CSIRO PUBLISHING
www.publish.csiro.au/journals/ajz
Australian Journal of Zoology, 2003, 51, 341–355
Extensive evaluation of faecal preservation
and DNA extraction methods in
Australian native and introduced species
Maxine P. PiggottA,B and Andrea C. TaylorA
A
B
School of Biological Sciences, Monash University, Vic. 3800, Australia.
To whom correspondence should be addressed. Email: [email protected]
Abstract
We evaluated and compared sixteen combinations of commonly used storage and extraction methods for
faecal DNA from two Australian marsupial herbivores, two marsupial carnivores and an introduced
carnivorous mammal. For all species the highest amplification and lowest genotyping error rates were
achieved using dried faeces extracted via a surface wash followed by spin column purification. The highest
error rates were seen in the two Dasyurus spp. and the lowest in Vulpes vulpes. The rates observed for each
species were incorporated into computer simulations to identify the number of PCR replicates required to
achieve accurate genotyping of DNA isolated via the optimised protocol. Three replicates per sample were
sufficient for V. vulpes, Thylogale billardierii and Petrogale penicillata. However, further replicates may be
required for marsupial carnivores, as their faeces yielded DNA that amplified substantially less often and
less reliably, for all preservation and extraction methods tested, than did the other species. Although pilot
studies remain vital for evaluating the feasibility of non-invasive sampling prior to undertaking any in-depth
study the availability of a thoroughly tested storage and DNA extraction combination protocol known to be
optimal for five different species should make that process much simpler.
ZO03012
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Introduction
Non-invasive genetic sampling has the potential to provide substantial and novel
information about species that are difficult to study by traditional means, particularly in
Australia where most native species are nocturnal, shy, cryptic, rare, threatened or a
combination of these. DNA recovered from faeces in particular offers an opportunity to
greatly enhance molecular genetic studies as samples are usually easily found in the field
and are often the only indication of a species’ presence (Kohn and Wayne 1997). Faecal
DNA analysis can determine species as well as the identity and gender of the individuals in
a population (Kohn and Wayne 1997; Taberlet et al. 1999; Piggott and Taylor 2003). The
resulting genotypic data can further be used to analyse patterns of relatedness, population
structure and phylogenetic relationships (Hoss et al. 1992; Kohn et al. 1995; Fernando et al.
2000; Garnier et al. 2001; Vigilant et al. 2001; Banks et al. 2002; Lucchini et al. 2002).
A substantial drawback of using faeces is the low quantity and quality of DNA they
usually contain, which typically results in high rates of genotyping error (Taberlet et al.
1999; Ernest et al. 2000; Smith et al. 2000). Although it is preferable to extract DNA from
faeces that are freshly collected, as was possible in a survey of a common wombat
(Vombatus ursinus) population (Banks et al. 2002), the species of interest may be found
only in remote locations, or may require a sampling strategy over several weeks. Thus, the
most crucial aspects of successfully using faeces for DNA analysis are to ensure that (1) the
storage protocol minimises degradation of the DNA, and (2) the extraction process recovers
DNA of sufficient quality and quantity for subsequent PCR analysis. A wide range of
approaches to both storage and extraction has been employed in published studies (Frantzen
© CSIRO 2003
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M. P. Piggott and A. C. Taylor
et al. 1998; Flagstad et al. 1999; Gerloff et al. 1999; Bayes et al. 2000; Ernest et al. 2000;
Farrell et al. 2000; Fernando et al. 2000; Constable et al. 2001; Murphy et al. 2002).
However, few have performed a comprehensive evaluation of the most appropriate methods
and combinations, in a variety of species. This makes it exceedingly difficult for
researchers to make informed decisions about which methods to use, particularly when
approaching a new species or collection situation. Access to a protocol that has been shown
to optimise recovery of faecal DNA for a range of taxonomic groups or ecological types
(e.g. herbivore v. carnivore), taking into account some of the known sources of variation in
intra-specific quality of faecal DNA (e.g. individual and dietary variation) would therefore
be exceedingly valuable. This motivated us to evaluate and compare storage and extraction
methods for faeces from a range of Australian species for which we are interested in
applying faecal DNA-based abundance estimates. These were two marsupial herbivores
(the Tasmanian pademelon, Thylogale billardierii, and brush-tailed rock-wallaby, Petrogale
penicillata), two marsupial carnivores (the spotted-tailed quoll, Dasyurus maculatus, and
eastern quoll, D. viverrinus), and an introduced carnivore (the red fox, Vulpes vulpes).
Our first aim was to evaluate combinations of a range of storage and DNA-extraction
methods that have been utilised in previous faecal DNA studies. For this we used faeces
from captive T. billardierii, from which a large number of fresh samples could easily be
collected. Storage methods included dehydration, either by air-drying (Flagstad et al. 1999;
Farrell et al. 2000) or alcohol treatment (Gerloff et al. 1999; Bayes et al. 2000; Fernando
et al. 2000; Constable et al. 2001), freezing (Ernest et al. 2000), and saturation in DET
buffer (Frantzen et al. 1998). Extraction methods included Dynabeads (Flagstad et al.
1999) and a Qiagen spin column method (Qiagen DNeasy Kit; cat. #369506) with different
initial steps: homogenisation of the faeces (Kohn et al. 1995; Wasser et al. 1997; Goossens
et al. 2000; Jensen-Seaman and Kidd 2001), scraping the surface of the faeces (Kohn et al.
1999) and surface-washing to remove intestinal cells from the mucosal layer of the faeces
(Flagstad et al. 1999). A subset of storage and extraction methods that provided the most
superior PCR template were then applied to faeces from P. penicillata, V. vulpes,
D. maculatus and D. viverrinus, collected both in captivity and/or in the wild. Finally, we
investigated the reliability of genotyping in each of these species using the
storage/extraction method combination that gave the highest amplification rate.
Methods
Comprehensive testing of storage and extraction methods on T. billardierii faeces
Sampling and preservation
Four preservation and four extraction methods were initially tested on fresh faeces collected from a
captive T. billardierii population held at Monash University. In total, 160 fresh faeces were collected over
several days from the feeding area, by raking and cleaning the area daily to ensure faeces were less than
24 hours old. Forty faeces were preserved using each of the following methods:
(1)
(2)
(3)
(4)
frozen in plastic ziploc bags at –20°C;
stored in 70% ethanol (1 g : 3 mL) in screw-top vials (Gerloff et al. 1999);
dried at room temperature in paper bags; and
stored in DETs buffer (2 g : 1 mL) in screw-top vials (Frantzen et al. 1998).
DNA extraction
Extraction blanks were included for all extractions to test for contamination. All samples were extracted
within a week of collection, having been stored by one of the methods described above. The 40 samples
from each preservation method were split into four groups of 10 samples, each of which was subjected to
one of the following DNA-extraction procedures:
Faecal DNA methods in Australian mammals
Aust. J. Zoology
343
Method A: Surface wash followed by Qiagen spin column purification (DNAeasy Kit: cat.
#69506). DNA extraction was performed using the protocol modified from Deuter et al. (1995). The
faeces were placed in a sterile plastic bag with a volume (between 2 and 4 mL) of SLP buffer (500 mM
Tris-HCl pH 9.0, 50 mM EDTA, 10 mM NaCl) (Deuter et al. 1995) that allowed recovery of approximately
1 mL following the wash. The exterior of the sample was gently washed and the wash was then removed
and placed in a 10-mL plastic tube. This was vortexed, incubated at 70°C for 10 min and centrifuged at 5000
rpm for 5 min The supernatant was transferred to a new tube, to which 500 µL of potato flour–SLP mixture
(50% w/v) was added, followed by vortexing, incubation at room temperature for 1 min and centrifugation
for 10 min at 5000 rpm. This supernatant was transferred to a new tube, to which 40 µL of proteinase K
(25 mg mL–1) and 1 mL of AL buffer was added. This was vortexed and incubated overnight at 55°C. Then
1 mL of 100% ethanol was added to the lysate and vortexed. DNA was purified from the lysate using Qiagen
spin columns, according to the manufacturer’s instructions, except that prior to the elution step, the spin
columns were incubated for 5 min at 37°C. DNA was eluted into 300 µL of AE buffer then stored at –20°C.
Method B: Surface scrape followed by Qiagen spin column purification. A sterile razor blade was used
to remove approximately 500 mg of the outer layer of the faeces, which was placed in an Eppendorf tube
with 1.5 mL of SLP lysis buffer. The sample was vortexed, incubated at 70°C for 10 min and incubated
overnight in a 37°C shaking water bath. It was then centrifuged at 13 000 rpm for 2 min, after which the
supernatant was transferred to a new tube. The protocol then followed Method A from, and including, the
addition of the potato flour–SLP mixture.
Method C: Homogenisation followed by Qiagen spin column purification. Whole faeces were
homogenised in 4–5 mL SLP buffer in a 10-mL tube, vortexed for 1 min and incubated overnight in a 37°C
shaking water bath. The tubes were then centrifuged for 10 min at 5000 rpm and the supernatant was
transferred to a new tube. The protocol then followed Method A from, and including, the addition of the
potato flour–SLP mixture.
Method D: Surface wash followed by Dynabeads purification. DNA extraction was performed using
a protocol modified from Flagstad et al. (1999) using Dynabeads DNA Direct (Dynal AS, Oslo, Norway).
Faeces were placed in separate sterile plastic bags and washed with 400 µL phosphate-buffered saline (PBS)
and the supernatant was then handled with 200 µL of Dynabeads according to Rudi et al. (1997). Prior to
the elution step, the microfuge tubes were incubated for 5 min at 37°C and the DNA was then eluted in
40 µL of TE, and diluted for PCR as described in Flagstad et al. (1999).
PCR amplification
Our previous (unpublished) work had shown that, of eleven macropod microsatellite loci that amplified
well from P. penicillata tissue DNA (Browning et al. 2001), those with alleles below 200 bp performed the
best on T. billardierii and P. penicillata faecal DNA. Three of these, developed for the allied rock-wallaby
(P. assimilis) (Pa 385, Pa 55, Pa 597: Spencer et al. 1995), were used to assess the effectiveness of each
storage/extraction combination for T. billardierii faeces. For each of the 10 faecal extracts, three PCRs
(Navidi et al. 1992; Taberlet et al. 1996) were carried out for each microsatellite locus, giving a total of 90
PCRs per storage/extraction combination.
PCRs were carried out on a MJ Research PTC-100 thermal cycler, and contained 8 µL of DNA extract,
75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 2.0 mM MgCl2, 200 µM dGTP, dTTP and
dCTP, 20 µM dATP, 0.05 µL [α33P]-dATP at 1000 Ci/mmol, 0.02% BSA (MBI), 8 pmoles of each primer
and 0.5 units of Taq polymerase (MBI) in a total volume of 15 µL. Annealing temperatures given in Spencer
et al. (1995) were used with 40 cycles for all PCRs. PCR products were electrophoresed through a 6%
polyacrylamide sequencing gel and visualised by autoradiography. Allele sizes were scored against an Aor T-terminating M13 control sequencing reaction size marker.
Comparison of storage/extraction methods in a range of species
Sampling, preservation and extraction
Ninety fresh faeces were collected from captive populations of each of P. penicillata (Healesville
Sanctuary, Victoria, Australia), V. vulpes (Keith Turnbull Research Institute, Victoria, Australia) and D.
maculatus (Healesville Sanctuary, Victoria, Australia). Pens and feeding areas for all animals were cleaned
daily so faeces were known to be less than 12 hours old when collected. Ten faeces were used for each of
nine storage/extraction method combinations (as above, but excluding DETs storage and Dynabeads
extraction, which performed poorly in the trials – see Table 1). Ten fresh faeces were collected from captive
D. viverrinus (Pearcedale Conservation Park, Victoria, Australia). These were stored by drying and
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M. P. Piggott and A. C. Taylor
extracted using the Qiagen surface wash method only, due to low availability of samples and initial results
obtained from D. maculatus faeces.
PCR amplification
For each species, amplification success at six microsatellite loci was used to assess the effectiveness of
each storage/extraction method. PCR conditions were as described above, with 3 replicates performed for
each sample. The loci used for P. penicillata were the three used in the T. billardierii trials, along with Pa
297 (also developed for P. assimilis) and two developed for the tammar wallaby (M. eugenii) (Me 17 and
Me 14: Taylor and Cooper 1998). Annealing temperatures as described in Spencer et al. (1995) and Taylor
and Cooper (1998) were used with 40 cycles for all PCRs. For D. maculatus and D. viverrinus, six primer
pairs developed for Dasyurus spp. (Firestone 1999), were used in 40 PCR cycles employing annealing
temperatures as described in Firestone (1999). Six microsatellites designed for Canis familiaris and used
previously by Lade et al. (1996) on V. vulpes tissue DNA were amplified with annealing temperatures as
described in Lade et al. (1996), using 35 cycles for all PCRs. These were DB1, DB3, DB4, DB6 (Holmes
et al. 1993), OB and C213 (Ostrander et al. 1993).
Evaluation of genotyping accuracy
Two approaches were used to evaluate the reliability of faecal genotypes from the preservation and
extraction method combination that resulted in the highest proportion of samples amplifying:
Comparison with tissue genotypes
Tissue samples were taken from the ears of five captive P. penicillata and V. vulpes individuals and ten
wild-trapped D. maculatus animals using a 2-mm biopsy punch, and placed in vials containing 100%
ethanol. DNA was extracted using the ‘salting out’ method (Sunnucks and Hales 1996). Genotyping was
performed as above for six microsatellite loci, except 1 µL (approximately 50 ng) of biopsy DNA was used
in a 10-µL PCR. Faecal samples from the same individuals (collected from enclosures or traps) were
preserved by drying, extracted within seven days using the Qiagen surface wash method, and genotyped as
above.
Replicate PCRs
Fresh faeces were collected from captive animals housed separately to ensure that they came from
different individuals. This was considered important so that genotyping errors could be quantified across
diverse genotypes, as would be encountered in a population survey. One pellet was collected from each of
10 captive V. vulpes, T. billardierii and D. viverrinus, and 5 captive D. maculatus and P. penicillata. Fresh
faeces were collected from each of 5 wild D. maculatus trapped at Werrikimbee National Park (New South
Wales, Australia) by Dr Gerhardt Körtner, and each of five P. penicillata trapped at Tidbinbilla Nature
Reserve (Australian Capital Territory, Australia) by Geoff Underwood. All faeces were stored dry and
extracted one week later using the Qiagen surface wash method. Eight replicate PCRs (Navidi et al. 1992;
Taberlet et al. 1996) at each of six microsatellite loci were performed on each extract, as described above,
except that PCR reactions contained 4 µL of DNA extract and 10 pmoles of each primer, and had a total
volume of 20 µL.
Simulation of the number of replicate PCRs required for accurate genotyping
The Gemini program, version 1.2.0 (Valière et al. 2002) was used to simulate the genotyping process using
the optimised preservation and extraction method, in order to estimate the ideal number of replicate PCRs
required to obtain accurate genotypes and quantify the rate of accepting incorrect genotypes. Using the
PCR Repetition Batch module we carried out 100 replicate simulations using the observed allele
frequencies from a known population of each species to simulate a population of 30 individuals. The same
allele frequencies for a population of P. penicillata were used for T. billardierii, as no population data were
available for this species. The populations used in this simulation for each species were: T. billardierii and
P. penicillata: Watagans Bowman, New South Wales (Mark Eldridge, unpublished data), V. vulpes: Phillip
Island, Victoria (Lade et al. 1996), D. maculatus: Werrikimbee National Park, New South Wales (Maxine
Piggott, unpublished data) and D. viverrinus: Gladstone, Tasmania (Firestone et al. 2000). Ten individuals
were sampled with replacement from that population and genotyping of these 10 samples was simulated
using four multiple tubes criteria: an allele had to be present in 2 of 3, 3 of 3, 3 of 5, and 5 of 8 replicate
PCRs. For each species, we introduced genotyping errors (both false alleles and allelic dropout) from the
Faecal DNA methods in Australian mammals
Aust. J. Zoology
345
species-specific rates observed in 480 PCRs in the pilot study (see above). For loci that failed to amplify in
either quoll species, the observed genotyping error rate from loci that amplified successfully was used.
Results
T. billardierii trials to identify optimal storage/extraction method
Data on amplification success achieved using the different storage and extraction methods
trialled for T. billardierii faeces are presented in Fig. 1. The best combination of
preservation and extraction methods was to store them dry, and extract them using the
Qiagen surface wash method. This resulted in a 100% total amplification rate (from 30
PCRs) (Fig. 1). The surface wash extraction method also worked well for faeces that were
frozen (80% amplification; Fig. 1) but not for those stored in ethanol (57%; Fig. 1). The
Qiagen homogenisation extraction method gave lower amplification rates than the surface
scrape and surface wash methods for all storage methods (31–52%; Fig. 1). All
combinations involving DETs storage and Dynabeads extraction performed poorly in
amplification trials (<40%; Fig. 1), so neither of these were used in subsequent trials.
Comparison of storage/extraction methods across species
Nine combinations of preservation and extraction method resulted in a 50% or greater
amplification rate for T. billardierii faeces, so these were all trialled on V. vulpes,
P. penicillata and D. maculatus faeces. Faecal DNA of Dasyurus spp. gave relatively low
amplification and high genotyping error rates no matter how faeces were stored or extracted
(Table 1). The combination of dry storage and Qiagen surface wash extraction that was
optimal for T. billardierii proved also to be superior for V. vulpes, P. penicillata and D.
maculatus (Table 1). Freezing the faeces prior to a surface wash extraction was almost as
successful for V. vulpes and P. penicillata faeces (Table 1). For V. vulpes and P. penicillata,
in all cases where a sample successfully amplified following drying or freezing and Qiagen
surface wash extraction, all three PCR replicates from that sample gave the same genotype,
for all loci.
Amplification Rate (%)
100
80
60
Surface Scrape
40
Surface Wash
20
Dynabeads
Homogenisation
0
Frozen Ethanol Dried
DETs
Preservation Method
Fig. 1. Percentage of samples that amplified from Thylogale billardierii DNA obtained
from faeces subjected to different combinations of preservation and extraction methods.
All but the Dynabeads extraction method was carried out using a Qiagen kit. Bars
represent amplification rates from 90 PCRs for three microsatellite loci, averaged over 10
extracts, for each treatment. Error bars represent variances across the 10 extracts.
Extraction method
Surface scrape
Surface wash
Homogenisation
Surface scrape
Surface wash
Homogenisation
Surface scrape
Surface wash
Homogenisation
Preservation
method
Frozen
Ethanol
Dried
075
100
060
076
063
057
073
086
056
090
100
075
089
093
071
090
100
075
P. penicillata
Amplification
Matching
rate (%)
genotypes
(%)
076
100
063
060
066
058
070
088
061
091
100
071
094
092
078
090
100
074
V. vulpes
Amplification
Matching
rate (%)
genotypes
(%)
36
65
33
30
40
42
26
61
34
75
88
65
75
65
62
75
86
70
D. maculatus
Amplification
Matching
rate (%)
genotypes
(%)
–
45
–
–
–
–
–
–
–
–
86
–
–
–
–
–
–
–
D. viverrinus
Amplification Matching
rate (%)
genotypes
(%)
Percentage of samples that amplified and percentage of successfully amplifying samples that gave a matching genotype for all three replicate PCRs, for
faecal DNA extracts prepared using different preservation and extraction methods
Results are from 180 PCRs consisting of 3 replicates over 6 microsatellite loci in 10 extracts. –, indicates that the preservation and extraction method was not tested on this
species
Table 1.
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Faecal DNA methods in Australian mammals
Aust. J. Zoology
347
Table 2. Number of times faecal genotype matched biopsy genotype for 5 individuals of each of
Vulpes vulpes and Petrogale penicillata from 18 PCRs consisting of 3 replicates over 6 microsatellite
loci, and 10 individuals of Dasyurus maculatus from 12 PCRs consisting of 3 replicates over 4
microsatellite loci for each faecal extract
Species
V. vulpes
P. penicillata
D. maculatus
1
2
3
18/18
18/18
10/12
18/18
18/18
09/12
18/18
18/18
08/12
Number of matching replicates
4
5
6
7
18/18
18/18
09/12
18/18
18/18
10/12
–
–
11/12
–
–
8/12
8
9
10
–
–
11/12
–
–
9/12
–
–
7/12
It proved difficult to carry out a surface wash on ethanol-stored faeces as, unlike frozen
and dried faeces, these broke apart and did not produce a clear wash. There also was
evidence of genotyping errors from such extracts, as not all three replicates matched for
some samples. The Qiagen surface scrape method performed relatively well for V. vulpes
and P. penicillata (60–76% amplification rates; Table 1) but there was some evidence of
genotyping errors. The Qiagen homogenisation method was the least successful for these
two species, and also performed particularly poorly for D. maculatus (33–42%
amplification; Table 1).
Accuracy of faecal genotypes
Comparison of tissue and faecal genotypes
Using the optimised storage/extraction combination, genotypes from the three replicate
faecal DNA PCRs at each of six microsatellite loci clearly matched those obtained from
tissue samples from the five captive V. vulpes and five wild P. penicillata individuals known
to have produced these samples (Table 2, Fig. 2). Conversely, the three replicate PCRs from
the faecal DNA samples collected from 10 wild D. maculatus individuals did not
consistently match the genotypes obtained from their tissue samples (Table 2). As discussed
below, only 4 loci amplified in D. maculatus.
Eight replicate PCRs
The second method of examining reliability of genotypes using the dried preservation
and Qiagen surface wash extraction combination involved carrying out 80 replicate PCRs
(8 replicates per sample) for 6 microsatellite loci – a total of 480 PCRs per species. For
V. vulpes, 100% of faecal DNA extracts amplified and yielded matching genotypes for all
eight replicate PCRs (Table 3). Genotypes matched consistently for all six microsatellite
loci with no evidence of false alleles or allelic dropout (Table 3). For T. billardierii, most
(91.5%) faecal DNA extracts amplified and 89.35% of these yielded matching genotypes
for all eight replicate PCRs (Table 3), with a total genotyping error of 2.07%. Genotyping
error rates ranged from 0 to 4.5% for different extracts/genotypes (Table 3, Fig. 3). These
figures were 95.2% and 90.86%, respectively for P. penicillata (Table 3, Fig. 3). Thus, less
than 5% of all extracts gave inconsistent results for P. penicillata. All genotyping errors
were from wild individuals with one wild animal sample (Individual 10) contributing
3.22% to the total genotyping error rate for the 10 P. penicillata animals (Fig. 3, Table 3).
Eight replicate PCRs were not sufficient to accurately genotype this sample (Sample 10;
Fig. 3), which had a total genotypic error rate of 33.3% and the lowest amplification rate
across loci and replicates (79%). If this sample is excluded, only 1.12% of extracts gave the
incorrect genotype with a range of 0–6% (compared with 0–33%; Table 3).
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M. P. Piggott and A. C. Taylor
Fig. 2. Autoradiograph showing allele patterns for the microsatellite locus OB
derived from template DNA isolated from tissue and scats from five Vulpes
vulpes individuals. The first lane for each individual is the tissue sample (T)
followed by three replicate faecal samples (S).
Table 3. Amplification and genotyping error rates across a total of 480 replicate PCRs (48 PCRs
for each extract) for 10 faecal extracts from Vulpes vulpes, Petrogale penicillata, Thylogale
billardierii, Dasyurus viverrinus and Dasyurus maculatus, prepared by the optimal method
Average total rates are based on 6 loci for Vulpes vulpes, Petrogale penicillata and Thylogale billardierii,
4 loci for Dasyurus maculatus and 5 loci for Dasyurus viverrinus
Species
V. vulpes
P. penicillata
T. billardierii
D. maculatus
D. viverrinus
Average total
amplification rate
(%)
Range
(%)
Average total
genotyping error
(%)
Range
(%)
100
95.20
91.50
59
35.25
100–100
79.16–100
85–100
21.87–84.37
10–95
0
4.34 (1.12)A
2.07
14.11
9.79
0
0–33.3 (0–6.0)A
0–4.50
0–34.81
0–37.50
A
Following removal of one sample from a wild animal (Individual 10 in Fig. 3) that contributed 3.22%
of the total genotyping error.
Of the six loci available, only three (1.3, 3.3.1 and 3.1.2) amplified successfully in
D. maculatus, while one (4.4.10) amplified in fewer than 50% of extracts and the remaining
two (3.3.2 and 4.4.2) failed to amplify for any sample (Table 4). Only 59% of faecal DNA
extracts amplified, and nearly 45% of these yielded matching genotypes for all eight
replicate PCRs (Table 3). Thus, 14.12% of extracts gave inconsistent results (Table 3,
Fig. 3). Wild D. maculatus samples produced substantial genotyping error rates
(14.58–34.81%), while the success with captive animals was generally better, with only
0–12.5% error rates (Fig. 3).
Faecal DNA methods in Australian mammals
Percentage
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Aust. J. Zoology
120
(a)
100
100
80
80
60
60
40
40
20
20
(b)
0
0
1
120
Percentage
349
2
3 4
5
6
7 8
1
9 10
120
(c)
100
100
80
80
60
60
40
40
20
20
2
3 4
5 6
7
8 9 10
2
3
5 6
7
8
(d)
0
0
1
2
3 4
5
6
7 8
9 10
Individual
Total amplification rate
1
4
9 10
Individual
Total error rate
Fig. 3. Total amplification rate across 48 PCRs (8 replicates per locus) at six microsatellite loci that
amplified, and the proportion that gave incorrect genotypes for (a) ten captive Thylogale billardierii
individuals, (b) five captive (1–5) and five wild (6–10) Petrogale penicillata individuals, (c) five captive
(1–5) and five wild (6–10) Dasyurus maculatus individuals, and (d) ten captive Dasyurus viverrinus
individuals. Error bars represent variance across loci.
Comparable results were obtained for D. viverrinus, in which only 35.25% of PCRs
amplified (from a total of 5 loci, as locus 4.4.2 failed to amplify for any sample), and a total
genotyping error rate of 9.79% was seen (Table 3, Fig. 3). In D. viverrinus two loci (3.3.1
and 1.3) amplified in more than 80% of samples, but three others (3.3.2, 3.1.2 and 4.4.10)
amplified in only a small subset, with a bias towards those from the extract from Individual
4 (Table 4). That sample also gave the highest total amplification rate for 5 loci (95%,
Fig. 3). The genotypic error rate varied from 0 to 37.5% for the 10 extracts (Table 3, Fig. 3).
Simulated number of replicate PCRs required for accurate genotyping
The percentage of correct genotypes obtained from the simulation using each of four
multiple tubes criteria is presented in Table 5. For P. penicillata, the simulation used
genotyping error rates observed from nine faecal samples, as the one from Individual 10
was excluded (see above). For V. vulpes, P. penicillata and T. billardierii three replicates are
sufficient as the chance of obtaining the wrong genotype was low (Table 5). Using the
criterion that an allele had to be present in only two out of three replicates to be accepted
leads to a lower probability of falsely rejecting heterozygotes, compared with only
accepting an allele if it is present in three out of three. If allelic dropout occurs in one of the
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Aust. J. Zoology
Table 4.
M. P. Piggott and A. C. Taylor
Amplification rates and number of matching replicates for 4 and 5 microsatellite loci for
10 Dasyurus maculatus and 10 Dasyurus viverrinus individuals respectively
FA: failed to amplify
Locus
Allele size range
(base pairs)
1.3
3.3.1
3.3.2
4.4.2
4.4.10
3.1.2
080–110
091–145
108–148
070–110
179–217
143–169
D. maculatus
Number
Matching
amplified
replicates
50/80
63/80
FA
FA
11/80
65/80
44/50
51/63
–
–
10/11
48/65
D. viverrinus
Number
Matching
amplified
replicates
49/80
70/80
08/80
08/80
FA
06/80
46/49
61/70
08/8
08/8
–
06/6
Table 5. Percentage of correct genotypes from Gemini simulations for Vulpes vulpes, Petrogale
penicillata, Thylogale billardierii, Dasyurus viverrinus and Dasyurus maculatus from an allele present
in 2 of 3, 3 of 3, 3 of 5 and 5 of 8 replicate PCRs
Species
2/3
V. vulpes
P. penicillata
T. billardierii
D. viverrinus
D. maculatus
100
099.67
098.95
083.40
084.50
Percentage of correct genotypes
3/3
3/5
100
098.89
097.15
073.30
074.0
100
100
100
092.70
092.40
5/8
100
100
100
096.10
094.50
three replicates the genotype will be falsely recorded as a homozygote instead of a
heterozygote under the more stringent criterion. Therefore, the two out of three replicate
criterion is a more reliable genotyping protocol, as reflected in Table 5. For D. maculatus
and D. viverrinus, up to eight replicates would be required for accurate genotyping from a
population with the high rates of genotyping errors observed in this pilot study (Table 5).
Discussion
This study is the first to investigate and evaluate faecal preservation and DNA-extraction
methods in a range of species with different life-history traits that might affect faecal
microsatellite genotyping success, particularly in the Australian context. Although other
studies have investigated a limited number of methods in one or two species (e.g. baboons:
Frantzen et al. 1998; sheep and reindeer: Flagstad et al. 1999; sun bear and black bears:
Wasser et al. 1997; grey and harbour seals: Reed et al. 1997; brown bears: Murphy et al.
2002), a comparison of methods over a range of species with differing dietary preferences
and/or faecal composition in the one laboratory has not been carried out to date.
Importantly, this study identified a single, relatively straightforward protocol that optimised
faecal microsatellite genotyping success for representatives of both marsupial and placental
carnivores, as well as marsupial herbivores.
The optimal method involved air-drying the faeces in paper bags (which may
incidentally be the most convenient field storage method), followed by a surface wash to
collect cells that were then subjected to a DNA-extraction process culminating in Qiagen
spin column purification. Faeces that were frozen prior to extraction by this method worked
almost as well for the red fox and macropodids, as did fresh faeces from another marsupial
herbivore, the common wombat (V. ursinus), in a previous study (Banks et al. 2002). Faeces
Faecal DNA methods in Australian mammals
Aust. J. Zoology
351
collected from captive southern hairy-nosed wombats (Lasiorhinus latifrons) and either
frozen for two weeks or extracted on the day of collection also performed very well using
the surface wash method (identical genotypes were obtained across three replicate
genotypes from 10 extracts: Sam Banks, Monash University, personal communication).
Preserving faeces in ethanol or DETs was less effective than drying or freezing for all
of the species investigated. This is in contrast to the experience of Frantzen et al. (1998) and
Murphy et al. (2002) using the same four storage methods for baboon and bear faeces
respectively. For baboon faeces, DETs solution was the most effective for preserving
nuclear DNA (Frantzen et al. 1998), while for bears ethanol-preserved samples performed
the best in evaluations of faeces stored for one week to six months (Murphy et al. 2002).
Murphy et al. (2002) used 90% ethanol compared with the 70% ethanol we used and thus
increasing the ethanol concentration may improve DNA preservation and amplification
success for the species discussed in this study. However, bear faeces are larger and require
a different extraction strategy (homogenising the whole sample and removing a subsample:
Murphy et al. 2002) compared with the smaller faeces produced by the species in this study
such that the whole sample can be used in the extraction process. Both Frantzen et al.
(1998) and Murphy et al. (2002) acknowledged that storing faeces dry is more feasible for
remote field collections, which we have also experienced.
As concluded in this study, Flagstad et al. (1999) recommended storing faeces dry prior
to extraction, and that a surface wash is a crucial first step for obtaining higher
amplification and lower genotyping error rates because it results in a clean supernatant
containing large numbers of intestinal cells. It was suggested that the dry and compact
nature of ruminant faeces facilitates the washing of cells from the sample surface, and that
this protocol will be suitable for other animals with a high-fibre diet (Flagstad et al. 1999).
This was certainly our experience with macropods, which share these characteristics, and
it is likely that the optimal method identified here will be superior for marsupial herbivores
in general.
Flagstad et al. (1999) also found that whole-sample homogenisation was vastly inferior
to surface washing for faeces of both reindeer (Rangifer tarandus) and domestic sheep
(Ovis aries). We similarly found that homogenisation of faeces, regardless of preservation
method, resulted in substantially lower amplification and higher replicate mismatching
rates than did surface washing or surface scraping. In accordance with this, the surface
wash extraction appears to be most effective when the faeces remains intact. This may have
contributed to the low success of faecal genotyping from D. maculatus and D. viverrinus,
whose faeces often formed a slurry at the bottom of the bag during the Qiagen surface wash.
In comparison, faeces from the other study species remained solid, and produced a clearer
wash supernatant. Previous studies that have involved crushing or homogenising faeces
have suffered relatively high rates of genotyping errors (e.g. mountain lions, 8%: Ernest et
al. 2000; wolves, 18%: Lucchini et al. 2002) but comparative data are not available to
conclude that a surface wash protocol would have resulted in fewer errors. In any case,
extraction options are limited by the size of the faecal sample produced by large animals.
Although the same combination of storage and extraction methods proved optimal for
each species in this study, there were substantial differences between species in the success
of faecal DNA recovery and microsatellite genotyping. Faeces from captive foxes that were
fed a relatively uniform diet amplified 100% of the time, with no genotyping errors
(although a pilot study will be necessary to quantify the genotyping error rate in wild
populations). Macropodid faeces performed almost as well, but marsupial carnivore faeces
provided markedly less reliably amplifiable DNA for all preservation and extraction
352
Aust. J. Zoology
M. P. Piggott and A. C. Taylor
methods tested, suggesting that gross aspects of the diet (i.e. carnivory v. herbivory) are not
the sole determinants of faecal genotyping success. Although the surface wash aimed to
target epithelial cells on the outside of the faeces and thus avoid inhibitors present inside
the faeces, quoll faeces did not remain intact during this process. It is therefore possible that
large amounts of PCR-inhibitory substances were released during extraction. It is known
that complex polysaccharides, numerous types of bacteria and food-degradation products
can inhibit PCR (Monteiro et al. 1997). Further purification steps were carried out but did
not provide a better result (M. Piggott, unpublished data). The composition of faeces
collected from wild D. maculatus animals was quite variable, and they often consisted
almost entirely of hair and bone fragments. Faecal DNA from captive individuals fed on a
uniform diet of chicken performed slightly better than that from wild animals, even though
their faeces still fell apart during the surface wash. This suggests that faecal composition
may be an important factor in amplification success and genotyping reliability for
Dasyurus spp.
Alternatively, DNA may be in low quantities in quoll faeces, or may be particularly badly
degraded. Although the amount of DNA was not quantified, it is possible that low amounts
of template DNA contributed to the lower amplification rates and much higher levels of
genotyping errors. Morin et al. (2001) reported an inverse relationship between amount of
DNA template and allelic dropout: samples with less than 26 pg DNA were unusable, and
template amounts in the range of 26–100 pg, 101–200 pg and >200 pg required seven, four
and two PCR replicates respectively for accurate genotyping. This finding is consistent
with earlier indications that 56 pg of template DNA was a critical threshold (Taberlet et al.
1996).
The low amplification rate for quoll faecal DNA may also be related to the microsatellite
markers available for these species. Our study was restricted to the use of the six markers
developed by Firestone (1999) despite the fact that some of them performed poorly on
faecal DNA. In particular, loci 3.3.2, 4.4.2 and 4.4.10 amplified from none, or only a small
proportion of the faecal extracts. Locus-specific amplification success was apparently not
determined by PCR product size in these species (Table 4) (contra Banks et al. 2002, who
found amplification success to be negatively related to product size for common wombat
faecal DNA), since primer pairs 3.3.2 and 4.4.2 yield products below 150 bp. For the other
species used in this study, a larger pool of loci was available to enable selection of those that
amplified well in the target species in general, and from faecal DNA in particular. Thus,
while further optimisation of extraction methods for quoll faeces may help increase the
quantity and quality of recoverable DNA, development of new markers may also be
required.
Variation in amplification and genotyping error rates amongst extracts was observed,
particularly for the two marsupial carnivore species. Interestingly, faecal extracts from wild
individuals typically amplified as well as those from captive ones, but with higher
genotyping error rates, suggesting that diet or health of wild individuals may affect DNA
quality (faeces from wild individuals were collected fresh from the trapped animal, so
storage differences are not a factor). Previous studies have reported a wide variation in the
percentage of samples that yielded no DNA or low amounts of amplifiable DNA (e.g.
mountain lions, less than 25%: Ernest et al. 2000; bears, 80%: Taberlet et al. 1997; coyotes,
39%: Kohn et al. 1999). At least for orang-utans and bears, species for which a small
portion of the faecal sample is extracted, it has been suggested that such variation may be
due to an uneven distribution of cells in faeces (Kohn et al. 1995; Goossens et al. 2000). It
is possible that the variation observed amongst extracts in the current study was due to
Faecal DNA methods in Australian mammals
Aust. J. Zoology
353
differences between faeces in the distribution of cells on the outside versus the inside of the
scat. If so, this may be due to differences between individuals per se (as reported for
orang-utans: Goossens et al. 2000) or to components of their diet. Multiple faecal pellets
from each of several individuals in captivity and in the wild would be required for this issue
to be adequately addressed. Observed variation in genotyping success amongst extracts led
Goossens et al. (2000) to conclude that at least two samples per individual, at least three
extracts per sample and three PCRs per extract, were required to ensure accurate
genotyping. However, multiple samples from the same individual will not be possible in
situations where faecal donors are anonymous, i.e. when faeces cannot be assigned to
individuals by methods other than genotyping.
Our findings match those of previous studies (Taberlet et al. 1996; Ernest et al. 2000;
Goossens et al. 2000; Banks et al. 2002) in demonstrating that single PCR reactions from
single faecal samples should not be relied upon to provide accurate genotypes. We
recommend a minimum of three PCR replicates for genotyping populations of V. vulpes,
P. penicillata and T. billardierii, although the genotyping error rates specific to a population
and/or laboratory should be quantified prior to commencing a full study. Although Gemini
simulations suggested that a single PCR was sufficient to accurately genotype V. vulpes
with the genotyping error rates observed in this pilot study, only captive animals were used
and this result may not reflect the genotyping error rates to be expected in wild populations.
The high genotyping error rates observed for both Dasyurus spp. in the pilot study, and the
accordingly higher likelihood of obtaining the wrong genotype in the genotyping
simulation, indicate that three PCR replicates is not sufficient and eight replicates may be
required.
Because of the great variability in success of faecal DNA genotyping reported in the
literature, it is highly recommended that researchers carry out a pilot study prior to
implementing a full study. Factors that may influence success include intrinsic ones like
individual, season and diet, over which the researcher has minimal control. However,
extrinsic sources of variation in success (storage and extraction of faeces) may be
minimised by the use of the protocol we found to be optimal for the species studied here,
or for other species that produce small faeces that can be easily handled during the
extraction process.
Acknowledgments
Thanks go to the staff at the Keith Turnbull Research Institute, Frankston, and at Healesville
Sanctuary for their assistance. Dr Gerhardt Körtner and Shaan Gresser are thanked for
providing samples from wild D. maculatus individuals. Natasha Czarny is also thanked for
obtaining samples from D. viverrinus individuals from Pearcedale Conservation Park.
Thanks go to Geoff Underwood at Tidbinbilla Nature Reserve for supplying samples from
P. penicillata individuals. Sam Banks is thanked for his assistance in developing faecal
DNA-extraction protocols and interpreting Gemini results. This research was funded by an
Australian Research Council SPIRT grant, and M. Piggott is supported by an APAI
Scholarship from the Australian Research Council and was carried out under Monash
University Animal Ethics Permit No. BSCI/2000/09.
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Manuscript received 26 February 2003; accepted 14 October 2003
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