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List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Rogell, B., Hofman, M., Eklund, M., Laurila, A. and Höglund J.
The interaction of multiple environmental stressors affects
adaptation to a novel habitat in the natterjack toad Bufo
calamita. (Accepted for publication in Journal of Evolutionary
Biology)
II
Rogell, B., Thörngren, H., Palm, S., Laurila, A. and Höglund J.
Genetic structure in peripheral populations of the natterjack
toad, Bufo calamita, as revealed by AFLP. (Submitted
manuscript)
III
Rogell, B., Eklund, M., Thörngren, H., Laurila, A. and Höglund
J. The effect of selection, drift and genetic variation on life
history trait divergence among insular populations of natterjack
toad, Bufo calamita. (Manuscript)
IV
Rogell, B., Thörngren, H., Laurila, A. and Höglund J. Fitness
costs associated with low genetic variation are reduced in a
harsher environment in amphibian island populations.
(Submitted manuscript)
V
Rogell, B., Berglund, A., Laurila, A, and Höglund J. Population
divergence of life history traits in the endangered green toad
Bufo viridis: implications for the support release program.
(Manuscript)
Cover illustration by Jonas Andersson
The following papers were written during the course of my doctoral studies
but are not a part of the present dissertation:
Rogell, B., Gyllenstrand, N. and Höglund J. 2005. Six polymorphic
microsatellite loci in the natterjack toad, Bufo calamita. Molecular Ecology
Notes, 5, 639-640.
Rudh, A., Rogell, B. and Höglund J. 2007. Non-gradual variation in the
strawberry poison frog Dendrobates pumilio: genetic and geographic
isolation suggests a role for selection in maintaining polymorphism.
Molecular Ecology, 16, 4284-4294.
Rudh, A., Rogell, B., Håstad, O. and Qvarnström A. Rapid population
divergence linked with co-variation between degree of aposematic coloration
and sexual display. (Submitted manuscript)
Richter-Boix, A., Teplitsky, C., Rogell, B. and Laurila A. Local selection
modifies phenotypic divergence among Rana temporaria populations in the
presence of gene flow. (Submitted manuscript)
Rogell, B., Laurila, A and Höglund J. Asymmetric interspecific
competition between endangered toad species. (Manuscript)
Contents
Introduction ..................................................................................................... 7
Fragmentation, population size and genetic diversity ................................ 7
Fitness and genetic diversity ...................................................................... 7
Inbreeding environment interactions .......................................................... 8
Adaptations and constraints of adaptability ............................................... 9
Adaptations ............................................................................................ 9
Adaptive constraints: genetic variation................................................ 10
Adaptive constraints: genetic correlations ........................................... 11
Life in peripheral regions ......................................................................... 11
Objectives of thesis .................................................................................. 12
Methods ........................................................................................................ 13
Study species and populations.................................................................. 13
Methodology ............................................................................................ 15
Paper I .................................................................................................. 15
Paper II ................................................................................................ 16
Paper III ............................................................................................... 17
Paper IV ............................................................................................... 18
Paper V ................................................................................................ 18
Results and discussion .................................................................................. 20
Adaptation to a peripheral habitat (paper I) ............................................. 20
Peripheral population structure (paper II) ................................................ 21
Adaptations within a peripheral habitat (paper III) .................................. 22
Environment dependent inbreeding depression (paper IV) ......................... 23
Local adaptations and support releases (paper V) .................................... 24
Conclusions ................................................................................................... 25
Sammanfattning på svenska .......................................................................... 27
Acknowledgements ....................................................................................... 32
References ..................................................................................................... 35
Introduction
Fragmentation, population size and genetic diversity
The current fragmentation of habitats due to increased human activities
decreases population sizes and increases the degree of isolation amongst
populations. Being one of the great conservation concerns of our time, the
consequences of inhabiting a fragmented landscape have received intense
interest from conservationists. A main feature of small and isolated
populations is that they are expected to lose genetic variability due to drift
(Gillespie 1998). The speed at which genetic diversity is being lost depends
mainly on two factors, the effective population size and the degree of
isolation to other populations (Falconer & Mackay 1996; Gillespie 1998),
leaving small and isolated populations with little genetic variation
(Frankham et al. 2002; Höglund 2009). Issues regarding fragmented habitat
are emphasised in organisms characterized by poor dispersal abilities
(Thomas 2000), such as amphibians (Beebee 2005). Indeed, previous
amphibian studies have found profound population structure and among
population variation in neutral genetic variation (Johansson et al. 2007;
Rowe et al. 2000; Rudh et al. 2007).
Fitness and genetic diversity
In small and isolated populations, genetic drift is likely to unveil the effects
of genetic load. Similarly, these populations may foster inbreeding via
mating of related individuals. Since both drift and inbreeding increase
homozygosity in the population, they are frequently associated with fitness
costs i.e. inbreeding depression (Keller & Waller 2002). Indeed, several
studies have shown that inbreeding depression, caused by increased
expression of recessive deleterious alleles due to lack of genetic variation
within individuals or populations, affects fitness both in natural and
experimental populations (Allendorf and Luikart 2007; Frankham et al.
2002; Höglund 2009; Keller & Waller 2002). The link between loss of
fitness and loss of neutral genetic diversity is well-established (Spielman et
al. 2004). Today it is widely accepted that the preservation of genetic
diversity is crucial for the long-term biodiversity conservation efforts
(Frankham et al. 2002; Höglund 2009). Thus, conservational strategies
7
should, when possible, take genetic aspects into consideration, especially if
the population is likely to be divided into subpopulations with low migration
rates between them. Amphibians are arguably the most threatened group of
organisms today with a third of the known species being classified as
endangered (Stuart et al. 2004). The causes of the still ongoing declines are
in many cases diseases and habitat destruction (Stuart et al. 2004).
Amphibians generally have low dispersal capacity and thus a well defined
population structure (Beebee 2005). Previous studies found inbreeding
depression in the natterjack toad (Rowe and Beebee 2003) and in two
European Rana species (Ficetola et al. 2007; Johansson et al. 2007).
Inbreeding environment interactions
The fitness losses associated with increased homozygosity cannot be
assumed to be constant across environments (Armbruster & Reed 2005). For
example, several studies have shown that inbreeding depression may interact
with environmental conditions (Armbruster & Reed 2005). A meta-analysis
examining the expression of inbreeding depression under stressful and
benign conditions found that inbreeding depression increased under stressful
conditions in 76 % of the 34 studies (Armbruster & Reed 2005).
Emphasizing the importance of these findings, a simulation study found that
a positive interaction between inbreeding depression and environmental
stress would decrease the persistence time of a population with 17.5 - 28.5 %
(Liao & Reed 2009). Inbreeding depression is rather frequently examined
under benign laboratory conditions and thus the effects of inbreeding
depression may be underestimated. In order to estimate persistence times of
small and endangered populations, it is important to estimate the effect of
inbreeding depression. The risk of underestimating inbreeding depression
under natural conditions is thus clearly a concern for conservation biologists.
However, many studies which have attempted to explore environmental
effects on inbreeding depression have used biologically irrelevant stressors
such as rare chemicals. Moreover, some studies have shown that inbreeding
depression is reduced under stressful conditions, making general conclusions
difficult (Armbruster & Reed 2005; Chen 1993; Pray et al. 1994; Waller et
al. 2008). The relevant issue of how inbreeding depression is expressed
under natural, or stressful, conditions remains thus largely unresolved.
8
Adaptations and constraints of adaptability
Adaptations
There is abundant evidence that optimal trait values of life history traits
change with environmental variation, with adaptation to local conditions
reported in a large number of organisms (Conover & Schultz 1995; Huey et
al. 2000; Roff 2001). However, the relative importance of natural selection
versus neutral genetic drift as a source of evolutionary change is still debated
in evolutionary biology. In order to disentangle the relative contributions of
genetic drift and selection to among population divergence, local adaptations
are frequently studied through the comparison of population differentiation
indexes for quantitative traits (QST) and neutral genetic markers (FST). A
discrepancy between the two indices in this context is interpreted as proof
for natural selection (Merilä & Crnokrak 2001). Previous meta-studies of
studies based on FST - QST comparisons (Leinonen et al. 2008; Merilä &
Crnokrak 2001) have reported two major patterns. First, QST tends to exceed
FST, suggesting a prominent role for divergent selection between populations.
Secondly, QST and FST tend to be correlated suggesting that both neutral
markers and quantitative traits may be affected by drift, but or alternatively
that there are spatial autocorrelations between both genetic divergences and
the environmental parameters to which adaptations are likely to occur.
Moreover, since natural environments are inherently variable in time and
space, predictions of how organisms respond to novel environmental
conditions are of central interest for the evolutionary and ecological sciences.
Organisms facing environmental change may respond in two ways: by
migrating to new areas or by adapting to the new conditions. A population
where the organisms are constrained in their responses will go extinct if the
environmental change exceeds their tolerance limits. It is clear that all species
do not harbor the adaptive potential to cope with rapid environmental changes
within their native range (Hoffmann et al. 2003; McCarty 2001), and
evidence shows that, for example, climate change is frequently associated
with changes in species distributions (Davis et al. 2005; McCarty 2001;
Stewart & Lister 2001). Thus, even after migration, species will face novel
environments and will need to adapt. However, migration to new areas is
often associated with the entrance into novel habitats where the selective
regimes are likely to differ from that the organisms have experienced before
(Davis et al. 2005; Phillips et al. 2006). Hence, one of the most crucial
questions for understanding the organismal responses to environmental
change is: what limits the adaptive potential of a species?
9
Adaptive constraints: genetic variation
Both theoretical (Kimura 1957) and empirical (Heuch 1980) studies have
predicted that effective population size affects the effectiveness of an
evolutionary response to a selective pressure, thus affecting the relative
importance of drift and selection. Though poorly understood, the interactions
between drift, selection and population size remains of crucial importance
for conservation biology as it questions the future of species at the brink of
extinction. Population size has a direct effect on adaptability as the
importance of stochastic sampling errors, such as genetic drift, increases
with smaller sample size. Genetic drift thus becomes the predominant
evolutionary force in small populations at the cost of non-random processes
such as natural selection (Kimura 1957). However, besides having a direct
effect on adaptability, small population size also influences adaptive
potential through secondary genetic effects. There are three main reasons for
this change; first, increased genetic drift will mediate the loss of potentially
advantageous alleles and thus delimit the plausible ranges beyond which
evolutionary potential is constrained (Lande 1995). Secondly, the potential
for evolutionary change is commonly estimated as the proportion of the total
phenotypic variance that is due to additive genetic variance, i.e. heritability
(Falconer & Mackay 1996). Heritability is dependent on additive genetic
variation, environmental variation and variation due to genetic/allelic
interactions. Strong drift may induce inbreeding depression through the
fixation of deleterious alleles which in turn may inflate environmental
variance through an increase in developmental instability (Levin 1970;
Sheldon et al. 1964; Whitlock & Fowler 1999), resulting in a constrained
heritability. Finally, a selective response may be delimited due to inbreeding
depression being more pronounced under the stressful conditions to which
adaptation be most beneficial (Armbruster & Reed 2005; Willi et al. 2006).
However, drift may also increase additive genetic variance and thus inflate
heritability due to the loss of dominance and/or epistatic effects and the
modification of covariances amongst epistatic, dominance and additive
variances (reviewed in Van Buskirk & Willi 2006). Theoretically, the strong
dominance and epistatic effects needed to yield increased additive genetic
variance during strong drift are most likely to be found in traits that are
strongly associated with fitness, for example life history traits (Crnokrak &
Roff 1995). These expectations were confirmed by Van Bushkirk and Willi
(2006) who found in a meta-analysis that while non-fitness traits lost
additive genetic variance linearly with the inbreeding coefficient, fitness
related traits generally followed a hump shaped distribution. It has been
argued that the increased additive genetic variance may facilitate an adaptive
response (Bryant et al. 1986; Goodnight 1987). However, as some of the
mechanisms yielding increased additive genetic variance during strong drift
10
are likely to be associated with increased expression of deleterious alleles
and the decay of co-adapted alleles, the increased additive genetic variances
may be of limited importance for adaptive processes (Barton & Turelli 2004;
Bryant et al. 1986; Van Buskirk & Willi 2006; Willi et al. 2006; Zhang et al.
2004).
Adaptive constraints: genetic correlations
Despite standing genetic variation for a trait, the evolutionary response to a
selective pressure may be constrained by genetic correlations between the
selected traits if the traits are selected antagonistically with respect to their
correlation, for example if selection favors high values of two traits that are
negatively genetically correlated (Björklund 1996; Cheverud 1984; Etterson
& Shaw 2001; Lande 1979, 1980). In nature, organisms are likely to
experience multiple selective pressures and since selection occurs on
individual phenotypes, the evolutionary responses of different traits are nonindependent. This is emphasized by the fact that life history traits in general
have complex developmental interactions and that pleiotropy (i.e. a single
gene affects multiple traits) is frequent (Rose 1983). Since genes producing
genetic correlations consistent with selection are subjected to congruent
selection from both traits, they should quickly become fixed. Thus genetic
correlations due to antagonistic pleiotropy should be common in nature
(Rose 1983). Consequently, at evolutionary equilibrium, genetic variation
may be retained by antagonistic pleiotropy producing trade-offs between
traits (Rose 1983). Hence, studies of single traits and selective factors may
severely bias the estimates of adaptations and adaptive potential (Björklund
1996; Chenoweth & Blows 2008; Etterson & Shaw 2001; Lofsvold 1988;
Via & Lande 1985). Although the complexity of natural systems makes it
difficult to identify the environmental stressors constituting selective
pressures in a population, divergence between populations or species and
their quantitative genetic properties can be used to reconstruct historical
selective pressures (Lande 1976, 1979). Using such methods, previous
studies have shown that selection on complex phenotypes frequently occurs
antagonistically with the genetic correlations (Merilä & Björklund 2004).
Life in peripheral regions
Geographical patterns of genetic variability do not only depend on
fragmented habitats but also on historical colonisation patterns where gradual
dispersal combined with selection to novel environmental conditions have
decreased genetic variability. For example, northern taxa frequently exhibit
patterns of diminishing genetic variability with increasing distance from their
glacial refuge. From a conservation viewpoint, it is important to distinguish
11
between genetic impoverishment due to recent fragmentation/bottlenecks and
genetic impoverishment due to historic colonisation patterns. As the rate of
inbreeding is likely to affect the effectiveness of selection against deleterious
alleles negatively, purging of these alleles is expected to occur during slow
colonisations, but not in recent fragmentation (Swindell & Bouzat 2006b). In
a study by Ficetola et al. (2007) fitness in Rana latasti was explained by loss
of genetic variability due to recent fragmentation but not by postglacial
colonisation effects on genetic variability. However, it is likely that these
factors will interact, as many species are less abundant in areas close to the
physiological limit of their distribution area due to the less frequent
occurrence of suitable habitats (Hoffmann & Blows 1994). The conservation
of fringe populations may thus be more dependent on factors resulting from
fragmentation.
Objectives of thesis
The objective of my thesis was to examine selection, genetic drift and their
consequences on fitness in naturally fragmented amphibian populations.
These issues may be difficult to study in natural populations, and natural
fragmentation may provide the possibility to study long-term effects of
fragmentation in natural populations. Moreover, since the natterjack toad
inhabits two very different habitats in Sweden, one typical and one highly
specific (see the description of study species) the Swedish populations of the
natterjack toad (Bufo calamita) provide an excellent opportunity for studying
adaptation to multiple environmental stressors. My objective was to:
1. Investigate the extent and constraints of local adaptation in larval
life histories in the natterjack toad populations of Skåne and
Bohuslän in a common garden experiment.
2. Examine neutral genetic population structure within the Bohuslän
archipelago.
3. Examine the relative influences of genetic drift and selection of
larval life history traits within the Bohuslän archipelago.
4. Examine inbreeding depression and the environmental dependency
of inbreeding depression in natterjack toad populations in the
Bohuslän archipelago.
5. Examine local adaptations in green toad Bufo viridis populations in
southern Scandinavia and how putative local adaptations may
affect the success of a support release project.
12
Methods
Study species and populations
I examined northern fringe populations of two toad species, the natterjack
toad (Bufo calamita, Fig. 1) and the green toad (Bufo viridis, Fig. 2). The
natterjack toad is endemic to Europe and is distributed in Western Europe,
ranging from Portugal to Great Britain and northwards to southern
Scandinavia and east to the Baltic countries (Beebee 1983). In Europe, the
green toad has a mostly eastern distribution, stretching from southern
Scandinavia in the north to Germany in the west, Italy in the south and
eastwards to central Asia. It also occurs in North Africa (Arnold & Ovenden
1978). Both the natterjack toad and the green toad live on the northern fringe
of their distribution area in Sweden and have become red listed (Gärdenfors
2005). Here the natterjack toad occurs in two disjunct distribution areas: in
southernmost Sweden (henceforth Skåne) and on the west coast (henceforth
Bohuslän, both areas in Fig. 3). These two areas differ in important breeding
pond characteristics (Andrén & Nilson 1985). In general, the toads breed in
sparsely vegetated, shallow temporary ponds throughout their northern range
(Beebee 1983), and while the natterjack toads in Skåne occur in this general
habitat (Fig 4), the toads in Bohuslän inhabit an atypical habitat on off-shore
islands (Fig 5). As compared to the general habitat in Skåne, the breeding
ponds in Bohuslän are generally smaller, shallower, more temporary and a
large proportion of the ponds are saline (Andrén & Nilson 1984, 1985). A
large scale phylogeographic study of the natterjack toad showed that the
Swedish west coast population clustered together with other northern
populations inhabiting the species’ general habitat (Rowe et al. 2006). This
suggests that the general habitat is the ancestral habitat from which toads
dispersed into the specific conditions in Bohuslän. The Swedish green toad
population inhabits a similar habitat to the natterjack toad in Skåne, and the
two species are often sympatric here. However, the Swedish green toad
population is restricted to the far south and is absent in the Bohuslän
archipelago. Though the poor osmoregulatory capacity of amphibian larvae
generally makes them inapt to saline conditions (Boutilier et al. 1992), the
natterjack toad is one of the few anurans that may breed in, and locally adapt
to, brackish environments (Andrén & Nilson 1984; Beebee 1983; GomezMestre & Tejedo 2003, 2004). The green toad is, as the natterjack toad, also
found in estuarine habitats and though local adaptations to salinity have not
13
been examined in the green toad, it is known to have a better salinity
tolerance than the natterjack toad (Wells 2007).
Fig. 3. Map over the sampled natterjack toad populations in paper I. Bohuslän: 1
Buskär, 2 Fågelskär, 3 Måseskär, 4 Oxskär, 5 Altarholmen. Skåne: 6 Järavallen, 7
Falsterbo, 8 Vårhallarna, 9 Ravlunda, 10 Åhus. The green toad only inhabits coastal
regions in the far south of Sweden, i.e. Skåne.
14
Fig. 1 Natterjack toad (Bufo calamita)
Fig. 4 Skåne breeding pond
Fig. 2 Green toad (Bufo viridis)
Fig. 5 Bohuslän breeding pond
Methodology
Paper I
Due to the major differences between their habitats in Sweden, northern fringe
populations of the natterjack toad (Bufo calamita) provide an excellent
opportunity for studying adaptation to multiple environmental stressors.
In this paper, I investigated the extent and constraints of local adaptation in
larval life histories in the natterjack toad populations of Skåne and Bohuslän
in a common garden experiment using two temperature (20 and 27°C) and
three salinity treatments (0, 3.6 and 6.2‰). Based on my characterization of
the larval habitat in terms of pond size and salinity in the two areas I
predicted that the Bohuslän populations should be locally adapted to higher
pond desiccation risk and salinity. Although many amphibians show plastic
responses to desiccation risk in terms of shortened development time (e.g.
Newman 1988; Denver 1997; Laurila et al. 2002), comparative studies have
shown that adaptive variation to desiccation risk among (e.g., Gomez-Mestre
& Buchholtz 2006) and within (Lind & Johansson 2007) species is found in
the constitutive (i.e. mean) development times rather than in their plasticity.
Consequently, I chose to measure the mean larval period in different
15
environments instead of the plastic response to desiccation. Also, anuran
larvae exhibit high plasticity to temperature (McDiarmid & Altig 1999) and
amphibian studies, as well as studies on other species, have shown that
reaction norms to environmental stressors may differ between biologically
relevant temperatures (Broomhall 2004; Crain et al. 2008). Consequently, I
included temperature treatments in my experiment even though temperature
differences between the areas are relatively minor (Andrén & Nilson 1985).
Finally, since anurans in general perform poorly in saline conditions, I
expected the plastic responses to saline conditions to be non-adaptive. Nonadaptive plastic responses (here used as defined by Ghalambor et al. 2007, a
plastic response that moves trait values away from a favored optimum) are
frequently found to be costly, and selection may thus favor more stress
tolerant genotypes, for example genotypes with low intrinsic growth rates
(Arendt 1997 and references therein). I investigated these potential
constraints of adaptation by calculating genetic correlations between trait
means and their non-adaptive plastic responses to salinity, as well as
between putatively adaptive trait values and survival in saline treatments.
Paper II
In this paper I examined neutral genetic structure in the Bohuslän natterjack
toad population. When assessing population structure and levels of genetic
variation it is important to choose a genetic marker system that works on an
appropriate spatial and temporal scale. In general, studies where the
populations have been isolated for shorter periods of time require more
polymorphic markers than do studies on a larger time scale. For example
mitochondrial DNA may be an appropriate choice when assessing large
scale phylogeographic patterns (e.g. Avise 2000), while more polymorphic
loci such as microsatellites can be a better choice on a smaller scale.
Quantifications of fine-scaled genetic structure are often limited by lack of
statistical power (Pemberton 2004), and the possibilities of assessing
population-wide levels of genetic variation within and among peripheral
populations, characterized by low levels of genetic variation, may be
particularly restricted (Zeisset & Beebee 2001). Indeed, my previous
attempts to quantify genetic variation within and among the Bohuslän
natterjack populations using microsatellite markers were impeded by lack of
genetic variation yielding low statistical power, emphasising the need to
develop another methodology suitable for assessing genetic variation in
these genetically impoverished populations.
Here I examined the genetic population structure and molecular genetic
variation in a subset of the Bohuslän natterjack toad populations using
Amplified Fragment Length Polymorphism (AFLP). Although dominant
16
genotypic data is generated, a large number of polymorphic markers can be
derived with this marker. Compared to co-dominant marker systems such as
microsatellites, which typically are studied using fewer but more informative
loci, AFLP may often provide equally robust estimates of genetic variation
when migration rates are low (Mariette et al. 2002). In fact, this technique
has been proven to have statistical power enough to pick up even fine-scale
differentiation (Bensch & Åkesson 2005), and inferences of population
structure derived from co-dominant and dominant markers have generally
been found to be congruent (reviewed in Nybom 2004). The aims of this
study were to asses the population structure and relative amounts of genetic
variation at a subpopulation scale in the Bohuslän natterjack toad. Due to the
nature of the habitat these populations inhabit, I expected to find a distinct
population structure over a relatively small spatial scale.
Paper III
The two main forces behind microevolutionary change are natural selection
and genetic drift. However, in addition to drift and selection, quantitative trait
values can also be affected by inbreeding depression (Lynch et al. 1999), a
factor often ignored in studies of population divergence. The Swedish west
coast populations of natterjack toad (Bufo calamita) provide an excellent
system to investigate local adaptation among genetically impoverished
populations. In this area, the toads inhabit approximately 30 small rocky
offshore islands and the populations inhabiting the islands differ in their
amount of genetic variation. The breeding ponds in the west coast are atypical
for the natterjack toad and consist of shallow rock-pools which run a high
desiccation risk and exhibit high variance in salinity (Andrén & Nilson 1984,
1985; Rogell et al. submitted). My previous studies found that high
desiccation risk in the breeding ponds has selected for shorter larval period
and higher growth rate in natterjack tadpoles (Rogell et al. submitted). In this
paper, I examined the relative contributions of drift, selection and neutral
genetic variation on life history trait divergence amongst insular populations
of the natterjack toad. This was done by using QST - FST comparisons of traits
known to exhibit local adaptations in anurans. In order to examine if
population wise trait values were associated with environmental properties or
population wise genetic variation, population-wise trait values were
correlated to environmental quantifications o selective importance and
population- wise neutral genetic variation. Desiccation of breeding ponds is a
strong selection pressure in larval natterjack toads within the Bohuslän area
(Andrén & Nilson 1984; Rogell et al. submitted). In order to quantify the
desiccation risk of individual rock-pools, I estimated water volume of the
pool. This is a reliable predictor of desiccation risk, with smaller ponds
having a higher probability for complete desiccation (Altermatt et al. 2009).
17
Paper IV
In this paper, I studied inbreeding depression in Bohuslän natterjack toads in
a factorial common garden experiment using six populations with different
amounts of genetic variation and three biologically relevant temperature
treatments. The islands are located relatively close to each other with pairwise distance between populations ranging between 0.5 and 40 km. I
predicted that the populations with lower genetic variation have lower
survival and that the fitness costs associated with low genetic variation are
more pronounced in the more stressful treatments. I used a factorial design
with three temperature treatments, one with a variable temperature (outdoors)
and two constant temperature treatments (27 ± 0.5 °C and 19 ± 0.5 °C). There
were six tadpoles from each family in each treatment. The temperature
treatments were chosen to resemble natural breeding-pond conditions. The
cold temperature treatment (19 °C) is close to the mean temperature in natural
breeding ponds in Sweden (mean temperature in breeding ponds in southern
Sweden, an area with similar temperature conditions is 19.3°C; (Andrén &
Nilson 1985). The warm temperature treatment (27 °C) mimics the warm
conditions during a dry spell when many of the breeding ponds are likely to
desiccate (Beebee 1983). Desiccation of ponds is a major selection pressure
in the present populations (Andren and Nilsson 1984; B. Rogell, unpublished
data). The outdoor treatment was arranged in a sunny location in a fenced
area within the University campus in order to capture the variable thermal
conditions experienced by the toads in their natural breeding ponds. Variable
thermal conditions can be more stressful for anurans than stable conditions
(Niehaus et al. 2006). The cold treatment was located in a shelf system in a
climate-controlled room. The warm and the outdoor treatments were both
arranged in six large tanks (1.1 × 1.1m filled to water depth of 15 cm, water
volume 180 l). In the warm treatment the tanks were placed in a laboratory
room (19 °C) and equipped with aquarium heaters to keep the temperature at
the desired level. The water volume in the large tanks was similar to that in a
typical natterjack breeding pond in Bohuslän populations (median pond
volume 250 l, B. Rogell et al. unpublished data) and, in the outdoor treatment,
prevented unrealistically high temperature fluctuations in the tadpole vials.
Ambient air temperatures during the experiment were obtained from the
Institute of Geosciences situated 500 m from the experimental setup.
Paper V
The aim of this study was to investigate possible implications of local
adaptations relevant for the Swedish conservation program for the green toad
(Bufo viridis) in Sweden. This was done through quantification of two
environmental parameters of known selective importance to amphibian larva
water temperature and salinity. Salinity and temperature were chosen due to
18
the explicit ad hoc assumptions that these are likely to differ amongst my
study populations. I further examined larval life history traits, of known
selection importance, and their responses to different thermal and saline
treatments. Temperature affects metabolism and thus the rate of
development, with colder temperatures leading to lowered metabolism and
slower development. To compensate for the cooler environment tadpoles
from cool habitat generally compensate by growing faster than conspecifics
from warm areas when reared in a common environment (Arendt 1997;
Conover & Schultz 1995). Anurans generally perform poorly in saline
conditions (Boutilier et al. 1992), however, the green toad is one of the few
anurans known to inhabit estuarine habitats (Duellman & Trueb 1994; Wells
2007). Additionally, both temperature and salinity are selective pressures
known to yield local adaptations in anurans, for example the closely related
natterjack toad (Bufo calamita) has been shown to locally adapt to saline
habitats (Gomez-Mestre & Tejedo 2003, 2004).
If my study populations are locally adapted to their native environment, I
expected tadpoles from cold localities to have a higher growth rate than
tadpoles from warmer localities. Similarly I expect tadpoles from saline
environments to have a higher salt tolerance in terms of survival and less
change in reaction norms to saline conditions.
19
Results and discussion
Adaptation to a peripheral habitat (paper I)
In accordance with my hypothesis that the Bohuslän toads have adapted to
the high desiccation risk in their breeding ponds, they exhibited a shorter
larval period and a faster growth rate as compared to their conspecifics in
Skåne. This pattern is expected in time constrained populations and is likely
to be selected against in less time-constrained environments (Laurila et al.
2008; Lind & Johansson 2007 and references therein). As expected, salinity
imposed a considerable environmental stress to the larvae. This was most
apparent in survival, which was lower in the saline treatments, but there was
also a slight prolonging effect on the larval period in the high saline
treatment. Prolongation of the larval period is a stress related symptom in the
natterjack toad larvae (Gomez-Mestre et al. 2004; Griffiths 1991; Griffiths et
al. 1991). However, despite inhabiting a more saline environment, the
Bohuslän toads showed signs of stronger environmental stress with
increasing salinity as indicated by several lines of evidence. First, Bohuslän
toads had a steeper decline in survival as salinity increased. Second, they
grew more slowly with increasing salinity than toads from Skåne. Third,
Bohuslän toads tended to have a disproportionally longer larval period in the
6.2‰ saline treatments, as indicated by the nearly significant area × salinity
interaction. Finally, with increased salinity levels, Skåne toads
metamorphosed at a disproportionally larger size than Bohuslän toads. While
the mechanistic explanation for these results is currently unclear, three lines
of evidence suggest that salinity tolerance trades off with growth and
development rate in natterjack toads. First, we found positive correlations
between mass at metamorphosis and survival in both salinity treatments in
Bohuslän, where families with the largest larvae in freshwater had higher
survival in saline conditions. This pattern was not present in Skåne toads.
Second, in Bohuslän the families with the longest larval period in the high
salinity treatment also had the highest survival, suggesting that families with
high salinity tolerance (and slow development) can be selected against in
desiccating ponds. Third, in both areas, growth and development rates were
more negatively affected by saline conditions in the fast-growing and –
developing families than in the slow growing families
To conclude, my results suggest that colonization of the novel rock-pool
habitat by the natterjack toad has involved evolution of faster growth and
20
development rates due to the increased desiccation risk of the habitat.
However, although they inhabit a more saline habitat, the Bohuslän toads
had a poorer performance in the saline treatments. Interestingly, I found
indirect evidence for a trade-off between salinity tolerance and development
and growth rates as indicated by the higher survival of large and slowly
developing toads in the saline treatments as well as the negative genetic
correlations between life history trait (especially growth rate) means and the
non-adaptive plastic effect of salinity on the trait. Adaptive potential is
crucial for a population’s persistence in the face of environmental change
(Davis et al. 2005). Measuring the selective regime in a novel or changing
environment is difficult, and future studies on microevolutionary change
should consider the role of multiple environmental stressors and the nonindependence of their evolutionary responses.
Peripheral population structure (paper II)
We found a well defined genetic structure among the local natterjack toad
populations in the archipelago of Bohuslän with intermediate to high
pairwise PT values and a global FST of 0.157. Though the structure analysis
was not congruent between the K and the ln(K) estimators used to assess
the number of independent genetic clusters, the increase of standard
deviation of ln(K) at K = 8 supports the idea that each of the seven sampled
islands represent a local population (cf. Evanno et al. 2005).
In addition, we found no isolation by distance which, combined with the
distinct population structure, implies that even a short distance over the sea
acts as an impenetrable dispersal barrier and emphasises the importance of
genetic drift over gene flow in this system (cf. Hutchison & Templeton
1999). My analysis instead suggests a hierarchical structure, with the islands
FÅ and MÅ constituting one "genetic group", while the rest of the
populations constitute another. The reason behind this pattern is unclear, but
we note that FÅ and MÅ are closely located (ca. 1 km), and it is possible
that their relative genetic similarity could be explained by some recent
(otherwise unusual) immigration or colonisation event.
My results are in line with a previous study showing that the natterjack toad,
despite its (for amphibians) rare capacity to inhabit estuarine habitats
(Beebee 1983; Gomez-Mestre & Tejedo 2003), is a poor disperser over sea
(Rowe et al. 2000). In contrast, a previous allozyme study on the common
toad B. bufo in a similar skerry habitat, but in the less saline Baltic sea,
reported a much lower FST value of 0.019 (Seppä & Laurila 1999),
emphasising the importance of saline ocean as a dispersal barrier for
anurans. We also found strong differences in genetic variation among the
21
populations, which together with the variation in pair-wise PT values
suggests large variation in local effective population sizes and (or) different
demographic histories such as founder effects during colonisation. It is
widely accepted that low genetic variation has negative effects on the
viability of a population and is expected to limit its adaptability (Lande
1995; Frankham et al. 2002). Hence, differences in genetic variation should
be taken into account when planning conservation strategies for the Swedish
natterjack toad populations. For example, support releases could be
considered if genetically impoverished populations decline in size (Madsen
et al. 1999). In the case of Bohuslän natterjacks, local populations with high
levels of genetic variation within the same archipelago would be the most
immediate choice for source populations.
Additionally, since extinction risk may increase due to interactions between
inbreeding depression and environmental stress (Liao & Reed 2009), a more
complete assessment of threats to the Bohuslän natterjack populations is
needed, including both genetic and ecological information. For example,
complementary studies focusing on estimation of current local effective
population sizes from genetic and demographic data would be informative
for the future conservation of these populations (e.g. Jorde & Ryman 1995).
Finally, conventional population genetic markers such as microsatellites may
be impeded by low statistical power when assessing genetic structure and
variation in systems of populations characterised by generally low genetic
variation. Although AFLP is a method with certain limitations (yielding
dominant), my results show that the use of AFLP may nevertheless facilitate
population genetic studies of peripheral populations.
Adaptations within a peripheral habitat (paper III)
We found a low degree of population differentiation between quantitative
traits in populations which were relatively strongly diverged in neutral
genetic markers. All QST estimates were smaller or of the same magnitude as
FST, and none of them were significantly different from zero. As QST < FST
indicates uniform selection, and QST FST indicates that selection cannot be
distinguished from genetic drift, my results suggest that either drift or
uniform selection explain the divergence in quantitative traits. Larval life
history traits are often under selection in amphibians (Altwegg & Reyer
2003; Gomez-Mestre & Buchholz 2006; McDiarmid & Altig 1999; Palo et
al. 2003), and, in the present case, these traits are locally adapted to the
archipelago environment (Rogell et al. submitted). It thus seems unlikely
that these traits would be selectively neutral. Additionally, although both
temperature treatment and population origin explained a significant portion
of the total phenotypic variation, there was remarkably little variation among
22
the populations and very few population × treatment interactions. This is
especially the case if we compare the present study to other population-level
studies on the same traits in anuran larvae. While the high QST values
reported in literature suggest a predominant role for divergent natural
selection, this pattern may partly be due to publication bias (Leinonen et al.
2008; Whitlock 2008). I argue that the results of this study suggest that
uniform selection is likely to account for the small among-population
divergence in larval life history traits of the natterjack toad, hence
contradicting the majority of previous studies. This is further emphasized by
the fact that these traits are commonly affected by divergent selection among
anuran populations, even at small spatial scales (Johansson et al. 2007; Lind
& Johansson 2007; Marangoni & Tejedo 2008). Additionally, our results
suggest that inbreeding depression or low adaptive potential due to drift have
played a role in shaping the divergence pattern among the local populations,
and potentially increased the variation among the populations.
Environment dependent inbreeding depression (paper IV)
I found lower survival in the outdoor treatment, which was the most natural
thermal environment as compared to the two stable temperature treatments,
suggesting that the more natural thermal conditions constitute a more
stressful environment for tadpoles. Since inbreeding depression is frequently
found to increase with environmental stress (Armbruster & Reed 2005), our
original prediction was that fitness costs associated with low genetic
variation are stronger in the most stressful treatment. Contrary to the
expectation, I found that fitness costs associated with low genetic variation
were more pronounced in the benign (cold and warm temperature
treatments) environments. I did not find significant of genetic variation in
the warm treatment, however, this may result from the limited statistical
power in that treatment. Indeed, the results actually suggest a steeper slope
in the warm than in the cold treatment. These analyses suggest that while
fitness costs associated with low genetic variation were present in our
analysis, they were not aggravated under the more stressful outdoors
conditions.
To conclude, contrary to the general findings, I found fitness costs associated
with low genetic variation under benign but not under stressful conditions.
This result may be explained that the higher environmental stress masks the
fitness costs. Since fragmentation of natural habitats is likely to result in both
higher environmental stress and loss of genetic diversity (Willi et al. 2007),
environment-dependent inbreeding depression can have considerable
conservation implications. Moreover, if purging of deleterious alleles is
important in reducing inbreeding depression under stressful conditions, the
23
interaction between inbreeding depression and stress may be more
detrimental in recently fragmented habitats where purging has not had the
opportunity to act (Ficetola et al. 2007; Swindell & Bouzat 2006a). Clearly,
more research on the role of interaction between inbreeding and
environmental stress is needed in order to evaluate the consequences of
habitat fragmentation and environmental stress on natural populations.
Local adaptations and support releases (paper V)
I found that the localities with present green toad populations, as well as
localities with extinct populations, differ in both thermal conditions and
salinity. The salinity in the ponds was measured at the beginning of the
breeding season and is likely to increase later during the season due to water
evaporation. At the localities where ponds are fed by groundwater (all but
Utklippan and Brantevik) water salinities are likely to be more constant than
localities were ponds are fed only by rain. As expected, both temperature
and salinity affected larval performance significantly. High temperatures led
to a higher survival, a higher growth rate, shorter larval period, lowered
weight at metamorphosis and a higher amount of spinal deformation.
Salinity imposed a considered stress to the larval green toads as evident by
decreased survival in more saline conditions, and the induction of nonadaptive (i.e. plastic responses away from an optimal (Ghalambor et al.
2007) plastic response. The larvae in saline treatments grew slower, had
longer larval periods, had smaller mass at metamorphosis and increased
degree of scoliosis as when compared to larvae in freshwater treatment. To
conclude, I found that the different localities where green toad are either
present, historically present, or introduced differ in temperature and salinity,
both environmental parameters putatively important for green toad fitness.
The experimental part of the study indicated that the Scandinavian green
toad populations are diverged in important fitness traits and that it may be
speculated that some of these divergences are adaptive. I recommend the
green toad conservation program to acknowledge this trait divergences and
the possibility that it is adaptive. For further support releases, efforts should
be made to find source populations similar (in respect of habitat) to the
localities to which they will be introduced. Moreover, the effect of warmer
rearing conditions should be noticed during the rearing of larvae for support
releases. Previous anuran studies have shown that smaller metamorphosis
size is associated with poorer performance during the early terrestrial stages
(Altwegg & Reyer 2003; Berven 1990; Scott 1994), and that warmer and
more saline conditions may thus yield unfit juveniles. However, I was not
able to perform crosses for logistical and ethical reasons and I have thus not
controlled for the possible presence of a maternal effect. The results should
thus be interpreted with caution.
24
Conclusions
To conclude, I have found that the natterjack toad populations in the
Bohuslän archipelago are adapted to the specific habitat they inhabit with a
higher growth rate and a shorter larval period. These adaptations are likely to
be counter selected in a less time constrained environment and the
conservation of the species within its Swedish range should acknowledge the
presence of these local adaptations. For example, translocation between the
two areas of occurrence may swamp the populations with genotypes with
low fitness. Interestingly, the trait values likely to be favored in a habitat
with high desiccation risk had low fitness in saline conditions. The Bohuslän
toads were thus, though inhabiting a more saline habitat, less tolerant to
saline conditions.
Within the Bohuslän habitat the toad populations on the different islands
were highly isolated. There was no evidence of isolation by distance,
indication that sea is a robust dispersal barrier. Importantly, the populations
varied greatly amongst each other in amount of genetic variation. This
variance in genetic variation explained survival of the tadpoles under benign,
but not under stressful thermal conditions. Thus, though there seems to be
inbreeding depression within some of the Bohuslän populations, it is unclear
how this inbreeding depression affects the populations under natural
conditions.
The low degree of among population divergence in larval life history traits is
most likely explained by uniform selection over the populations. However,
there were correlations between the population mean weight at
metamorphosis and the genetic variation of the population. Though a large
mass at metamorphosis is generally considered to be beneficial for fitness
during early terrestrial stages, the mechanisms behind these correlations
remains obscure. One possibility is that inbreeding depression might have a
direct effect on the trait values, a scenario that might limit the adaptive
potential of the populations.
My studies regarding the putative presence of local adaptations in the
support release program of the green toad revealed that the green toads were
diverged in a selectively important larval life history trait. However, though
some this divergence was in the direction expected by natural selection
25
within the particular habitat, I could not draw a rigid conclusion regarding
the presence or absence of local adaptations on this species. However,
further conservation efforts of this species should acknowledge the potential
presence of local adaptations.
At range margins delimited by physiological constraints, populations are
often more scattered than in more central regions (Hoffmann & Blows
1994). The increased isolation may yield an increased loss of genetic
variation and populations in peripheral regions may be thus be relatively
more threatened by fitness losses associated with low genetic variation than
populations in central regions. Moreover, populations on the species
physiological limits are likely to be more affected by environmental stress
than more central populations (Hoffmann & Blows 1994). Though the
increased environmental stress may require specific adaptations to the
peripheral habitats, the lack of genetic variation in these regions may
potentially constrain local adaptation. In this thesis I have found local
adaptations within peripheral regions and a rather distinct population
structure in the natterjack toad. I thus argue that the Bohuslän natterjack toad
populations and the Utklippan green toads should be viewed as evolutionary
significant units and that their conservation should be prioritised (Fraser &
Bernatchez 2001).
26
Sammanfattning på svenska
Små och isolerade populationer förväntas förlora genetisk variation, och
eftersom förlusten går snabbare ju mindre och mer isolerad populationen är,
är detta ett mycket aktuellt problem då arters livsmiljöer delas upp i en allt
högre grad. Detta skapar flera små populationer snarare än få stora, vilket
kommer att resultera i en ökad förlust av genetisk variation. Populationer
med låg genetisk variation riskerar problem med inavelsdepression d.v.s.
reduktion av livskraften hos individerna i populationen. Detta har påvisats
hos många organismer på flera olika plan, t.ex. mindre kullstorlekar,
försämrad överlevnad och försämrad förmåga att utstå stressfulla miljöer.
Populationsuppdelningar är ett aktuellt problem för amfibier då de har en
begränsad rörelseförmåga jämfört med många andra djur. Förutom en direkt
påverkan på populationers livskraft, så är det även möjligt att
anpassningsförmågan minskar i inavlade populationer.
En populations förmåga att anpassa sig till lokala miljöförutsättningar är en
viktig ekologisk faktor, då arters livsmiljöer sällan är helt homogena. De
selektionstryck som ger upphov till lokala anpassningar sker oftast på så
kallade kvantitativa egenskaper, t.ex. vikt, längd, tillväxthastighet och
tålighet mot olika miljöfaktorer. En kvantitativ egenskap t.ex. vikt består av
två komponenter, en genetisk och en miljömässig. Det är den genetiska
komponenten som förs vidare till nästkommande generationer och därmed
möjliggör evolution av egenskapen i fråga. Mängden genetisk variation för
kvantitativa egenskaper är därför mycket viktigt för anpassningsförmågan
hos organismer. Anpassningsförmåga begränsas dock även av att egenskaper
inte evolverar oberoende av varandra. T.ex. längd och vikt hos människor är
positivt korrelerande och selektion på en av dessa karaktärer kommer att
generera en förändring, inte enbart i den selekterade egenskapen, utan även i
den korrelerade egenskapen. I fall att den kombination som vore optimal på
de olika egenskaperna står i kontrast till vad som är möjligt givet deras
korrelationer, t.ex. låg vikt och lång längd hos människor, kommer den
evolutionära utgången bli en avvägning av de relativa selektionstrycken. Det
är dock oklart hur dessa teoretiska förväntningar beter sig i naturliga
populationer. Ökad kunskap om vilka effekter låg genetisk variation och
korrelerade egenskaper har på lokal anpassningsförmåga är viktigt för att
kunna planera bevarandeåtgärder såsom stödutplanteringar mera effektivt,
27
och för att öka förståelsen för vilka faktorer som påverkar isolerade
populationer.
Amfibier är bra studieorganismer för dessa frågor eftersom de har en
begränsad rörelseförmåga jämfört med många andra djur och därmed en mer
definierad populationsstruktur. Detta innebär att enskilda amfibiepopulationer ofta beter sig som enskilda enheter och därmed ofta skiljer sig i
mängd genetisk variation. De lägger stora mängder ägg vilket medför att de
kan studeras utan att populationerna påverkas nämnvärt. Amfibier är också
mycket lämpliga som försöksdjur i ekologiska och laboratorieexperiment.
I Sverige förekommer strandpadda (Bufo calamita) och grönfläckig padda
(B. viridis) på den norra gränsen för arternas respektive utbredningsområden.
Strandpaddan och den grönfläckiga paddan är båda fridlysta och har minskat
kraftigt under de senaste decennierna. Medans den grönfläckiga paddan
enbart förekommer i södra Sverige (Skåne och Blekinge) har strandpaddan
två utbredningsområden i Sverige, dels i Skåne, Blekinge och Halland och
dels i Bohusläns skärgård. I Skåne förekommer strandpaddan i ungefär
samma habitat som den grönfläckiga paddan, betade strandängar med grunda
dammar. I Bohusläns skärgård förekommer strandpaddan på cirka trettio
små, steniga öar i det yttre skärgårdsbandet. Bohusläns skärgård är en
mycket speciell miljö för strandpadda. Öarna karaktäriseras av en större
påverkan av havet än andra strandpaddspopulationer utsätts för samt en
större risk för uttorkning av lekvatten än de habitat strandpaddan bebor i
övriga delar av sitt norra utbredningsområde. Tidigare spanska studier har
visat på lokala anpassningar i salttolerans och att salt är en viktig selektiv
faktor i de populationer som utsätts för högre salthalt. De paddor som bebor
öarna i Bohusläns skärgård bebor ett mycket fragmenterat habitat, något som
bör leda till skillnader mellan populationer i genetisk variation. Den isolering
som troligtvis finns mellan populationerna kan också ge upphov till lokala
anpassningar. Det finns ett starkt bevarandebiologiskt intresse i att
undersöka dessa skillnader samt hur de påverkar livskraften och
anpassningsförmågan hos populationerna.
I den här avhandlingen har jag studerat lokala anpassningar, genetisk
variation och inavelsdepression i svenska populationer av strandpadda. Mer
specifikt undersöktes lokala anpassningar till två selektionstryck (uttorkning
och salttolerans) som skiljer sig mellan Skåne och Bohuslän, genetisk
populationsstruktur i Bohuslän, lokala anpassningar inom Bohuslän samt
vilken effekt olika mängd genetisk variation har på yngelöverlevnad i olika
temperaturer.
Resultaten visar att strandpaddorna som bebor Bohusläns skärgård är lokalt
anpassade till högre uttorkningsrisk. De hade dock en sämre salttolerans än
28
de paddorna i Skåne som bebor ett mindre salt habitat. Dessa skillnader kan
troligtvis förklaras av att de egenskaper som är knutna till en förbättrad
uttorkningstolerans (kortare yngelperiod och högre tillväxt hastighet) är
negativt korrelerade till hur egenskaperna påverkas i salta miljöer. D.v.s. de
yngel som växte långsammast och hade längst yngelperiod var de som
påverkades minst av salinitet. Dessutom var överlevnad positivt korrelerad
till yngelperiod, d.v.s. de yngel som hade längs yngelperiod i sötvatten var
de som hade bäst överlevnad i bräckvatten. Detta tyder på att selektion på
kortare yngelperiod och snabbare tillväxt kan ha minskat salttoleransen i
Bohuslänspopulationerna av strandpadda.
Inom Bohusläns skärgård visade resultaten att strandpaddorna har betydligt
mindre genetisk variation än sina kontinentala släktingar vilket troligtvis
beror på att de har genomgått perioder då populationsstorlekarna varit små
under den gradvisa koloniseringen av perifera habitat. Detta är ett vanligt
mönster och har visats hos många av de organismer som koloniserat
Skandinavien efter istiden. Paddor som lever på olika öar var dels mycket
isolerade från varandra och dels var paddorna inte mer genetiskt lika paddor
på öar som ligger nära, än paddor på öar på längre avstånd. Detta indikerar
att paddor har en dålig spridningsförmåga även över korta sträckor hav.
Öarna hade även mycket varierande nivåer av genetisk variation. Eftersom
storleken på populationerna inte är korrelerad till genetiska variationen så
beror den genetiska variationen troligen på det ursprungliga antalet paddor
som koloniserade ön eller senare fluktuationer i populationsstorlek. Med de
populationsstorlekar som finns idag är den genetiska driften troligtvis relativt
låg. Det är också sannolikt att öpopulationerna av strandpadda har startats av
ett fåtal individer, vilket också leder till minskad genetisk variation.
I de temperaturbehandlingar vi använt var överlevnaden lägre hos yngel i
den temperaturbehandling som var designad att imitera naturliga
temperaturförhållanden. Detta tyder på att naturliga temperaturförhållanden
är mer stressfulla för ynglen än de stabila temperaturer vi använt i
laboratoriet. I de två övriga temperaturbehandlingarna (de stabila
temperaturerna) var överlevnaden hos yngel korrelerad till mängden genetisk
variation, vilket indikerar inavelsdepression i de populationer som har låg
genetisk variation. Att den genetiska variationen inte enbart är korrelerat till
överlevnad i de mindre stressfulla temperaturbehandlingarna tyder på att
inavelsdepression inte accentueras i stressfulla miljöer vilket är ett relativt
ovanligt resultat.
Inom Bohusläns skärgård verkar den divergens som finns mellan
populationer främst förklaras av stabiliserande selektion, d.v.s. att samtliga
populationer utsätts för liknande selektionstryck. Därmed finns det inga
bevis för förekomst av lokala anpassningar inom västkustpopulationerna. Ett
29
anmärkningsvärt resultat var att populationernas medelvikt var negativt
korrelerad till deras neutrala genetiska variation. Generellt sett brukar en stor
vikt hos juvenila amfibier ge en hög livskraft under den första tiden som
landlevande, något som skulle innebära att de inavlade ynglen har bättre
förutsättningar. Det är oklart vad detta resultat beror på, men det är möjligt
att inavelsdepression påverkar vikt, vilket kan leda till att de inavlade
populationerna kan ha mindre möjligheter att svara på ett eventuellt
selektionstryck.
Resultaten angående populationsstruktur, genetisk variation, inavelsdepression och lokala anpassningar hos strandpaddan har viktiga
implikationer för bevarandet av de svenska populationerna av strandpadda.
De lokala anpassningar som finns mellan Skåne och Bohuslän är viktiga att
ta hänsyn till under bevarande åtgärder t.ex. kan utplanteringar mellan de
olika habitaten (Skåne och Bohuslän) vara en nackdel för artens bevarande
då de är anpassade till just den miljön de lever i. Inom Bohuslän bör hänsyn
tas till att en del populationer har lägre genetisk variation samt en
väldefinierad populationsstruktur. Även om det är svårt att tolka huruvida
inavelsdepression påverkar strandpaddorna i naturen bör en
försiktighetsprincip gälla, och de populationer som har låg genetisk variation
bör övervakas för att försäkra att de inte minskar. Det bör även noteras att i
fall att de börjar minskar i storlek bör utplanteringar göras från populationer
med mer genetisk variation. Detta bör dock enbart göras i yttersta nödfall då
man inte säker kan utesluta att de olika populationerna är lokalt anpassade
inom Bohusläns skärgård.
Utöver arbetet med strandpaddorna behandlar även avhandlingen lokala
anpassningar hos grönfläckig padda och hur de stödutplanteringar som utförs
av denna art kan påverka bevarande arbetet av arten. Återintroduktioner och
stödutsättningar är vanliga metoder för att bevara hotade djurarter. Dessa
metoder är dock kontroversiella eftersom utsättningsdjuren kan ha lokala
anpassningar som inte lämpar sig för den nya miljön. Om dessa
missanpassningar sprider sig i populationen kan det i längden utgöra ett
ytterligare hot mot den redan hotade populationens fortlevnad. I det här
projektet vill vi undersöka vilken roll lokala anpassningar har spelat i ett
utplanteringsprojekt av grönfläckig padda. Den grönfläckiga paddan är enligt
den svenska rödlistan klassad som akut hotad i Sverige. Många populationer
har minskat kraftigt eller försvunnit på senare år. I Sverige finns i nuläget
endast ett fåtal populationer med mer än en handfull reproducerande
individer. För att säkra den grönfläckiga paddans fortlevnad i Sverige har
utsättningar av ägg, yngel och unga paddor gjorts på flera platser. Resultaten
har dock varit under förväntan. En bidragande orsak till att utsättningarna
inte gett de förväntade resultaten kan vara att utsättningsmaterial tagits från
Limhamns Kalkbrott, en mycket speciell miljö med hög temperatur och höga
30
saltkoncentrationer. Det specifika habitatet kan ha selekterat för lokala
anpassningar till den grad att de klarar sig sämre i normala svenska habitat.
Efter ett experiment där vi födde upp yngel av grönfläckig padda från fyra
lokaler i Sverige och Danmark i två olika temperaturer drog vi slutsatsen att
de olika populationerna har divergerat i yngelegenskaper. En del av dessa
divergenser i den riktning man förväntar om populationerna besitter olika
lokala anpassningar. T.ex. var yngel från ön utklippan mer salttoleranta än
andra yngel. Yngel från Eskilstorps ängar, ett habitat med en hög risk för
uttorkning, växte snabbare och hade en kortare larvperiod än yngel från
andra lokaler. Även om indicier antyder att dessa divergenser är adaptiva är
dock inte möjligt att dra säkra slutsatser angående detta. Det fortsatta
bevarandearbetet bör ta hänsyn till att de svenska grönfläckiga paddorna är
divergerade i selektivt viktiga karaktärer och att divergenserna kan vara
adaptiva. I fall att stödutplanteringar ska utföras är det optimala att ta
utplanteringsdjuren från populationer som bebor ett habitat så likt
målhabitatet som möjligt.
31
Acknowledgements
It is a weird feeling finishing something that has taken this time and effort,
and I feel quite sentimental. Since this started as an interest that grew into
this work-like situation, I have many to thank. When I was very young and
gained an interest in herpetology, I got much help and inspiration from older
members of the herpetological societies. I specifically want to mention Anna
and Göran Sahlén. If it was not for all your support I would probably have a
decent job by now and be less of a geek. I wouldn’t want that… When
attending Spånga upper secondary school I met many friends to share and
develop interests with, thanks Ekan, Valle, Packe, Alex, Jossan, Olivia,
Petter, Simon K, Axel, Vide, Crille, Peps, Morgan and Trollet!
Thanks Johan W for explaining that everything else than Uppsala would be a
stupid choice for my studies, it was good! Thanks Anders, Andreas, Jonas
and Karin for being fun friends since I moved to Uppsala. Simon K, thanks
for being great hushippie, you know what I want to write! Höhöhö… And the
supervisors! Thanks Jacob for giving me this opportunity, for heaps of
support, serious and perhaps not so serious discussions. Anssi, thanks for all
help and comments, and for great support in general and specifically during
the last months… I have learned a lot and had a lot of fun when working with
both of you! Thanks Marianne for being so friendly and helpful, I would
probably have forgotten something serious without your help. The members
of the frog group were a fun bunch to share the basement with during the
summer when everyone else seemed to do things that, at the time, appeared to
make more sense, like nothing for example. So thanks Emma, Alex, German
(I hope we can put my misunderstanding regarding Spain’s economic
situation behind us at some point), Bea, Ane, Markus, Fredrike, Fredrik,
Attila, Ana, Celine, Katja and Sandra H! Also, thanks to Per, Hampus,
Jonas and Johan for all the help with endless water changes... Thanks
Manuel, Hanna, Maarten and Axel for your great exam works, it was really
nice working with you! Thanks Robert E for teaching me molecular lab
work, I am happy I had a teacher like you and not like me... And thanks to
Robert M and Gunilla for your help and company in lab.
Thanks Sara B for always being a good friend, making sure I don’t forget
things and that I end up in the right place at the right time and so on… and
32
for letting me copy your psychotically well structured lecture notes as
undergraduates!
Thanks Jonas for all the help in lab, online image improvement, and for
drawing the cover! Your (and peters) online support with images and file
conversions during the last month is probably what saved my computer from
a rapid exit throughout the window. The work related trips were a nice
addition to the work, thanks the Galapagos gang (Emma, Tanja, Thomas,
Pär, Lars, Peter, the boring guide and the macho guide) and the Guyana
crew Jobs Kalle and Martin. Thanks Andreas for the Panama trips. It was
extremely interesting and great fun! Thanks also for all constructive
criticism and innovative suggestions for improvement on all aspects of life.
You are a top of the line consigliere! Thanks Martin E for all help with
statistics! You probably haven’t noticed this, but I’ve learned heaps of
statistics from you and it is by far the best cooperation I’ve started after
seven beers at snärran. Thanks Stefan P for your contribution on the paper
II, it helped a lot. Markus, Andreas, Axel and Thomas (sorry for all pea
soup), were great office mates, thanks for all laughs and that you put up with
that my desk cover tended to expand over the neighbouring desk resembling
an amoeba in feeding mood.
All the field work, thanks to Valle (I didn’t put you mug shot on the cover,
sorry), Ekan, Petter, Sinom H, Rastajohan, Rille, Andreas, Britta and
Mårten for joining me in field, it was always fun! And a special thanks to
Valle, Petter, Torvald and Mårten who volunteered during spare time!
And to Sara and Petter for supplying us with everything we needed during
fieldwork in Skåne: somewhere to sleep, nice company, guidance and
explanations of the local manners, headlamp batteries, maps, beer etc. Also,
thanks to Valle, Mårten, Fiskjossan, Tonje and all innocent bystanders at
Kristineberg for an unforgettable (?) grande finale of field work.
Thanks everyone that have inhabited the fika room during the last 5 years
and made this place a great working place (popbio, natgen zooeko). Thanks
also to all fun people I have met at EBC, Mirjam, Amber, Richard,
Sandra S and her horndog, Kate, the Spanish funky bunch (Nanet and
Santi), Pia and Jonas and Phillip and the other limnos, Simmone, Alexei,
Maria, Omar, Alejandro, Vendela. Thanks Paolo for all severely
competitive pingpong games and for our statistics discussions! The car thing
is special to me. Thanks Fiskjossan for keeping my fading dream of a
drivers licence before 30 alive! Also, thanks to Jobs Karl Larsson for
teaching me the essentials of car mechanics. I now know which direction
you twist a screw to make it lose, which direction you twist it to make it
tight, as well as the consequences of confounding these directions.
33
Perhaps the greatest thanks of all, thanks to my parents Marianne and Gert
and my sister Annika for all help, encouragement and support in all ways
even though you put up with more than your fair share of weird incidences.
The wall paper got eaten by caterpillars, the floor got burned, giant beetles,
trillions of flies, some rats and a (small) python escaped, giant moths were
rooming the house and that’s just the things you know about…
Many toad experts have helped and improved my work substantially, and I
would specifically want to thank Mats Wirén, Claes Andrén, Owe
Törnqvist, Lars Briggs and Jan Pröjts. I got comments that greatly
improved the manuscripts from Mats B, German O, Anna Q, Amber R,
Andreas R, Sara B, Torsten N K, Alexei M, Gustaf G, Alex R, Mårten H
and Martin L. Sandra S commented on the thesis summary. Thanks again!
Andreas Rudh (Fig. 3), Hanna Thörngren (Fig. 4) and Simon Hultby
(Fig. 5) contributed pictures to the thesis.
If you feel forgotten and know me you’re probably not surprised. Sorry!
34
References
Allendorf, F., and W. Luikart, G. 2007. Conservation and the Genetics of
Populations. Blackwell Publishing.
Altermatt, F., V. I. Pajunen, and D. Ebert. 2009. Desiccation of rock pool
habitats and its influence on population persistence in a Daphnia
Metacommunity. PLoS ONE 4:e4703.
Altwegg, R., and H. U. Reyer. 2003. Patterns of natural selection on size at
metamorphosis in water frogs. Evolution 57:872-882.
Andrén, C., and G. Nilson. 1984. Breeding pool characteristics and
reproduction in an island population of natterjack toads, Bufo calamita
Laur., at the Swedish west coast. Amphibia-Reptilia 6:137-142.
Andrén, C., and G. Nilson. 1985. Habitat and other environmental
characteristics of the natterjack toad (Bufo calamita Laur) in
Sweden. British Journal of Herpetology 6:419-424.
Arendt, J. D. 1997. Adaptive intrinsic growth rates: An integration across
taxa. Quarterly Review of Biology 72:149-177.
Armbruster, P., and D. H. Reed. 2005. Inbreeding depression in benign and
stressful environments. Heredity 95:235-242.
Arnold, N., and D. Ovenden. 1978. Field guide to the reptiles and
amphibians of Britain and Europe. HarperCollins Publishers,
London. UK.
Avise, J. C. 2000. Phylogeografy: The history and formation o species.
Harvard University Press, Cambridge, Massachusetts.
Barton, N. H., and M. Turelli. 2004. Effects of genetic drift on variance
components under a general model of epistasis. Evolution 58:
2111-2132.
Beebee, T. J. C. 1983. The natterjack toad. Oxford university press, Oxford,
England.
Beebee, T. J. C. 2005. Conservation genetics of amphibians. Heredity
95:423.
Bensch, S., and M. Åkesson. 2005. Ten years of AFLP: why so few animals?
Molecular Ecology 14:2899-2914.
Berven, K. A. 1990. Factors affecting population fluctuations in larval and adult
stages of the wood frog (Rana sylvatica). Ecology 71:1599-1608.
Björklund, M. 1996. The importance of evolutionary constraints in
ecological time scales. Evolutionary Ecology 10:423-431.
Boutilier, R. G., D. F. Stiffler, and D. P. Toews. 1992. Exchange of respiratory
gases, ions, and water in amphibious and aquatic amphibians. M. E.
Feder and W. W. Burggren, eds. Enviromental physiology of the
amphibians. Univ. of Chicago press, Chicago, USA.
35
Broomhall, S. D. 2004. Egg temperature modifies predator avoidance and
the effect of the insecticide endosulfan on tadpoles of an Australian
frog. Journal of Applied Ecology 41:105-113.
Bryant, E. H., S. A. McCommas, and L. M. Combs. 1986. The effect of an
experimental bottleneck upon quantitative genetic-variation in the
housefly. Genetics 114:1191-1211.
Chen, X. F. 1993. Comparison of inbreeding and outbreeding in
hermaphroditic Arianta arbustorum (L) (land snail). Heredity
71:456-461.
Chenoweth, S. F., and M. W. Blows. 2008. Q(st) meets the G matrix: The
dimensionality of adaptive divergence in multiple correlated
quantitative traits. Evolution 62:1437-1449.
Cheverud, J. M. 1984. Quantitative genetics and developmental constraints
on evolution by selection. Journal of Theoretical Biology 110:
155-171.
Conover, D. O. and E. T. Schultz. 1995. Phenotypic similarity and the
evolutionary significance of countergradient variation. Trends in
Ecology & Evolution 10:248-252.
Crain, C. M., Kroeker, K. and B. S. Halpern. 2008. Interactive and
cumulative effects of multiple human stressors in marine systems.
Ecology Letters 11:1304-1315.
Crnokrak, P., and D. A. Roff. 1995. Dominance variance - associations with
selection and fitness. Heredity 75:530-540.
Davis, M. B., R. G. Shaw, and J. R. Etterson. 2005. Evolutionary responses
to changing climate. Ecology 86:1704-1714.
Denver, R. J. 1977. Proximate mechanisms of phenotypic plasticity in
amphibian metamorphosis, American Zoologist 37:172-184.
Duellman, W. E., and L. Trueb. 1994. Biology of amphibians. The John
Hopkins Univerity Press. London.
Etterson, J. R., and R. G. Shaw. 2001. Constraint to adaptive evolution in
response to global warming. Science 294:151-154.
Evanno, G., S. Regenaut, and J. Goudet. 2005. Detecting the number of
clusters using the software STRUCTURE: a simulation study.
Molecular Ecology 14:2611-2620.
Falconer, D., and T. Mackay. 1996. Introduction to Quantitative Genetics,
4th edn. Longman, Essex.
Ficetola, G. F., T. W. J. Garner, and F. De Bernardi. 2007. Genetic diversity,
but not hatching success, is jointly affected by postglacial
colonization and isolation in the threatened frog, Rana latastei.
Molecular Ecology 16:1787-1797.
Frankham, R., Ballou, J.D., Briscoe, D.A 2002. Introduction to conservation
genetics. Cambridge University Press. Cambridge, UK.
Fraser, D. J., and L. Bernatchez. 2001. Adaptive evolutionary conservation:
towards a unified concept for defining conservation units. Molecular
Ecology 10:2741-2752.
Gärdenfors, U. 2005. Rödlistade arter i Sverige. SLU Publikationsservice
36
Ghalambor, C. K., J. K. McKay, S. P. Carroll, and D. N. Reznick. 2007.
Adaptive versus non-adaptive phenotypic plasticity and the potential
for contemporary adaptation in new environments. Functional
Ecology 21:394-407.
Gillespie, J. H. 1998. Population genetics, a concise guide. The John
Hopkins Unversity Press, London.
Gomez-Mestre, I., and D. R. Buchholz. 2006. Developmental plasticity
mirrors differences among taxa in spadefoot toads linking plasticity
and diversity. Proceedings of the National Academy of Sciences of
The United States Of America 103:19021-19026.
Gomez-Mestre, I., and M. Tejedo. 2003. Local adaptation of an anuran
amphibian to osmotically stressful environments. Evolution
57:1889-1899.
Gomez-Mestre, I., and M. Tejedo. 2004. Contrasting patterns of quantitative
and neutral genetic variation in locally adapted populations of the
natterjack toad, Bufo calamita. Evolution 58:2343-2352.
Gomez-Mestre, I., M. Tejedo, E. Ramayo, and J. Estepa. 2004.
Developmental alterations and osmoregulatory physiology of a
larval anuran under osmotic stress. Physiological and Biochemical
Zoology 77:267-274.
Goodnight, C. J. 1987. On the effect of founder events on epistatic genetic
variance. Evolution 41:80-91.
Griffiths, R. A. 1991. Competition between common frog, Rana temporaria,
and natterjack toad, Bufo calamita, tadpoles - the effect of
competitor density and interaction level on tadpole development.
Oikos 61:187-196.
Griffiths, R. A., P. W. Edgar, and A. L. C. Wong. 1991. Interspecific
competition in tadpoles - growth-inhibition and growth retrieval in
natterjack toads, Bufo calamita. Journal of Animal Ecology
60:1065-1076.
Heuch, I. 1980. Loss of incompatibility types in finite populations of the
heterostylous plant Lythrum salicaria. Hereditas 92:53-57.
Hoffmann, A. A., and M. W. Blows. 1994. Species borders - ecological and
evolutionary perspectives. Trends in Ecology & Evolution 9:
223-227.
Hoffmann, A. A., R. J. Hallas, J. A. Dean, and M. Schiffer. 2003. Low
potential for climatic stress adaptation in a rainforest Drosophila
species. Science 301:100-102.
Höglund, J. 2009. Evolutionary Conservation Genetics. Oxford University
Press, Oxford, UK.
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. Serra. 2000.
Rapid evolution of a geographic cline in size in an introduced fly.
Science 287:308-309.
Johansson, M., C. R. Primmer, and J. Merila. 2007. Does habitat
fragmentation reduce fitness and adaptability? A case study of the
common frog (Rana temporaria). Molecular Ecology 16:2693-2700.
37
Jorde, P. E., and N. Ryman. 1995. Temporal allele frequency change and
estimation of efficient population size in populations with
overlapping generations. Genetics 139:1077-1090.
Keller, L. F., and D. M. Waller. 2002. Inbreeding effects in wild populations.
Trends in Ecology & Evolution 17:230-241.
Kimura, M. 1957. Some problems of stochastic-processes in genetics.
Annals of Mathematical Statistics 28:882-901.
Lande, R. 1976. Natural-selection and random genetic drift in phenotypic
evolution. Evolution 30:314-334.
Lande, R. 1979. Quantitative genetic-analysis of multivariate evolution,
applied to brain - body size allometry. Evolution 33:402-416.
Lande, R. 1980. The genetic covariance between characters maintained by
pleiotropic mutations. Genetics 94:203-215.
Lande, R. 1995. Mutation and conservation. Conservation Biology 9:
782-791.
Laurila, A., B. Lindgren, and A. T. Laugen. 2008. Antipredator defenses
along a latitudinal gradient in Rana temporaria. Ecology 89:
1399-1413.
Laurila, A., S. Karttunen, and J. Merilä. 2002. Adaptive phenotypic
plasticity and genetics of larval life histories in two Rana temporaria
populations. Evolution 56:617-627.
Leinonen, T., R. B. O´Hara, J. M. Cano, and J. Merilä. 2008. Comparative
studies of quantitative trait and neutral marker divergence: a metaanalysis. Journal of Evolutionary Biology 21:1-17.
Levin, D. A. 1970. Developmental instability and evolution in peripheral
isolates. American Naturalist 104:343-&.
Liao, W., and D. H. Reed. 2009. Inbreeding-environment interactions
increase extinction risk. Animal Conservation 12:54-61.
Lind, M. I., and F. Johansson. 2007. The degree of adaptive phenotypic
plasticity is correlated with the spatial environmental heterogeneity
experienced by island populations of Rana temporaria. Journal of
Evolutionary Biology 20:1288-1297.
Lofsvold, D. 1988. Quantitative genetics of morphological-differentiation in
Peromyscus .2. Analysis of selection and drift. Evolution 42:54-67.
Lynch, M., M. Pfrender, K. Spitze, N. Lehman, J. Hicks, D. Allen, L. Latta,
M. Ottene, F. Bogue, and J. Colbourne. 1999. The quantitative and
molecular genetic architecture of a subdivided species. Evolution
53:100-110.
Madsen, T., R. Shine, M. Olsson and H. Wittzell. 1999. Conservation
biology – restoration of an inbred adder population. Nature 402:
34-35.
Marangoni, F., and M. Tejedo. 2008. Variation in body size and
metamorphic traits of Iberian spadefoot toads over a short
geographic distance. Journal of Zoology 275:97-105.
Mariette, S., V. Le Corre, F. Austerlitz and A. Kramer. 2002. Sampling
within the genome for measuring within population diversity: tradeoffs between markers. Molecular Ecology 11:1145-1156.
38
Nybom, H. 2004. Comparison of different nuclear DNA markers for
estimating interaspecific genetic diversity in plants. Molecular
Ecology 13:1143-1155.
Pemberton, J. 2004. Measuring inbreeding depression in the wild: the old
ways are the best. Trends in Ecology & Evolution 19:613-615.
Rogell, B., M. Hofman, M. Eklund, A. Laurila, and J. Höglund. The
interaction of multiple environmental stressors affects adaptation to
a novel habitat in the natterjack toad Bufo calamita. Submitted
Seppä, P. and A. Laurila. 1999. Genetic structure of island populations of the
anurans Rana temporaria and Bufo bufo. Heredity 82:309-317.
Sheldon, B. L., D. E. Finlay, and J. M. Rendel. 1964. Effect of
homozygosity on developmental stability. Genetics 49:471-484.
Spielman, D., B. W. Brook, and R. Frankham. 2004. Most species are not
driven to extinction before genetic factors impact them. Proceedings
of the National Academy of Sciences of the United States of
America 101:15261-15264.
Stewart, J. R., and A. M. Lister. 2001. Cryptic northern refugia and the
origins of the modern biota. Trends in Ecology & Evolution 16:608.
Stuart, S. N., J. S. Chanson, N. A. Cox, B. E. Young, A. S. L. Rodrigues, D.
L. Fishman, and R. W. Waller. 2004. Status and trends of amphibian
declines and extinctions worldwide. Science 306:1783-1786.
Swindell, W. R., and J. L. Bouzat. 2006a. Ancestral inbreeding reduces the
magnitude of inbreeding depression in Drosophila melanogaster.
Evolution 60:762-767.
Swindell, W. R., and J. L. Bouzat. 2006b. Reduced inbreeding depression
due to historical inbreeding in Drosophila melanogaster: evidence
for purging. Journal of Evolutionary Biology 19:1257-1264.
Thomas, C. D. 2000. Dispersal and extinction in fragmented landscapes.
Proceedings Of The Royal Society of London Series B-Biological
Sciences 267:139-145.
Van Buskirk, J., and Y. Willi. 2006. The change in quantitative genetic
variation with inbreeding. Evolution 60:2428-2434.
Via, S., and R. Lande. 1985. Genotype-environment interaction and the
evolution of phenotypic plasticity. Evolution 39:505-522.
Waller, D. M., J. Dole, and A. J. Bersch. 2008. Effects of stress and
phenotypic variation on inbreeding depression in Brassica rapa.
Evolution 62:917-931.
Wells, K. D. 2007. The ecology and behavior of amphibians. The University
of Chicago Press, Chicago.
Whitlock, M. C. 2008. Evolutionary inference from QST. Molecular
Ecology 17:1885-1896.
Whitlock, M. C., and K. Fowler. 1999. The changes in genetic and
environmental variance with inbreeding in Drosophila
melanogaster. Genetics 152:345-353.
Willi, Y., J. Van Buskirk, and A. A. Hoffmann. 2006. Limits to the adaptive
potential of small populations. Annual Review of Ecology Evolution
And Systematics 37:433-458.
39
Willi, Y., J. Van Buskirk, B. Schmid, and M. Fischer. 2007. Genetic
isolation of fragmented populations is exacerbated by drift and
selection. Journal of Evolutionary Biology 20:534-542.
Zeisset, I. and T. J. C. Beebee. 2001. Determination of biogeographical
range: an application of molecular phylogeography to the European
pool frog Rana lessonae. Proceedings of The Royal Society of
London Series B-Biological Science 268:933-938.
Zhang, X.-S., J. Wang, and W. G. Hill. 2004. Redistribution of gene
frequency and changes of genetic variation following a bottleneck in
population size. Genetics 167:1475-1492.
40
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