Biosecurity in the new millennium: DNA barcoding provides a reliable, rapid, and cost effective method of forest pest identification
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1 Biosecurity in the new millennium: DNA barcoding provides a reliable, rapid, and cost effective method of forest pest identification Shelley L. Ball & Karen F. Armstrong Centre for Advanced Bio-protection Technologies, Lincoln University, Canterbury, New Zealand. Bio-Protection is a national centre incorporating staff from Lincoln University, Massey University, AgResearch, Crop & Food Research and seven other collaborator institutes that conducts world class research and development to meet the biosecurity and pest management needs of New Zealand s plant based primary industries and natural ecosystems.
2 Exotic Pest Species rapid & reliable identification is critical
3 Limitations of Morphological Identification variation within species (polymorphism, sexual dimorphism) lack of variation between species (cryptic species) keys only for a certain gender or life stage high degree of expertise required dwindling pool of taxonomic expertise
4 Molecular Genetic Diagnostics PCR-RFLP Advantages: ease population level identification Disadvantages: coarse grained (no quantitative information) not anticipatory no longer cost/time effective
5 DNA barcoding ~ 650 base pairs mtdna (COI) Protein coding rapidly evolving
6 The Barcoding Approach 1. Create profile (reference) database 2. To identify an unknown: add unknown sequence to profile sequences create a Neighbour-joining (NJ) tree using K-2-P distances profile sequence with which unknown matches most closely (smallest genetic distance)
7 Purpose Test efficacy of DNA barcoding in identification of selected pest lepidoptera: species identification population identification?
8 Lepidoptera: Lymantriidae Lymantria & Orgyia Lymantria dispar (Gypsy Moth)
9 Testing the efficacy of COI identifications 6 species Lymantria 3 species Orgyia 1 species Perina 1 species Teia Total of 87 specimens ( unknowns ), across 11 species Included specimens from different geographic regions & populations
10 The Barcoding Approach 1. Create profile (reference) database 37 species 13 genera 4 families (Lymantriidae, Noctuidae, Notodontidae, Arctiidae).
11 Results 100% success rate (87/87) most species showed very little haplotype diversity a few species showed substantial genetic divergence between populations
12 Lymantria monacha Korea (Sora) Lymantria monacha Korea (Sora) Lymantria monacha Korea (Sora) Lymantria monacha Korea (Sora) Lymantria monacha Korea (Sora) Lymantria monacha Poland (Polo) Lymantria monacha Poland (Polo) Lymantria monacha Czech Lymantria monacha Czech Lymantria monacha Czech Lymantria monacha Czech Lymantria monacha Poland Lymantria monacha Czech Lymantria monacha Poland (Polo) Lymantria monacha Czech Zale unilineata Zale duplicata Lymantria lucescens Japan (Hons) Lymantria fumida Japan (Iwat) Lymantria fumida Japan (Hons) Profile Unknown Sequence divergence = 0.2% Little haplotype diversity
13 Lymantria atemeler Thailand (Kamp) Lymantria atemeler Thailand (Kamp) Melanchra assimilis Melanchra adjuncta Feralia comstocki Feralia jocosa Lymantria mathura Japan (Hent) Lymantria mathura Japan (Hut) Lymantria mathura Korea (Sora) Lymantria mathura Korea (Sora) Lymantria mathura Korea (Sora) Lymantria mathura Korea (Yong) Furcula cinerea Furcula modesta Teia anartoides Teia anartoides X W = 0.3% X W = 0.5% X B = 4% sequence divergence between groups Orgyia pseudotsugata Canada (BC) Orgyia vetusta USA CA) Orgyia vetusta USA (CA) Orgyia vetusta USA (CA)
14 Orgyia thyellina Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma Canada (ON) Orgyia leucostigma USA (MD) Orgyia leucostigma USA (MD) Orgyia detrita USA (FL) Orgyia antiqua Czech Orgyia antiqua Czech Orgyia antiqua Czech Orgyia antiqua Czech Orgyia antiqua Czech Orgyia leuschni USA (AZ) Orgyia antiqua Canada (ON) Orgyia antiqua Central Europe Orgyia antiqua Canada Orgyia antiqua Canada (ON) Orgyia antiqua Canada ON) Orgyia antiqua Canada (ON) Orgyia antiqua Canada (ON) Orgyia antiqua Canada (ON) Orgyia antiqua Canada (ON) Orgyia antiqua Canada (ON) Orgyia antiqua Canada (ON) X W =0.1% sequence divergence X W = 0.3% sequence divergence X B = 0.9% sequence divergence
15 Lymantria xylena Taiwain (TauY) Lymantria xylina Taiwan (Kuan) Lymantria dispar hok Japan (Hokk) Lymantria dispar Japan (Hokk) Lymantria dispar hokk Japan (Hokk) Lymantria dispar Korea (Wont) Lymantria dispar China (Beij) Lymantria dispar Canada (ON) Lymantria dispar Canada(ON) Lymantria dispar Russia (Sak) Lymantria dispar Russia (Sak) Lymantria dispar Japan (Iwat) Lymantria dispar Japan (Iwat) Lymantria dispar Japan Lymantria dispar Canada (ON) Lymantria dispar Japan Lymantria dispar Japan Lymantria dispar Japan Lymantria dispar Japan (Iwat) Lymantria dispar Japan (Iwat) Lymantria dispar Japan (Iwat) Lymantria dispar Korea (Won) Lymantria dispar Korea (Won) Lymantria dispar Korea (Won) Lymantria dispar Japan (Iwat) Lymantria dispar USA (WV) Lymantria dispar France (Hagu) Lymantria atemeler Thailand (Kamp) Lymantria atemeler Thailand (Kamp) Melanchra assimilis Melanchra adjuncta Feralia comstocki Feralia jocosa X W = 0.7% X W = 0.5% X B = 2.7%
16 Some limitations. how do we define species limits? can t detect hybridization for some taxa, little population-level information may not resolve sibling species
17 Conclusions 100% success rate (87/87) easy, accessible, rapid relatively inexpensive
18 Intraspecific Interspecific Sequence Divergence
19 How do we define successful identification? Furcula cinerea Furcula modesta Lymantria fumida Japan (Iwat) Unknown moth Lymantria xylena Taiwain (TauY) Lymantria dispar hok Japan (Hok) Lymantria dispar Japan (Iwat) Profile sequence Unknown Lymantria lucescens - Japan (Hons) Zale unilineata Zale duplicata Lymantria bantaizana - Japan (Iwat) Acronicta morula Perina nuda - Taiwan Schizura unicornis Schizura badia L fumida (Iwat) Unkown = 0% sequence divergence Unknown L. xylena = 3% sequence divergence LeHepialidae
20 How do we define successful identification? Furcula cinerea Furcula modesta Lymantria fumida Japan (Iwat) Lymantria fumida Japan (Hons) Lymantria xylena Taiwain (TauY) Lymantria dispar hok Japan (Hok) Profile sequence Unknown Lymantria dispar Japan (Iwat) Lymantria lucescens - Japan (Hons) Zale unilineata Zale duplicata Lymantria bantaizana - Japan (Iwat) Acronicta morula Perina nuda - Taiwan Schizura unicornis Schizura badia LeHepialidae
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