Biological Conservation Vol. 83, No. 1, pp.
43-54, 1998
PII: S0006-3207(97)00045-
ELSEVIER
GENETIC STRUCTURE, METAPOPULATION PROCESSES A N D
EVOLUTION INFLUENCE THE CONSERVATION STRATEGIES
FOR TWO E N D A N G E R E D FROG SPECIES
Don A. Driscoll
Department of Zoology, University of Western Australia, Nedlands, Perth, WA 6907, Australia.
(Received 27 June 1996; accepted 21 January 1997)
Abstract
The survival and continued evolution of a species is a
pivotal tenet of conservation biology. Therefore, we need
to understand the factors affecting survival and evolution
of species to conserve them adequately. In this study I use
allozyme electrophoresis to investigate the metapopula-
tion structure and evolutionary processes that operate
within the endangered frog species Geocrinia alba and
G. vitellina. Genetically, G. alba and G. vitellina are
highly subdivided. A number of intraspecific genetic
groups can be recognised, although even within these
groups there are significant differences in allele frequen-
cies among populations. These differences imply that
migration between populations is likely to be extremely
restricted, if it occurs at all. The intraspecific genetic
patterns suggest an evolutionary history of population
bottlenecks followed by range expansion. Therefore, in
the short term neither species exists as a metapopulation.
However, at a larger time scale, migration, extinction and
recolonisation may be central to the evolution and survival
of both species. Maintenance of these processes is a
challenge to wh&h conservation managers must rise for
the criteria of long-term survival and evolution to be met.
© 1997 Published by Elsevier Science Ltd
Keywords: genetic structure, conservation, evolution,
metapopulation, phylogeography, frog.
INTRODUCTION
A central goal of conservation biology is to ensure the
survival and continued evolution of a species (Allendorf
and Leary, 1986; Vrijenhoek, 1989; Woodruff, 1989).
Metapopulation studies have emphasised the potential
importance of dispersal, colonisation and local extinction
in determining the likelihood of species survival (e.g.
Hanski, 1991; Hanski and Gilpin, 1991). Harrison (1991,
1994) stressed that most species may not exist in the
precarious balance between local extinction and recolo-
nisation as described in the classic metapopulation
43
© 1997 Published by Elsevier Science Ltd
All rights reserved. Printed in Great Britain
I 0006-3207/98 $19.00 + 0.00
(Levins, 1970). However, investigation of colonisation,
extinction and migration remains central to any conser-
vation strategy for species that are spatially subdivided.
For example, Warren (1994) suggested that habitat loss
could disconnect remaining fragments of the marsh fri-
tillary Eurodryas aurinia and thereby pose a major
threat to the survival of the butterfly. Stepping stones
between populations were needed to maintain the
metapopulation of a skipper butterfly, Hesperia comma
(Thomas and Jones, 1993). Laan and Verboom (1990)
suggested that dispersal was crucial in compensating
for local extinctions of frog communities. Sj6gren
(1991) found that both re-colonisation and the rescue
effect (Brown and Kodric-Brown, 1977) were central to
the regional persistence of the pool frog Rana lessonae
in northern Sweden. In contrast, Templeton et al.
(1990) suggested that there was no dispersal between
populations of collared lizards Crotaphytus collaris, so
any local extinctions would be effectively permanent
since recolonisation was unlikely. These insights into
migration, extinction and colonisation are necessary for
species conservation.
The continued evolution of a species can only be
ensured if one has some knowledge of the evolutionary
processes that influence it. Those processes can be
investigated by examination of intraspecific genetic
structuring. For instance, Ellsworth et al. (1994) inves-
tigated genetic structuring of white-tailed deer Odocoi-
leus virginianus by using mtDNA, and found that the
pattern of differentiation was consistent with vicariant
processes associated with sea-level changes. In a study
of allozyme and morphological differentiation in the
alpine snail Arianta arbustorum, Arter (1990) concluded
that the snails became extinct in alpine areas in the past
and may have recolonised from lower altitudes using
streams as corridors. Arter (1990) also found that the
geographic structuring of morphological traits was
influenced by natural selection. Thus, both selection in
different environments and historic biogeographic
processes were important in moulding the population
structure and phenotype of A. arbustorum. Eguiarte
44
et al. (1992) found very low levels of genetic differentia-
tion amongst widely dispersed samples of a tropical
palm, Astrocaryum mexicanum, and inferred that the
population structure consisted of large, outbreeding
populations. The authors concluded that evolution
amongst tropical trees may therefore result from natural
selection leading to specialisation in narrow niches.
Specialisation can occur because taxa with wide powers
of dispersal are able to reach niches which may be geo-
graphically restricted, yet they can maintain a large
effective population size which favours natural selection
over random processes (Dobzhansky, 1950). If A. mex-
icanum had existed as many small, isolated populations
then the alternative evolutionary process of genetic drift
and consequent non-adaptive speciation would have
been favoured. Eguiarte et al. (1992) suggested that a
small number of large populations is needed to maintain
the palm's genetic diversity, and to avoid inbreeding.
This strategy would also maintain the evolutionary
processes operating within A. mexicanum.
Conservation strategies can therefore benefit from
evolutionary studies. When evolutionary processes that
operate within a species are identified, the essential
requirements for those processes to continue can be
incorporated in the conservation plan. Requirements
may include the maintenance or reconstruction of cor-
ridors for dispersal, the reservation of areas likely to be
important during changes in geographic range, the
maintenance of existing gene flow patterns, or the con-
servation of representative areas of an influential selec-
tion gradient.
N .....
I 0 30
~0 Scale (Kin)
m
o
lat. 35
Fig. 1. Distributions of the four species in the Geocrinia rosea complex.
D. A. Driscoll
The Geocrinia rosea complex (Roberts et al., 1990)
consists of four frog species with allopatric distributions
in the south-west of Australia (Fig. 1). Geocrinia alba
and G. vitellina have the most restricted ranges of the
four species and are the focus of this study. G. vitellina
falls into the 'vulnerable' category of the International
Union for the Conservation of Nature (IUCN) because
it has a small distribution and only occurs alongside six
creeks (criterion D) (IUCN, 1994). G. alba is critically
endangered due to its restricted distribution, fragmented
populations and recent local extinctions (criteria B1, B2,
IUCN, 1994). Both taxa are considered to be 'threa-
tened fauna' by the Western Australian Department of
Conservation and Land Management (CALM, 1991;
Burbidge and Wyre, 1993) and are the subjects of
recovery plans (Wardell-Johnson et al., 1995). Adults
range in size from 19 to 30mm (average 23.8mm).
Males call from frog-sized depressions beneath leaf litter
or moss in the peaty soil of broad drainage lines.
Females lay eggs inside the burrow and there the eggs
develop through to metamorphosis. There is no free-
swimming tadpole stage (Roberts et al., 1990). Suitable
breeding habitats are discontinuously distributed
throughout the landscape, so it is possible that these
species form a classic metapopulation (Levins, 1970). In
addition, substantial genetic structuring may have
evolved if dispersal between sites has been restricted.
In this study, I describe the genetic structure of G. alba
and G. vitellina by using allozyme electrophoresis. In
a preliminary genetic study, Driscoll et al. (1994)
reported substantial genetic substructuring amongst
~.~ Warren
tea
 Conservation genetics o f two endangered frog species 45

populations of G. alba, and discussed the implications
for reintroduction strategies. The data presented here
include, and expand upon, the earlier data set and are
used to address the following questions:
(1) What is the relationship between migration rate,
extinction and colonisation? Do G. alba and
G. vitellina each have a metapopulation structure?
(2) What is the best strategy for conserving their
genetic resources?
(3) What evolutionary processes are important, and
therefore, what conservation measures should be
taken to ensure the continued evolution of these
species?
METHODS
Sampling
Geocrinia alba
Twenty-three populations of G. alba from throughout
the species' range were sampled for genetic analysis
(Fig. 2, latitudes and longitudes given in Results).
Populations 1, 2, 4, 9, 10, ll, 14, 15, 17, 18, 20, 22, and
23 were sampled in 1992, and populations 3, 5, 6, 7, 8,
12, 13, 16, 19, and 21 were sampled in 1993. All frogs
sampled were calling males, and were captured during
the breeding season. Up to three toes were taken from
each frog, with no more than two toes removed from
each foot. Innermost fingers ('thumbs') were not taken.
All frogs were returned alive to their point of capture.
High recapture rates suggested that toe clipping had a
minimal impact on frog survival (Driscoll, 1996). Toes
were stored in liquid nitrogen in the field before being
transferred to a freezer (-70°C) at the University of
Western Australia. Electrophoresis was conducted within
six months of collection. For large populations (> 30
calling males), at least 30 frogs were sampled (see
Results for list of sample sizes). For populations smaller
than this, all males that were calling on the night of sam-
pling were collected. Samples taken near the peak of the
breeding season represent 80-90% of the total number of
adult males in those small populations (Driscoll, 1996).
Geocrinia vitellina
Toes from 28 to 44 calling males were collected at each
of seven sites in 1992 (Fig. 3, latitudes and longitudes
given in Results), using the same techniques as for G. alba.

Electrophoresis
D ? o
Horizontal starch-gel electrophoresis was used to inves-
tigate genetic structuring (modified from Richardson
et al., 1986; Murphy et al., 1990). Thirty-six enzyme
o .~ JooO "~ ~- ,_
systems were screened, and 12 had scorable activity,
3 ~ P /
representing 16 presumptive loci (Table 1). Alleles were
assigned letters, beginning with the allozyme which
migrated fastest towards the anode. The same lettering
o ,e'"~,m
j%a' ~~ To oo ~- 6 system was used for each species and follows the
nomenclature of Murphy et al. (1996).

0 2 "y---" W \

f r o m W e n d yy R d ~" ¢ ] ., ~ I' " ~ x. ~,

Fig. 2. Distribution of Geocrinia alba, and sites sampled for
genetic analysis. Numbered squares = sample sites (G. alba
present); • , G. alba present 1994; o, G. alba absent; O, former
G. alba sites, presumed extinct.
Z CY--x-"
Fig. 3. Distribution of Geocrinia vitellina, and sites sampled
for genetic analysis (squares). W, Wendy Road; M, Metacrinia
Creek; H, Hut Pool; G, Geo Creek; N, Spearwood Creek
North; S, Spearwood Creek South; C, Crinia Creek; O,
G. vitellina absent; • (and squares), G. vitellina present.
46 D. A. Driscoll

Table 1. Enzyme systems used in the electrophoretic study of Two-dimensional scaling was used to provide a visual
G. alba and G. vitellina TEB = tris-EDTA-borate; TM = indication of genetic structuring within each species,
tris-maleate and the reality of any structuring was then verified
Enzyme Locus Buffer and described by examination of the allele frequency
data. Thus, two-dimensional scaling provided an
Leucyltyrosinepeptidase Ltp TEB
Lpp TEB overview of genetic structuring which was difficult to
Leucylprolinepeptidase
Leucylglycylglycinepeptidase Lgg TEB envisage by using the allele frequencies alone. The
Glycerol-3-phosphate dehydrogenase G-3-pd TEB original data set was then used either to support and
Glucose-6-phosphate isomerase Gpi TM further characterise the structuring, or to identify spur-
Phosphoglucomutase Pgm TM ious or ambiguous structuring that might have been
Malate dehydrogenase Mdh I TM
Phosphogluconate dehydrogenase Pgd TM generated in deriving the two-dimensional plot. Toge-
Malic enzyme Me 1 TM ther, these approaches gave a better understanding of
Me 2 TM the patterns within the data set than either could give
Aspartate aminotransferase Aat 1 TM alone.
Aat 2 TM Chi-squared analyses were used to test for differences
Lactate dehydrogenase Ldh 1 TM
Ldh 2 TM in allele frequency amongst populations within sub-
Isocitrate dehydrogenase Idh 1 TM groups of each species. This was performed as an indi-
Idh 2 TM cation of the cohesiveness of each of the subgroups.
Rare alleles were added to a more common allele on six
occasions to permit unbiased chi-squared values to be
Analysis calculated (no expected value < 1, no more than 20%
of expected values < 5) (Zar, 1984). Rare alleles inclu-
The overall magnitude of differentiation within each ded Gpi(d) (G. alba population 20), Ldh(a) (G. alba
species was assessed by using allele frequencies to population 15) and Gpi(a) (four eastern G. vitellina
calculate Wright's (1978) Fst, with the methods of Weir populations). Unbiased chi-squared values could not be
and Cockerham (1984). Jack-knifing over loci was used calculated for Mdh, Lpp and Ldh-2 in G. vitellina
to obtain standard deviations. Fst is an estimate of the because the variation at these loci consisted of one rare
proportion of the total genetic variance that is due to allele in a single population.
differences amongst populations, and indicates the Allele frequencies, heterozygosities (direct count),
extent to which they have differentiated. I used two- F-statistics and chi-squared analyses were calculated
dimensional scaling in conjunction with the tables of using BIOSYS-1 release 1.7 (Swofford and Selander,
allele frequencies to examine genetic structure amongst 1981; Swofford, 1989).
populations. Multidimensional scaling can highlight
both hierarchical and linear geographic structuring,
whereas clustering methods, such as UPGMA, impose a RESULTS
hierarchy regardless of whether or not one exists
(Lessa, 1990). For this reason I have used multidimen- Geocrinia alba
sional scaling in preference to clustering methods. Two- Four of the 16 loci examined for G. alba were poly-
dimensional scaling takes a matrix of distance measures morphic (Lgg, G-3-pd, Ldh, Gpi). All of the populations
between populations, and attempts to place all of the were fixed for the following alleles: Ltp(a), Lpp(b),
populations into a two-dimensional space in a way that Pgm(b ), Mdh(b), Pgd(e), Me-l (c), Me-2(c), Aat-I (b),
represents all of the distance measures between all Aat-2(b), Ldh-l(a), Idh-l(c), Idh-2(b). The variable
populations. An iterative process of changing the rela- loci indicated that G. alba is highly genetically sub-
tive positions of populations is used to improve the two- divided, with a weighted mean Fst value of 0.444
dimensional model. Improvements are assessed by test- (SD = 0.096). The high Fst value reflects small-scale
ing the change in stress levels with each move. Stress is differentiation amongst populations, and broader sub-
the square root of the differences between the optimally- division into loose clusters of populations.
transformed data and the squared distances, as a pro- The small-scale differentiation is highlighted by some
portion of the total sum of squares, and represents how remarkable juxtapositions of allele frequency differences
well the two-dimensional plot fits the original data. (e.g. populations 14 and 18 at all four loci, populations
Stress levels vary between zero and one, with stress 10 and 11 at the Gpi and G-3-pd loci; Table 2). The large
levels approaching zero as the fit improves. A Eucli- differences across the range of the species are empha-
dean-distance matrix was calculated by considering each sised by a fixed difference at the G-3-pd locus between
allele as a separate variable. This is analogous to using the two southern populations of G. alba (populations 22
the abundance of different species to calculate a distance and 23) and the two northern populations (populations
measure between sites with different plant or animal 1 and 2; Table 2). Interestingly, only population 20 has
assemblages. In this way, the frequency of occurrence of Gpi(d) (Table 2). This allele occurs in all populations of
each allele was incorporated into the distance measure. G. vitellina.
 Table 2. Allele frequencies for 23 populations of G. alba at the four variable loci, and mean heterozygosity (H) with standard errors
sw sw sw sw sw sw c c c c c c ne ne ne ne ne ne ne ne ne o o
Group
15 16 13 14 11 1 4 23 5 9 2 3 19 20 17 21 22 8 18 6 7 12 10
Pop.
340652 340854 340745 340752 340532 340735 340814 340830 340755 340838 340620 340632 340645 340440 340528 340405 340410 340328 340225 340035 340212 340940 341015
Lat.
1150620 1150538 1150548 1150735 1150738 1150855 1150818 1150910 1150940 1151110 1150912 1150940 1151118 1150950 1151100 1150405 1150950 1151010 1150930 1150705 1150819 1150858 1150852 g~
Long.
(N) 30 9 32 30 15 59 29 23 36 15 25 30 17 30 30 15 17 30 18 16 14 8 7
Lgg
b 0.43 0~50 0.48 0,22 0-03 0.10 0.74 0.89 0-49 0,37 0.52 0.68 1.00 1-00 1.00 1.00 1.00 1.00 1.00 1-00 t-00 0.25 0-21
d 0-57 0.50 0-52 0.78 0.97 0-90 0.26 0.11 0.51 0-63 0-48 0.32 0.00 0.00 0-00 0,00 0-00 0.00 0.00 0-00 0.00 0.75 0.79
G-3-pd
a 0.83 0.89 0-78 0.50 0.43 0.38 0.54 0.37 0.21 0.40 0.26 0.46 0.03 0.27 0.63 0.90 0.82 0.71 0.89 1.00 1.00 0.00 0.00
b 0.17 0.11 0-22 0.50 0.57 0.62 0.46 0.63 0.79 0.60 0.74 0-54 0.97 0.73 0,37 0.10 0.18 0.29 0.1t 0-00 0.00 t-00 1-00
Gpi
a 0.92 1.00 0-80 0,88 0.30 0.74 0.14 0,09 0.00 0.00 0-36 0.07 0-00 0.00 0-00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00
b 0.08 0.00 0.20 0.12 0-70 0.26 0,86 0.91 1.00 0.63 0.64 0.93 1.00 1.00 1-00 1.00 1.00 0.98 1.00 1.00 1.00 1.00 1.00 ff
d 0-00 0.00 0.00 0.00 0-00 0.00 0.00 0.00 0-00 0.37 0.00 0.00 0.00 0-00 0.00 0,00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 o~
Ldh- 2
a 0.00 0-00 0-02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0-00 0.00 0.00 0-00 0.00 0-00 0.00 0-00 0.00 0-00 0.00 0-00
b 0.42 0.83 0-75 0.85 0.77 0.93 0.55 0.50 0.99 0.93 1-00 0.98 1.00 1.00 1.00 1-00 1.00 1.00 1-00 1.00 1.00 0-06 0-93
c 0.58 0.17 0.23 0.15 0-23 0-07 0.45 0.50 0.01 0.07 0-00 0.02 0.00 0.00 0.00 0,00 0.00 0.00 0.00 0.00 0.00 0,94 0.07

H 0.071 0-042 0-074 0.065 0-058 0.058 0-093 0-090 0-061 0.100 0-068 0.062 0-004 0.029 0.021 0-013 0.022 0.015 0.014 0 0 0.023 0.018
SE 0.034 0.028 0.034 0.030 0.034 0.034 0.044 0.046 0.043 0.055 0.038 0.037 0.004 0-029 0.021 0.012 0.022 0.013 0.014 0 0 0.017 0-012

'Group' indicates the genetic cluster to which each population belongs (see Fig. 4): sw = south-western group; c = central group; ne = north-eastern group; o = outliers. 'Pop.' indicates population
number (see Fig. 2)
48 D. A. Driscoll

2.5 (Table 2): the range of allele frequencies in populations

o 22 from the central and south-western groups overlap sub-
2 stantially. Gpi(b) has a very low frequency in all of the
south-western populations, with the exception of popu-
1.5 • 23
lation 9 (Table 2). Lgg(d) occurs at a frequency of 0-5
• 12 or more in all of the south-western populations, but it
1
a 17 also has a frequency above 0.5 in two populations of the
e~ * 7 t, 19 central group (Table 2).
o 0.5
t, 20 • 9 The results of the chi-squared tests emphasise the
8 "1118 410
.e 0 extent of divergence within each of the genetic clusters
•4 • 14 (Table 3). There are significant (p < 0.001) differences in
•5 allele frequencies amongst the populations of each
-0.5 • 3,6 1116
1,2 group at each of the variable loci (Table 3).
-1
• 15
• 13
Geocrinia vitellina
-1.5 Four of the loci examined were polymorphic (Lgg, Lpp,
• 21
Gpi, Me-l), and three individuals were found to be het-
-2 i i - - i 1
erozygous at one of three other loci (Mdh, Lpp, Ldh-2;
-2 -1 0 1 2 Table 4). Ldh-l(a), Me-2(c), G-3-pd(b), Pgm(b),
Dimension 1 Pgd(b), Aat-l(b), Aat-2(b), ldh-l(d), Idh-2(b) were
fixed in all populations.
Fig. 4. Two-dimensional scaling of Euclidean distance for 23
G. alba populations. Population numbers are indicated G. vitellina exhibits a similar magnitude of substruc-
(cf. Fig. 2). Populations fall into loose clusters: 0 , north- turing to G. alba, with a weighted mean Fst of 0-302
eastern group; A, central group; i , south-western group; (SD = 0.133) over a maximum distance of only 4 km.
0 , 0 , outliers. Stress = 0.088. Two-dimensional scaling (Fig. 5) suggests that the
seven populations do not fall into distinct clusters.
However, the geographic position of each population is
Two-dimensional scaling (Fig. 4) suggests that popu- partially reflected in the genetic structure. The three
lations are not randomly distributed throughout the western populations, Wendy Road (W), Metacrinia
two-dimensional space, and that most populations fall Creek (M) and Hut Pool (H), fall towards one end of
into dispersed groups. Populations along the north- the two-dimensional space, and the remaining four
eastern edge of the species' range clump together at the populations fall towards the opposite end. This rela-
left of Fig. 4 (populations 1, 2, 3, 4, 5, 6, 7, 8, and 12). tionship is reflected in differences in allele frequencies
Populations 7 and 12 are separated from the other
north-eastern populations. However, the separation is Table 3. Chi-squared tests for homogeneity o f allele frequencies
driven by divergence at only one locus (G-3-pd, Table 2). within three groups of G. alba and two groups of G. vitellina
Populations 7 and 12 share the allelic characteristics populations
which define this group. None of the populations in the
Locus No. of alleles ;(2 d.f. p
north-eastern group have Lgg(d), whereas all other
populations do. In addition, the north-eastern popula- Central G. alba
tions are fixed for Gpi(b) and Ldh(b), with the excep- Lgg 2 34-3 5 < 0.001
G-3-pd 2 19.3 5 0-002
tion of one heterozygous frog at the Gpi locus in Gpi 2 47.3 5 < 0.001
population 4 (Table 2). The north-eastern populations Ldh-2 2 103.4 5 < 0.001
also tend to have very low heterozygosity (Table 2). In
South-western G. alba
contrast, the adjacent populations in the central part o f Lgg 2 52.2 5 < 0.001
the species' range have relatively high heterozygosity G-3-pd 2 55.8 5 < 0.001
(H > 0.05). Gpi 2 57.0 5 < 0.001
Populations 22 and 23 in the south are genetic outli- Ldh-2 2 64-2 5 < 0.001
ers (Fig. 4), due to the absence of variation at the G-3-pd North-eastern G. alba
and Gpi loci (Table 2). Population 22 is particularly G-3-pd 2 152.3 8 <0.001
divergent because it has an unusually high frequency of Eastern G. vitel/ina
Ldh(c) (Table 2). Ltp 2 10.0 3 0.019
The remaining populations could be divided into two Lgg 2 13,0 3 0.005
groups based on Fig. 4: central populations 10, 11, 17, Gpi 2 25.8 3 < 0-001
Me-1 2 24.2 3 <0-001
18, 19, 20, and south-western populations 9, 13, 14, 15,
16, 21 (cf. Figure 2). However, there are no consistent Western G. vitellina
allelic differences between these groups. The distribution
Ltp 2 26.4 2 <0.001
Gpi 3 40.3 4 < 0-001
of the G-3-pd and Ldh loci do not resemble this pattern
 Conservation genetics of two endangered frog species 49

1 DISCUSSION
G•
0.5 Migration rates
e- M•
N • Current rates of migration of both species must be
0 H" S• vanishingly small. The genetic differences throughout
W•
the ranges of G. alba and G. vitellina are very large,
-0.5 especially given the small distances between populations
C • (maxima of 18 km and 4 k m respectively). Taking the
-1 f r
sampling scale into account, the size of this differentia-
-2.5 -2 - 1.5 -1 - 0.5 0 0.5 1 1.5 tion is as large as for the most genetically subdivided of
D~nsion I plethodontid salamanders, a group renowned for the
Fig. 5. Two-dimensional scaling of Euclidean distance for magnitude of genetic differentiation (Larson et al.,
all G. vitellina populations. Population letters are indicated 1984). Assuming that natural selection has little influ-
(cf. Figure 3). Stress = 0.0008. ence over allele frequencies, these enormous genetic dif-
ferences suggest that current levels of gene flow are
approximately zero, since even one migrant per genera-
(Table 4). The four eastern populations have Lgg(a), tion can prevent fixation or near fixation of alternative
and three of the four have Me-l(b), while these two alleles (Slatkin, 1987; Slatkin and Barton, 1989). A pre-
alleles are absent from the western populations. In cise value for the rate of dispersal cannot be calculated
addition, the western populations have high frequencies (e.g. following Wright, 1931), due to the historical
of Gpi(a) relative to its frequency in the eastern pop- associations amongst populations (see discussion below)
ulations. However, significant variation within the (Larson et al., 1984; Whitlock, 1992). The conclusion
eastern and western populations at the four most-vari- that individuals of both species do not disperse far from
able loci (Table 3) has resulted in all seven populations their natal swamp is consistent with a m a r k - r e c a p t u r e
being broadly scattered in the two-dimensional plot study of G. alba and G. vitellina (Driscoll, 1997).
(Fig. 5). Driscoll (1997) found that 95% of adult male frogs were

Table 4. Allele frequencies for seven populations o f G. vitellina at seven variable loci. Mean heterozygosities with standard errors
are also shown. Sample sizes (N) were reduced in two populations at the Ltp and Me-I loci; other loci had the same sample size (shown
for Lgg). N = Spearwood Ck North, S = Spearwood Ck South, G = Geo Ck, C = Crinia Ck, H = Hut Pool, M = Metacrinia Ck,
W = Wendy Rd

Pop. N S G C H M W
Lat. 340405 340495 340450 340510 340522 340515 340455
Long. 1151845 1151850 1151810 1151858 1151730 1151718 1151615
Lgg
(N) 34 44 44 30 30 32 30
a 0.18 0.10 0.01 0.13 0.00 0.00 0.00
b 0.82 0.90 0-99 0.87 1.00 1.00 1.00
Ltp
(N) 34 39 44 30 30 22 30
a 0.37 0-56 0-41 0-58 0.63 0.71 1.00
b 0.63 0.44 0.59 0.42 0-37 0.30 0-00
Gpi
a 0.00 0.13 0.03 0.02 0.55 0.91 0.90
b 0.69 0.57 0-49 0-90 0-32 0.03 0.00
d 0.31 0.31 0.48 0-08 0-13 0.06 0-10
Me-1
(N) 28 44 44 20 30 32 30
b 0.20 0.08 0.30 0.00 0.00 0.00 0.00
c 0-80 0.92 0-71 1.00 1-00 1.00 1.00
Mdh
a 0.99 1.00 1.00 1.00 1.00 1-00 1.00
b 0-02 0-00 0.00 0.00 0-00 0.00 0.00
Lpp
a 0.00 0.00 0.00 0.00 0.00 0.00 0.02
b 1.00 1.00 1.00 1.00 1.00 1-00 0.98
Ldh-2
b 1-00 1-00 1.00 1.00 1.00 1.00 0.98
c 0.00 0.00 0.00 0.00 0.00 0-00 0.02

H 0.07 0.07 0.07 0.05 0.05 0.03 0-02

SE 0.03 0.04 0.04 0.03 0-03 0.02 0.01
50 D. A. Driscoll
displaced less than 20 m over one year, while the maxi-
mum displacement was 50 m.
Genetic structuring
Geocrinia alba
Slatkin and Arter (1991) pointed out that spatial genetic
patterns could be produced by gene flow, spatially
varying natural selection, or historic associations
amongst populations. These patterns can be obscured
by genetic drift and mutation (Slatkin and Arter, 1991).
I will argue that the third option, historic associations,
is the most likely explanation for genetic structuring in
G. alba.
Contemporary patterns of gene flow are inadequate
to explain the genetic structure. Significant differences in
allele frequencies within genetic groups (Table 3) sug-
gest that gene flow is not holding genetic groups toge-
ther. Migration rates are likely to be extremely small,
both within and between groups.
Natural selection is also unlikely to have a major
influence over the distribution of allele frequencies in G.
alba, given that the abrupt changes in allele frequencies
do not correspond with any changes in environmental
parameters. All populations fall within the same cli-
matic zone (Wardell-Johnson and Roberts, 1993); and,
although there are three different landforms within the
range of G. alba (Tille and Lantzke, 1990), they do not
correspond with the spatial pattern of genetic variation.
Although there is no evidence in support of the possible
influence of natural selection, it is impossible to rule out
the effects of a very subtle selection regime acting in
combination with low rates of dispersal (Slatkin, 1985)
or some past regime of natural selection.
When levels of both natural selection and gene flow
are low, any genetic structuring within a species may be
a consequence of the species' biogeographic history
(Arter, 1990). Thus it is likely that historic patterns of
dispersal, combined with genetic change, have led to the
existing genetic structure of G. alba.
The biogeographic scenario is most graphically por-
trayed by the north-eastern group. The genetic similari-
ties amongst the nine populations in the north-eastern
group of G. alba imply that the members of the group
originated from a common ancestral population. A
likely biogeographic scenario includes either: (1) range
contraction into a refuge population, followed by range
expansion; (2) long-distance dispersal, followed by
range expansion; or, (3) rapid colonisation and range
expansion from a more centrally located population.
Changes in geographic distribution may have been
driven by climatic fluctuations. Rainfall and tempera-
ture have fluctuated throughout history, on time-scales
from decades to millions of years (Sturman and Tapper,
1996). It is feasible that climatic fluctuations have driven
range changes in G. alba. However, since we do not
know what magnitude of climate change may affect the
distribution of Geocrinia, there is no basis for predicting
which historic climatic changes have allowed range
changes and subsequent divergence. It is also not possi-
ble to predict the timing of divergence using the allo-
zyme data. Thorpe (1982) noted that different loci can
diverge at different rates, and the rate of divergence can
vary through time, making it difficult to allocate a time
since divergence based on genetic distance. Electrophor-
esis also suffers from a 'saturation' effect because only a
maximum of one substitution per enzyme can be detected,
and there is a finite number of bands that can be resolved
on a gel. Measures of genetic distance derived from pro-
tein electrophoresis may therefore be non-linear (Thorpe,
1982). What is more, the rate of divergence through
genetic drift is inversely related to the effective popula-
tion size. Therefore local populations may diverge at
different rates depending on their population size and
the amount of genetic variation present to begin with
(Witter and Carr, 1988). Johnson (1989) argued that we
need to know the history of populations to determine if
divergence indicates time, or some other process. Johnson,
(1989) suggested that molecular clocks are likely to be
accurate to within 2 million years. This error could be
up to several orders of magnitude higher than the actual
time of divergence amongst G. alba populations. These
difficulties present insuperable barriers to placing a
time-scale on divergence between major genetic groups
within this species, using the current data set.
Driscoll (1996) argued that, for G. rosea, divergence
in geographic isolation was a more likely explanation of
genetic structure than was recent immigration or long-
distance dispersal. G. rosea has a similar genetic struc-
ture to G. alba, and it occurs in similar habitat and is
morphologically similar to G. alba (Wardell-Johnson
and Roberts, 1991, 1993). Therefore, historic range-
changes and divergence in isolation may also explain the
formation of the north-eastern group in G. alba.
The same evolutionary processes are likely to have
influenced divergence throughout the range of G. alba.
The central and south-western groups identified in the
two-dimensional scaling may therefore represent an
historic bifurcation within G. alba. However, inspection
of the allele frequencies shows that the boundaries of
these groups are poorly defined. Genetic drift may have
obscured any original patterns. Barendse (1990) and
Preziosi and Fairbairn, (1992) interpreted a lack of
geographic pattern in allozyme data as indicative of the
effects of genetic drift amongst isolated populations.
The two southern populations of G. alba (populations
22, 23) may be the remnants of a clade distinct from the
rest of the species. This group may have been charac-
terised by the lack of variation at the G-3-pd and Gpi
loci. However, with only two populations exhibiting
these characters, it is uncertain whether they have lost
the same alleles independently, or are phylogenetically
related. This contrasts with the north-eastern group,
where nine populations share the same characters,
making it improbable that drift alone would produce
such uniformity.
 Conservation genetics of two endangered frog species
Driscoll et al. (1994) reported that population 12 had
Gpi(d), an allele characteristic of G. vitellina, and sug-
gested there had been gene flow between the two species.
However, with our current understanding of their dis-
tribution and migration abilities, it seems impossible
that movement between G. alba and G. vitellina popu-
lations could occur under present conditions. Historic
changes in distribution of the two species must have
occurred for this genetic exchange to have taken place.
Alternatively, Gpi(d) in G. alba may not be homologous
with Gpi(d) in G. vitellina. The fact that this allele has
not migrated to nearby populations again attests to the
sedentary nature of this species.
Geocrinia vitellina
The amount of genetic variation partitioned amongst
populations of G. vitellina is at least as large as that
found in G. alba, considering that the Fst value of 0-3
occurred amongst seven populations over a distance of
4 km. Like G. alba, G. vitellina also shows genetic struc-
turing based on the geographic locations of popula-
tions. It is therefore possible that the same processes of
divergence in isolation and patterns of historic migra-
tion that operated in G. alba have also led to the
observed genetic patterns in G. vitellina.
However, it is possible that mutations may have
arisen within one eastern population and traversed the
very short distances between the other two creeks, fol-
lowed by diffusion throughout those populations. While
this alternative process may account for spatial genetic
structuring at this small scale, the fact that G. vitellina
still has substantial genetic structuring demonstrates
that diffusion of alleles cannot account for more wide-
spread structuring of the magnitude seen in G. alba, or
in G. rosea and G. lutea (Driscoll, 1996).
Do G. alba and G. vitellina exist as metalmpulations?
From the preceding discussion of genetic structure and
dispersal rates, it appears that there may be long-term
cycles of range changes overlying the short-term pattern
of relatively isolated populations. Therefore, neither
species is likely to exist as a metapopulation in the tra-
ditional sense, with frequent population turnover
(Levins, 1970). Migration rates between populations are
so low that any local extinctions are unlikely to be
countered in the short term by recolonization. Sarre
(1995) came to a similar conclusion for the gecko
Oedura reticulata. Separate populations exhibited dif-
ferences in the frequency of mitochondrial-DNA hap-
lotypes, indicating low levels of gene flow and a low
probability of an effective metapopulation (Sarre, 1995).
In addition, in Geocrinia, the formation of a classic
metapopulation is unlikely due to spatial correlation of
fluctuations in population size (Driscoll, 1996). Corre-
lated spatial fluctuations are inimical to the formation
of a stable equilibrium between colonisation and
extinction (Harrison and Quinn, 1989; Hanski and
Woiwood, 1993; Lahaye et al., 1994).
51
Although in the short term each population is auton-
omous, I argued above that, in the long term, there has
been a process of isolation and extinction followed by
migration and recolonisation. Therefore, G. alba and
G. vitellina could be considered to have non-equilibrium
metapopulations (Harrison, 1991). This model involves
long-term deficits or surpluses of colonisations com-
pared with extinctions, resulting in range changes over
time. I argued above that range changes could have
been driven by climatic fluctuations. Therefore, non-
equilibrium metapopulation dynamics could be deter-
mined by the expansion and contraction of suitable
habitat related to climatic oscillations. This model is
consistent with the contention of Thomas (1994) that
most metapopulations in nature are driven by changes
in the suitability of habitat, rather than by stochastic
extinction and re-colonisation of permanently suitable
sites. The metapopulation structure of the pool frog
Rana lessonae also supports a non-equilibrium meta-
population model that is driven by deterministic chan-
ges in the suitability of habitat (Sjrgren Gulve, 1994).
Implications for conservation
Conservation of genetic resources
Although there are firm theoretical grounds for arguing
that genetic variation is needed for the long-term survi-
val of a species (Frankel and Soulr, 1981; Allendorf and
Leafy, 1986; Lande and Barrowclough, 1987; Vrijenhoek,
1989, 1994), several authors have noted that species
with low levels of genetic variation are thriving (Men-
zies, 1991; May, 1994; Hitchings and Beebee, 1996).
Lacy (1987) and Avise (1994) noted the difficulty of
predicting the importance of loss of variation for any
particular species.
In view of the contrasting evidence regarding the
importance of genetic variation, the safest strategy is to
maintain as much genetic variation as possible in a state
that approximates the natural situation (Vrijenhoek,
1989; Woodruff, 1989). For Geocrinia, this will mean
ensuring the survival of the maximum number of popu-
lations, because so much of the variation is held as dif-
ferences between populations. The natural subdivision
of Geocrinia is fortuitous from the point of view of
conserving genetic diversity. Maintaining many small
populations is an effective way of preventing allelic loss
from the species as a whole. Different populations will
become fixed for different alleles through genetic drift,
thereby maintaining more of the variation in the long
term than could be held in a smaller number of large
populations (Simberloff, 1988; Henry et al., 1991). This
is true, providing that the small populations do not
become extinct, which would result in loss of unique
genetic variants (Templeton et al., 1990).
Conservation of Evolutionary Processes
The likely biogeographic history of G. alba and
G. vitellina (and G. rosea and G. lutea; DriscoU, 1996)
52 D. A. Driscoll
suggests that contractions and expansions of geographic
range may be a natural phenomenon, and that they play
an important role in the evolution of this group of spe-
cies. Therefore, to maintain the evolutionary processes
of G. alba and G. vitellina, range changes need to be
incorporated into management plans. While human-
induced causes of population decline should be identi-
fied and halted or reversed, managers will need to accept
natural local extinctions as an important part of the
ongoing evolution of these two species.
In addition, for range expansion to take effect, unoc-
cupied swamps need to be available, and there needs to
be suitable habitat between sites through which frogs
can migrate. It is certain that historic periods of frog
migration have taken place through natural vegetation,
since clearing for agriculture only began with the arrival
of Europeans to the area in the 1830s (McDermott,
1928). It is less certain whether migration will occur
over cleared paddocks. Oldham (1985) found that pas-
ture was a substantial barrier to dispersal in young
toads, Bufo bufo. Furthermore, range expansion may
depend on the expansion of breeding habitat. G. rosea is
known to breed away from creek lines in the wetter
parts of its range, where it uses rotting logs as burrow
sites (Main, 1965; Wardell-Johnson and Roberts, 1991;
personal observations). This behaviour suggests that
suitable breeding habitat may expand into natural
vegetation during wetter climatic periods, possibly con-
necting formerly discontinuous breeding sites. If native
vegetation has been cleared, then breeding habitat (rot-
ting logs) will no longer be available, preventing the
frog populations from colonising the landscape.
Given that natural recolonisation is unlikely in the
short term, reintroductions or population augmenta-
tion may be a necessary strategy in the face of any
human-induced declines or extinctions. Translocation
programmes need to be carefully planned because
translocation of conspecifics can alter natural patterns
of genetic variation (Leberg et al., 1994), and break up
genomes adapted to local conditions (Leary et al.,
1993). Given the interesting intraspecific evolutionary
patterns in G. alba and G. vitellina, any translocations
should avoid altering the existing genetic structure. The
source and site of release of frogs need to be as close as
possible, and the stocks should fall within the same
major genetic group. Cowie (1992) noted that the
extinction of Partula, a speciose group of terrestrial
snails, represented a lost opportunity to study specia-
tion on islands. Obscuring the evolutionary patterns
within Geocrinia species would represent a similar loss.
Maintaining the natural system differs from the
approach to conservation taken by Templeton et al.
(1990), Templeton (1991) and Vrijenhoek et al. (1985).
These authors recommended mixing individuals from
genetically different populations to reduce the effects of
inbreeding depression, and to enhance the ability of
populations to adapt to the local environment. The best
decision can only be made on a species-by-species basis.
The risk of losing important evolutionary features of a
species through mixing genomes needs to be weighed
against the risk of species extinction if those practices
are not implemented. It is not known whether popula-
tions of G. alba or G. vitellina suffer from inbreeding
depression, or from an inability to adapt because they
lack genetic variation. Most populations have levels of
heterozygosity that are typical of amphibians (Nevo,
1978), so it is unlikely that inbreeding depression is a
threat to either species. Given this, we can afford to
allow natural processes to continue operating, even if it
means that some populations suffer inbreeding.
Evolutionary history and continuing evolutionary
processes are important characteristics of a species.
Therefore, in the same way as we endeavour to conserve
the morphological and behavioural integrity of species,
we should also ensure that these less-obvious features
are maintained. It remains to be seen whether or not our
bureaucrats and politicians have the necessary foresight
and will to manage processes that occur on a time-scale
orders of magnitude longer than their own. However,
their willingness to do so will determine if we are able to
conserve species in their entirety, including their evolu-
tionary processes. If we cannot, and those natural
processes are terminated, then species in the wild will
be rendered impoverished shadows of their former
selves.
ACKNOWLEDGEMENTS
I am indebted to Dale Roberts and Grant Wardell-
Johnson for the practical support they provided during
the study, and for their valuable comments on earlier
drafts of this paper. Mike Johnson provided useful
advice regarding the analysis and interpretation of allo-
zyme data and his criticism of previous drafts of this
paper were appreciated. Other people who commented
on previous drafts include Penny Atkinson, Dorian
Moro, Jacqui Richards, Kelley Whitaker and the mem-
bers of the genetics discussion group. I thank Mike
Johnson, Michelle Stuckey and Caroline Fuery for gel
lessons. This project was funded by the Australian
Nature Conservation Agency through the Geocrinia
Recovery Team, the Department of Zoology, University
of Western Australia, and a Post Graduate Research
Award provided by the Australian Government. I also
thank the WA Department of Conservation and Land
Management and many private landholders for permits
and access to swamps.
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