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!!"#""$ !%&'%"##$&#'( )*)))"(&+%# ! ""# "$""% &% % '&&()& * +&( ,-( ""#(. & % ( ( // (((#01#1!!210!"1 ( &% &* % & & *&% (3 &*&* & 45 6 7& 5(8&$87 * && 9887 & * & &5& 98887 &%% % % * & &5& 9 8:7 &%% % % & & % %(:78& & % & 67 ()& & &5 45 & 5 && (;* & & &&* *& &% & & ( )& * 5 & % * & &% ( < & &5& & & * & %% & % ( = * & & 5 & & % ( ;* * & %% & (8 & 45 % * &5% * ()& & 5 & ( 8 & & 8% & & & &&& & & & ()& &% *&& &5* &g (% & &% % %%l & %+ % & && >( ! " % " "%&'()* "+ ?-4@, ""# 8/!1/ 2 8-#01#1!!210!"1 $$$$1"0A#!6& $BB(5(BCD$$$$1"0A#!7 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. 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