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Using museum specimens to identify MOTUs in larval scarab beetles

Using museum specimens to identify MOTUs in larval scarab beetles. Andrew Mitchell , NSW DPI Kelly Rigg, Charles Sturt University Gus Campbell, NSW DPI Tom Weir, CSIRO Entomology A. Raman, Charles Sturt University. The barcoding bottleneck.

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Using museum specimens to identify MOTUs in larval scarab beetles

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  1. Using museum specimens to identify MOTUs in larval scarab beetles Andrew Mitchell, NSW DPI Kelly Rigg, Charles Sturt University Gus Campbell, NSW DPI Tom Weir, CSIRO Entomology A. Raman, Charles Sturt University

  2. The barcoding bottleneck... • What is the most time-consuming part of the DNA barcoding process? • Most expensive? • Most difficult (requiring specialist knowledge?) • Acquiring expertly identified specimens

  3. ...and how to squeeze through it • Go collecting in a collection • Constraints: • Museum policies on “destructive” sampling • DNA preservation? • Can one routinely recover DNA barcodes from 30-40 year old insect material? • If yes...

  4. The study group – scarab beetles • Scarab larvae are pests of sugarcane, pastures... • Conserved morphology • Long life cycles (1-2 years) • Different spp. need diff. management strategies • Identification of larvae is crucial • Adults generally do not cause direct damage • Anoplognathus adults feed on eucalypts • Rural tree decline in Australia

  5. Our Grand Plan  X X • Collect larvae • Collect adults • Barcode both • Ahrens et al. (2007) Mol. Phylogenet. Evol. • 3’-half of COI gene (Jerry – Pat)

  6. PCR primer development • Folmer primers not very successful • A few rounds of primer design needed • Degenerate (Folmer) primers, with M13 tails • Lepidoptera, Hemiptera, various other insects • Redesigned the downstream primer 3 codons further downstream • Bark beetles & others • Considerable time & effort

  7. Plan B • Collect larvae • Barcode larvae

  8. Results: Highly variable: p-distance = 32% within a family (vs. half that between insect orders) Base composition bias minimal (60% AT versus 70-80% in other insects) Identified 30 MOTUs Best strategy for identifying the MOTUs? Focus on one genus at a time Youngest specimens Larvae only ~100 sequences 667 bp COI

  9. PCR strategy • Aim: Determine the effects of age and amplicon size • on PCR success • Designed sets of degenerate, M13-tailed primers to • target a range of sizes of amplicons: • 667, 340, 327, 238, 140, 92 bp each

  10. Museum specimens n = 240 Mean age = 37 years

  11. Effect of amplicon length on PCR “PCR success” = DNA band (of expected size) (not the same as sequencing success!)

  12. Current data set Added short sequences (238-329 bp) for ~70 adults

  13. p-distances within 0.4% between 1.7-2.7% within 0.8% 2.6% Summary With exceptions to be noted, max p-distance within = 1.3% min p-distance between = 3.0% max p-distance = 19.1% Anoplognathus only NJ tree, K2P +bootstrap

  14. 92 bp alignments 5’-section 3’-section

  15. Conclusions • DNA barcoding “works” in scarabs • COI is variable enough that mini-barcodes also will distinguish among species • Use of 30-40 yr old museum specimens is a feasible option in this group • Will this hold true for specimens from museums in the tropics? • The barcode standard should allow registration of short sequences as reference barcode

  16. Acknowledgements Funding:

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