The Effects of Population Bottlenecks on
Multilocus DNA Variation in Robins
S. L. Ardern, D. M. Lambert, A. G. Rodrigo, and I. G. McLean
The effects of bottlenecks on genetic variation in two wild populations of New Zealand robins (Petrolca auatralia auatralls) were examined using multilocus minisateiiite DNA probes. In each case the size and timing of the bottlenecks were known,
together with the location of the source populations. The two founder events occurred In 1973 and both Involved five or fewer Individuals. Parameters of multilocus
DNA profile variation, specifically average percent difference (APD), heterozygoshty,
numbers of polymorphic loci, and numbers of novel restriction fragments present,
were used to measure levels of minisateiiite DNA variation in the two sets of source
and translocated robin populations. New statistical methods are described that test
the significance between the APDs of source and bottlenecked populations. An
overall trend toward reductions In minisateiiite DNA variation through bottleneck
events was observed. However, despite establishment from only one or two founding pairs, moderate levels of variation were maintained In both bottlenecked populations. These results Indicate that catastrophic losses of multilocus DNA variation, like single-locus variation, should not be regarded as an inevitable consequence of founder events and population bottlenecks.
From Ecology and Evolution and the Centre for Conservation Biology, School of Biological Sciences, University ol Auckland, Auckland, New Zealand (Ardern,
Lambert), the Department o{ Ecology, Massey University, Palmerston North, New Zealand (Lambert), Kingett Mitchell and Associates Ltd., Takapuna, Auckland,
New Zealand (Rodrigo), and the Zoology Department,
Canterbury University, Chrlstchurch, New Zealand
(McLean). S L Ardern is now In the Veterinary Clinical
Sciences Department, Faculty of Veterinary Science,
Massey University, Palmerston North, New Zealand. D.
M. Lambert Is now in the Department of Ecology, Massey University, Private Bag 11-222, Palmerston North,
New Zealand. A. G. Rodrigo Is now Senior Research Fellow In the Department of Microbiology, University of
Washington School of Medicine, Seattle, Washington.
This work was supported by grants from the Research
Committees of the University of Auckland and Canterbury University to D.M.L and S.LA., and I.G.M., respectively. We would like to thank E. C. Young, J. L
Craig, J. Robins, D. Towers, and C. C. Holmes for valuable comments on the manuscript.
Journal of Heredity 1997^8:179-186; 0022-1503/97/15.00
The genetic consequences of population
bottlenecks and founder events are of fundamental Importance to both evolutionary
and conservation biology. Both fields of
research are rooted in population genetic
theory which predicts that genetic variation may be lost through population bottlenecks as a result of founder effects and
random genetic drift (Lande 1980; Maruyama and Fuerst 1984,1985; Nei et al. 1975).
Among evolutionary biologists, several authors have advanced the theory of genetic
"revolutions" and rapid speciation as a result of founder effects (Carson 1975; Carson and Templeton 1984; Mayr 1954; Templeton 1980). However, the conditions under which population bottlenecks and
founder effects are likely to result in speciation have been difficult to predict using
current theory (Spencer 1995). Therefore
the extent to which these phenomena explain speciation remains a subject of evolutionary debate (Lambert and Spencer
1995). The accepted theory of conservation genetics concerning population bottlenecks is that losses of genetic variation
occurring In small populations increase a
species' susceptibility to extinction, as reduced levels of genetic variation are considered to result In short-term decreases
in fitness (Inbreeding depression) and a
long-term lack of adaptive flexibility (Frankel and Soule 1981; Franklin 1980; Lande
and Barrowclough 1987; O'Brien et al.
1985; Schonewald-Cox et al. 1983; Simberloff 1988).
Although theoretical studies in popula-
tion genetics (e.g., Nel et al. 1975; Wright
1978) have advanced important predictions, surprisingly few authors have provided empirical evidence of the effects of
documented bottlenecks on genetic variation (e.g., empirical studies by Berlocher
1984; Schwaegerle 1979; Taylor and Gorman 1975). Several studies have reported
decreased levels of genetic variation following founder effects in colonizing populations [e.g., mynas (Acridotheres tristis;
Baker and Moeed 1987); pitcher plant
(Sarracenis purpurea ; Schwaegerle 1979)].
The empirical results are variable however. Increased levels of genetic variation
have been detected in other species after
contractions In population size. For example, an Increase in haplotype number
was reported after a bottleneck event in a
laboratory population of Hawaiian Drosophila siluestris (Carson and Wisotzkey
1989). Again, contrary to theoretical expectation, an increase in average allozyme
heterozygosity In dispersed populations
of the Laysan finch (Tetespiza cantans)
was observed following founder events
(Fleischer et al. 1991).
Clearly, even for single-locus genetic
variation, current evidence suggests some
disparity between theory and practice.
Additional analyses of genetic variation in
populations where bottlenecks have been
well documented will aid the continuing
development of evolutionary theory and
the appropriate application of genetic
principles In conservation management.
Here we examine changes in levels of mul-
179
NEW ZEALAND
tilocus DNA variation in two bottlenecked
populations of New Zealand robins (Petroica australis australis) and their respective source populations after 20 years of
separation. A set of two new randomization tests, based on a fixed covariance
model (FCM) and an independent assortment model (1AM), are described and applied, to determine the significance of differences between levels of genetic variation In source and bottlenecked populations.
NORTH ISLAND
Materials and Methods
/
SOUTH ISLAND
Figure 1. Location of New Zealand robin populations involved in the founder events of 1973.
Source
Population
Bottlenecked
Population
5 robins
1973
12O-15Oha
1973
N-400
56ha
1993
N-400
1973
N=5
5 robins
KAIKOURA
S2
1973
o_
ALLPORTS
B2
16ha
24Oha
mi
~ 80-100
1993
N-320
1223.
N-35
1973
N=5
1993
N-60
Figure 2. Details of the robin population bottleneck events showing changes in population sizes (N represents
estimated population sizes).
1 8 0 The Journal of Heredity 199788(3)
Study Sites
The robin populations on Motuara and Allports Islands were both established in
1973 by transfers of five birds from Nukuwaiata Island and Kalkoura, respectively
(Flack 1974, 1978) (Figures 1 and 2). For
clarity, hereafter we will refer to the populations on Nukuwaiata and Motuara Islands as SI (source population 1) and Bl
(bottlenecked population 1), respectively,
and the populations at Kaikoura and on
Allports Island as S2 (source population 2)
and B2 (bottlenecked population 2), respectively. SI is likely to have maintained
a large population size since the time of
the transfer. In contrast, S2 is smaller, and
fluctuations in population size at this
mainland location appear to be more common than In island populations (Flack
1979). However, there is the potential for
immigration from adjacent populations in
the mainland S2 population, whereas we
have presumed that the island SI population is "closed."
Although five individuals were translocated in each case examined here, B2 appears to have been founded by two pairs,
as one male disappeared soon after release (Flack 1974). It is possible that a single pair founded the Bl robin population,
as a high degree of infertility was recorded
for one pair during the first season on the
Island. The remaining female was not seen
after the transfer (Flack 1974). Island size
has restricted the B2 population to a maximum of approximately 30 pairs, while the
larger habitat occupied by Bl currently
supports approximately 160 pairs (Maloney 1991).
Tissue Collection, DNA Extraction, and
Digestion
Blood was obtained from randomly sampled robins of the populations of interest
in 1992 and 1993. Birds were caught in
hand nets, using Tenebrio molitor larvae as
bait. Blood was collected in hematocrits
following venipuncture of the brachlal
vein using a heparinized 13 mm, 27 gauge
needle, and subsequently stored at -80°C
(Ardern et al. 1994).
Total genomic DNA was extracted from
whole blood beginning with a cell lysis
step. Small volumes (20 n.1) of whole blood
were resuspended in 400 p.1 of lysing buffer (144 mM NrL,Cl, 10 mM NH4HCCg, followed by centrifugation at 1000 g for 10
min to pellet cells. The supernatant was
removed and replaced with 400 p.1 of SET
buffer (0.1 M Tris-HCl pH 8.0, 0.01 M NaCl,
1.0 mM EDTA) to which SDS and proteinase K were added at final concentrations
of 0.5% and 0.5 mg/ml, respectively. After
overnight incubation at 65°C, high molecular weight DNA was extracted twice with
phenol-chloroform-isoamyl (25:24:1 v/v),
once with phenol, once with chloroformisoamyl (24:1), and finally dissolved in
ddH2O after precipitation with 2.5 vol of
ethanol and 0.1 vol of 5 M NaOAc pH 5.2.
Approximately 20 jtg DNA was digested
overnight with Hae\\\ (15 units) using the
manufacturer's (BRL) buffer in the presence of 4 mM spermldine trihydrochloride
and 0.1 mg/ml BSA. Samples were incubated an additional 1-2 h after addition of another five units of restriction enzyme.
DNA Electrophoresls, Transfer, and
Hybridization
DNA fragments (3.5 jtg Hae\\\ digested
DNA/sample) were resolved in a 0.8% agarose gel in TBE running buffer (1.34 M Tris,
749 mM boric acid, 25.5 mM EDTA). Gels
were run at 54 V for approximately 58 h.
A sample from a control animal was included on every gel to allow standardization of hybridization and wash conditions
across gels. Molecular weight markers (1
kb ladder/W/ndlll cut I DNA, BRL) indicated the distance run and sizes of restriction
fragments analyzed on all gels. "Cold" molecular weight markers (2.5 ng 1 kb ladder,
7.5 ng Hindttl cut X DNA; following Birkhead et al. 1990) were also included In
loading dye.
After electrophoresis the DNA was depurinated in 0.25 M HC1 for 15 min, denatured in 0.5 M NaOH, 1.5 M NaCl for 1 h,
and finally neutralized by two 15 min
washes in 1.5 M NaCl, 0.5 M Tris pH 7.2, 1
mM EDTA. The DNA was transferred to a
nylon membrane (Hybond-N, Amersham)
by Southern blotting in 6x SSC (0.15 M
NaCl, 0.015 M sodium citrate) overnight.
After blotting, membranes were washed
for several minutes in 6X SSC, air dried,
and the DNA was permanently bonded to
the membrane by baking for 2 h at 80°C.
The human-derived minisatellite sequences 33.15, 33.6 (Jeffreys et al. 1985),
pV47-2 (Longmire et al. 1990), and internal
lane marker (1 kb/X. ///ndlll cut DNA) were
labeled with a- n P by random priming
(Amersham multiprime kit) and used as
probes. All membranes were hybridized
sequentially with pV47-2, 33.15, 33.6, and
the internal lane marker. Membranes were
prehybridlzed at 55°C (33.6, pV47-2, internal lane marker) or 61°C (33.15), in 0.25 M
Na2HPO4 pH 7.2, 1 mM EDTA pH 8.0, 7%
SDS, for approximately 2 h before addition
of the radioactively labeled probe. Following a 12-16 h period of hybridization at
prehybridization temperatures, filters
were washed to final stringencies of 5x
SSC, 0.1% SDS (pV47-2, internal lane marker) or 0.5x SSC, 0.1% SDS (33.15, 33.6). Autoradiography was carried out at -80°C
with one intensifying screen for 1-10 days.
Before rehybridization filters were stripped
in 0.4 M NaOH at 45°C for 30 min, followed
by two neutralization washes (15 min
each) in 0.1 x SSC, 0.1% SDS, 0.2 M Tris pH
7.5.
DNA Fingerprint Analyses
Bands of molecular weight greater than 8
kb, 5 kb, and 4 kb were scored for profiles
produced with 33.6, pV47-2, and 33.15, respectively. No overlapping fragments were
observed in the regions scored except in
DNA profiles produced with 33.15 and 33.6
for Bl robins [proportion of fragments detected by both probes was calculated as
0.02 ± 0.03 (SD)]. Wherever overlap occurred fragments were scored only once.
Bands of similar mobility were considered to be shared between individuals if
they differed less than twofold in intensity.
Bin sizes (ranges within which the centers
of bands must fall in order to be classified
as being the same) varied with the molecular weight range being scored and were
assigned according to curvature observed
for internal lane marker fragments across
gels. Bin sizes of 1 mm and 2 mm were
used for fragments x > 6 kb and 6 kb > x
> 4 kb, respectively.
Genetic variation within populations
was estimated using all pairwise comparisons of individuals in each population.
Comparisons were made between individuals on the same gel only. Four within population gels were scored (/V = 15 Nukuwaiata; N = 17 Motuara; N = 12 Kaikoura;
and N = 14 Allports). Pairwise difference
values [percent difference (PD)] were calculated as
= [FJ(F, +
100
where F& is the number of fragments
which differed between two individuals a
and b, and F, and Fb equal the number of
fragments scored in DNA profiles of each
Individual, respectively (Gilbert et al.
1991; Packer et al. 1991). The average of
all pairwise difference values [average
percent difference (APD)] was used for
each population as an index of within-population genetic variation (Baker et al.
1993; Gilbert et al. 1990; Wayne et al.
1991).
Heterozygosity was calculated for each
population according to the formula in
Stephens et aL (1992, Equation 5)
where A and k are the number of scorable
bands on the gel and the frequency of occurrence of the ftth band, respectively.
The proportion of fixed restriction fragments and the number of novel or different restriction fragments observed were
also examined as indicators of genetic
variation within populations. The computer program "Thumbprint" (Marshall 1992)
performed calculations of APDs and standard deviations, heterozygosity, and other
statistics such as numbers of fixed restriction fragments.
Statistical Analyses
Standard statistical methods cannot be
used to test the significance of differences
in APDs between two populations because
of the nonindependence and lack of normality of genetic data derived from all
pairwise comparisons of individuals
(Dietz 1983; Lynch 1990). T tests, for example, are likely to be too liberal in the
analysis of statistical significance of differences in genetic variation between populations, as the number of pairwise comparisons inflates sample sizes, exceeding
the actual number of individuals sampled.
With DNA fingerprint data, nonindependence may also occur as a result of linkage
or linkage disequilibrium, which has been
documented in studies of a number of species (e.g., Brock and White 1989; Hanotte
et al. 1992; Jeffreys and Morton 1987). To
avoid the problems relating to nonindependence of the data, the statistical significance of differences in APDs between
source and bottlenecked populations was
tested here using two randomization procedures that are based on the bootstrap
resampling method of Efron and Gong
(1983).
The two randomization techniques in-
Ardern et al • Population Bottlenecks and Multitocus DNA Variation 1 8 1
corporate different assumptions regarding
the degree of independence between fragments in a DNA fingerprint. The first procedure generates bootstrapped pseudosamples with the same number of individuals as in the original sample, by sampling
individual DNA profiles with replacement.
In the construction of a pseudosample all
bands in each DNA fingerprint are therefore treated as if they are linked, hence
this procedure is called the fixed covariance model (FCM). The second randomization procedure generates pseudosamples by sampling each different fragment
detected in the original DNA fingerprints
with replacement across all individuals.
The status of each unique fragment in the
original sample (I.e., presence or absence
of the fragment) is sampled with replacement across all individuals, until all individuals in the pseudosample have been
Identified as either possessing or not possessing that particular fragment. This
method—the Independent assortment
model (1AM)—assumes that all fragments
in the DNA fingerprints of the original sampled individuals are independent of each
other and therefore free to assort independently.
These two models represent the extreme ends of the spectrum between complete linkage of all loci and complete independence among loci. Testing against
both models frees us, to some extent, from
identifying where in the spectrum our system lies. If both models allow us to reach
the same conclusion, then it is safe to assume that this conclusion is not dependent on levels of nonindependence and
linkage.
To test whether the level of genetic variation in one population (measured as
APD) is significantly different from that in
another population, the null distribution
of differences that can occur as a result of
sampling (and therefore of chance) must
be constructed. The statistic of interest is
the difference between the APDs (D,^ of
the two populations under consideration.
During the FCM and IAM randomization
procedures, pseudosamples are generated
for each population from the original data.
These pseudosamples are used to construct the null distribution from which the
statistic of interest can be calculated. If
the difference in APDs between pseudosamples is greater than the observed difference between populations in the original sample (D^) less than 5% of the time,
we reject the null hypothesis that there is
no difference between the populations (or
that any difference is due to chance).
1 8 2 The Journal of Heredity 1997 88(3)
Table 1. Statistical significance of differences In
genetic variation (D^s) within source (SI, SZ)
and bottlenecked (Bl, B2) robin populations
based on P values obtained using the fixed
covarlance model (FCM) and independent
assortment model (JAM) randomization
procedures
Populations
compared
/"values
Probe
FCM
1AM
Overall
slgnlficance
Sl-Bl
33.15
33.6
pV47-2
All probes
combined
.0
.04
.0
.0
.0
.01
.0
.0
S
S
S
S
S2-B2
33.15
33.6
pV47-2
All probes
combined
.04
.72
.075
.125
.02
.6
.05
.005
S
NS
NS
NS*
Overall significance Indicates where P < .05 lor both
FCM and 1AM.
* Where one model Is significant, we accept a strong
trend toward significance.
The following procedure is used to estimate the sampling (null) distribution of
D^j between two populations:
1. Calculate the Z)ipd between the two
populations.
2. Generate two pseudosamples for
population 1 using the FCM.
3. Calculate the D^, between the two
pseudosamples. Call this D*^.
4. If the D*Bpa is equal to or greater than
the Dmpa in the original population, then
add one to a count variable C.
5. Repeat steps 2-5 a large number of
times. In this analysis steps 2-5 were repeated 100 times.
6. Express C as a percentage of the
number of times steps 2-5 were repeated.
7. Repeat steps 2-6 using population 2
as the original data set.
8. Average the values of C (expressed as
a percentage) obtained for population 1
and population 2. If this average (reported
as the P value) is less than 5% (P < .05),
then the observed difference (D^) is significantly different from zero at the 5% level.
The randomization procedure described
above was repeated using the FCM and
IAM, since it is likely that independent assortment occurs at least among some fragments in a DNAfingerprint.The FCM tends
to give a more conservative indication of
statistical significance (Table 1). Levels of
genetic variation In the two populations
were Judged to be significantly different
when the proportion of pseudosamples
with D*^ greater than D^, In the original
populations was less than 5% for both the
FCM and IAM. Similarly, when both models
indicated that D*^ was greater than D^,
in more than 5% of pseudosamples, the
null hypothesis that there is no difference
between the two populations was not rejected.
To determine whether the average decrease in genetic variation measured within both bottlenecked populations compared with source populations was significantly different from zero (P < .05) an
ANOVA with a linear contrast was performed on PDs of independent pairwise
comparisons. The null hypothesis under
test was that there was no significant difference between source and bottlenecked
populations, according to the following
model:
(APDsl - APDB0 +
= 0
where SI, Bl, S2, and B2 refer to the robin
populations of Nukuwaiata Island, Motuara Island, Kaikoura, and Allports Island,
respectively.
Unpaired two-tailed t tests were used to
test the statistical significance (P < .05) of
differences in mean numbers of fragments
detected per individual in source versus
bottleneck populations. Discovery curves
(showing the cumulative sampling of novel fragments with increasing numbers of
fragments scored) were produced for preand postbottleneck populations in order
to assess whether the numbers of different fragments detected in each population
were likely to reflect real differences rather than sampling artifacts.
Results
The average percent difference between
DNA fingerprints of robins in both bottlenecked populations was lower than in
source populations for all probes except
33.6, for which a higher APD was recorded
among B2 robins than among S2 robins
[B2 = 46.3 ± 21.6 (SD); S2 = 43.5 ± 18.6
(SD); Table 2]. Standard deviations indicate overlap of these measures in some
cases. The difference observed in APDs
for the S2-B2 comparison was significant
for probe 33.15 (P = .04, FCM; P = .02,
1AM; Table 1), although not significant
when data for all probes were combined
(P = .125, FCM; P = .005, IAM; Table 1). In
contrast, for the Sl-Bl comparison, differences in population genetic variation for
all probes were statistically significant,
yielding a significant overall difference in
within-populatlon genetic variation for
robins on these islands (P = .00, FCM; P
= .00,1AM; all probes combined; Table 1).
Table 2. Summary statistics describing minUateUite DNA variation detected within source (51, S2) and
bottlenecked (Bl, B2) robin popnlations using probes 33.15, 33.6, pV47-2, and all probes combined
Population
(sample
size)
Average
heterozygoslty
Mean number
of fragments/
Individual
(SD)
Total
fragments
scored In
population
Proportion
of fragmerits fixed
Probe
APD(SD)
33.15
33.6
pV47-2
All probes
combined
62.9(12.2)
51.6(16.2)
48.9(14.6)
55.4 (8.7)
0.73
0.65
0.54
13.5 (2.6)
10.3 (2 0)
7.9 (2.2)
31.6(4.0)
47
24
24
95
0
0
0.04
0.011
•)
Bl
(N = 17)
33.15
33.6
pV47-2
All probes
combined
46.3(11.9)
42.5 (14.6)
37.6(11.5)
39 4 (7.3)
0.62
0.59
0.35
12.6 (2.1)
9.7 (2.7)
8.6 (1.3)
30.9 (2.8)
31
19
15
65
0
0
0.13
0.031
3
o
S2
(N = 12)
33.15
33.6
pV47-2
All probes
combined
54.2 (12.2)
43.5 (18.6)
47.2 (13.8)
49.2(9.1)
0.62
0.58
0.52
15.4 (2.8)
9.1 (1.8)
8.0(1.5)
32.5 (3.7)
44
20
22
86
0.02
0
0.05
0.023
B2
(N = 14)
33.15
33.6
pV47-2
All probes
combined
44.7 (10.8)
46.3(21.6)
38.5(13.1)
43.1 (8.9)
0.59
0.61
0.40
12.6 (1.5)
8.9 (2 2)
7.6(1.6)
29.2 (2.9)
30
19
16
65
0
0
0.125
0.031
SI
(W= 15)
Results of the ANOVA indicated that the
trend toward reduced levels of genetic
variation (APDs) within bottlenecked populations averaged over both translocation
events (data from all probes combined)
approached statistical significance (P =
.056). When data for each probe were analyzed separately, the average decrease in
genetic variation in bottlenecked compared with source populations was statistically significant for probes 33.6 (P =
.034) and pV47-2 (P = .010), but not for
33.15 (P = .147) for which the largest number of fragments were scored.
As expected (Stephens et al. 1992), heterozygosities calculated for bottlenecked
robin populations reflected APD values,
tending to be lower than values for their
respective source populations (Bl = 0.52
compared with SI = 0.65; B2 = 0.54 compared with S2 = 0.59).
In their analysis of the bottleneck effect
on allozyme variation, Nei et al. (1975)
suggested that numbers of alleles per locus represent an important measure of genetic variation in addition to the traditional measure of heterozygosity. In studies of
minisatellite DNA variation, the number of
novel DNA fragments of different molecular weights found in a population similarly
represents another source of information
in addition to the calculations of APD and
heterozygosity. Larger numbers of different fragments were detected in both
source populations compared with bottlenecked populations (Table 2; Figure 3).
The plateau observed especially in "discovery curves" of bottlenecked popula-
tions (Figure 3), suggests that the reduced
number of different fragments present in
these populations is likely to reflect a real
difference rather than the effect of inadequate sample sizes. However, it is unlikely
that all rare fragments will have been detected in the populations examined, given
the sample sizes used. In addition to fewer
different fragments detected in bottlenecked populations, a larger proportion of
fragments were fixed in these populations
compared with source populations (proportions of fixed fragments in Bl and B2
= 0.031, SI = 0.011, S2 = 0.023; all probes
combined; Table 2).
Mean numbers of fragments per individual were generally not significantly lower
for the bottlenecked populations, despite
decreased levels of heterozygosity. For
probe pV47-2 the observed mean of 8.6
(±1.3) fragments per individual for Bl robins was similar to the value for the SI population (7.9 ± 2.2), and the observed difference was not statistically significant (P
= .2172). The only significant difference
was observed for profiles produced with
33.15 for the S2-B2 combination (Figure 4;
mean numbers of fragments per individual
equaled 15.4 ± 2.8 and 12.6 ± 1.5, respectively; P = .0092).
Discussion
Moderate Levels of Genetic Variation
Retained Despite Severe Population
Bottlenecks
Moderate levels of minisatellite DNA variation were detected in botUenecked pop-
C
t
C
0
100
200
300
400
500
600
Number of fragments sampled
I
S
0
100
200
300
400
500
600
Number of fragments sampled
Figure 3. Discovery curves for source and bottlenecked populations of robins showing cumulative numbers of new fragments detected with Increasing number of fragments sampled, using results of probes
33.15, 33.6, and pV47-2 combined. (A) Sl-Bl comparison (B) S2-B2 comparison. (Circles and squares represent Individuals of bottlenecked and source populations, respectively.)
ulations of South Island robins despite the
severity of both founder events involving
five birds or less. Estimates of genetic variation among robins of Motuara and Allports Islands contrast with very low measures reported in several other species, including naked mole-rats (Heterocephalus
glaber, APD between 1.0 and 6.0; Reeve et
aL 1990), Channel Island foxes (Urocyon littoralis; APD between 0 and 25.3; Gilbert et
ai 1990), and the Gir forest lion (Panthera
leo; APD of 2.6; Gilbert et aL 1991).
Several authors have emphasized that a
sudden population contraction followed
by rapid recovery to large population size
does not necessarily have profound consequences on the reduction of genetic
variation (e.g., Lande and Barrowclough
1987; Net et aL 1975). Although the rarest
Ardern et al • Population Bottlenecks and Mulblocus DNA Variation 1 8 3
Icb
23.1
KaiVoura(S2)
Allports (B2)
10.2
8.1
6.1
5.1
4.1
33.15
33.15
Figure 4. Multllocus DNA fingerprints of New Zealand robins from Kalkoura (S2) and Allports Island (B2). DNA
profiles are of //aelll-dlgested DNA hybridized with the mlnlsatelllte probe 33.15.
alleles are expected to be lost during
founder events, a substantial proportion
of variation, such as heterozygosity, can
be maintained. In the bottlenecked Allports and Motuara Islands robin populations studied here, approximately 65-75%
of the restriction fragments present in
each respective source population have
been retained through the founding event
and subsequent population growth (Figure 3). The moderate levels of minisatellite variation detected among robins of
these islands appear to be indicative of
the likely rapid increases in population
size possible in both newly established
populations after the founder events.
Although the two source populations
displayed higher levels of genetic variation relative to bottlenecked populations,
robins of Nukuwaiata Island and Kaikoura
themselves exhibited lower levels of minisatellite DNA variation than commonly reported in birds (e.g., Burke and Bruford
1987; Hanotte et aL 1992). Two possible
explanations for this can be considered.
First, the species may be characterized by
relatively low levels of minlsatellite DNA
variation. This explanation seems unlikely
however, since higher levels of variation
have been recorded for putative nonrelatives of a large, outbred, mainland population of the North Island subspecies P. a.
longipes (Ardern SL et al., unpublished
data). Alternatively, lower levels of variation at the minisatellite loci of Nukuwaiata
Island and Kaikoura robins may reflect his-
1 8 4 The Journal of Heredity 1997.88(3)
torical population contractions at these
sites. While this explanation is speculative, it is consistent with our knowledge of
the fluctuations in population size of mainland robins at Kaikoura, and the role of
founder effects, isolation, and drift in the
establishment of island populations.
Trend Toward the Loss of Minlsatellite
DNA Variation Through Bottleneck
Events
An overall trend toward loss of minisatellite DNA variation estimated as APD was
observed through bottleneck events for
the two pairs of source and bottlenecked
robin populations studied. Other characteristics of DNA variation examined, including heterozygosity, numbers of polymorphic loci, and numbers of novel or different restriction fragments present, also
suggested a tendency toward reduced genetic variation In the bottlenecked populations. The number of different fragments
found in a population appeared to be a
particularly sensitive indicator of reductions in genetic variation through bottleneck events (Figure 3).
The mean number of fragments detected per individual did not differ significantly between source and bottlenecked populations. Although a decrease in fragments
scored per individual is expected with increasing homozygosity (Lynch 1990), this
trend was also not well documented in
studies of the bottlenecked Crater lion (P.
led) population (Gilbert et al. 1991) or the
endangered Isle Royale gray wolf (Canis lupus; Wayne et al. 1991). It may be that the
magnitude of the changes in minisatellite
DNA variation observed did not allow detection of changes in numbers of fragments scored per individual. This hypothesis is supported by observations of extremely low levels of minlsatellite variation detected among Chatham Island black
robins (P. traversi; Ardern and Lambert, in
press), which were associated with low
mean numbers of fragments scored per individual.
The effect of the bottleneck event on average percent difference appeared to vary
in the two cases examined. Generally a
greater decrease in genetic variation was
apparent among Bl robins compared with
the reduction in variation observed
among B2 robins. The apparently greater
loss of minisatellite variation observed
among the former reflects the higher levels of genetic variation measured in its
source population (SI) than in S2. Comparable levels of genetic variation were
detected among robins in each bottlenecked population (P = .25, FCM; P = .09,
LAM; all probes combined). Factors potentially affecting the changes in genetic variation associated with the two bottleneck
events Include genetic variation In source
populations, number of effective founders
and their genetic contributions, population size in the bottlenecked populations,
and random drift. The influence of these
factors on observed changes in genetic
variation are discussed In the next section.
Random genetic drift is considered to
be the predominant factor affecting genetic changes in small populations over time
(e.g., Franklin 1980). Indeed, random genetic drift appeared to be a major determinant of the genetic changes observed in
colonizing populations of starlings (Sturnus vulgaris; Ross 1983) and mynas (Acridotheres tristis; Baker and Moeed 1987) in
New Zealand, both established in the mid
to late 1880s. In the robin bottleneck
events examined here it is possible that
the changes in genetic variation resulted
largely from stochastic sampling events or
random drift occurring during and after
the transfers to Allports and Motuara Islands.
The degree of loss of genetic variation
due to founder effects and random drift
depends on both the number of founding
individuals, and their genetic composition
(Nei et al. 1975; Taylor and Gorman 1975).
The greater loss of variation from Bl compared with B2 may be partly attributable
to the likely establishment of the former
by a single effective breeding pair, and the
latter from two pairs, potentially representing a larger proportion of the original
genetic variation in the source population.
In other respects the greater loss of
variation detected In the Sl-Bl bottleneck
event was unexpected. If equivalent proportional losses of the variation present in
each source population occurred as a result of the founder effect, and population
size increased steadily in both bottlenecked populations after transfer, a higher
level of minisatellite variation would have
been anticipated among Bl than B2 robins. This expectation is based on the apparently higher levels of variation in SI
and the potential generation of more novel
variation by mutation among Bl robins,
where the current population size is approximately five times greater than in B2
on Allports Island. Because mutation rates
among minisatellite DNA loci are very high
in most species [mutation rates per meiotic event: e.g., humans, 1-4 x 10~3 (Jeffreys et al. 1985); indigo buntings, 1.1 x
10"2 (Westneat 1990), including robins
[3.273 x 10"3 (Ardern et al., unpublished
data)], It is likely that new variation would
have arisen from mutation in the 20 years
since the bottleneck events.
We cannot discount the possibility that
a greater loss of variation has passed undetected through the S2-B2 bottleneck
event. As estimates of the levels of minisatellite DNA variation in source populations at the time of the transfers, current
measures are confounded by the continuing processes of population genetic
change. Because of the greater instability
and smaller population size of S2 (Flack
1979), the effects of drift (and potentially
loss of genetic variation) in this population may be more significant than in the
SI population.
Our results (see Table 2) contrast with
those reported by Gilbert et al. (1990) on
the Channel Island fox (U. littoralis) in
which low within-island multilocus DNA
variation was detected (APDs ranged from
0.0-25.3%), while between-island variation
was high (43.8-84.4%). In fact, these authors recorded that foxes from each of the
six islands within the Channel Island
group off California could be distinguished
by the presence of diagnostic restriction
fragments. In the case of the robin populations investigated here, no such diagnostic fragments were found and the withinisland APDs for robin populations were all
higher than the maximum within-island
value reported by Gilbert et al. (1990).
These authors suggested that their result
is due to the rapid fixation of RFLPs in
small isolated populations which has outpaced the generation of new variation by
recombination. This finding is surprising
since minisatellite DNA characteristically
has very high rates of mutation, and our
results suggest a considerable amount of
multilocus DNA variation passes through
even a severe bottleneck such as one or
two breeding pairs. Moreover, the fox populations have been isolated for 800-11,500
years and current population sizes range
from 490 to 2,120, with the first record of
an island fox being 16,500 years ago.
Changes in single-locus genetic systems
as a result of population bottlenecks have
been well characterized theoretically and
there is some evidence of patterns of genetic change in both laboratory and natural populations. Changes in quantitative
characters are substantially more complex
and much more difficult to predict. For example, heritabillty and additive genetic
variation in eight morphological traits generally increased in experimentally bottlenecked populations of house flies (Musca
domestica; Bryant et al. 1986). Minisatellite
DNA profiles may provide an intermediate
system to investigate changes in the genetics of natural populations. Multilocus
systems are more complex than single-locus ones because they are potentially affected by factors such as the degree of
linkage. Interestingly, our results suggest
that patterns of changes, similar to those
expected in single-locus genetic systems,
have occurred as a result of population
bottlenecks in New Zealand robins. For example, we recorded a substantial reduction in minisatellite DNA variation, but
moderate levels of variation still remained
In the bottlenecked populations. Conversely, very low levels of variation were
detected in multilocus minisatellite DNA
profiles of the closely related Chatham Island black robin (Petroica traversi; Ardern
and Lambert, in press). In fact our studies
have shown that this species has among
the lowest levels of minisatellite variation
reported in wild populations of birds. All
existing individuals of this species are descended from a single breeding pair in the
early 1980s and population size has been
very low (not more than 30 birds; Butler
and Merton 1992) for approximately the
last 100 years. This result again parallels
that generally expected for single-locus genetic systems.
In the future the most informative studies of founder events and their effect on
genetic variation will be those conducted
prospectively, with sampling of source
populations and founding individuals at
the time of establishment of new populations. The results of this study clearly suggest a trend toward the loss of minisatellite DNA variation through bottleneck
events. In addition, despite establishment
from only one or two founding pairs, moderate levels of variation were maintained
in both bottlenecked populations. Therefore catastrophic losses of multilocus DNA
variation should not be regarded as an inevitable consequence of founder events
and population bottlenecks.
References
Ardem SL and Lambert DM, 1996. Is the black robin In
genetic peril?. Mol Ecol 6:21-28.
Ardem SL, McLean IG, Anderson S, Maloney R, and
Lambert DM, 1994. The effects of blood sampling on
the behavior and survival of the endangered Chatham
Island black robin Petroica traoerzi. Conserv Blol 8:857862.
Baker CS, Gilbert DA, Welnrlch MT, Lambertsen R, Calambokidls J, McArdle B, Chambers GK, and O'Brien SJ,
1993. Population characteristics of DNAfingerprintsIn
humpback whales Megaptem novaeangliae J Hered 84:
281-290.
Baker AJ and Moeed A, 1987. Rapid genetic differentiation and founder effect In colonising populations of
common mynas Aaidotheres trislus Evolution 41:525538.
Berlocher SH, 1984. Genetic changes coinciding with
the colonization of California by the walnut husk fly
Rhagolelis completa. Evolution 38506-918.
Birkhead TR, Burke T, Zann R, Hunter FM, and Krupa
AP, 1990. Extra-pair paternity and Intraspeclfic brood
parasitism in wild zebra finches Taeniopygia guttata, revealed by DNAfingerprinting.Behav Ecol Socloblol 27:
315-324.
Brock MK and White BN, 1989. Application of DNA fingerprinting to the recovery program of the endangered
Puerto Rican parrot. Proc Natl Acad Sci USA 89:1112111125.
Bryant EH, McCommas SA, and Combs LM, 1986. The
effect of an experimental bottleneck upon quantitative
genetic variation In the house fly Genetics 114:11911211.
Burke T and Bruford MW, 1987. DNAfingerprintingin
birds. Nature 327:149-152.
Butler D and Merton D, 1992. The black robin, saving
the world's most endangered bird. Oxford: Oxford University Press.
Carson HL, 1975. The genetics of speclation at the dlplold level. Am Nat 109:73-92.
Carson HL and Templeton AR, 1984. Genetic revolutions in relation to speclation phenomena: the founding
of new populations. Annu Rev Ecol Syst 15:97-131.
Carson HL and Wlsotzkey RG, 1989. Increase in genetic
variance following a population bottleneck. Am Nat
134*68-673.
Dleti EJ, 1983. Permutation tests for association between two distance matrices. Syst Zool 32:21-26.
Efron B and Gong G, 1983. A leisurely look at the bootstrap, the (ackknlfe, and cross-validation. Am Stat 37:
Flack JAD, 1974. Chatham Island black robin. Wildlife
5:25-34.
Flack JAD, 1978. Interlsland transfers of New Zealand
Ardem et al • Population Bottlenecks and Multilocus DNA Variation 1 8 5
black robins. In. Endangered birds: management techniques for the preservation of threatened species
(Temple SA, ed). Madison, Wisconsin: University of
Wisconsin Press; 365-372.
Rack JAD 1979. Biology and ecology of the South Island
robin. In: Ecology of Kowhal Bush, Kalkoura Mauri Ora
2 (Hunt DM and Gill BJ, eds). Christchurch, New Zealand: University of Canterbury Biological Society; 2226.
Relscher RC, Conant S, and Morin MP, 1991. Genetic
variation In native and translocated populations of the
Laysan finch Telespiza cantons. Heredity 66:125-130.
Frankel OH and Soule ME, 1981. Conservation and evolution. Cambridge: Cambridge University Press.
Franklin R, 1980. Evolutionary changes In small populations. In: Conservation biology: an evolutionary-ecological perspective (Soule ME and Wilcoy BA, eds).
Sunderland, Massachusetts: Slnauer 135-149.
Gilbert DA, Lehman AN, O'Brien SJ, and Wayne RK,
1990. Genetic fingerprinting reflects population differentiation In the California Channel Island fox. Nature
344:764-767.
Gilbert DA, Packer C, Pusey AE, Stephens JC, and
O'Brien SJ, 1991. Analytical DNAfingerprintingIn lions:
parentage, genetic diversity, and kinship. J Hered 82:
378-386.
Hanotte 0, Bruford MW, and Burke T, 1992. Multllocus
DNA fingerprints In gallinaceous birds: general approach and problems. Heredity 68:481-494.
Jeffreys AJ and Morton DB, 1987. DNA fingerprints of
dogs and cats. Anlm Genet 18:1-15.
Jeffreys AJ, Wilson V, and Theln SL, 1985. Hypervariable 'mlnlsatelllte' regions In human DNA. Nature 314:
67-73.
Lambert DM and Spencer HG, 1995. Speclation and the
recognition concept: theory and application. Baltimore,
Maryland. Johns Hopkins University Press.
Lande R, 1980. Genetic variation and phenotyplc evolution during allopatric speclation. Am Nat 116463479.
1 8 6 The Journal of Heredity 1997:88(3)
Lande R and Barrowclough GF, 1987. Effective population sizes, genetic variation, and their use in population management. In: Viable populations for conservation (Soule ME, ed). Cambridge: Cambridge University
Press.
Longmlre JL, Kraemer PM, Brown NC, Hardekopf LC,
and Deaven LL, 1990. A new multHocus DNA fingerprinting probe: pV47-2. Nucleic Acids Res 18:1658.
Lynch M, 1990. The similarity Index and DNA fingerprinting. Mol Blol Evol 7:478-484.
Maloney RF, 1991 Enemy recognition and response in
New Zealand robins. (Masters thesis). Christchurch,
New Zealand: Canterbury University.
Marshall S, 1992. Thumbprint v2. Wellington, New Zealand: Victoria University
Mamyama T and Fuerst PA, 1984. Population bottlenecks and nonequillbrium models in population genetics. I. Allele numbers when populations evolve from
zero variability. Genetics 108:745-763.
Mamyama T and Fuerst PA, 1985. Population bottlenecks and nonequillbrium models In population genetics. II. Number of alleles In a small population that was
formed by a recent bottleneck. Genetics 111:675-689.
Mayr E, 1954. Change of genetic environment and evolution. In: Evolution as a process (Huxley J, ed). London. Allen and Unwin; 157-180.
Nel M, Mamyama T, and Chakraborty R, 1975. The bottleneck effect and genetic variability In populations.
Evolution 29:1-10.
O'Brien SJ, Roelke ME, Marker L, Newman A, Wlnkler
CA, Meltzer D, Colly L, Evermann JF, Bush M, and Wlldt
DE, 1985. Genetic basis for species vulnerability In the
cheetah. Science 227.1428-1434.
Packer C, Pusey AE, Rowley H, Gilbert DA, Martenson
J, and O'Brien SJ, 1991. Case study of a population bottleneck: lions of the Ngorongoro crater Conserv Blol 5:
219-230.
Reeve HK, Westneat DF, Noon WA, Sherman PW, and
Aquadro CF, 1990 DNA "fingerprinting" reveals high
levels of Inbreeding In colonies of the eusocial nakedmole rat. Proc Natl Acad Sci USA 87:2496-2500.
Ross HA, 1983. Genetic differentiation of starling Stwnus vulgaw. aves populations In New Zealand and
Great Britain. J Zool Lond 201J51-352.
Schonewald-Cox CM, Chambers SM, Mac Bryde B, and
Thomas L, 1983. Genetics and conservation. London:
Benjamin Cummlngs.
Schwaegerle KE, 1979. Genetic variability and founder
effect In the pitcher plant Sarracema purpurea L. Evolution 33:1210-1218.
Slmberioff D.1988. The contribution of population and
community biology to conservation science. Annu Rev
EcolSyst 19:473-511.
Spencer HG, 1995. Models of speclation by founder effect: a review. In: Speclation and the recognition concept: theory and application (Lambert DM and Spencer
HG, eds). Baltimore, Maryland: Johns Hopkins University Press; 141-156.
Stephens O, Gilbert DA, Yuhkl N, and O'Brien SJ, 1992.
Estimation of heterozygoslty for single-probe multllocus DNA fingerprints. Mol Blol Evol 9:729-743.
Taylor CE and Gorman GC, 1975. Population genetics
of a "colonising" lizard: natural selection for allozyme
morphs In Anolis grahami. Heredity 35:241-247.
Templeton AR, 1980. The theory of speclation via the
founder principle. Genetics 94:1011-1038.
Wayne RK, Lehman N, Glrman D, Gogan PJP, Gilbert DA,
Hansen K, Peterson RO, Seal US, Elsenhawer A, Mech
LD, and Krumenaker RJ, 1991. Conservation genetics of
the endangered Isle Royale gray wolf. Conserv Blol 5.
41-51.
Westneat DF, Sherman PW, and Morton ML, 1990. The
ecology and evolution of extra-pair copulations In
birds In: Current ornithology, vol. 7 (Power DM, ed).
New York: Plenum; 331-369.
Wright S, 1978. Evolution and the genetics of populations, vol 4, Variability within and among natural populations. Chicago: University of Chicago Press.
Received January 2, 1996
Accepted July 29, 1996
Corresponding Editor: Rodney Honeycutt