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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

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
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
and how to squeeze through it
...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...
the study group scarab beetles
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
our grand plan
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)
pcr primer development
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
plan b
Plan B
  • Collect larvae
  • Barcode larvae
slide10

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

pcr strategy
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
museum specimens
Museum specimens

n = 240

Mean age = 37 years

effect of amplicon length on pcr
Effect of amplicon length on PCR

“PCR success” = DNA band (of expected size) (not the same as sequencing success!)

current data set
Current data set

Added short sequences (238-329 bp) for ~70 adults

slide17

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

slide18

92 bp alignments

5’-section

3’-section

conclusions
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