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

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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

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...and how to squeeze through it collecting in a collection

Go

Constraints: Museum policies on destructive sampling DNA preservation? Can one routinely recover DNA barcodes from 30-40 year old insect material? If yes...

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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

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Our Grand Plan

1.Collect larvae 2.Collect adults X 3.Barcode both X

Ahrens et al. (2007) Mol. Phylogenet. Evol. 3-half of COI gene (Jerry Pat)

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PCR primer development

Degenerate (Folmer) primers, with M13 tails
Lepidoptera, Hemiptera, various other insects

Folmer primers not very successful A few rounds of primer design needed

Redesigned the downstream primer 3 codons further downstream

Bark beetles & others

Considerable time & effort

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Plan B

1.Collect larvae 2.Barcode larvae

1027

L 26 10 4 L 65

8L 82 2L 83 840 L 83 8L

su A poro

L 021 3 1101 L 1020 L 101 10 9 L 0 10 6 12 L L

1 82

s 650

Results:
L 1 L 01 053 L 11 0 0 0 1 1 1 0 0 L 10 2 1014 04 12 L 00 L 0 L9 L

10 38

L 1043 L

10 41

101

5L

10 39

102

9L

97 5 973 L L 976 L 8 L 97

81 6L

903

65 5L

666

96 9L

965 L L 905 4L 90
L

73 6L

737 L

901 L L 900

97 7

891 L

7L 89

9 LL 896 89
L 898

1033 L 1035 L

1034

31 10

0.05

885 L

103 7L L 1030

829 827 82 L 6L

836 L
104
1040 10 L 42 L 4L

102

5L

981

Highly variable: p-distance = 32% within a family (vs. half that between insect orders)
7L 88 81 81 7 4L L
e ia an sm 6 72

979 L

us di ho Ap

ta

L 71 99 68 L 972 L
970 L

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

Lep Lepid idiota iota n ne 65 3L ega gto 66 ato rira 0 ia66 52 A 51

98 L 984 5L
1L 98 3L 99

9L 990 L 73 73 1L

L 8 65 Lhop 5R ea m 99 agnic ornis 661

Rh op ea m ag ni co rn is 66 2

Larvae only ~100 sequences 667 bp COI

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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

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Museum specimens
n = 240 Mean age = 37 years
80 70

Number of specimens

60 50 40 30 20 10 0 <20 25 30 35 40 45 50 >50 Age in years

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Effect of amplicon length on PCR

90 80 70 60 50 40 30 20 10 0 0 200 400 Target seq. length 600 800

PCR success (%)

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

100 90 80 70 60 50 40 30 20 10 0 92 140 238 327 340 667 Length of target seq. (bp)

PCR success rate (%)

<20 25 30 35 Age 40 45 50 >50

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Current data set

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

2.6%

Summary With exceptions to be noted,

p-distances
within 0.4% between 1.7-2.7% within 0.8%

max p-distance within = 1.3%

min p-distance between = 3.0% max p-distance = 19.1%

Anoplognathus only
NJ tree, K2P +bootstrap

68 97 48

A pallidicollis 1564 A pallidicollis 1566

A pallidicollis 1563 A rugosus 1613

46

98

A rugosus 1614

A aeneus 1319 13 98 A aeneus 1320 A punctulatus 1599 4 96 A punctulatus 1598

92 bp alignments
96

85 A porosus 1585 A porosus 1590 1010 L 1005 L 1003 L 1011 L 1019 L 1012 L 1022 L 37 A porosus 650 A porosus 1586 A porosus 1591 1021 L

73 A punctulatus 1164 38 2 38 A punctulatus 1596

A smaragdinus 1178 A multiseriatus 1355 A macleayi macleayi 1348 21 A prasinus 1162

A porosus 1592

89

A prasinus 1593 A prasinus 1594

1006 L 1013 L

93 1027 L 37 29 A viriditarsis 1643 A viriditarsis 1640

21

1020 L 1014 L 1004 L

A viriditarsis 1642
1009 L

70

A montanus 1726
A boisduvalii 1325

69 A viridiaeneus 1185 2 49 A viridiaeneus 1186

17

A chloropyrus 1333 40 1026 L A chloropyrus 1335 23 A macleayi macleayi 1348 654 L 9 A brevicollis 1327 60 A olivieri 1368

39 A montanus 1727 41 A montanus 1352 A montanus 1353 51 A porosus 1585 A porosus 1590 1021 L A porosus 1592 12 1004 L 1005 L 1010 L 74 3 1012 L A porosus 1591 A porosus 1586 1009 L 63 1022 L A porosus 650 1013 L 1019 L 1011 L 1003 L 1014 L 1006 L 1020 L A boisduvalii 1325 44 34 6 23 A chloropyrus 1333 A chloropyrus 1335 1026 L 654 L A nebulosus 1739 32 59 A brevicollis 1327 A olivieri 1368 86 A olivieri 1740 A narmarus 1359 67 68 A narmarus 1358 A narmarus 1730 A narmarus 1360 A velutinus 1634 A flindersensis 1343 A velutinus 1184 37 A velutinus 1632 A velutinus 1633 99
33 3 30 94 6 7 3 8 21 7 7

16

A olivieri 1740 94 A rugosus 1613 8 A rugosus 1614 48 A narmarus 1730 A narmarus 1359 78 A narmarus 1358 A narmarus 1360 A nebulosus 1739

41

A pallidicollis 1566 81 A pallidicollis 1563 36 A pallidicollis 1564

A smaragdinus 1178 A prasinus 1593 78 A prasinus 1162 73 A prasinus 1594 A montanus 1352 63 99 A viridiaeneus 1185 A viridiaeneus 1186 A montanus 1727 66 A montanus 1353 33 A montanus 1726 97 A aeneus 1319 31 A aeneus 1320 A multiseriatus 1355 A viriditarsis 1640 32 53 72 A punctulatus 1164 A punctulatus 1596 A punctulatus 1599 A punctulatus 1598 99 A brunnipennis 1331 A brunnipennis 1332 A velutinus 1182 A velutinus 1632 A viriditarsis 1642 1027 L A viriditarsis 1643

99 A velutinus 1184

A velutinus 1182 A velutinus 1183 A velutinus 1630 A parvulus 1577

2 1 2 2 A flindersensis 1343 A velutinus 1634 A velutinus 1633 A velutinus 1630

99 75 16 18

A parvulus 1570 A parvulus 1573

8 A velutinus 1183 60 A abnormis 1317 59 75 22 A abnormis 1315 A abnormis 1650 A abnormis 1649 A parvulus 1570 73 A parvulus 1573 61 A parvulus 1577 A concolor 1337 99 A concolor 1338 A concolor 1336 7 17 A aureus 1323 821 L 99 66 40 7 2 2 836 L 840 L 832 L 828 L 838 L 827 L

A aureus 1323 A brunnipennis 1331 99 A brunnipennis 1332 35 A concolor 1336

11

99 A concolor 1338 A concolor 1337 A abnormis 1317 40 10 97 A abnormis 1650 A abnormis 1649 A abnormis 1315 28 836 L 827 L

5-section
0.01

79 62

821 L 832 L 828 L 1 1 840 L 826 L

3-section
0.02

3 838 L 9 829 L

4 826 L 11 829 L

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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

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Acknowledgements
Funding: