Content uploaded by Steven Miller
Author content
All content in this area was uploaded by Steven Miller on Jan 04, 2016
Content may be subject to copyright.
ORIGINAL PAPER
Survivors or reinvaders? Using genetic assignment
to identify invasive pests following eradication
James C. Russell •Steven D. Miller •
Grant A. Harper •Hamish E. MacInnes •
Malcolm J. Wylie •Rachel M. Fewster
Received: 10 January 2009 / Accepted: 18 September 2009 / Published online: 4 October 2009
ÓThe Author(s) 2009. This article is published with open access at Springerlink.com
Abstract When new individuals from a pest species
are detected following eradication, identifying
whether the new individuals are survivors from the
eradication attempt, or reinvaders from another pop-
ulation, is important for management practices. Pearl
Island (512 ha) in New Zealand was the first island in
the world on which simultaneous eradication of all
three invasive rat species was attempted. Rats were
detected again 9 months after the eradication opera-
tion. We use genetic assignment methods to discrim-
inate between survivor and reinvader hypotheses. All
rats found on Pearl Island after eradication were likely
to be reinvaders from an adjacent population on much
larger Stewart Island (174,600 ha), suggesting that
rats were swimming to the island at a rate much
greater than anticipated, but that the original
eradication was successful. Adequate genetic signal
was obtained from opportunistically collected sam-
ples, making the method feasible for conservation
managers with limited time and resources.
Keywords Conservation Invasion
Island Microsatellite New Zealand
Rattus Reinvasion
Introduction
Invasive species are now a component of almost
every ecosystem on earth (Vitousek et al. 1997;
McKinney and Lockwood 1999), and are regarded as
an important driver of species extinction (Blackburn
et al. 2004). Eradication of invasive pests is the
ultimate goal of pest management in terrestrial and
marine systems (Myers et al. 2000; Simberloff 2003;
Thresher and Kuris 2004), and is now a common
management tool on islands (Donlan et al. 2003).
However, pest individuals may reappear after the
eradication attempt. If a long time has elapsed since
eradication it may be assumed that this is an
independent reinvasion, but when pests are rediscov-
ered promptly it might not be clear whether the new
individuals are survivors from the eradication
attempt, or reinvaders (Abdelkrim et al. 2007). This
has important management implications, because
failed eradications may necessitate a change of
J. C. Russell S. D. Miller R. M. Fewster
Department of Statistics, University of Auckland,
Private Bag 92019, Auckland, New Zealand
J. C. Russell H. E. MacInnes
School of Biological Sciences, University of Auckland,
Private Bag 92019, Auckland, New Zealand
G. A. Harper M. J. Wylie
Department of Conservation, Southland Conservancy
Office, P.O. Box 743, Invercargill, New Zealand
J. C. Russell (&)
Department of Environmental Science, Policy, and
Management, University of California, Berkeley,
CA 94720, USA
e-mail: jrussell@berkeley.edu
123
Biol Invasions (2010) 12:1747–1757
DOI 10.1007/s10530-009-9586-1
protocol for the future (Abdelkrim et al. 2005), or
alternatively, a revision of biosecurity measures to
prevent reinvasion (Russell et al. 2008b).
Genetic techniques are now affordable and widely
used in ecology (Selkoe and Toonen 2006), and
include assignment methods for assigning individuals
to a source population (Davies et al. 1999). Assign-
ment methods compute the probability of finding the
genotype of a given individual within each of a
sampled set of populations, and can be used to select
or exclude populations as possible sources for the
individual (Manel et al. 2005). These methods have
important applications in conservation (e.g. Manel
et al. 2002; Berry et al. 2004; Robertson and
Gemmell 2004). For invasive pest species following
eradication, assignment methods can be used to select
between the hypotheses of survival or reinvasion.
The invasive rat species Rattus exulans,
R. norvegicus, and R. rattus are among many pest
species now routinely eradicated from islands
(Howald et al. 2007), but rat eradication attempts
remain uncommon where there are multiple pest species
or high reinvasion risk. R. norvegicus are known to be
capable swimmers, and are considered the most likely
invaders of offshore islands in New Zealand (Russell
et al. 2005), and reinvading rats can be difficult to detect
at low densities (Russell et al. 2008a).
Pearl Island (512 ha; 47°110S, 167°420E; Fig. 1)
was the first island in the world on which simultaneous
eradication was attempted for all three invasive rat
species (Clout and Russell 2006). Rats were eradicated
during July and August 2005 by a standard aerial
poison baiting program using brodifacoum 20R pellets
(Animal Control Products Ltd, Whanganui, New
Zealand). Low levels of reinvasion by swimming from
the adjacent Stewart Island population were expected
across Whale Passage and so four post-eradication
monitoring lines each 1 km long were established on
the north-eastern slopes by Whale Passage. Monitoring
lines consisted of a combination of kill-traps, bait-
stations, and monitoring tunnels. Monitoring took
place every 3 months after eradication.
No sign of rodents was detected until early May
2006, 9 months after eradication, when a rat was
observed in the camp site. No sign of rodents in any
monitoring devices had been found at this time,
highlighting the difficulty of detecting rats at low
density. Five rats were then caught in May 2006 (4 R.
norvegicus, 1R. rattus). All rodent monitoring to this
point was confined to north-east Pearl Island adjacent
to Whale Passage, so it remained unknown whether
rats were widespread across the island or confined to
this vicinity. In July eight more rats (unidentified
species) were found in detection devices. A total of
13 rats had now been caught, consisting of both
R. norvegicus and R. rattus and both sexes. No young
rats indicative of recent breeding were caught.
Whether the post-eradication rats were survivors of
the eradication attempt, reinvaders across Whale
Passage from Stewart Island, or a combination of
both, was unknown. By April 2007, an R. norvegicus
population was confirmed across the entire island.
Under current rat eradication protocols, there is a
management expectation of complete eradication
success. A known eradication failure would instigate
costly investigation and revision of protocols. We
Fig. 1 Pearl Island, north-east Port Pegasus, Stewart Island
1748 J. C. Russell et al.
123
aimed to evaluate if genetic assignment methods could
successfully discriminate between post-eradication
survivors or re-invaders. Such a method is relevant
for guiding ongoing invasive rat management on Pearl
Island, and in other similar management scenarios.
Materials and methods
Study site
Pearl Island lies in north-east Port Pegasus, Stewart
Island. At its closest point to Stewart Island it is
separated by a 225 m channel across Whale Passage,
which lies parallel to the coast of Pearl Island for
about 800 m. The average water temperature around
Stewart Island throughout the year is 12°C. Vegeta-
tion on the island is described by Harper (2006). Pearl
Island is rarely visited by people or boats, and there
are no easy landing points around the island. Boats
occasionally pass through Whale Passage as they
enter Port Pegasus. On neighbouring Stewart Island,
all three species of invasive rat are present. Most
common is R. rattus, while R. norvegicus and
R. exulans are restricted to specific habitats (Harper
et al. 2005; Russell and Clout 2004).
Field sampling
Rat tissue samples were acquired from the pre-
eradication Pearl population (12 R. norvegicus and
11 R. rattus) the adjacent Stewart Island population (9
R. norvegicus and 8 R. rattus), and the post-eradication
Pearl individuals (10 R. norvegicus and 1 R. rattus). All
rat tissue samples were stored in 70% ethanol at room
temperature. For the pre-eradication Pearl population,
samples were gained opportunistically from a study of
habitat use by all three rat species shortly before the
eradication in June 2005 (Harper 2006). This sample
size is constrained, as no further pre-eradication rats
can now be sampled. For post-eradication Pearl
individuals, tissue samples were available from 10
R. norvegicus and 1 R. rattus, comprising the first five
rats caught in May 2006, three of eight rats caught in
July 2006, and three more rats caught in August and
September 2006. The three unidentified rats in this
sample were identified as R. norvegicus by our DNA
analysis. For the Stewart Island population, rats were
obtained from September to November 2006 on the
region of Stewart Island adjoining north-west Whale
Passage, for the purpose of this study.
An additional 31 tissue samples of R. rattus were
available from three other locations on Stewart Island,
all captured in 2005 and 2006: 14 samples from Tupari;
11 from Rakeahua; and 6 from Ackers Point. These
locations are, respectively, 18, 25, and 45 km from the
region adjoining Whale Passage. We use these samples
to give a wider context to our results when identifying
possible source populations for some rats.
Genetics
DNA was extracted using the DNeasy Tissue Kit
(Qiagen, Hilden, Germany). Eleven microsatellite
markers characterised for R. norvegicus genome
mapping were used on both rat species (Jacob et al.
1995; D10Rat20, D11Mgh5, D12Rat76, D15Rat77,
D16Rat81, D18Rat96, D19Mit2, D20Rat46, D2Rat234,
D5Rat83, D7Rat13). To avoid physical linkage,
markers were chosen on different chromosomes.
Each forward locus primer was labelled with fluo-
rescent dyes before amplification by polymerase
chain reaction (PCR). PCR was performed in 10 ll
volumes, containing 10 ng DNA, 0.1 lM of forward
primer labelled with 50fluorescent labels, 0.2 lMof
reverse primer, 0.2 lM of each dNTP, 0.2 units
Platinum Taq DNA polymerase (Invitrogen, Carls-
bad, USA), and 19reaction buffer with 1.5 mM
MgCl
2
. PCR products were pooled with Genescan
400HD [ROX] size standard for a single run using an
ABI Prism 3730 Genetic Analyzer capillary electro-
phoresis system (Applied Biosystems, Foster City,
USA). Amplification size was scored using GENESCAN
ANALYSIS 3.7 and GENOTYPER 3.7.
Samples with missing data were amplified and
scored a second time to identify missing values where
possible.
Statistical analysis
For the samples from the pre-eradication Pearl Island
and Stewart Island populations, we calculated allele
frequencies, observed and expected heterozygosities,
and mean number of alleles per locus as summaries
of genetic diversity within the two hypothesised
source populations. Our assignment methods rely on
assumptions of Hardy–Weinberg proportions and no
linkage disequilibrium, so we checked these in each
Using genetic assignment to identify invasive pests following eradication 1749
123
population using Fisher’s exact tests. We estimated
F
IS
and F
ST
, with 95% confidence intervals from
10,000 bootstraps across loci, following Weir (1996).
We did not treat the post-eradication rats as a
population, because each individual could have been
either a survivor or a reinvader. All calculations were
done in R(Version 2.4.0).
The small sample sizes raised concerns that the
observed differentiation between the assignment
populations (i.e. the Pearl Island rats pre-eradication,
and the rats from mainland Stewart Island) might arise
through sampling variability alone even if the popu-
lations were genetically identical. To test for the
possibility that the assignment populations were not
genetically differentiable, we applied the global test
for population differentiation using the log-likelihood
Gstatistic (Goudet 1995) over 10,000 randomisations,
available in the program FSTAT (Version 2.9.3.2).
For genetic assignment analysis, we used GENE-
CLASS2 (Piry et al. 2004), implementing the Bayesian
assignment criterion of Rannala and Mountain (1997).
This criterion was recommended by Cornuet et al.
(1999) as the best of a set of assignment criteria, and
they specifically demonstrated high assignment accu-
racy for samples as small as ours (n=10). For each
post-eradication Pearl Island rat, the criterion gives
posterior probabilities of finding the rat’s genotype in
the pre-eradication Pearl Island population (survivor
hypothesis), and of finding the rat’s genotype in the
Stewart Island population (reinvader hypothesis).
We present the assignment results graphically,
using a scatterplot of the log posterior probabilities
for the two assignment populations. The graphical
method is a powerful tool for assessing the different
hypotheses. The two-dimensional scatterplot displays
the clustering in the samples and the orders of
magnitude spanned by the posterior probabilities. This
information is lost by compressing the two-dimen-
sional points into the one-dimensional score values
provided by GENECLASS2 (Eq. 4 of Piry et al. 2004). If
there are missing data at some loci, however, the log-
posterior probabilities of all individuals are not directly
comparable (Piry et al. 2004), so they can only be
plotted on the same graph if an imputation algorithm is
used. Missing data occur frequently in genetic analy-
ses, especially when opportunistic samples include
specimens that were not fresh when preserved in
ethanol, and methods for coping with missing data are
necessary.
To construct the log posterior probabilities for an
individual with missing data, we calculated the log
posterior probabilities based on only the loci that were
complete for the individual in question, along with the
corresponding probabilities using this reduced set of
loci for all individuals with complete data records. We
then ranked the probabilities for the individual in
question among the probabilities for the other indi-
viduals. We used the rank of the individual based on
the incomplete data to impute the missing value for its
log posterior probabilities in the complete-data graph.
This was achieved by generating an empirical density
of the complete-data log posterior probabilities for all
complete-data individuals. The imputed value for the
individual with missing data was given by the same
quantile in the complete-data distribution as its
incomplete log probability achieved in the incom-
plete-data distribution. Imputing missing values is
used only for the visual presentation of results, and
does not alter any numerical summaries of the
analysis or significance values.
To investigate the accuracy of using imputation for
low levels of missing data, we present results from
two passes at genotyping the samples: the first with
up to 4% of missing data, and the second after most
of the missing loci had been successfully retyped,
leaving only 0.5% missing data.
To investigate quantitatively whether post-eradi-
cation individuals could plausibly have derived from
each of the assignment populations, we used the
algorithm of Paetkau et al. (2004), as implemented in
GENECLASS2. This algorithm provides percentile
rankings of each individual’s log posterior probabil-
ities in a distribution of log posterior probabilities
simulated to represent the assignment population.
Here we did not impute missing values. For a given
assignment population, the output percentile gives the
rank of the individual’s log posterior probability
among 100,000 individuals simulated to have ‘real’
genotypes from this population. High values indicate
good fit to the assignment population, while values of
about 0.05 and less suggest that the rat has a marginal
or poor fit to the population. The percentile rankings
can be interpreted as P-values against the hypothesis
that the individual originates from the population in
question. We used 100,000 simulations to achieve
consistent percentiles to two decimal places. The
output from this algorithm complements the visual
plots, and should give similar conclusions.
1750 J. C. Russell et al.
123
Results
A total of 51 rat samples of the two species of interest
were collected from the pre-eradication and post-
eradication Pearl Island populations, and from the
adjacent Stewart Island population. Trapping success
was \10% per 100 trap nights. Harper (2006)
describes the pre-eradication distribution of all three
rat species on Pearl Island. Table 1shows samples
collected and the pattern of missing data from each of
the two genotyping sessions. In the first session for
R. rattus, all genotypes were missing for one locus and
for one individual, so this locus and this individual
were excluded from the results for this session,
leaving 3.3% missing data (18/540). In the second
session previous missing data (including the entire
missing locus and individual) were retyped success-
fully, leaving 0.5% missing data (3/561). All numer-
ical results reported use the genotype data from the
second session.
Microsatellite data were polymorphic with mean
numbers of alleles per locus between 3.1 and 4.0, and
heterozygosity between 0.49 and 0.60, for pre-
eradication Pearl and Stewart Island populations.
Genetic differentiation was found between pre-erad-
ication Pearl Island and the adjacent Stewart Island
population for both species (F
ST
[0.05; Table 2),
including alleles exclusive to single populations. No
significant departure from Hardy–Weinberg equilib-
rium (P[0.05) and only minor levels of linkage
disequilibrium between some locus pairs (3/181,
P\0.05) were found in populations.
The global test for population differentiation
provided very strong evidence that the level of
genetic differentiation observed between the pre-
eradication Pearl Island and Stewart Island samples
did not arise by chance alone (P\0.0001 for both
R. norvegicus and R. rattus). This gives confidence
that the observed genetic differentiation is real for the
purposes of discriminating between the survivor and
reinvader hypotheses.
Figures 2and 3show the log posterior genotype
probability plots, using the imputation algorithm for
missing data as featured in Table 1. For each rat, the
log posterior probability of finding its genotype in the
pre-eradication Pearl population (‘survivor hypothe-
sis’) is plotted against the log posterior probability of
finding its genotype in the Stewart Island population
(‘reinvader hypothesis’). High values on either axis
indicate a good fit of the rat to the corresponding
population. The plots show all samples of known
origin (pre-eradication Pearl and Stewart Island
samples) as well as those of unknown origin (post-
eradication Pearl samples), so that the placement of
Table 1 Numbers of rat samples from Pearl Island before the eradication (June 2005), from the adjacent Stewart Island population
(September–November 2006), and from Pearl Island after the eradication attempt (May–September 2006)
Genotyping session 1 Genotyping session 2
Pearl Island Stewart Island After eradication Pearl Island Stewart Island After eradication
R. norvegicus
Samples 12 9 10 12 9 10
Loci 11 11 11 11 11 11
Missing data 1 4 10 0 1 0
Missing individuals 1 2 3 0 1 0
Percentage missing 0.8 4.0 9.1 0 1.0 0
R. rattus
Samples 10 8 1 11 8 1
Loci 10 10 10 11 11 11
Missing data 0 3 0 0 2 0
Missing individuals 0 3 0 0 2 0
Percentage missing 0 3.8 0 0 2.3 0
For each of two genotyping sessions, subsequent rows give the number of loci employed, the number of individual 9loci missing
data imputed in the scatterplots, the number of individuals missing data and the percentage missing data
Using genetic assignment to identify invasive pests following eradication 1751
123
the unknown samples can be seen with respect to that
of the known samples. A leave-one-out procedure is
used to place the samples of known origin, so that they
also serve to cross-validate the assignment procedure.
Rats below the solid diagonal lines in the plots
have greater posterior probability of occurring in the
pre-eradication Pearl population than occurring in the
Stewart Island population, and rats above the solid
lines have greater posterior probability of occurring
in the Stewart Island population. Points lying outside
the dashed diagonal lines have over nine times
greater posterior genotype probability from one
population than the other. Under the Bayesian
assignment criterion of Rannala and Mountain
(1997) implemented in GENECLASS2, all points out-
side the dashed lines have a GENECLASS2 assignment
score of 0.9 or above for the favoured population, and
0.1 or below for the other population, except for
small discrepancies that might arise from using the
imputation algorithm. The range of scores from 0.1 to
0.9 covers the very narrow region of the log posterior
plot contained within the dashed lines, and corre-
sponds to posterior probabilities within a single order
of magnitude of each other. This is put into context
by the two-dimensional plot which covers about 20
orders of magnitude, revealing that a seemingly high
score of 0.9 is in fact barely discriminative between
the two hypotheses.
None of the 10 R. norvegicus caught on Pearl
Island following eradication were grouped geneti-
cally with the pre-eradication Pearl population
(Fig. 2). Similarly, the single post-eradication
R. rattus specimen was not grouped with the pre-
eradication Pearl population (Fig. 3). The pre-eradi-
cation Pearl Island specimens were grouped closely
together for both species, indicative of a strong
genetic signal from the island populations. Stewart
Island rats were more dispersed but nearly always had
posterior probabilities to the left of the Pearl Island
rats. Sensitivity analysis, leaving one locus out at a
time, demonstrated robustness in these groupings
(results not shown). Based on these plots, we believe
that all individuals trapped on Pearl Island after the
eradication were reinvaders.
The graphical findings corroborate the percentile
rankings of Paetkau et al. (2004) given by GENE-
CLASS2 (Table 3). These rankings are P-values
against the corresponding survivor or reinvader
hypothesis, and should not be interpreted as mem-
bership probabilities. For all post-eradication rats,
there was strong evidence against the survivor
hypothesis (P\0.01 in every case; Table 3). Evi-
dence that the reinvading rats derived from the
Table 2 F-statistics for pre-eradication Pearl Island and adjacent Stewart Island rat populations
R. norvegicus Pearl Stewart R. rattus Pearl Stewart
Pearl -0.11
(-0.21, -0.02)
0.20
(0.12, 0.30)
Pearl 0.03
(-0.07, 0.19)
0.05
(0.01, 0.11)
Stewart -0.02
(-0.25, 0.25)
Stewart 0.20
(0.04, 0.34)
Diagonal values are F
IS
, off-diagonal are F
ST
. 95% bootstrap confidence intervals across loci are indicated in brackets below each
estimate
Table 3 Percentile probability rankings (P-values) for post-
eradication rats detected on Pearl Island under the ‘survivor’
and ‘reinvader’ hypotheses
Rat ID Species Date caught Survivor Reinvader
S1 rattus May 0 0.02
N1 norvegicus May 0 0.01
N2 norvegicus May 0.01 0.45
N3 norvegicus May 0 0.25
N4 norvegicus Jun 0 0.05
N5 norvegicus Jul 0 0.08
N6 norvegicus Jul 0 0.65
N7 norvegicus Jul 0 0.22
N8 norvegicus Aug 0 0.02
N9 norvegicus Sep 0 0.10
N10 norvegicus Sep 0 0.29
Bold font indicates no evidence against the corresponding
source population for 5 of the 11 rats ([0.10 by Paetkau et al.
2004.). Values between 0.01 and 0.10 indicate poor to marginal
fit to the corresponding population, and values of 0 indicate
very poor fit or very strong evidence against the corresponding
hypothesised source population
1752 J. C. Russell et al.
123
Fig. 2 Log posterior probability plot for R. norvegicus from
Pearl Island before and after eradication, and on adjacent
Stewart Island. Crosses indicate post-eradication rats to be
assigned. Plots show aresults from genotyping session 1,
where the imputation algorithm was applied with 4% missing
data involving 6 individuals (Table 1); and bresults from
genotyping session 2, where the imputation algorithm was used
for one missing locus of one Stewart Island individual (marked
with a tag on plot b). Points below the solid diagonal line have
greater posterior probability of belonging to Pearl Island than
to Stewart Island. Points outside the dashed diagonal lines
have over nine times greater posterior probability of belonging
to one population than the other
Fig. 3 Log posterior probability plot for R. rattus from Pearl
Island before and after eradication, and on adjacent Stewart
Island. The cross indicates the post-eradication rat to be
assigned. Plots show aresults from genotyping session 1,
where the imputation algorithm was used for three missing loci
of three individuals (Table 1); and bresults from genotyping
session 2, where the imputation algorithm was used for two
missing loci of two Stewart Island individuals (marked with
tags on plot b). Plot binvolves one extra locus and one extra
individual missing from plot a. Points below the solid diagonal
line have greater posterior probability of belonging to Pearl
Island than to Stewart Island. Points outside the dashed
diagonal lines have over nine times greater posterior proba-
bility of belonging to one population than the other
Using genetic assignment to identify invasive pests following eradication 1753
123
Stewart Island population was more ambiguous, with
only five of the post-eradication R. norvegicus
samples given percentile rankings greater than 0.10
(Table 3). The ambiguous fit is likely to be due to
small sample sizes, and interpretation is aided by the
graphical results. Figure 2b shows that the remaining
five post-eradication R. norvegicus had scores on the
vertical axis that were comparatively low for the
Stewart Island population, but still clearly grouped
together with Stewart Island rats. By contrast, Fig. 3b
shows that the single post-eradication R. rattus was
not grouped with either the Stewart Island or the
Pearl Island samples.
We widened the spatial sampling scale for Stewart
Island using data from another study to see if the single
R. rattus could have come from a more distant
geographical location. Figure 4shows the results of
augmenting the Stewart Island sample by 31 extra
samples, from three locations between 18 and 45 km
distant from Whale Passage. All conclusions from
Fig. 3remain the same when the larger sample from
Stewart Island is used in Fig. 4. In particular, the small
sample (n=8) from the Whale Passage region of
Stewart Island appears to capture much of the range
and extent of the larger Stewart Island sample
(n=39). The pre-eradication Pearl Island population
retains its genetic distinctiveness. The single post-
eradication R. rattus sample remains close to the lower
extreme of Stewart Island rats, but it now appears
consistent with the general profile from Stewart Island.
Comparison of plots (a) and (b) in Figs. 2and 3
demonstrates that the imputation algorithm has
worked effectively, with substantive conclusions
remaining the same from the first genotyping session
to the second. The transition from Fig. 3a to b is more
strongly affected by the addition of an extra locus and
an extra individual than by the imputation algorithm.
Discussion
We have shown that genetic assignment methods can
enable conclusive discrimination between survivor
and reinvader hypotheses following an eradication
attempt. Even with small opportunistic samples,
excluding the survivor hypothesis with a high level
of confidence was possible. Percentile rankings under
the survivor hypothesis were zero in nearly every case,
and graphical results showed that none of the post-
eradication rats appeared to associate with the pre-
eradication Pearl Island population. The strong genetic
signal from the pre-eradication Pearl Island rats
allowed for clear differentiation between Pearl and
Stewart Island populations. This is an important result
for managers who generally do not have the resources
for extensive genetic sampling and profiling.
The majority of rats were positioned outside the
dashed diagonal lines on Figs. 2and 3, indicating that
their GENECLASS2 assignment scores exceed 0.9 in
favour of one population or the other. The graphs
show that a GENECLASS2 assignment score of about
0.9 can be misleading, because it might be interpreted
as strong evidence in favour of the corresponding
assignment population, whereas in reality the evi-
dence is weak or absent. Posterior probabilities that
are within the same order of magnitude for both
populations do not present strong evidence in favour
of either, when seen in the context of about 20 orders
of magnitude in the overall span of the posterior
probabilities (about 10
-25
–10
-5
; Figs. 2,3). For
example, posterior probabilities from genuine pre-
eradication Pearl rats cover about five orders of
magnitude (10
-10
–10
-5
) when assigned to their
correct population, and despite this they are reason-
ably tightly clustered on the graph. Additionally, it is
possible for individuals to gain high GENECLASS2
Fig. 4 Log posterior probability plot for R. rattus when 31
additional samples from three dispersed locations on Stewart
Island are included in the Stewart Island sample. Diagonal
lines are as for Fig. 2
1754 J. C. Russell et al.
123
scores in favour of one population when the visual
results suggest that their fit to both populations is
poor. An example is the post-eradication R. rattus
sample (Fig. 3b), which gains an assignment score of
1.0 in favour of the Stewart Island population, but
does not appear to be associated with this population
from these data. Information is lost by compressing
the two-dimensional points into a single score value,
while the graphical presentation of results retains this
information.
The graphical method provides a more informative
and more interpretable output than numerical sum-
maries, and can simplify decision making for manag-
ers. Its disadvantage is the need for imputation in
order to display all individuals on the same plot.
However, we have shown that imputation is effective
for low levels of missing data (\5% in Fig. 2). We
suggest that the benefit of the visual presentation of
results outweighs the disadvantage of applying impu-
tation for low levels of missing data. Imputation
should only be used where missing data are sparse and
distributed at random among samples and loci. If an
entire locus fails for one or more of the populations, as
happened in our first genotyping session for R. rattus,
the locus should be excluded from the analysis.
The majority of R. norvegicus gained moderate to
high percentile rankings under the hypothesis that
they were reinvaders from the adjacent population on
Stewart Island (Table 3). The remaining R. norvegi-
cus, and the single R. rattus, gained ambiguous
percentile rankings for the adjacent Stewart Island
population. Graphical results suggested that these rats
were nonetheless consistent with the Stewart Island
population profile. The hypothesis of reinvasion by
swimming across Whale Passage is supported by the
ecological evidence that rats were only detected on
the part of Pearl Island closest to Whale Passage and
most accessible by swimming. An alternative expla-
nation is that rats were transported to Pearl Island by
boat after the eradication. However, Pearl Island is a
protected reserve in a remote location, and is rarely
visited, having no easy landing spots. It seems most
likely that all post-eradication rats were reinvaders by
swimming across Whale Passage. For the R. rattus
sample, Fig. 4demonstrates that the larger Stewart
Island R. rattus population is genetically diffuse and
spatially homogeneous. The R. rattus reinvader could
therefore have been geographically close to Pearl
Island despite its observed genetic profile.
Immediate post-eradication monitoring is not usu-
ally undertaken for rat populations, but was con-
ducted on Pearl Island because of the perceived risk
of reinvasion. Survivors of rat eradications are
plausible, especially in large populations where
occasional individuals might withstand LD99 doses
of poison (Airey and O’Connor 2003). The Allee
effect suggests low-density survivors may fail to
locate each other for breeding (Courchamp et al.
2008), but alternatively there is evidence that rats
adapt their behaviour at low densities to overcome
this effect (Russell et al. 2005). As larger and riskier
eradications are undertaken, with higher probabilities
of survivors or reinvasion, immediate post-eradica-
tion monitoring will become increasingly necessary.
The genetic differentiation observed between the
two possible source populations suggests they were
isolated to some degree prior to eradication, despite the
high reinvasion rate after the eradication. This might be
explained by an incumbent advantage, whereby resi-
dent animals repelled invaders prior to eradication
(Granjon and Cheylan 1989). Genetic methods of
assigning post-eradication individuals would have
been less successful if there had been more extensive
population mixing prior to eradication.
For this study, the genetic differentiation was
evident even with small, opportunistically collected
samples. Small samples may generate spurious allele
frequency distributions, or fail to detect rare alleles
which are important for assignment, however the
analysis methods used here are designed to incorpo-
rate variability due to sample size, and to prevent
population exclusion due to unsampled alleles.
Cornuet et al. (1999) found by simulation that the
Bayesian assignment criterion yielded over 80%
correct assignment with samples of size n=10, ten
loci, and F
ST
around 0.1, for the much more difficult
task of distinguishing between ten source popula-
tions. Our data have similar characteristics and only
two source populations.
Sampling prior to the eradication was essential for
the genetic assignment method. We urge all pest
managers undertaking eradication to collect a repre-
sentative spatial sample of pre-eradication individuals
(Abdelkrim et al. 2007), something which is not
currently included in best-practice protocols. Genetic
labwork need not be undertaken unless a reinvasion
occurs, in which case the genetic costs can defray the
much higher costs of remedial action with unknown
Using genetic assignment to identify invasive pests following eradication 1755
123
focus. To minimise effects of genetic drift, source
populations are best sampled as soon as post-eradi-
cation individuals are detected, from as many differ-
ent source locations as possible. In some cases it
might not be possible to identify the exact reinvasion
source, but it might still be possible to exclude other
sources. In our example, exclusion of the survivor
hypothesis was the result most fundamentally useful
to managers.
Acknowledgments This research was funded by New
Zealand Department of Conservation Science Advice
Funding. James Russell and Steven Miller were supported by
Top Achiever Doctoral scholarships from the NZ Tertiary
Education Commission, and Rachel Fewster and Hamish
MacInnes were supported by a Royal Society of New
Zealand Marsden grant. Thanks to Matt Hare, Richard
Clayton, Brent Beaven, Miriam Ritchie, Finn Buchanan and
Clint Brown of the New Zealand Department of Conservation
for their support. Andy Cox, Andy Roberts and two anonymous
referees provided valuable feedback on earlier versions of the
manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which
permits any noncommercial use, distribution, and reproduction
in any medium, provided the original author(s) and source are
credited.
References
Abdelkrim J, Pascal M, Calmet C, Samadi S (2005) Importance
of assessing population genetic structure before eradica-
tion of invasive species: examples from insular Norway
rat populations. Conserv Biol 19:1509–1518
Abdelkrim J, Pascal M, Samadi S (2007) Establishing causes of
eradication failure based on genetics: Case study of ship
rat eradication in Ste. Anne archipelago. Conserv Biol 21:
719–730
Airey AT, O’Connor CE (2003) Consumption and efficacy of
rodent baits to Norway rats. DOC Science Internal Series
148. Department of Conservation, Wellington
Berry O, Tocher MD, Sarre SD (2004) Can assignment tests
measure dispersal? Mol Ecol 13:551–561
Blackburn TM, Cassey P, Duncan RP, Evans KL, Gaston KJ
(2004) Avian extinction and mammalian introductions on
oceanic islands. Science 305:1955–1958
Clout MN, Russell JC (2006) The eradication of mammals
from New Zealand islands. In: Koike F, Clout MN, Ka-
wamichi M, De Poorter M, Iwatsuki K (eds) Assessment
and control of biological invasion risks. IUCN, Gland,
Switzerland and Cambridge, U.K., and Shoukadoh Book
Sellers, Kyoto, Japan, pp 127–141
Cornuet J-M, Piry S, Luikart G, Estoup A, Solignac M (1999)
New methods employing multilocus genotypes to select or
exclude populations as origins of individuals. Genetics
153:1989–2000
Courchamp F, Berec L, Gascoigne J (2008) Allee effects in
ecology and conservation. Oxford University Press, Oxford
Davies N, Villablanca FX, Roderick GK (1999) Determining the
source of individuals: multilocus genotyping in nonequi-
librium population genetics. Trends Ecol Evol 14:17–21
Donlan CJ, Tershy BR, Campbell K, Cruz F (2003) Research
for requiems: the need for more collaborative action in
eradication of invasive species. Conserv Biol 17:1850–
1851
Goudet J (1995) FSTAT (Version 1.2): a computer program to
calculate F-Statistics. J Hered 86:485–486
Granjon L, Cheylan G (1989) Le sort des rats noirs (Rattus rattus)
introduits sur une ı
ˆle, te
´ve
´le
´par radio-tracking. Comptes
Rendus de l’Acade
´mie des Sciences, se
´rie III 309:571–575
Harper GA (2006) Habitat use by three rat species (Rattus spp.)
on an island without other mammalian predators. N Z J
Ecol 30:321–333
Harper GA, Dickinson KJM, Seddon PJ (2005) Habitat selec-
tion by three rat species (Rattus spp.) on Stewart Island/
Rakiura, New Zealand. N Z J Ecol 29:251–260
Howald GR, Donlan CJ, Galva
´n JP, Russell JC, Parkes J, Sa-
maniego A, Wang Y, Veitch CR, Genovesi P, Pascal M,
Saunders A, Tershy B (2007) Invasive rodent eradication
on islands. Conserv Biol 21:1258–1268
Jacob HJ, Brown DM, Bunker RK, Daly MJ, Dzau VJ,
Goodman A, Koike G, Kren V, Kurtz T, Lernmark A
˚,
Levan G, Mao Y-P, Pettersson A, Pravenec M, Simon JS,
Szpirer C, Szpirer J, Trolliet MR, Winer ES, Lander ES
(1995) A genetic linkage map of the laboratory rat, Rattus
norvegicus. Nat Genet 9:63–69
Manel S, Berthier P, Luikart G (2002) Detecting wildlife
poaching: identifying the origin of individuals with
Bayesian assignment tests and multilocus genotypes.
Conserv Biol 16:650–659
Manel S, Gaggiotti OE, Waples RS (2005) Assignment meth-
ods: matching biological questions with appropriate
techniques. Trends Ecol Evol 20:136–142
McKinney ML, Lockwood JL (1999) Biotic homogenization: a
few winner replacing many losers in the next mass
extinction. Trends Ecol Evol 14:450–453
Myers JH, Simberloff D, Kuris AM, Carey JR (2000) Eradi-
cation revisited: dealing with exotic species. Trends Ecol
Evol 15:316–320
Paetkau D, Slade R, Burden M, Estoup A (2004) Genetic
assignment methods for the direct, real-time estimation of
migration rate: a simulation-based exploration of accuracy
and power. Mol Ecol 13:55–65
Piry S, Alapetite A, Cornuet J-M, Paetkau D, Baudouin L,
Estoup A (2004) GENECLASS2: a software for genetic
assignment and first-generation migrant detection. J Hered
95:536–539
Rannala B, Mountain JL (1997) Detecting immigration by
using multilocus genotypes. Proc Natl Acad Sci USA
94:9197–9201
Robertson BC, Gemmell NJ (2004) Defining eradication units
to control pests. J Appl Ecol 41:1042–1048
Russell JC, Clout MN (2004) Modelling the distribution and
interaction of introduced rodents on New Zealand off-
shore islands. Glob Ecol Biogeogr 13:497–507
1756 J. C. Russell et al.
123
Russell JC, Towns DR, Anderson SH, Clout MN (2005)
Intercepting the first rat ashore. Nature 437:1107
Russell JC, Beaven BM, MacKay JWB, Towns DR, Clout MN
(2008a) Testing island biosecurity systems for invasive
rats. Wildl Res 35:215–221
Russell JC, Towns DR, Clout MN (2008b) Review of rat
invasion biology: implications for island biosecurity.
Science for Conservation 286. Department of Conserva-
tion, Wellington
Selkoe KA, Toonen RJ (2006) Microsatellites for ecologists: a
practical guide to using and evaluating microsatellite
markers. Ecol Lett 9:615–629
Simberloff D (2003) Eradication - preventing invasions at the
outset. Weed Sci 51:247–253
Thresher RE, Kuris AM (2004) Options for managing invasive
marine species. Biol Invasions 6:295–300
Vitousek PM, D’Antonio CM, Loope LL, Rejmanek M,
Westbrooks R (1997) Introduced species: a significant
component of human-caused global change. N Z J Ecol
21:1–16
Weir BS (1996) Genetic data analysis II: methods for discrete
population genetic data. Sinauer Associates, Sunderland
Using genetic assignment to identify invasive pests following eradication 1757
123
