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Conservation Genetics 3: 191–196, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands. 191
Detecting a genetic bottleneck in Gilbert’s Potoroo (Potorous gilbertii)
(Marsupialia: Potoroidae), inferred from microsatellite and mitochondrial
DNA sequence data
Elizabeth A. Sinclair1,2∗, Brian Costello3, Jacqueline M. Courtenay4& Keith A. Crandall2
1Department of Zoology, University of Western Australia, Nedlands, W.A. 6907, Australia; 2Department of
Zoology, Brigham Young University, Provo, UTAH 84602, USA; 3School of Biological Sciences, University of New
South Wales, Sydney, N.S.W. 2052, Australia; 4School of Natural Sciences, Edith Cowan University, Mt Hawley,
W.A. 6050, Australia (∗Corresponding author: E-mail: es88@email.byu.edu)
Received 26 June 2001; accepted 20 August 2001
Key words: bottleneck, effective population size, microsatellites, mtDNA, Potorous gilbertii
Abstract
Gilbert’s Potoroo is Australia’s most critically endangered marsupial, known from a single population in the
Two Peoples Bay National Park in Western Australia. We present results from a study of genetic variation in
microsatellite and mitochondrial DNA. Mean heterozygosity at five microsatellite loci was 49.3%, and the amount
of mtDNA variation was extremely low (π= 0.0004). There was evidence for a bottleneck in both sets of markers,
and this was consistent with a demographic decline. Effective population size was estimated using two different
models of mutation for microsatellites (Ne= 243 and 362). The results from this study highlight the concern for
the long-term survival of this species.
Introduction
Gilbert’s Potoroo, Potorous gilbertii, is considered
to be the most critically endangered marsupial in
Australia (Maxwell et al. 1996). This species was
regarded as ‘presumed extinct’ (Seebeck et al. 1989),
until December 1994, when it was rediscovered in the
Two Peoples Bay National Park, Western Australia
(Sinclair et al. 1996). A study of phylogeneticrelation-
ships among extant congenerics indicated Gilbert’s
Potoroo should be given full species status (Sinclair
and Westerman 1997), and may be more closely
related to P. tridactylus, based on karyotype (Sinclair
et al. 2000).
P. gilbertii has probably survived at Two Peoples
Bay for similar reasons as the noisy scrub-bird (Atrich-
ornis clamosus), a species that was also rediscovered
in this area after being believed extinct (Webster
1962). Since 1970, management of the park has
focused on exclusion of fire from the noisy scrub-bird
areas. The successful implementation of this policy
has seen A. clamosus thrive in the last twenty years
(see Orr et al. 1994), and this may have helped P.
gilbertii. The very thick unburned vegetation has prob-
ably also provided some protection from introduced
predators. Heinsohn (1966) suggested that the absence
of the European fox, Vulpes vulpes, was a major
contributing factor to the continuing high numbers of
P. tridactylus in Tasmania. Regular fox baiting began
within the park in 1988. The subsequent reduction in
fox numbers may have allowed the wild population to
partially recover, but the impact of feral cats in the
area is unknown. The extremely small number of P.
gilbertii (10 captive, 17 wild) presently known from
the park suggests that this population may have been
through a recent demographic, and possibly a genetic
bottleneck. We collected data from microsatellite and
mitochondrial DNA to examine levels of variation, test
for evidence of a genetic bottleneck, and estimate the
effective population size in P. gilbertii. The presence
of a bottleneck is inferred from a significant deviation
from equilibrium expectations for microsatellite gene
192
diversity, the effective population size, structure of the
mtDNA cladogram, and genetic diversity estimates.
Methods and analyses
Tissue samples were collected from 17 wild caught
and 8 captive individuals, some of which were born
in captivity (n = 3, Table 1). DNA was extracted from
ear tissue samples using standard phenol/chloroform
extraction and isopropanol precipitation (Sambrook et
al. 1989). Genotypes were scored from 25 individuals
for five microsatellite loci originally isolated from P.
longipes (Luikart et al. 1997). Reverse primers in four
loci (P2, P3, P13, P22) were fluorescently labelled and
the remaining locus (P10) was end-labelled with γ33P-
ATP. PCR reactions contained 1×Geneamp PCR
buffer II, 2 pmol each primer, 1.25–2.5 mM mgCl2,
0.5 units AmpliTaq Gold (Perkin Elmer), 2 µlof1/10
dilution of template DNA, and water to 10 µl. Tth
plus (Biotech) was used for the end-labelled γ33P-
ATP primers. Reactions were performed in a PTC-100
thermal cycler (MJ Research Inc) at 94 ◦C3min,
followed by 94 ◦C 20 s, 50–54 ◦C 60 s (except P22,
55–52 ◦C touchdown, 0.5 ◦C/cycle), 72 ◦C45s,for
40 cycles, and 72 ◦C 10 min. For those reactions that
incorporated fluorescently labelled primer, products
were run on an ABI 377, and scored using the program
ABI GenotyperTM. 550bp of mtDNA control region
were PCR amplified and sequenced in both directions
from 23 individuals using Mt15996L (Campbell et al.
1995) and macTDKD (Pope et al. 1996). PCR reac-
tions were identical to those published in Sinclair (in
press). Sequence alignment was made using ClustalX
(Thompson et al. 1997), with some adjustment by eye.
Allele frequencies and heterozygosities were
generated using GENEPOP version 3.1a (Raymond
and Rousset 1995). Exact tests for Hardy-Weinberg
equilibrium were performed for each locus (Rousset
and Raymond 1995), using the algorithm of Louis
and Dempster (1987), as there were four or fewer
alleles at each locus. For the nucleotide sequence
data, genetic diversity was estimated using π
(Tajima 1983) and θ(Kuhner et al. 1995, 1998). π
was estimated using Matrix 2.0 (D. Posada, program
available at http://bioag.byu.edu/zoology/crandall_lab/
programs.htm). The computer program Fluctuate 1.3
was used to estimate theta, where θ=Neµ,Neis
the inbreeding effective population size and µis the
mutation rate per site per generation.
Detection of a bottleneck, using microsatellite
data, was an estimate of effective population size
(Ne) from heterozygosity (H) and the mutation rate
(µ), given that the heterozygosity was estimated for
P. gilbertii, and we used a mutation rate of 10−3
(Weber and Wong 1993). Estimates were made under
an infinite allele model (IAM, Crow and Kimura
1970) and a stepwise mutation model (SMM, Ohta
and Kimura 1973). The computer program Bottleneck
(Cornuet and Luikart 1996) was used to detect whether
there was a recent reduction in effective population
size. In a bottlenecked population, gene diversity will
be higher than that expected at equilibrium. Gene
diversity was estimated under three models, SMM,
IAM, and the two-phase model (TPM) of Di Rienzo
et al. (1994), which may be closer to the true model
of mutation for most loci. The proportion of alleles
attributed to SMM under the TPM was 90%, with
a variance of 4 (equal to the maximum number of
repeat differences among alleles). Ten thousand simu-
lation replicates were conducted under each model of
mutation.
Results
Microsatellite polymorphism was low (Table 2),
varying between two to four alleles per locus with
observed heterozygosities between 8.3 and 78.3%
(overall H = 49.3%). Exact tests for Hardy-Weinberg
equilibrium showed no significant excesses or defi-
cits of heterozygotes. The generally lower hetero-
zygosities in P. gilbertii than P. longipes may be the
result of using heterologous primers, as P. gilbertii has
considerably more variation at nuclear allozyme loci
(Seebeck and Johnston 1980; Sinclair and Westerman
1997). The effective population size was estimated
as 243 (IAM) and 362 (SMM). For the Bottleneck
analysis, Wilcoxon sign-rank tests were significant,
indicating a slight excess in gene diversity under all
three models: SMM, TPM, and IAM (p= 0.03125,
0.04688, 0.04688, respectively). These p-values did
not change with alternative proportions of alleles
attributed to the SMM (range 80–95%) or variance
(2–10) under the TPM.
Unambiguous sequence data were obtained for
521bp of control region from 23 P. gilbertii indi-
viduals. Ambiguity codes were required for some
samples at 29 positions. No further tests were
undertaken to assess if these ambiguous sites may
reflect heteroplasmy or nuclear copy. Complete
193
Table 1. Mitochondrial haplotypes and microsatellite genotypes for P. gilbertii. Animals currently in the wild (W) or
in captivity (C). Trap locations are given
Individual Sex Captive/ Mother Microsatellite loci MtDNA
ID wild P2 P3 P13 P22 P10 haplotype
1 F C 144/144 130/130 99/101 127/127 163/163 PgH1
2 M W 142/144 130/130 95/97 127/127 163/163 –
3 M C 142/142 130/130 95/97 125/127 163/163 PgH1
4 F C 142/144 130/130 97/99 127/127 153/163 PgH1
5MW
∗––––––
6 M C 142/144 130/138 99/99 127/127 155/155 PgH1
7 M C 4 142/142 130/130 97/97 127/127 153/163 PgH1
8 (20) M W 142/144 130/130 99/99 127/127 153/163 PgH1
9 M W 142/144 130/130 95/99 127/127 153/163 PgH1
10 F C 144/144 130/130 97/99 127/127 153/155 PgH1
11 M C 10 142/144 130/130 97/99 127/127 153/163 PgH1
12 F W 144/144 138/138 99/99 127/127 153/155 –
13 M W 144/144 130/138 95/97 127/127 153/155 PgH1
14 M W∗––––––
15 F W∗––––––
16 M W∗––––––
17 F W 142/144 130/130 99/99 127/127 – PgH1
18 F C (born) 1 142/144 130/138 99/101 127/127 155/163 PgH2
19 F C (born) 10 142/144 130/130 95/99 127/127 153/163 PgH1
21 F W 144/144 130/138 95/99 127/127 155/163 PgH1
22 M W 144/144 130/138 95/99 127/127 155/163 PgH1
23 F W 144/144 138/138 97/99 – – PgH1
24 M W 142/144 130/138 95/97 127/127 155/163 PgH1
25 F W 144/144 130/138 95/99 127/127 153/163 PgH1
26 M W 142/144 130/138 95/99 127/127 153/163 PgH3
27 F C 144/144 138/138 97/99 127/127 153/153 PgH1
28 M C (born) 10 142/144 130/130 97/99 127/127 153/163 PgH1
29 F C (born) 17 144/144 130/138 95/99 127/127 153/163 PgH1
30 F W 144/144 138/138 99/99 125/127 153/163 PgH2
W∗= trapped, but no tissue sample collected.
sequences have been submitted to Genbank (accession
numbers AF421774–AF421788). Overall nucleotide
diversity among unambiguous sequence positions was
extremely low (π= 0.0004, range = 0.0000–0.0021).
The Fluctuate estimate of θwas considerably higher
(θ= 0.016), indicating higher diversity in the past or
a biased estimate due to small sample sizes. Three
unique haplotypes were identified that differed by a
single base, one of which was very common (PgH1,
frequency = 20).
Discussion
Genetic evidence for a bottleneck was observed in
both the microsatellite and mitochondrial data sets.
Significant evidence for a bottleneck at the microsatel-
lite loci was based on the estimates for effective
population size and the Bottleneck analysis. Estimates
of effective population size were close to an order of
magnitude larger than the number of currently known
individuals. This pattern is typical of a recent bottle-
neck, where the census population is considerably
lower than the inbreeding effective population size
(Crandall et al. 1999 e.g. Gerber and Templeton 1996).
This result may also be observed when an incorrect
mutation rate is used or when the true population is
larger than the number of individuals that have been
trapped so far. However, given that their distribution
is very patchy within the National Park, and that a
large portion has been surveyed, it is unlikely that
194
Table 2. Allele frequencies, observed heterozygosities, and
expected heterozygosities under Hardy Weinberg equilibrium for
P. gilbertii. Sample sizes are given in parentheses
Locus Alleles (named by size in bp) HeHo
and their frequencies
P2 142 144
(25) 0.320 0.680 0.444 0.480
P3 130 138
(25) 0.660 0.340 0.458 0.360
P13959799101
(25) 0.220 0.240 0.500 0.040 0.656 0.760
P22 125 127
(24) 0.042 0.958 0.082 0.083
P10 153 155 163
(23) 0.348 0.196 0.457 0.646 0.783
Mean 0.457 0.493
±SE 0.104 0.131
there is a large undetected population. The models
used to calculate Neboth assume neutrality of loci,
and that populations are at equilibrium. In P. gilbertii,
it is likely that at least the second of these assump-
tions has been violated. However, we suggest that the
higher effective population size estimate is consistent
with a recent demographic bottleneck: that is, gene
diversity measures are elevated relative to the equi-
librium expectations due to a loss of rare alleles.
Explicit testing for a bottleneck (significant result)
also supports this conclusion.
Effects of a recent bottleneck are more likely
observed in the mitochondrial DNA due to its haploid
nature and maternal inheritance (e.g. Snowbank and
Krajewski 1995; Mundy et al. 1997). The number of
unique P. gilbertii haplotypes (n = 3) and variable sites
was extremely low (n = 2/550; although it is not known
if further variation may be indicated at those sequence
positions that could not be fully resolved). This
level of variation is comparable to other Australian
marsupials which have been through extreme demo-
graphic declines (e.g., the numbat, Myrmecobius
fasciatus; n = 9/663bp, 1.4%; Fumagali et al. 1999).
Similar patterns are seen in island populations relative
to mainland populations (e.g., the Thevenard Island
mouse, Leggadina lakedownensis, which has no vari-
ation among 16 control region sequences; Moro et al.
1998). It is difficult, however, to distinguish between
a species having naturally low levels of variation
and a significant loss in variation as the result of a
bottleneck, particularly when there is no large extant
population (or suitable museum material) with which
to make comparisons. An alternative approach is to
compare estimates of πand θto gain insight into the
species’ population history (see Templeton 1993). The
coalescent estimator of genetic diversity (θ= 0.016)
was considerably larger than the Tajima estimator (π
= 0.0004), implying historically higher diversity for
P. gilbertii. This estimate likely reflects a time when
this species had a wider distribution and larger popula-
tion size. Given Ne=θ/4µ, if we use the estimates
for historical and present genetic diversity, and a µof
2%/Myr (Wilson et al. 1985), then Neis estimated as
2000 and 50 respectively, also consistent with a recent
bottleneck.
Here, we have presented multiple lines of evidence
for a genetic bottleneck in P. gilbertii. Although it is
difficult to determine the census size, it has no doubt
suffered a significant demographic decline since the
arrival of Europeans in the early nineteenth century.
The effects of this bottleneck are fairly extreme in
the mitochondrial DNA, and occur to a lesser (but
significant) extent in the nuclear microsatellite loci.
This pattern is expected under conditions of a recent
bottleneck (as illustrated in Figure 9, Wilson et al.
1985).
Implications for conservation
Estimating effective population size is important
for conservation of endangered species since small
populations may lose variation very quickly (Lande
and Barrowclough 1987; Nunney and Elam 1994).
Our estimates of inbreeding effective population
size from microsatellite data are higher than the
minimum recommended for short-term viable popula-
tions (effective population size of 50; Franklin 1980).
Given this, our higher estimates of Neindicate that this
species still has considerable variation at nuclear loci
relative to the census population. Therefore, our goal
should be to increase the census population to main-
tain a variance effective population size of at least 500,
thus preventing further erosion of genetic variation.
For this to be achieved, a successful captive breeding
program is required that incorporates assessments of
genetic variation on mating pairs.
Potorous gilbertii is essentially showing a pattern
of that observed in island populations (also see
Eldridge et al. 1999; Sinclair, in press). However, the
195
major advantage of islands as refuges for threatened
or endangered species is that many of the processes
that cause declines or extinctions on the mainland are
usually absent from them (Dickman 1992). Here, we
have a species that shows similar genetic effects to
island populations (a small, geographically isolated
population), but with the added pressures associ-
ated with extinctions on the mainland. Long-term
survival of P. gilbertii in the wild is not promis-
ing. It will be dependent on an integrated approach
involving continued preservation of the Two Peoples
Bay National Park as a conservation area, continued
control of non-native predators, success of the captive
breeding program, surveying for new populations,
maintenance of remaining genetic diversity, and
possible translocation of animals into formerly occu-
pied habitats.
Acknowledgments
Thank you to Bridget Hyder, Adrian Wayne, Leigh
Whisson, Wes Manson, Jeff Middleton, Sarah Vetten,
and Alan Danks for their help in the field; Bill
Sherwin for the use of his laboratory at the Univer-
sity of New South Wales; David Posada for advice
on analyses; members of the Potoroo Recovery Team;
Mike Johnson, Jack Sites, and Bill Sherwin for
their constructive comments on earlier drafts of this
manuscript. The Gilbert’s Potoroo Recovery Program
is funded by Environment Australia, Edith Cowan
University, and the Department of Conservation and
Land Management (CALM). Funding for this project
was provided by the University of Western Australia,
ALCOA of Australia, and an Australian Post-graduate
Research Award to EAS.
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