDWBC-3666 G. aceriana 08-JDWBC-3665 British Columbia, Canada 08-JDWBC-3664 08-JDWBC-3663 08-JDWBC-3662 08-JDWBC-3661 08-JDWBC-3660 08-JDWBC-3659 08-JDWBC-3658 08-JDWBC-3657 overwintering site of 08-JDWBC-3656 early instar larva PFC-2006-1970 PFC-2006-0907 Figure 3. Species identification report and Dun-08-282 Figure 1. Light trapping for nocturnal moths. Dun-08-281 neighbour-joining trees from BOLD-ID. Dun-08-280 feeding site of mature 08-JDWBC-3656 larva in spring Biosurveillance for NIS 1% CGUKLB402-09 G. aceriana England • 31 species and 16% of all moths captured Figure 5. Neighbour-joining tree of COI Methods were NIS. sequences and geographic origin of samples for Gypsonoma species. Larval • moths sampled with UV lights (Fig. 1) • two NIS, Argyresthia pruniella and samples are denoted with italics. • a single leg was removed and barcoded Dichelonia histrionana (Fig. 4), were new using standardized procedures (Fig. 2) introduction records for North America (deWaard et al. 2009) • two NIS, Paraswammerdamia lutarea and Building DNA reference Prays fraxinella recorded for the first time libraries from western Canada DNA barcoding identifies unknown species by DNA barcoding provides an efficient and rapid comparing their COI sequences to reference means of assessing large samples. It enhances sequences derived from reliably identified both species recognition and the detection of species sampled from museum collections. new NIS by: Development of the sequence libraries is done Figure 2. Sampling • minimizing valuable specialist time; in collaboration with taxonomic specialists. tissue from moths for barcoding. • detecting species at low density Two examples of reference library develop- ment follow. DNA barcoding and forest biosecurity (cont’d) Building DNA reference Lymantria spp. results (cont’d) Literature cited libraries • 142 COI haplotypes identified across all deWaard, J.R., Schmidt, B.C., Anweiler, G.G. and Humble, 36 species L.M. 2008. First Canadian records of Lampropteryx suffumata ([D. & Schiff.], 1775) (Geometridae: 1. Lymantria tussock moths • no haplotypes shared between species Larentiinae). J. Ent. Soc. Brit. Columbia 105: 19–25. Lymantria includes serious quarantine & • 91 COI haplotypes within L. dispar deWaard, J.R., Landry, J.-F., Schmidt, B.C., Derhousoff, J., McLean, J.A. and Humble, L.M. 2009. In the dark in a forest pests [e.g. gypsy moth (L. dispar), pink • haplotypes of Asian subspecies L. dispar large urban park: DNA barcodes illuminate cryptic and gypsy moth (L. mathura) and nun moth (L. asiatica and L. dispar japonica cluster introduced moth species. Biodiversity and Conservation monacha)] 18: 3825–3839 separately from the European and North deWaard, J.R. 2010. Forest biomonitoring, biosecurity and • Species are often transported globally as American subspecies L. dispar dispar DNA barcoding. Ph.D.Dissertation. University of dormant egg masses on vessels and cargo British Columbia, Forest Sciences Department. • allows rapid identification of “Asian https://circle.ubc.ca/handle/2429/30496 • DNA reference library constructed for the gypsy moth” recovered from deWaard, J.R., Mitchell, A., Keena, M.A., Gopurenko, D., identification of 36 Lymantria spp. monitoring programs Boykin, L.M., Armstrong, K.F., Pogue, M.G., Lima, J., (deWaard et al. 2010a) Floyd, R., Hanner, R.H. and Humble, L.M. 2010a. 2. Geometridae Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of DNA reference library was developed for the biosecurity concern. PLoS ONE 5: e14280. 349 spp. of Geometridae in British Columbia deWaard, J.R., Schmidt, B.C.S., and Humble, L.M. 2010b. (deWaard 2010). DNA barcoding flags the first North American records of a Eurasian moth, Eupithecia pusillata (D. & Schiff., • specimens from 8 museum collections 1775) (Lepidoptera: Geometridae). J. Entomol. Soc. sampled, databased and imaged; DNA Brit. Columbia 107:1–7. extracted and COI sequenced Floyd, R., Lima, J., deWaard, J., Humble, L. and Hanner, R. 2010. Common goals: policy implications of DNA • all data is publicly available from the barcoding as a protocol for identification of arthropod Barcode of Life Data Systems (BOLD) pests. Biological Invasions 12 (9): 2947-2954 Hebert, P.D.N., Cywinska, A., Ball, S.L. and deWaard, J.R. (Ratnasingham and Hebert 2007) in 2003. Biological identifications through DNA barcodes. project ‘GOBCL – Geometridae of BC Proc. of the Royal Soc. of London B 270: 313–321 Library’ Humble, L.M., deWaard, J.R. and Quinn, M. 2009. Delayed recognition of the European poplar shoot Results borer, Gypsonoma aceriana (Duponchel) (Lepidoptera: • 2392 COI sequences generated from 400 Tortricidae) in Canada. J. Ent. Soc. Brit. Columbia 106: 61–70. species in 125 genera Ratnasingham, S. and Hebert, P.D.N. 2007. BOLD: The • 374 (93.2%) of the species could reliably Barcode of Life Data System be distinguished with barcodes (http://www.barcodinglife.org). Molecular Ecology Notes 7: 355–364. • only 27 species (6.8%) had undifferent- iated or overlapping barcodes Acknowledgments • both a new NIS (Fig. 7) and a new native The contributions of all of the co-authors of the species for Canada were detected by publications listed above are gratefully barcoding museum collections (deWaard acknowledged. We also acknowledge funding support from the Ontario Centres of Excellence Figure 6. Maximum likelihood tree for 36 et al. 2008, 2010b) research program, the Ontario Ministry of species of Lymantria constructed with the Agriculture, Food and Rural Affairs, the Canadian barcode region of the COI gene. The number Food Inspection Agency and the Canadian Forest of specimens sampled per species is noted in Service. Additional support was provided by a parentheses (after deWaard 2010). Forest Investment Account - Forest Science Program Student Grant, Canadian Forest Service - Results Pacific Graduate Student Supplements and an NSERC Graduate Scholarship (to JRdW). This • 518 adult Lymantria from 35 research was also supported through funding to the countries barcoded (Fig. 6) Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics Institute), • barcode data led to morphological NSERC and other sponsors listed at and taxonomic re-evaluation of http://www.BOLNET.ca. specimens in two clusters (deWaard Figure 7. Neighbour-joining tree of the et al. 2010a) European species, Eupithecia pusillata, and For further information Please contact the authors (see below) for pub- • L. sp. nr. mathura (Fig. 6) now two closely related native spp., Eupithecia niphadophilata and E. interruptofasciata. All lications. More information on these and related considered to be L. subpallida feed on juniper (after deWaard et al. 2010b) projects can be obtained from the Canadian Centre for DNA Barcoding - http://www.ccdb.ca/ or the • L. nebulosa is a valid species Abbreviations: DE – Germany, FI – Finland, IT – Italy, CA –