375-380
Odonatologica 32(4):
December
The use of Odonata museum specimens in
I, 2003
questions
of molecular evolution
R.J. Lodge and J.R. Freeland
*
Department
of
Sciences,
Biological
MK7
Received
Studies
of
representing
be
variety
of
collections therefore
from
very
described
provide
a
potentially
(PCR)
allowed
DNA from
to 80 yrs old. It is
of
means
or
anticipated that
Keynes,
2003
from individuals
necessary individuals
may
geographicallyremote organisms. Museum
valuable resource,
and the
that
of DNA
target
regions
widespread use
in future these
taken from
museum
will be
of
amplified
techniques
and sequence
amplify,
techniques
of conservation
be
can
modifications to DNA extraction
single dragonfly legs
studies, including questions
Milton
Accepted January 20,
the authors to extract,
parts
Hall,
phylogenetics require samples
small amounts of tissue. Here
that have
and
Walton
Kingdom
populations. Collecting the
for seasonal, rare,
chain reactions
mitochondrial
odon.
and
spp.
problematic,particularly
polymerase
and
population genetics
a
United
/ Revised
2002
July 8,
Open University,
6AA,
are
portion
a
specimens
applied to
a
range
of
up
of
genetics.
INTRODUCTION
Successful extraction of DNA from
wide range of studies. First,
geographical
ranges than
can
museum
museums
be visited
samples
often house
be of great benefit
can
samples
individual researchers
by
duration, and they may therefore allow biogeographical studies
areas
than would otherwise be
varying
altered
time scales will add an
species distributions,
changes
over
alternative
important
conservation
time.Third, because
to
circumstances
capturing
provide
A number of studies
1994; MUNDY
*Author for
investigated. Second,
et
and
a more
on
on
grants
to cover
are
of limited
much
larger
concerned with
otherquestions that may involve
samples already exist, they
live individuals,
humane alternative to de
and
novo
birds and mammals (e.g. ROY
et
may
provide
therefore in
an
some
sampling.
al., 1994; TAYLOR
al., 1997) have used genetic information from
correspondence: J.R.Freeland@open.ac.uk
a
collected over
samples
dimension to studies that
genetics, and
museum
sampling
access to
to
from much broader
museum
et
al„
samples
to
376
R.J.
address
Lodge &
J.R. Freeland
questions of conservation genetics (see
However, insect
(CATERING
al., 2000). Here
et
of DNA from
dragonfly
have
samples
museum
also LAND WEBER, 1999 for
yet been used in
not
successful extraction and
we report on our
samples
museum
up
review).
comparable
a
manner
amplification
80 years old, taken from Anax junius
to
and Aeshna mixta.
METHODS AND RESULTS
In
developing these protocols,
number
of museums and
dried, adult
samples
and
we
used Anax
other collections
small,
junius
and Aeshna mixta
(see acknowledgements).
80
old,
up to
years
(<10
long)
mm
to minimise contamination risk, extractions from tissue with
were
done in
material
a
was
laminar flow hood
In
processed.
microcentrifuge
and vortex
in
were
al.
is
years old, and
a
which is
(2001),
a
dedicated
specific
Two methods ofextraction
ten
small, enclosed
a
addition,
single leg
this
to
generally dried, and
in
1.5ml
with
had formed
a
seldom if
multiple
fine
storage buffer),
existing protocol
a
cationic
ever
DTAB
concentrations aiding in the
When
required,
section),
K
was
where
a
was
K
added. The additional
small
quantities
digestion
proteinase
of muscle
nucleases from DNA, aids in the
yield
from these
incubation used by
et
The final modification that
to
the
hours,
incubation time
was
extended here
to
to
to
et
the
up
to
WATTS
samples
were
a
et
sample
al. (2001) short
to
are
placed
motorised
50% ofthe
homogenate
lyse
binds DNA in
details
(for
to
term
the cell
salt
high
as
was
of DNA
PCR, such
to
legs,
removes
thereby increasing
the
overnight
samples.
with
to rotate
yield
silica in
glassmilk
at room
original protocol.
of DNA
by providing
suspended
high
from other
as
for small
Proteinase K
effective than the
outlined in the
increase the
later in this
and thus the separation
proteins
more
see
48 hours if proteinase
required
was
collide and bind with the
separation
to
be
on newer
(BiolOl, 2001).The selective binding of DNA
RNA, and substances inhibitory
museum
at least
preserved.
of adhering
30 minutes
for DNA molecules
salt allows the
as
the DNA. The second cationic
digestion step
have been
improved yield
two
chaotrophic
such
samples
Single legs
extended from 20
proved
(2001)
temperature for
opportunities
to
ground using
selectively
included
was
K
may
This
al.
not
in which fresh
equipment
al., 2001 for details). The
for WATTS
as
that it
was
digestion
samples.
WATTS
a
acetone
in ethanol. In order
protocol by
of DNA from other cellular components (SAUNDERS, 1999),
DNA
or
of other cell components (SAUNDERS, 1999).
with incubation in DTAB which
only
et
until
nitrogen,
then added
advantage
separation
proteinase
air
yield (e.g. partial legs)
applied
was
then
forceps,
(prepared
detergent,
CTAB had the added
other
stored in DTAB buffer.
components and denature proteins, thereby releasing
detergent
and
necessary because
additions of liquid
powder.
DNA
extraction
(see WATTS
were
tubes and crushed with
eppendorf
pellet pestle
low
provided by
extraction method using DTAB and CTAB buffers
salting-out
are
were
included
area.
used. The first method
modification of the
and Geneclean (BiolOl) kit components
modifications
‘clean’
samples
different floor to the lab
used,
were
that
samples
samples stored
larval
relatively
room on a
pipettes
to this separate
were
a
The
The
more
silica matrix
concentrations of
proteins,
chitin and other
most
a
forms of
polysaccharides
DNA
extraction
technique for
(SAUNDERS, 1999), which may form
The second method of extraction
substantial
used for
proportion
samples
more
of these
than
samples.
years old. For
ten
samples (often
DNA
(BiolOl), which is designed for isolation of DNA from samples of bone, preserved
tissue,
animal
or
ultra-pure,
required
as
by-products.
half a
a
377
specimens
these
as
small
was
museum
some
of
a
48 hour
with the
proteinase
K
manufacturer)
EDTA
such
was
as
added to
20
the
calcium and
an
soaking
agarose
to
standard
heating
A and
was
protocol
glassmilk
binding.
oxidase 1 mtDNA PCR
protocols,
which
Samples
included
incubation
proteinase K) represents
1 -6
168
an
anionic
success
the maximum of
pi
0.5MEDTA,
distilled deionised
pi
by chelating
detergent,
when
15 hours
water.
divalent cations
to
solubilize cell
for younger
1-2 hours
museum
at room
products (354 base pairs long)
proteinase
(80°C). Sample
1 year old
samples,
was
temperature,
as
an
this
The third modification was towards the end
K
7
from Aeshna mixta,
(dehybemation A, A2,
digestion (prot. K.),
(using protocol
the oldest 4 eshna mixta
were
to
solution consisting of2
K and
to
labels show the extraction buffer used
step after initial
obtained.
soaking
of DNA from
separation
had much greater
proteins (MILLIGAN, 1998). Our second modification,
the likelihood of DNA
gel. Lane
a
and SDS,
alteration of the above
Fig. 1. Cytochrome
the manufacturers suggested
solution to inhibit DNases
extension of the incubation with
improved
in
mg/ml proteinase
magnesium
membranes and denature
echoing
using
in addition to incubation with
aid in the
digestion (as opposed
pi
10
to
adhering proteins. However, we
20
10% SDS,
K
proteinase
recommendedby the
pi
protocol. First,
dehybemation solutions,
with
samples
cell components and
did
used the Geneclean kit for Ancient
This kit contains instructions for DNA extraction
experimentation
pre-incubation
we
we
ultra-clean solutions (to furtherminimise contaminationrisk), but nevertheless
chemical denaturants in the kit’s
a
single leg)
sample (80
dried A. mixta.
as
years
for
B
or
and/or
DTAB)
an
sample 1,
old)
run
additional
with
on a
1%
and additions
10
min.
dehybemation
for which mtDNA sequence
378
R.J.
of the
protocol.
50-100
when
we
used
glassmilk
only
35
pi
point
worth
manufacturers include
case
of
an
to
dehybemation
solutions for the initial
proteins, capable
inhibiting
nuclease
of
on
lysis/denaturation
guanidine
which is
of
solutions varies between
dragonfly legs,
without the
we
and
the environment in which
additional
Success of DNA extractions
(mtDNA)
samples, particularly
obtained the best results from
suggested
strong
nuclear
activity (SAUNDERS, 1999). Dehybemation
demineralise bone tissue, combined with
sample preservation
a
and
dissolving cytoplasmic
EDTA based solution, which will inhibit DNases, and in the
aqueous
specific samples
two
yields
soak into the
detergent
a
cell membranes and other components (MILLIGAN, 1998; Bio 101,2001). The
of these
in
pellet
had higher DNA
about the Geneclean kit for Ancient DNA is that the
noting
two
chemical denaturant of
solution B is
we
by gentle pipetting.
solution A, is based
step. One of these, dehybemation
membranes and
the DNA
resuspending
for 1-2 seconds, but
vortexing
of elution solution, and allowed this solution
and then mixed
The other
J.R. Freeland
At this stage, manufacturers suggest
elution solution by
pi
Lodge &
cytochrome
with
respect
samples
dehybemation
were
to
lyse
efficacy
the method
to
kept.
solution A
For the
1),
(Fig.
detergent (A2) provided (BiolOl, 2001).
confirmed
was
oxidase
I
using
by amplifying
a
of mitochondrial
region
‘universal’ COI
Cl-J-1751
primers
’
(5 GG ATCACCTG ATATAGC ATTCCC3’) and Cl-N-2191 (5’CCCGGTAAAATTAAAATATAAACTTC3’)(PALUMBI
2-6
et
al., 1991). Amplification reactions included
pi DNA, IX PCR buffer with 1.5 mM MgCl
of each
primer,
amplification
and 1U
Taq
(Promega)
with 2 minutes
proceeded
program
200
(Promega),
2
polymerase
DNA
at
in
a
pM dNTPs, 0.5 pM
50
pi
95°C for 30 seconds, 50°C for 30 seconds and 72°C for 30 seconds, and
72°C for 5 minutes. An
-2191,
was
order
to
junius
verify
et
on
difficult/ low
large proportion
that the DNA
individuals
Wishart
A
data).
from A.
was
of these
junius
or
al., GenBank accession
no.
were
to
AF550581).
of
our success
354-490 bp
because
To
date,
aged
aged
we
samples
are
(accession
was
only
20-39 years old,
42% of 7
drop
as
fragments
high
a
are
as
study
of
aeshnid
numbers AF550515
that the
increasing
fragments
we
somewhat conservative estimate
generally
easier
to
amplify
from
in
success
most
levels of success, but this varied with the age
80-40 years old, 75% of the 8
samples
10-19 years and 74% of 65
samples
samples aged
lack of muscle tissue in very small larval
exoskeleton), although
therefore
samples aged
100% of the 6
2-9 years old. The
of 92 Anax
from the
3.65 % for A. junius,
noting
et
in
relatively large fragments.
have had reasonably
e.g.
are
small
relatively
old, degraded DNA than
of the
and
long,
Freeland
sequenced
AF429284) verified the sequences
The maximum sequence divergence
were
final step of
alignment
subpupilla
14% when the 5 confamilials were included. It is worth
amplified
subsequently
A. mixta. An
COL These sequences havebeen submitted to GenBank
to
a
yielding samples (J.R.
5 confamilials(including Aeshna
plus
The
internal to Cl-J-1751 andCl-N-
aeshnid-specific primer pair,
also used in nested PCR
al., unpublished
reaction.
95°C, followed by 40 cycles of
of the
rate
for the last age category may be due to the
samples (especially compared
recent
samples
were
to
preserved
the
toughened
in ethanol
as
DNA
opposed
Of the
to
air dried,
remaining
extraction
technique for
the
samples
effect on the percentageof successful
no
DNA kit methods,
although
bands upon PCR with
did
ranges
not
yield
from
amplify
a
over
these
were
not
K
(Fig.
digestion
For
minority.
generate
a
the
sample.
and storage had
did
or
Ancient
yield brighter
Some samples from all age
1).
amplification,
in the
quality of
preservation
with eitherthe CTAB
proteinase
20 years old
that did
sample
method of
amplifications,
any DNA upon PCR
largely unclear, although
may
exact
the addition of the
samples
379
specimens
may also affect the
preservation technique
dried adult
museum
for
reasons
example,
a
that remained
144bp fragment
345bp fragment (HANDT
et
al.,
1994).
DISCUSSION
These results suggest that
research
sampling
will
is
restricted.
highly
Museum
in
keeping samples
e.g. parts
of
aware
will
single
a
the respective
be
the
viable
source
of DNA for future
insect
leg,
was
not
sample
age
sequences from
preservation
very
allows the retention of
to
range) they
museum
DNA recovery.
small
quantities
large proportion
a
for further study (CATERING
should be able
35%
to
excess
of
genetics (ROY
et
can
et
samples
at
not
all
samples
reach their target number of desired
specimens. Degradation
al., 1994, CATERING
The
sample
samples (in the 10-80
of DNA
over
time
method may limit the molecular techniques available, but
have shown, the information obtained
of
of
2000). Researchers should
et al.,
factor in the likelihoodthat
a
kept
drying samples (with acetone)
preclude possible
obtained from
that, although they may have
current
particular sample
outlined above, this likelihood can be
be assured that
should
envelopes
museums
can
yield DNA, by obtaining approximately
year
are a
there is no guarantee that any
protocols
curators
with which useful sequence
ease
samples
Although
yield DNA, by following
reasonably high.
or
museum
odonates, and should be particularly useful for studies in which
on
be invaluable
to
as
regardless
of
many studies
studies of conservation
al., 2000).
ACKNOWLEDGEMENTS
K.F.
CONRAD,
GOODGER
P.
BRUNELLE
(Bitter
Insect
M.
MAY, K. SIMPSON,
(Natural History
(Nova
Lake National
Museum,
Scotia Museum),
Wildlife
B. LANDWER
London),
Refuge),
J.
V.
(Wilbur R.
C. BELLAMY
SCOTT
ACORN,
F.
(University
ORR,
Entomology Museum),
Natural
of Colorado
SPERLING,
Museum), B. KONDRATIEFF (Colorado State University), N.
M. McPEEK, R.
Enns
(Los Angeles
S. SHAW
D.
History Museum),
Museum),
(University
R. LARSEN
of
Wyoming
VON ELLENRIEDER, R. DUBOIS,
G. SPRANDEL.
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