Molecular Ecology Resources (2015) doi: 10.1111/1755-0998.

12380

Collecting in collections: a PCR strategy and primer set for

DNA barcoding of decades-old dried museum specimens
ANDREW MITCHELL
Australian Museum Research Institute, Australian Museum, 6 College Street, Sydney, NSW 2010, Australia

Abstract
Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions
about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding
projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that
purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods.
Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This
study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect
specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of
older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies
utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high-
throughput laboratory workflows. The strategy uses hemi-nested, degenerate, M13-tailed PCR primers to amplify
two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are
reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI
gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE
standard-compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of
specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification,
‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by
curation work in decades past.

Keywords: COI, DNA barcodes, DNA sequencing, insect

Received 6 October 2014; revision received 10 January 2015; accepted 13 January 2015

generally considered a suitable source of tissue for DNA

Introduction
In biological and especially in biodiversity sciences,
DNA barcode data in public databases are increasing
exponentially due to massive efforts in both Sanger and
Next Generation Sequencing techniques. Natural history
museums are uniquely placed to map the links between
these data and species names, represented by specimens
in their collections and taxonomic monographs in their
libraries, through DNA barcoding. Museum collections
may contain the bulk of a region’s described species,
sorted and identified by a succession of specialists over
many years. Most of the specimens were collected before
DNA sequencing became a mainstream occupation for
biodiversity science and were preserved with only mor-
phological characters in mind. The DNA in these dried
specimens degrades within a few years, and they are not
Correspondence: Andrew Mitchell, Fax: +61 2 9320 6011;
E-mail: Andrew.Mitchell@austmus.gov.au
sequencing studies beyond about 2 years (Bisanti et al.
2009) to 15 years of storage at ambient temperatures
(Hern
andez-Triana et al. 2014). Unfortunately, this
means that the vast majority of dried specimens, such as
pinned insects in most museums, remain untapped for
DNA sequencing studies. Indeed, almost all DNA bar-
coding studies published to date have had to incorporate
fieldwork to collect fresh material for DNA sequencing,
even when old material has been sequenced as well (e.g.
Hausmann et al. 2009). Notable exceptions include Strut-
zenberger et al. (2012) who sequenced 96 moth speci-
mens aged 79–157 years, by amplifying six short
overlapping PCR fragments for each sample, and Hebert
et al. (2013) who used a six-step PCR protocol to amplify
full or partial DNA barcodes from 31 585 specimens with
a median age of 28.9 years.
Recollecting specimens for DNA barcoding studies is
not only a time consuming and costly endeavour, it is
also highly unlikely to result in as broad and complete a

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2 A. MITCHELL
collection of taxa as that already accumulated in
museum collections over decades or centuries of collect-
ing effort. Ideally, one should be able to integrate the
sequencing of dried museum specimens into DNA bar-
coding studies in order to forge direct rather than
inferred links between specimens and DNA sequence
data. Sequencing older museum specimens would allow
one to fill the inevitable sampling gaps and capitalize on
the unpublished knowledge stored in collections through
the curation process, such as undescribed species already
recognized as different and separated in the collection,
awaiting formal description.
The advent of next generation sequencing (NGS) tech-
nologies holds much promise for sequencing of older
museum specimens (Pe~ nalba et al. 2014), although many
issues remain to be resolved, particularly in sequence
capture techniques and analytical challenges. Although
these technologies bode well for the future utility of old
museum material as a source of tissue for DNA sequenc-
ing, biodiversity researchers still need to rely on PCR-
based, first generation Sanger sequencing for data collec-
tion, as suitable techniques are not available to date. This
means that most museum specimens will continue to be
unsuitable for DNA barcoding studies for some time to
come, unless laboratory techniques can be developed
that extend the utility of dried museum specimens for
DNA sequencing studies.
This study first assessed whether simply amplifying
and sequencing shorter PCR fragments would provide a
practical solution to overcoming PCR failure in older
insect specimens, using a sample of Anoplognathus bee-
tles (Coleoptera: Scarabaeidae: Rutelinae) ranging in age
from 2 to 53 years old. Subsequently, a new PCR primer
set and a novel two-step PCR strategy are developed,
allowing routine, high-throughput PCR amplification
and Sanger DNA sequencing of 559 bp of the DNA bar-
code region of the mitochondrial COI gene of many
orders of insects, including Coleoptera, Lepidoptera,
Diptera, Hemiptera, Odonata and most likely other taxa.
This PCR amplification protocol greatly extends the util-
ity of pinned museum specimens for Sanger sequencing
from around 5 years old to at least 25 years old for com-
parable PCR success rates (99% success, i.e. 83 of 84 spec-
imens yielded sequence data) and even for the oldest
specimens utilized (at least 50 years old) albeit with
much lower success rates.
Materials and methods
Insect specimens
Two sets of specimens were utilized. The first set
comprised 133 specimens of Anoplognathus species
(Coleoptera: Scarabaeidae: Rutelinae) ranging in age
from 2–53 years old at the time of DNA extraction, with
mean and median ages of 32.8 and 34.0 years, respec-
tively. These specimens were used to test the effect of
PCR amplicon length and specimen age on PCR success.
The DNA extractions from this first set were depleted by
PCR experiments; therefore, a second leg was subse-
quently sampled from each specimen which had failed
to yield a PCR product of at least 300 bp (117 specimens)
and used to test the PCR reamplification strategy
described below. A further 24 beetles (Anoplognathus and
closely related genera) and 56 moths of the genera Adisu-
ra and Australothis (Lepidoptera: Noctuidae: Heliothinae)
were added to this second set of specimens (Table 1).
DNA extraction
One leg was removed below the coxa for smaller beetle
specimens and all moths, and below the femur for larger
beetle specimens, using forceps and microdissection scis-
sors and placed in a 1.5-mL microcentrifuge tube. To
ensure adequate penetration of DNA extraction buffer
into beetle leg muscles, each femur, tibia and tarsus of
beetles was cut into two or more pieces. Forceps and
scissors were sterilized between specimens by first wip-
ing with laboratory tissue, then dipping into 100% etha-
nol and flaming. DNA extractions were performed using
a GenEluteTM Mammalian Genomic DNA Miniprep Kit
(Sigma-Aldrich, Sydney) using the manufacturer’s rec-
ommended protocol, with DNA elution volumes
adjusted to 100 lL.
Testing PCR success by specimen age and amplicon size
Initial PCRs using primer pair LCO1490 – HCO2198 (Fol-
mer et al. 1994) were unsuccessful for all scarab beetle
samples but successful for a positive control lepidopteran
sample. To disentangle two possible factors causing this
PCR failure, that is primer/template mismatch and DNA
degradation, and ameliorate them, PCR primer pairs
were designed to target amplicons of decreasing length
from a broad range of insect taxa. Approximately 70
complete COI sequences, including published sequences
available on GenBank and unpublished data, were
aligned in BIOEDIT ver. 7.0.9 (Hall 1999). Taxa represented
in the alignment, in decreasing order of abundance,
included Lepidoptera, Diptera, Coleoptera, Hemiptera,
Hymenoptera and Orthoptera. The alignment was
scanned manually for regions within the COI-50 region,
containing both a high proportion of conserved sites and
a relatively high-GC content. Degenerate primers were
designed manually to match most sequences at such
sites, targeting six amplicons of 667, 329, 250, 199, 148
and 140 bp, excluding primers (Table 2). PCR was
performed on the first set of 133 scarab beetle DNA
© 2015 John Wiley & Sons Ltd
 COLLECTING IN COLLECTIONS: BARCODING OLD INSECTS 3

Table 1 Age distribution for specimens

Beetle set 1 Beetle set 2 Moth set 1 Moth set 2 used for PCR reamplification experiments
(Anoplognathus) (Anoplognathus) (Australothis) (Adisura)

n 117 24 23 33
n <25 years old 19 14 17 30
% <25 years old 16.2 58.3 73.9 90.9
Median 34 23.5 15 15
Mean 32.8 19.0 21.3 16.0
Std Dev 9.1 10.6 12.5 8.9
Minimum 4 2 4 2
Maximum 53 36 55 47

Table 2 Primer sequences and amplicons lengths for 6 amplicons

Amplicon length
Amplicon Primer sequence (50 -M13 tail separated (excluding primers)
number Primer Name from gene-specific sequence by a hyphen) (bp) Reference and notes

1 BC1Fm GTAAAACGACGGCCAGT-TCWACWA 667 Cho et al. (2008)

AYCAYAARGAYATYGG
1 Scar-3RDm CAGGAAACAGCTATGAC-AAAATRTA 667 Mitchell & Maddox (2010)
WACTTCDGGRTGNCC
2 BC1Fm (above) 329
2 AMbc5r1m CAGGAAACAGCTATGAC-GADARWG 329 This study, see Table 3.
GNGGRTANACDGTTC
3 Scar-1aFm GTAAAACGACGGCCAGT-AAYGTNA 250 This study
TYGTNACWGCHCAYGC
3 BC2Rm CAGGAAACAGCTATGAC-CCTAAAA 250 This study
TDGADGARAYHCCNGC
4 Scar-2F CTATCTTAATTGGWGGATTYGG 199 This study
4 BC2Rm (above) 199
5 Scar-3aFm GTAAAACGACGGCCAGT-GCHCCHG 148 Gopurenko et al. (2013)
AYATAGCNTTYCCNCG (but sequence reported
incorrectly in that study).
5 BC2Rm (above) 148
6 Scar-2F (above) 140
6 AMbc5r1m (above) 140

extractions for most of the six amplicons, to examine the
relationship between specimen age and the degree of
DNA degradation. A 2 lL aliquot of each PCR was elec-
trophoresed on a 1.5% agarose gel in TAE buffer, stained
with GelRedTM (Thermo Scientific, Scoresby, Australia)
and scored as successful if a DNA band was visible and
judged to be of sufficient concentration for DNA
sequencing, that is approximately 25 ng/lL. PCR prod-
ucts were sequenced only if a sequence had not previ-
ously been obtained for that region of the gene for that
sample. If a DNA sequence was determined not to
belong to the species that sample belonged to, the PCR
was subsequently rescored as unsuccessful.
PCR amplification strategy for recent specimens
Primer sequences are given in Table 3. The following
naming conventions were used for new primers: ‘AM’
denotes author; ‘bc’ denotes barcode; ‘0’, ‘5’ or ‘3’ denote
whether primers target the ends of the barcode region
(for amplifying the entire region), or the 50 -half or 30 -half
only; ‘f’ and ‘r’ denote forward and reverse; ‘1’ or ‘2’
denotes whether the primer is intended for use as a first
round (external) primer, or a second round (internal) pri-
mer for reamplification reactions; ‘m’ denotes 50 -M13 tail
incorporated into sequence.
The primer pair AMbc0f1m – AMbc0r1m was
designed to amplify the full-length barcode fragment,
yielding 667 bp of COI sequence for Lepidoptera, Cole-
optera, Hemiptera (Auchenorrhyncha), Diptera and Od-
onata. Primer AMbc0r2m is an alternative reverse strand
primer, binding 21 bp upstream of AMbc0r1m and yield-
ing a 646 bp amplicon. AMbc0r2m is a modified version
of JerR2m, previously used for Diptera (Bellis et al. 2013)
and Hemiptera (Gopurenko et al. 2013), although it
amplifies a broader range of insects, including the taxa

© 2015 John Wiley & Sons Ltd

4 A. MITCHELL
Table 3 Degenerate primers for amplification and sequencing of DNA barcode region of COI gene. All primers designed for this study,
unless noted
Primer sequence (50 -M13 tail separated from gene-specific sequence
Primer name* by a hyphen)
AMbc0f1m GTAAAACGACGGCCAGT-TCWACWAAYCAYAARRWTATYGG
AMbc0r1m CAGGAAACAGCTATGAC-AAAATRTAWACYTCDGGRTGNCC
AMbc0r2m CAGGAAACAGCTATGAC-CAAARAAYCARAAYARRTGYTG
AMbc5r1m CAGGAAACAGCTATGAC-GADARWGGNGGRTANACDGTTC
AMbc5r2m CAGGAAACAGCTATGAC-GTTCANCCNGTWCCWGCNCC
AMbc3f1m GTAAAACGACGGCCAGT-GCHCCHGAYATAGCNTTYCCNCG
AMbc3f2m GTAAAACGACGGCCAGT-TTYCCNCGRMTRAAYAAYATNAG
AMbc3r1m CAGGAAACAGCTATGAC-ARYATNGTRATNGCNCCNGC
miniScarFm GTAAAACGACGGCCAGT-TTYCCNCGRMTRAAYAAYATRAG
miniLepFm GTAAAACGACGGCCAGT-TTYCCNCGAATRAAYAAYATNAG
JerF2m GTAAAACGACGGCCAGT-CARCAYYTRTTYTGRTTYTTTGG
M13F GTAAAACGACGGCCAGT
M13R-pUC(-40) CAGGAAACAGCTATGAC
*Naming convention used: ‘AM’ denotes author; ‘bc’ denotes barcode; ‘0’, ‘5’ or ‘3’ denote whether primers target the end of the barcode
region (for amplifying the entire region), or the 50 -half or 30 -half only; ‘f’ and ‘r’ denote forward and reverse; ‘1’ or ‘2’ denotes whether
the primer is intended for use as a first round (external) primer, or a second round (internal) primer for reamplification reactions; ‘m’
denotes 50 -M13 tail incorporated into sequence.
sampled in this study. It was not used in this study but is
included here for completeness. An additional forward
primer, JerF2m, was designed at the same site as
Ambc0r2m to facilitate amplification of the 30 -half (non-
barcode fragment) of the COI gene when additional COI
data are required and it is simply a degenerate, M13-
tailed version of the primer ‘Jerry’ (Simon et al. 1994).
PCR amplification strategy for older specimens
PCR reamplification with hemi-nested primers has pro-
ven useful in amplifying low-copy number nuclear genes
(e.g. Mitchell et al. 2000) even when no PCR product is
Notes
Based on BC1Fm (Cho et al. 2008) but
incorporates the additional degeneracy
of BC1culicFm (Bellis et al. 2013).
Binds to the same site as LCO1490
(Folmer et al. 1994)
Based on Scar-3RDm (Mitchell & Maddox 2010).
Based on JerR2m from Bellis et al. (2013) but
more degenerate and one base shorter
on 50 -end (where the last 3 nt of M13
sequence now matches the COI template,
coincidentally). Sequence of JerR2m
reported incorrectly in Gopurenko et al. (2013).
Based on Scar-2RDm reported in
Gopurenko et al. (2013) but more degenerate.
Sequence of Scar-2RDm reported incorrectly
in Gopurenko et al. (2013).
Based on Scar-3aFm of Gopurenko et al. (2013)
but sequence reported incorrectly in
Gopurenko et al. (2013).
Combines the degeneracy of both miniScarFm
(designed for Coleoptera) and miniLepFm
(designed for Lepidoptera) so this single
primer can be used for both taxa.
Designed for Coleoptera only, see Materials
and methods.
Designed for Lepidoptera only, see Materials
and methods.
Degenerate, M13-tailed version of Jerry
(Simon et al. 1994) for amplifying 30 -half
of COI gene (not the DNA barcode fragment).
M13 forward sequencing primer
M13 reverse sequencing primer
detected by agarose gel electrophoresis, therefore, that
strategy was attempted in this study. Further degenerate,
M13-tailed PCR primers were designed for two overlap-
ping amplicons, each of approximately 320 bp (Fig. 1).
In combination, these amplicons yield a contiguous
559 bp fragment of the COI DNA barcode region, far
exceeding the 486 bp (i.e. 75% of 648 bp) required by the
BARCODE data standard (Hanner 2009). An additional
internally nested primer was designed for each of these
two amplicons, to facilitate specific reamplification of tar-
get DNA from initial PCRs.
The two-step amplification strategy is illustrated in
Fig. 1 and outlined below.
© 2015 John Wiley & Sons Ltd
 COLLECTING IN COLLECTIONS: BARCODING OLD INSECTS 5
Full barcode
amplicons
AMbc0f-1 m
AMbc0f-1 m
LCO1490 100 200
½ amplicons
89 bp
1st round
AMbc0f-1 m 329 bp
AMbc3f-1 m
½ amplicons
2nd round
58 bp
M13F 313 bp
AMbc3f-2 m
Fig. 1 Primer map and amplification strategy.
1 Two PCRs are performed using primer pairs
AMbc0f1m – AMbc5r1m and AMbc3f1m – AMbc3r1m,
which target overlapping 50 - and 30 -amplicons, respec-
tively.
2 The PCR products from step 1 above are used as the
DNA template for reamplification PCRs using primer
pairs M13F – AMbc5r2m and AMbc3f2m – M13R-pUC
(-40), respectively.
In the experiments reported here, primers miniS-
carFm and miniLepFm were used for scarab beetles and
moths, respectively, in place of AMbc3f2m, but the latter
primer was subsequently developed by incorporating
the degeneracy of both primers and has since been used
successfully to amplify both taxa.
Sterile technique and practical considerations
It is essential to practice robust sterile technique, use fil-
tered pipette tips and run sufficient DNA-free negative
controls to avoid cross-contamination of samples when
using the PCR reamplification protocol. PCR products
from primary (first round) amplifications usually are not
of sufficient concentration for visualization on an agarose
gel, besides which, opening the PCR tubes in the post-
PCR area of a general use PCR laboratory just increases
the chances of cross-contamination. It is prudent, there-
fore, not to run a diagnostic gel on the first round PCRs.
Instead, the plate is cooled and centrifuged to reduce
aerosols and opened only inside a UV-sterilized laminar
flow hood.
Primary (first round) PCR products are diluted 1:10
with PCR-grade water, and 1 lL of the diluted PCR
product is used as DNA template for the second round
of PCRs (reamplifications). To minimize time and con-
sumables (pipette tips), it is easiest to set up two new
© 2015 John Wiley & Sons Ltd
JerF2m
667 bp AMbc0r-1 m
646 bp AMbc0r-2 m
559
300 400 500 600 HC02198
AMbc5r-1 m
319 bp AMbc3r-1 m
AMbc5r-2 m
304 bp M13R-pUC (–40)
PCR plates for the secondary (reamplification) PCRs, a
dilution plate with 10 lL of sterile PCR-grade water and
the reamplification PCR Plate containing the PCR mas-
termix. Both plates are prepared in a separate pre-PCR
clean laboratory and sealed with strip caps before trans-
fer to the PCR hood for aliquoting of the DNA template.
Working in the PCR hood, only a single row or col-
umn of each plate is uncapped at a time, to minimize
cross-contamination. A multichannel pipette is used to
transfer 1 lL of primary PCR products from the primary
PCR plate to the dilution plate, where they are mixed by
pipetting out and in a few times. The now diluted 1 lL
contents of the pipette tips is then dispensed into the
corresponding row/column of the secondary (reamplifi-
cation) PCR plate containing mastermix.
PCR conditions
PCRs were prepared using an Invitrogen Platinum Taq
PCR kit (Life Technologies, Mulgrave, Australia). Each
15 lL reaction contained 19 PCR buffer, 2.8 mM MgCl2,
200 lM dNTP mix, 2 pmol each of forward and reverse
primers, 4 lL of genomic DNA extraction (or 1 lL of
1:10 diluted PCR product for reamplification reactions)
and 0.375 U (0.075 lL) of Platinum Taq DNA polymer-
ase. Thermal cycling was performed using an Eppendorf
Mastercycler ep gradient S PCR machine and consisted
of an initial 2 min at 94 °C, followed by a 40 cycles (35
cycles for reamplification reactions) of 30s at 94 °C, 40s
at 50 °C, 60s at 72 °C, a final 7 min extension at 72 °C
and storage at 10 °C. Secondary (reamplified) PCR prod-
ucts were electrophoresed on a 1.5% agarose gel and
visualized on a UV transilluminator. Successful PCRs
were sequenced only if the sequence of that gene region
had not previously been obtained for that DNA extrac-
tion.
6 A. MITCHELL

DNA sequencing was performed on an ABI3730xl,

and sequence trace files were checked for accuracy and
assembled into contigs using GENEIOUS v. 6.5 (Kearse et al.
2012). All DNA sequences were deposited in GenBank,
Accession nos KP688405 – KP688569.

Results

Amplification of the entire DNA barcode region from

recent material
PCR amplification of the full-length barcode fragment
(667 bp) with primer pair AMbc0f1m – AMbc0r1m was
successful for the vast majority of recent (1–3 years old)
and ethanol-preserved insects sampled. For specimens
more than 3 years old, it was more efficient to move Fig. 3 PCR reamplification success plotted as percentage of
directly to the reamplification strategy. samples from which ‘Either’ or ‘Both’ the 50 - or 30 - amplicons
were recovered for each age category.

PCR success of different age specimens for different

amplicon lengths
PCR success rates for the first set of beetle specimens are
summarized by specimen age and PCR amplicon size in
Fig. 2. High PCR success rates, generally more than 66%,
were seen for all amplicons for the specimens aged 0–
5 years, for all specimens regardless of age for the two
shortest amplicons, 140 and 148 bp, and for specimens
aged 6–20 years and amplicons 199 and 250 bp. All other
combinations yielded PCR success rates of <40%, and
most were <20%.
PCR reamplification protocol
Results of the PCR reamplification experiments are
shown in Fig. 3 and Table 4. Figure 3 shows PCR suc-
cess with success scored as ‘either’ fragment amplified
and ‘both’ fragments amplified, summarized by speci-
men age categories. Success rates were above 60% for all
100
90
80
PCR success (%)
age categories when scoring amplification of either frag-
ment as success, but when both fragments were consid-
ered (i.e. BARCODE standard compliance) success
ranged from 29% to 74%. Table 4 shows how many sam-
ples were successful for both 50 -amplicon and 30 -ampli-
con reamplification PCRs, vs. only one of the two
fragments or neither. Both amplicons could be reampli-
fied from 72% of all specimens up to 25 years old, but
only 36% of older specimens. When only one of the two
fragments could be amplified, it was always the 30 -half
amplification that failed for specimens up to 25 years
old. For older specimens, the 30 -half amplicon failed
three times more often than the 50 -half amplicon. The 50 -
half amplicon always amplified for specimens up to
25 years old, while neither amplicon could be amplified
for 23% of older specimens, or 15% of all specimens.
Discussion
PCR amplicons of up to 148 bp (excluding primers)
could be obtained from more than 80% all specimens up
to 40 years old, and the slightly shorter amplicon of
140 bp successfully amplified from 67% of older speci-
mens up to 51 years old. For all age categories except
specimens <5 years old, PCR success rates dropped dra-

70
60
50
matically as amplicon length increased above 148 bp.
40 These primers could therefore be used to obtain
30 sequence data from older specimens if all that was
20
10 required was enough sequence to match a particular
0 specimen to existing barcode BINs. However, use of such
0-5
6-20
20-25
26-30
small amplicons is not an efficient strategy to assembly
31-35
Specimen age (years) 36-40
>40 BARCODE standard-compliant data.
667 bp 329 bp 250 bp 199 bp 148 bp 140 bp
Although desiccation of insect specimens would
appear to stabilize genomic DNA sufficiently for about
Fig. 2 PCR success for specimen age and amplicon size classes. 2 years after death (Bisanti et al. 2009) to allow most

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 COLLECTING IN COLLECTIONS: BARCODING OLD INSECTS 7

Table 4 Recovery of half-length barcode amplicons using PCR reamplification strategy

Both amplicons Only 50 -half amplicon Only 30 -half amplicon Neither amplicon
recovered recovered recovered recovered

Beetles, ≤25 years old

Successful 26 11 0 0
n (total) 37 37 37 37
% successful 70.3 29.7 0 0
Moths, ≤25 years old
Successful 31 13 2 0
n (total) 47 47 47 47
% successful 66.0 27.7 4.3 0
All specimens, ≤25 years old
Successful 57 24 2 0
n (total) 84 84 84 84
% successful 67.9 28.6 2.4 0
Beetles, >25 years old
Successful 51 23 5 25
n (total) 104 104 104 104
% successful 49.0 22.1 4.8 24.0
Moths, >25 years old
Successful 3 1 0 5
n (total) 9 9 9 9
% successful 33.3 11.1 0 55.6
All specimens, >25 years old
Successful 54 24 5 30
n (total) 113 113 113 113
% successful 47.8 21.2 4.4 26.5

routine PCR applications, DNA damage continues to
accumulate over time to the point where it is too frag-
mented to be of any practical value for amplification of
PCR amplicons of a few hundred base pairs in length.
There appears to be little consensus in the literature
about when this point is reached, but the upper limit of
all estimates appears to be 15 years (Hern
andez-Triana
et al. 2014). In this study that age appeared to be no more
than 3 years, although that might reflect differences in
DNA extraction efficiency, or simply that too few speci-
mens in the 5–15 years age range were sampled in this
study. In practice, the vast majority of DNA barcoding
studies use only specimens that are <5 years old, as this
is the age range in which one can still recover the DNA
barcode regions (500–700 bp) in a single PCR amplicon
from the majority of specimens.
Few studies have reported successfully sequencing
large numbers of specimens more than a decade old. A
notable exception is the study by Hebert et al. (2013)
which used Lepidoptera specimens from the Australian
National Insect Collection with median ages comparable
to those used in this study. Hebert et al. (2013) produced
24 671 BARCODE compliant sequences from 41 650
specimens, that is 59% success. That required 134 140
PCRs, or 5.4 reactions per specimen, in a complicated
six-step cascade of reactions, requiring different subse-
quent PCR treatments depending on the success of each
previous PCR for each DNA extraction in a plate. The
protocol described in this study was successful in 70% of
specimens up to 25 years old, and 36% of older speci-
mens, or 58% of all the Lepidoptera sampled, regardless
of age, thus success rates are comparable. However, the
reamplification protocol reported here does not require
any hit picking as all samples in a DNA plate are sub-
jected to the same two-step protocol, and the primers
work on a much broader range of taxa, essentially all of
the five orders of insects tested to date.
Zuccon et al. (2012) designed a similar protocol for
decapod crustaceans, using two overlapping PCR frag-
ments of approximately 350 bp to obtain BARCODE
compliant data from specimens up to 40 years old with-
out resorting to PCR reamplification. However, the
authors did not provide an analysis of PCR success rates
by specimen age.
The BARCODE standard requires, among other
things, that sequences be contiguous over at least 75%
the length of the standard barcode marker for that taxon
(Hanner 2009). For animals, the standard region is a
648 bp fragment of the mouse COI gene (Hanner 2009),
so BARCODE compliance requires at least 486 bp of con-
tiguous COI sequence. An efficient strategy for assem-
bling BARCODE standard-compliant sequences from
smaller amplicons requires both sufficient overlap
between amplicons to be able to establish that they are

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8 A. MITCHELL
conspecific (i.e. to detect contamination when it occurs)
and amplicons long enough that only two are required
for a BARCODE compliant sequence. The amplicons
used in this study fulfil both criteria, with an 89 bp over-
lap between first round amplicons, a 58 bp overlap
between the second round reamplified amplicons and an
assembled contig length of 559 bp.
The reamplification strategy is most efficient for speci-
mens up to 25 years old, with both amplicons being
recovered from 77% of beetles and 67% of moths, and
the 50 -half amplicon being recovered from 95% of beetles
and 97% of moths in this age category. For specimens
26–55 years old, success rates dropped to 35% and 42%
for both amplicons, and 73% and 95% for either ampli-
con, for beetles and moths, respectively. So even for
specimens up to 55 years old the strategy usually pro-
vides enough data to match a specimen to a particular
BIN, if a full-length BARCODE compliant sequence can-
not be obtained. The high success rate obtained for PCR
of either amplicons, 73% and 95% for beetles and moths,
respectively, suggests that improved PCR primer design
might be all that is required to obtain similarly high PCR
and sequencing success rates for both amplicons, yield-
ing BARCODE standard-compliant sequences.
This PCR strategy therefore extends the age range for
dried, pinned museum specimens to be useful for obtain-
ing BARCODE standard-compliant data, from approxi-
mately 3 years (for the specimens sampled in this study)
out to at least 25 years with high PCR success rates, and
at least twice that age if one is satisfied with lower PCR
success rates, as reported in other studies (e.g. Hebert
et al. 2013). This means that specimens collected from the
late 1980s onwards remain useful today for DNA barcod-
ing. The 1980s and 1990s were very active periods for
insect collecting in Australia and resulted in a large
amount of material in museum collections that has been
expertly identified and curated. This study suggests that
most of this material is amenable to DNA barcoding. The
disadvantages of having to perform four times as many
PCRs and twice the amount of DNA sequencing are out-
weighed by the ready access to comprehensive collec-
tions that have been expertly identified and curated, and
in some cases even databased. Indeed, the author has
used the methods and specimens described in this study,
along with modest collections of recent material, to
obtain BARCODE compliant data for every species of
Anoplognathus and every species of Australian Heliothi-
nae (the data presented in this study forms part of these
data sets).
Hern
andez-Triana et al. (2014) emphasize the impor-
tance of primer choice on PCR success when working
with older specimens. In addition to the Coleoptera and
Lepidoptera taxa utilized for this study, the primers in
Table 3 have been successfully used on other families of
those orders, and on Diptera, Hemiptera and Odonata,
with similar success rates although with much smaller
sample sizes. While the new primers developed in this
study amplify a much broader range of taxa than the Fol-
mer primers (Folmer et al. 1994), there remains room for
improvement in primer design for the 30 -amplicon,
because PCR success rates are significantly lower for this
fragment than for the 50 -amplicon, despite the 10 bp
shorter length of the 30 -amplicon.
Initial PCR experiments using amplicons of different
lengths suggested that the DNA of specimens more than
about 20 years old was too fragmented to allow PCR
amplification of the required 329 bp amplicons, and yet,
reamplification of these PCR products with hemi-nested
primers was successful. Clearly, a small population of
DNA molecules of sufficient molecular weight must still
exist in the original DNA extraction, but it takes a first
amplification to increase their abundance to the point
where PCR with another primer pair can succeed. It is
worth noting also that reamplifying first round PCR
products with the same primers invariably results in a
smeared product when visualized on an agarose gel,
which does not produce a readable DNA sequence. The
use of an internally nested primer on at least one end of
each amplicon is key to a successful reamplification reac-
tion.
Contamination is a concern for any strategy relying of
reamplification of PCR products; however, the issue can
be minimized through strict separation of DNA extrac-
tion, PCR set up and post-PCR manipulation in the labo-
ratory, judicious use of negative controls, and other
considerations described above in Materials and meth-
ods. In addition, further analytical quality control mea-
sures are warranted. For example, it is unwise to trust
sequences derived from reamplified PCR products
where they are the only source of data for that species.
The data would be much more robust if it were con-
firmed by sequencing a second specimen, or amplicons
derived from different DNA extractions from the same
specimen, even if they are short fragments. To this end,
it is worth noting that as a last resort to obtain COI
sequence data from valuable specimens such as types,
one may use primers AMbc3f1m and AMbc5r1m to
obtain a PCR amplicon of 89 bp from almost any insect,
and if no PCR product is visualized it is worth trying to
reamplify that PCR product using a combination of an
M13 primer on one end and one of the internal primers
AMbc3f2m or AMbc5r2m on the other end.
Next generation sequencing (NGS) technology shows
promise for sequencing degraded DNA templates, espe-
cially for sequencing many genomic regions from a small
number of samples. However, the DNA capture tech-
niques which non-amplicon-based NGS methods rely
on, work only for very similar sequences with up to
© 2015 John Wiley & Sons Ltd
 COLLECTING IN COLLECTIONS: BARCODING OLD INSECTS 9
about 10% sequence divergence (Pe~ nalba et al. 2014). In
contrast, COI sequences may be more than 20% diver-
gent between congeneric beetles, for example in the
Anoplognathus beetles sampled in this study. Thus DNA
capture currently is impractical for DNA barcoding. San-
ger DNA sequencing therefore remains the method of
choice for building a DNA barcode library. Sanger
sequencing is much more accessible for the average biol-
ogist as it requires only basic PCR skills, but the disad-
vantage is that the target DNA molecules must be intact,
that is at least as long as the target PCR amplicons. As a
result, dried specimens such as pinned insects which are
more than a few years old are generally not considered
suitable for PCR.
One of the key factors distinguishing DNA barcoding
from molecular systematics and other endeavours is the
use of high-throughput laboratory workflows to realize
the economies of scale intrinsic to genomics (Mitchell
2008). However, conventional workflows involve PCR
amplification of the barcode standard gene regions in a
single reaction, requiring high molecular weight DNA as
starting material. This has limited the application of
DNA barcoding to specimens collected in the past few
years (generally 1–5 years) in the vast majority of studies
published to date (but see Hebert et al. 2013). As a result,
the rate limiting step in most DNA barcoding workflows
is the collection, curation and identification of new speci-
mens. Use of the protocol described here provides
researchers a fast and automatable route through this
bottleneck, opening up the masses of decades-old mate-
rial in museum collections to high-throughput DNA
analysis.
It is impractical and unrealistic to expect to be able to
recollect fresh material for most species, let alone find
the taxonomic experts capable of identifying freshly col-
lected material on the scale and in the time frames neces-
sary for high-throughput DNA barcoding. As of
September 2014, the Barcode of Life Data System (BOLD,
Ratnasingham & Hebert, 2007) had recorded species
level identifications for only 39% of its almost 3 million
animal COI sequences. This is a direct consequence of a
sampling strategy that preferentially utilizes fresh mate-
rial because it is more amenable to high-throughput
sequencing. If the DNA barcoding community is to
increase the proportion of formally identified sequences
on BOLD, it is essential that it takes full advantage of the
treasure trove of previously collected, curated and iden-
tified specimens held in the world’s natural history
museums. The methods described here extend the utility
of dry, pinned museum specimens in high-throughput
DNA analysis workflows from the 1 to 5 years of con-
ventional wisdom to at least 25 years, and somewhat
beyond. This allows DNA barcoders to go collecting in
collections. By DNA barcoding material that has already
© 2015 John Wiley & Sons Ltd
been identified by taxonomic specialists, past and pres-
ent, DNA barcoders can stand on the shoulders of
experts, codify their taxonomic knowledge, even if it has
yet to be formalized through species descriptions, and
make it available in an intrinsically digital format, with-
out having to repeat the identification work.
Acknowledgements
The author thanks Tom Weir, Marianne Horak and You Ning
Su of the Australian National Insect Collection (ANIC), CSIRO,
for the loan of beetle and moth specimens, and the Australian
Centre for Wildlife Genomics for technical support. Funding
was provided by the Australian Biological Resources Study.
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AM designed the PCR primers, performed the lab work,
analysed the data and wrote the manuscript.
Data Accessibility
Two spreadsheets are included as Supplementary data.
Data S1 (Supporting information) contains the data
for Fig. 2, the amplicon length experiment (‘0’ = PCR
failure, ‘1’ = PCR success).
Data S2 (Supporting information) contains sequencing
results for each of the DNA extractions for the PCR
reamplification protocol, and the age of each specimen at
the time of DNA extraction. Table 1, Table 4 and Fig. 3
are derived from the data in this spreadsheet.
DNA sequences derived from this study have been
deposited in GenBank (Accession nos: KP688405 –
KP688569) and in Dryad (http://dx.doi.org/10.5061/
dryad.6v4h5).
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Data S1. PCR results, amplicon length experiments.
Data S2. Sequencing results, reamplification protocol.
© 2015 John Wiley & Sons Ltd