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 Myr ago

1st scale insect “barcode” 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 PCR and sequencing paper published including sequences and stored DNA as part of holotype material Apiomorpha gullanae

A. munita

18S rDNA (nuclear SSU rRNA)

18S variation within a morphospecies

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

18S helix E10-1

18S NJ K2P+G

Cryptococcus

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

Why such low success with COI?

ATACGT COI rds≤ COII rds≤ mtDNA nucleotide frequencies (%)

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

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

Florida lac scale (= lobate lac scale) Case study

Paratachardina lobata

28S conserved regions only COI - 3’ region

28S D2 region for lac scales length of the region varies from

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

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?

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