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. NSW DPI 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. NSW DPI...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. NSW DPI 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

7. NSW DPI Our Grand Plan 1.Collect larvae 2.Collect adults 3.Barcode both Ahrens et al. (2007) Mol. Phylogenet. Evol. 3’-half of COI gene (Jerry – Pat)  X X

8. NSW DPI 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

9. NSW DPI Plan B 1.Collect larvae 2.Barcode larvae

10. Larvae only ~100 sequences 667 bp COI 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

12. NSW DPI 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

13. NSW DPI Museum specimens n = 240 Mean age = 37 years

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

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

17. p-distances within 0.4% between 1.7-2.7% within 0.8% NJ tree, K2P +bootstrap Anoplognathus only 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%

18. 92 bp alignments 5’-section 3’-section

19. NSW DPI Conclusions 1.DNA barcoding “works” in scarabs 2.COI is variable enough that mini-barcodes also will distinguish among species 3.Use of 30-40 yr old museum specimens is a feasible option in this group 1.Will this hold true for specimens from museums in the tropics? 4.The barcode standard should allow registration of short sequences as reference barcode

20. NSW DPI Acknowledgements Funding: