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DNA-barcoding on a different scale: challenges within Coccoidea Lyn Cook, The University of Queensland, Australia SIBI Scale Insect Barcode Initiative (Stellenbosch, Feb 2007) Outline • What are scale insects? • Why have they been a challenge? • Prospects for a universal scale insect barcode APHIDS SCALE INSECTS MY 210 - 16010Myr ago 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 A. munita A. munita 18S rDNA (nuclear SSU rRNA) 18S variation within a morphospecies PCR product for 5’ end of nuclear ssu rDNA (18S) 1160 bp 980 bp * single genus 720 bp 480 bp 360 bp 18S helix E10-1 18S NJ K2P+G Cryptococcus results of COI barcoding attempts COI 5’ COI 3’ tRNA numts barcode *mealybugs *armored scales *soft scales felt scales *archaeococcoids *stictococcoids other scales * major pest groups few no no few few no no many many some some few no some + +/- yes? +/- yes? yes? Why such low success with COI? mtDNA nucleotide frequencies (%) AT A C G T COI 69-85 23-42 8-17 6-19 28-47 3rds ≤ 97.5 30-58 2-19 0-5 27-54 COII 72-86 28-52 8-23 4-15 23-43 3rds ≤ 100 38-65 0-26 0-16 22-55 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 Alternatives *mealybugs *armored scales *soft scales felt scales archaeococcoids *stictococcoids other scales 18S most most most most most most most var. many x most most most most most 28SD2 most most many many many var. most most? most most most yes in many most Case study Florida lac scale (= lobate lac scale) Paratachardina lobata 28S COI - 3’ region conserved regions only 28S D2 region for lac scales length of the region varies from 703-1114 28S D2 Aligned using muscle no adjustment Analysed using NJ K2P India India Florida Christmas Is. Bermuda &, as of 8 weeks ago, Cuba 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? Acknowledgements CBOL SAFRANET NSF NSF-PEET ARC ABRS 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