1. DNA-barcoding on a different scale: challenges within Coccoidea Lyn Cook, The University of Queensland, Australia

2. SIBI Scale Insect Barcode Initiative (Stellenbosch, Feb 2007)

3. Outline What are scale insects? Why have they been a challenge? Prospects for a universal scale insect barcode

5. APHIDS SCALE INSECTS 210 - 160 Myr ago

10. 1st scale insect “barcode” 1995 - DNA extracted from new morphotype. Some ovarian tissue frozen at -70C. Cuticle slide mounted as voucher. Other specimens from same collection frozen or stored in 100% ethanol 1999 - PCR and sequencing 2003 - paper published including sequences and stored DNA as part of holotype material Apiomorpha gullanae

11. A. munita

12. 18S rDNA (nuclear SSU rRNA)

13. 18S variation within a morphospecies

14. PCR product for 5’ end of nuclear ssu rDNA (18S) 360 bp 480 bp 720 bp 980 bp 1160 bp * single genus

15. 18S helix E10-1

16. 18S NJ K2P+G

17. Cryptococcus

18. COI 5’ barcode COI 3’tRNAnumts *mealybugsfewmany+yes? *armored scalesnomany+/- *soft scalesnosome felt scalesfewsome+/-yes? *archaeococcoidsfew yes? *stictococcoidsno other scalesnosome * major pest groups results of COI barcoding attempts

19. Why such low success with COI?

20. ATACGT COI69-8523-428-176-1928-47 3rds≤ 97.530-582-190-527-54 COII72-8628-528-234-1523-43 3rds≤ 10038-650-260-1622-55 mtDNA nucleotide frequencies (%)

21. COI barcode region Taxa that amplify appear to have higher CG in 3rd positions than failures –not direct comparison Amino acid divergence of up to 8% in aphid crown Amino acid divergence of up to 55% within scale crown

22. 18Svar.28SD2var. *mealybugsmostmanymost *armored scalesmostx most? *soft scalesmost manymost felt scalesmost manymost archaeococcoidsmost manymost *stictococcoidsmost other scalesmost yes in many most Alternatives

23. Florida lac scale (= lobate lac scale) Case study

26. Paratachardina lobata

29. 28S conserved regions only COI - 3’ region

30. 28S D2 region for lac scales length of the region varies from 703-1114

31. 28S D2 Aligned using muscle - no adjustment Analysed using NJ K2P

32. India Florida Christmas Is. Bermuda &, as of 8 weeks ago, Cuba

33. Conclusions Proceed with non-COI barcode - 28S when successful 18S might be an alternative in “decision tree” -18S to assign specimen to group or direct ID - next step depends on grouping (e.g. COI barcode or COI 3’, 12S etc with group-specific primers) Attempt to find solution to COI barcode problem. Options include: more primer design mini COI barcode cocktails whole mt genome other?

34. SIBI, Stellenbosch, February, 2007 Participants: BB Normark, W Pieterse, PJ Gullan, I Millar, J Giliomee, D Downie, F Haas, C Eardley, LG Cook, A Rung, D Masinga, A Venter, D Muller, DR Miller, R Hanna Acknowledgements CBOL SAFRANET NSF NSF-PEET ARC ABRS