>>>> Gerlien Verhaegen & Martin Haase (1st cohort) <<<<

Thesis topic: Variable shell morphology as key for colonizing a wide spectrum of habitats?

State of the art: During phases of global change generalist species are predicted to have a higher survival chance than specialists. However, what constitutes a generalist, in particular with regard to the relative importance of phenotypic plasticity and genetic adaptation, is little understood. This question will be addressed here in the ovoviviparous New Zealand mud snail Potamopyrgus antipodarum occupying a wide range of fresh and brackish water habitats and exhibiting an extreme morphological variation. In this species, triploid, (almost) all-female parthenogenetic lineages have evolved repeatedly and successfully invaded other continents, emphasizing the species’ adaptive potential. The broad ecological niche has possibly been realized due to a high degree of phenotypic plasticity. Gastropod shell shape and size have been already related to environmental factors including current velocity and temperature. Therefore, this project focuses on the adaptive value of shell shape and size as well as on osmotolerance in P. antipodarum, disentangling phenotypic and genetic adaptation and the influence of the reproductive mode thereon.

Working hypotheses and work plan: We will relate adult shell size and shape of P. antipodarum, which has finite growth, as well as the number of brooded embryos to habitat characteristics in its native range. We will control for confounding factors including phylogeny, geography, ploidy, parasitism, predation risk, and population density. In a similar fashion, we will analyze parthenogenetic populations established in Europe assuming that clonal strains accumulate mutations and thus adapt differentially. These analyses will form the basis for the selection of differentially adapted populations for common garden experiments aiming at measuring reaction norms, estimating heritabilities through parent-offspring regressions, thus disentangling genetic from plastic effects. Snails will be exposed to different flow velocities, temperatures, and salinities and effects on shell size, shape, growth rate, fecundity and survival will be recorded. We expect sexual populations to show a higher degree of plasticity, and, assuming costs of phenotypic plasticity, trade-offs with fecundity.

>>>> Lisa Männer & Martin Haase (2nd cohort) <<<<

Thesis topic: Quantifying genetic and environmental effects in adaptation to different habitats in the morphologically variable New Zealand mud snail Potamopyrgus antipodarum

State of the art: During phases of global change, generalist species are predicted to have higher survival chances than specialists (BMC Evol Biol 13: 94). However, what constitutes a generalist, in particular with regard to the relative importance of phenotypic plasticity and genetic adaptation, is poorly understood (Mol Ecol 17: 20). Moreover, when it comes to questions of epigenetic gene regulation including parental effects, reserachers have only just started to have a inkling of its potential importance in the context of adaptation (TREE 31: 514; J Evol Biol 30: 1612). Invasive species are ideal models to dissect adaptation into its constituent mechanisms, as they have successfully colonised novel habitats. We have been working with the ovoviviparous New Zealand mud snail Potamopyrgus antipodarum occupying a wide range of fresh and brackish water habitats and exhibiting extreme morphological variation. In this species, polyploid, almost all-female parthenogenetic lineages have evolved repeatedly (J Evol Biol 8: 385) and these have invaded other continents (Zool Stud 53: 70), emphasising the species’ adaptive potential. The clonal reproduction of these lineages is an ideal prerequisite to study adaptation and to disentangle and quantify genetic and non-genetic effects on traits. Apart from being an emerging ecomorphological model, P. antipodarum is an established model species in ecotoxicology (Environ Toxicol Chem 22: 145) and for studying host-parasite co-evolution and the maintenance of sex (Am Nat 174 Suppl 1: S43).

Working hypotheses and work plan: This project contributes to RESPONSE by analysing the fitness consequences of phenotypic and evolutionary reactions to varying environmental conditions in a generalist freshwater snail with clonal as well as sexual reproduction.In the first project phase, we could show that shell morphology is adaptive with phenotypically plastic as well as genetic effects, and size being more environment-dependent than shape. In phases two and three, we aim at estimating the heritability of shell traits, i.e. at quantifying the relative contributions of genetic and environmental factors on the variation of shell morphology (2nd cohort), as well as the environmental sensitivity of selection on these traits (3rd cohort). These estimates will be based on common garden experiments in the laboratory as well as reciprocal transplant experiments between different habitat types. Factors to be manipulated in the laboratory include temperature, food, salinity or population density. The morphological analyses will be conducted in the framework of geometric morphometrics. Genetic and plastic effects will be disentangled by measuring reaction norms and estimating heritability through parent-offspring regressions in sexual populations, and by comparison of intra- and inter-lineage variation in asexual ones. We also intend to shed light on the mechanistic, epigenetic side of phenotypic plasticity by studying DNA-methylation patterns in experimental and natural populations from different habitat types and climatic regions, focusing on populations from Germany and the Iberian Peninsula in Europe, and the far North and the far South of New Zealand respectively. Finally, we plan to complement our analyses by screening native natural populations for genetic signatures of habitat related selection, again comparing samples from different habitat types and climatic regions. Both methylation and selection analyses will be conducted using NGS approaches. The resulting patterns will be mapped against the reference genome currently established by our external collaboration partner M. Neiman (Univ. of Iowa). Our investigations aim at understanding adaptation of P. antipodarum to different habitat types focusing on shell morphology as well as on the more general genetic and epigenetic footprints of selection and phenotypic plasticity, hence on the underlying mechanisms of adaptation to novel environments. For shell morphological traits, we will assess the importance of phenotypic plasticity for the ecological success of the species. We expect that variation in shape will be larger between than within lineages and mainly genetically controlled. Moreover, we expect that heritability of size will be considerably lower than that of shape given the apparent importance of phenotypic plasticity in the former. The genetic and epigenetic analyses will reveal possibly complementary patterns indicative of genetic adaptation and adaptation through phenotypic plasticity. Analysing the New Zealand field samples, we expect to find region-specific patterns that are irrespective of habitat and climate, climate-specific patterns that are irrespective of region and habitat, and habitat-specific patterns that are irrespective of climate and region. Finally, in the third project phase we aim to synthesise the results of all three project phases including modelling future, temperature imposed range limits in collaboration with project P. This shall lead to a comprehensive understanding of population persistence and range expansions in this ecologically successful generalist gastropod species.

>>>> Peter Schächinger & Martin Haase (3rd cohort) <<<<

Thesis topic: Winner in a changing world- reconstructing global expansion and invasive routes of the New Zealand mud snail Potamopyrgus antipodarum and the role of shell morphology

State of the art: During phases of global change generalist species are predicted to have a higher survival chance than specialists. Climate change will produce winners and losers, the former surviving with a higher probability because they can adapt to novel situations and/or track their niche more successfully, i.e. have a higher potential for dispersal, than specialists. We will work with the ovoviviparous New Zealand mud snail Potamopyrgus antipodarum occupying a wide range of fresh and brackish water habitats and exhibiting an extreme morphological variation. In this species, polyploid, all-female parthenogenetic lineages have evolved repeatedly and these have invaded other continents including Europe, emphasising the species’ adaptive potential. The genetic diversity of the clonal European lineages is very restricted compared to the native range. Although clonal lineages accumulate mutations and evolve as well, albeit slower, phenotypic plasticity is probably more important for their successful range expansion than genetic adaptation..

Working hypotheses and work plan: Historical and future range expansion of the New Zealand mud snail Potamopyrgus antipodarum. We will reconstruct phylogeographies for both New Zealand as well as European populations based on genomic approaches to be developed using the genomic resources established in the previous generations of this project as well as by our external collaboration partner Maurine Neiman (Univ. Iowa). The phylogeographic analyses will allow to estimate modes as well as spatial and temporal dimensions of dispersal and serve, in conjunction with climatic data, as the basis for modelling future trends, in particular in Europe. The genomic data will also serve for the detection of genetic signatures of selection. As the colonization of Europe does not date back more than 200 years and the spread is fairly well documented, we expect to be able to assess the importance of genetic diversity and selection – and in turn phenotypic plasticity – especially for the conquest of the continent. As the remarkable variation of shell morphology, which is of adaptive significance, has an important genetic component, we will also assess the role of size and shape in the phylogeographic overall picture.

>>>> Amanda Wiesenthal, Christian Müller & Jan-Peter Hildebrandt (1st cohort) <<<<

Thesis topic: Limits of physiological acclimation to salinity stress in Theodoxus.

State of the art: Molecular phylogeographic studies have revealed that Theodoxus fluviatilis (Gastropoda: Neritidae) has formed regional subgroups (lineages) in northern Germany which seem to occur either in freshwater or in brackish water (Mol Ecol (2005) 14: 4323; Mol Phylogenet Evol (2007) 42: 373). These lineages were assigned to different subspecies, although individuals from freshwater and brackish water habitats are indistinguishable with respect to shell size and patterning (J Conchol (2004) 38: 305) and are genetically uniform with respect to mitochondrial DNA markers (Mol Ecol (2005) 14: 4323; Mol Phylogenet Evol (2007) 42: 373). In their responses to salinity changes, however, snails from the two lineages do differ (Biol Zentralbl (1960) 5: 585; Estuar Coast Mar Sci (1978) 6: 409; J Comp Physiol B (2010) 180: 337). The higher salinity tolerance of the brackish water lineage enables these animals to thrive in areas of the Baltic Sea with salinities up to 18 ‰. These animals seem to avoid freshwater habitats such as lakes and streams connected to the Baltic Sea (J Conchol (2004) 38: 305). It is currently unclear whether high salinity tolerance is an original trait of this species (having broad reaction norms that may have been shifted by local adaptation) or a derived feature which was newly developed during re-colonization of the Baltic Sea from freshwater habitats after the last glaciation period. Using biochemical tests, we have confirmed that Theodoxus individuals from brackish water are much more capable of regulating cell volume by the accumulation of free amino acids in foot muscle tissue than individuals from freshwater. Comparisons of protein expression patterns of soluble foot muscle proteins using 2D-gel electrophoresis and silver staining revealed that some proteins seem to be regulated according to the physiological condition of the animal (phenotypic plasticity) while others are differentially expressed in a lineage-specific manner (indicating genetic differentiation).

Working hypotheses and work plan: Using animals from the two lineages collected at three different sites each, we will aim to answer the following questions: (1) Is there a shift in reaction norms in animals to different osmolalities due to plastic responses in biochemical traits? Test: Determine osmotolerance ranges of individuals which have been kept at different salinities for up to 8 weeks. (2) Is the range of osmotolerance inheritable? Test: Breeding of animals of one population over at least 2 generations under controlled conditions and testing the osmotolerance ranges (survival, sugar and amino acid accumulation) in parents and their offspring. (3) Are there qualitative or quantitative differences in mRNA / protein expression in animals of the two lineages which provide evidence for genetic differentiation? Test: N-terminal sequencing of proteins isolated from 2D-gels, comparison with data from transcriptome analyses, Western blotting, in situ-hybridisation for analyzing tissue-specific expression and function.

>>>> Jan Knobloch, Christian Müller & Jan-Peter Hildebrandt (2nd cohort) <<<<

Thesis topic: Molecular basis of phenotypic plasticity in osmoregulation in Theodoxus fluviatilis.

State of the art: Phylogeographic studies have revealed that Theodoxus fluviatilis (Gastropoda: Neritidae) has formed regional subgroups in northern Germany, which seem to occur either in freshwater or in brackish water (Mol Ecol 14: 4323; Mol Phylogenet Evol 42: 373). Individuals from freshwater (FW) and brackish water (BW) habitats are not easily distinguishable by parameters such as shell size or patterning (J Conchol 38: 305) and are genetically uniform with respect to mitochondrial DNA markers (Mol Ecol 14: 4323; Mol Phylogenet Evol 42: 373). In their responses to salinity changes, however, snails from the two subgroups do differ (Biol Zentralbl 79: 585; Estuar Coast Mar Sci 6: 409; J Comp Physiol B 180: 337). Moreover, freshwater animals show different survival rates in experiments with different medium salinities than those from brackish water. Thus, we have labelled the animals from limnic populations as FW-ecotype and the animals from brackish water habitats as BW ecotype. Theodoxus uses organic osmolytes (urea, amino acids) to balance cell volume during changes in medium salinity. We could recently show, however, that the pathways of organic osmolyte accumulation for cell volume regulation are different in animals of the two ecotypes, and may have different energetic requirements more or less limiting survival under stressful conditions (Wiesenthal et al., 2018). Other factors (inorganic ions, ion transporters, skin water permeability) may be relevant as well and are being considered in ongoing studies. These studies contribute to the general understanding of how osmoregulatory organs in animals are fitted to functional needs, either by subtle genetic adaptation or by phenotypic-plastic adjustments. The studies using individuals from different T. fluviatilis subgroups allow the identification of factors limiting or facilitating survival under unfavourable medium salinities. This may ultimately enable us to derive assumptions about population persistence under conditions of environmental change.

Working hypotheses and work plan: This project contributes to the framework of RESPONSE by studying the question: To which degree are the limits of osmotolerance observed in two ecotypes of the neretid snail Theodoxus fluviatilis based on genetic adaptation or phenotypic plasticity? Based on our previous results, we conclude that energetic constraints and contributions of other osmolytes (inorganic ions), their transporters and water permeability of the integument may be truly limiting factors for survival under osmotic stress. We will further investigate the reasons why reaction norms of FW- and BW-ecotypes cannot be made to match by stepwise acclimation of the animals to alien salinities. Comparison of protein expression patterns of foot muscle proteins (gel-based and gel-free; cooperation with the proteome analysis group headed by K. Riedel) in conjunction with the analysis of qualitative or quantitative differences in mRNA patterns (we have sequenced the Theodoxus transciptome during the initial period of this project) in animals of the two origins may reveal possible markers for processes or mechanisms of osmotolerance in this species. Understanding the physiology and the biology of this species is the essential prerequisite for understanding how populations may respond to the current environmental changes. In addition, we want to develop molecular markers that enable us to determine the genetic relatedness of individuals from different populations to better understand the current distribution pattern of the species in northern Germany (re-colonisation after the glacial period and adaptation of the FW- and BW-ecotypes). Our overall aim is to derive conclusions about how populations cope with changing salinities in-situ or through range shifts.

Press release from the Estonian University of Life Sciences (Cooperation with Daniel Herlemann)

>>>> Laura Fuchs, Christian Müller & Jan-Peter Hildebrandt (3rd cohort) <<<<

Thesis topic: Osmotolerance in the snail Theodoxus fluviatilis

State of the art: Phylogeographic studies have revealed that Theodoxus fluviatilis (Gastropoda: Neritidae) has formed regional subgroups in northern Germany which seem to occur either in freshwater or in brackish water (Mol Ecol 14: 4323; Mol Phylogenet Evol 42: 373). Individuals from freshwater and brackish water habitats are not easily distinguishable by parameters as shell size or patterning (J Conchol 38: 305) and are genetically uniform with respect to mitochondrial DNA markers (Mol Ecol 14: 4323; Mol Phylogenet Evol 42: 373). In their responses to salinity changes, however, snails from the two lineages do differ (Biol Zentralbl 79: 585; Estuar Coast Mar Sci 6: 409; J Comp Physiol B 180: 337). Moreover, freshwater animals show different survival rates in experiments with different medium salinities than those from brackish water. Theodoxus uses compatible osmolytes (amino acids) to balance cell volume during changes in medium salinity. We could recently show, however, that amino acid biochemistry is obviously not a strictly limiting factor for survival during osmotic stress. This indicates that other factors (inorganic ions, ion transporters, skin water permeability) are significantly contributing. Future studies are planned to identify factors limiting or allowing survival under unfavourable medium salinities and enable us to estimate which of them may be determined genetically or may respond plastically to changes in environmental conditions.

Working hypotheses and work plan: We will further investigate the reasons why reaction norms of freshwater- and brackish water-ecotypes of Theodoxus fluviatilis cannot be made to match by stepwise acclimation of the animals to alien salinities. Our hypothesis is that this is due to genetic differences between the ecotypes. Based on the preliminary results of our genome sequencing project (PacBio) in cooperation with the Institute of Clinical Molecular Biology (IKMB) we will perform DNA re-sequencing with individuals from either ecotype and from within populations to compare these sequences and identify genetic differences related to local adaptations.

>>>> Christine Reusch & Gerald Kerth (1st cohort) <<<<

Thesis topic: Costs, benefits and constraints of responses to recent climate change in bats.

State of the art: Recent studies have reported decreasing body size as a response to climate change in various taxa, a phenomenon termed ‘global shrinking’ (Nat. Clim. Change 1: 401). As body size and fecundity are often positively correlated, shrinking body size may lead to higher extinction risks. Interestingly, in a few species increasing body size in response to climate change has been observed, which may occur in species needing warm weather for growth, such as bats. However, the fitness implications of such poorly documented trends are currently unclear. Bats are particularly interesting for studying the influence of climate change on body size and its relations to individual fitness, because they are long-lived, of high conservation concern, and show a number of behaviours that allow them to cope with weather variation, e.g. social thermoregulation, torpor, and roost switching.

Working hypotheses and work plan: This project will study the morphological and behavioural responses to variation in weather conditions in three bat species. For Myotis bechsteinii, long-term field data and detailed genetic data are available for several populations, where all bats are individually marked with RFID-tags. We also possess comparable long-term data for a number of populations of Myotis nattereri and Plecotus auritus. In this project we will test whether (1) ambient temperature affects body size directly and indirectly via food availability (2) social factors such as group size also influence body size via social thermoregulation, and thus can buffer the effects of the weather and (3) body size has an effect on the fitness, in particular during extreme weather events. The study will be carried out in collaboration with the group of Prof. Dr. F. Schweitzer, ETH Zurich (social network analyses) and Dr. A. Scheuerlein, MPIDR Rostock (demography). The work plan includes (1) analyses of the long-term data on morphology, demography, behaviour, genetic composition of populations, and individual fitness, (2) experimental manipulations of roost quality to quantify behavioural responses to changing temperatures (3) use of parent-offspring regressions and more advanced methods such as “animal models” to estimate the heritability of body size (4) comparisons of species-specific responses among different study sites.

>>>> Carolin Mundinger, Caroline Schöner & Gerald Kerth (2nd cohort) <<<<         

Thesis topics: Relative importance of plastic and genetic responses to weather conditions in long-lived bats.

State of the art: Smaller body sizes in response to global warming have been described in various taxa (‘global shrinking’; Nat Clim Change 1: 401). As body size and fecundity are often positively correlated, shrinking body size may lead to declining populations and ultimately higher extinction risks. Interestingly, in a few species increasing body size in response to climate change has been observed (Nat Clim Change 1: 401), which may occur if warm weather is required for growth. However, the fitness implications of such poorly documented trends are unclear. Bats are long-lived, of high conservation concern and show behavioural
responses such as roost switching and social thermoregulation, as well as physiological responses such as torpor that allow them to cope with weather variation. Torpor, which is characterised by a reduction in metabolic rate and body temperature, allows bats to save energy during adverse conditions when food availability is low. At the same time, frequent use of torpor during the breeding season can slow down the growth of the juveniles. Evidence for possible cost-benefit trade-offs have also been found for behavioural responses to changing environmental conditions such as the timing of the departure from the hibernaculum in spring. Early departure increases the mortality risk in some years (Reusch et al. submitted), but may be beneficial in others when the food availability is high during warm weather conditions in late winter/early spring. In combination with their longevity and low annual reproductive output, such trade-offs and possible limits/costs of phenotypic plasticity make bats highly interesting for studying the potential impact of climate change on individual fitness and ultimately population/species persistence.

Working hypotheses and work plan: This project contributes to RESPONSE by studying fitness consequences of behavioural and life-history responses to varying weather conditions (proxy for climate change) in free-ranging, individualised populations of four long-lived bat species.Building upon the findings from the first funding period outlined above, our next research goals are: (1) to assess to which extent the observed fitness-relevant responses are caused by genetic factors or phenotypic plasticity (2^nd cohort PhD-topic); and (2) to which degree the relative impact of genetic versus plastic responses differs among populations of the same species as well as between species and how this may effect population persistence (3^rd cohort PhD topic, as well as two associated doctoral researchers, whose funding has already been secured). The aforementioned previous results underline the importance of individualised long-term data sets for studying fitness consequences of responses to environmental changes in long-lived species. The existing field and genetic data of up to 25 years will thus be combined with new (experimental) data collected during the envisaged second funding period to quantify the influence of weather conditions on the behaviour, morphology, reproductive success and survival of RFID-tagged bats belonging to four species (M. bechsteinii, M. daubentonii, M. nattereri, Plecotus auritus). In the recently started PhD-project (2^nd cohort), multigenerational family pedigrees in combination with animal models are used to measure the heritability of body size and other potential fitness-relevant traits such as departure timing from the hibernaculum or age at first reproduction. We will also perform roost selection experiments and manipulations of the micro-climate of roosts to test for the influence of roosting temperatures on body size of the juveniles growing up in these roosts. This will allow us to get further insights into the possible effects of global warming on the body size of our study species. In the second PhD-topic of the envisaged second funding period (3^rd cohort), we aim to test whether behavioural, demographic and morphological responses to varying weather conditions differ between syntopic bat species as well as among populations of the same bat species. We will analyse to which extent local environmental conditions and social factors influence the fitness consequences of the observed responses to weather conditions. Here, our ultimate goal is to understand to which degree the responses to environmental conditions vary within species (e.g., in replicated populations living in different regions of Germany, studied by the RTG-paid as well as associated doctoral researchers) and to use these data in order to model the fate of the studied populations under different global change scenarios, in collaboration with the project P.

>>>> Janis Wolf, Alexander Scheuerlein & Gerald Kerth (3rd cohort) <<<<

Thesis topic: Responses within and between populations of four bat species to climate change scenarios and consequences for population persistence.

Background: Decreasing body sizes in response to global warming have been described in various taxa (‘global shrinking’; Nat Clim Change 1: 401). As body size and fecundity are often positively correlated, smaller body size may lead to a reduction in population growth rate and ultimately to lower population size and higher extinction risk. Interestingly, in a few species increasing body size in response to climate change has been observed, which may occur when warm weather is required for growth. The fitness implications of such poorly documented trends are unclear. Bats are long-lived, of high conservation concern and show behavioural responses that allow them to cope with weather variation, such as roost switching and social thermoregulation, as well as physiological responses such as torpor. Torpor, which is characterised by a reduction in metabolic rate and body temperature, allows bats to save energy during adverse conditions when food availability is low. At the same time, frequent use of torpor during the breeding season can hamper growth of the juveniles. Evidence for possible cost-benefit trade-offs have also been found for behavioural responses to changing environmental conditions such as the timing of the departure from the hibernaculum in spring. Early departure increases mortality risk in some years, but may be beneficial in others, when food availability is high during warm weather conditions in late winter/early spring. In combination with their longevity and low annual reproductive output, possible limitations and costs of phenotypic plasticity, as well as underlying trade-offs, make bats a prime candidate to study the potential impact of climate change on individual fitness and population persistence. 
For more than 25 years, our group has been studying group-living and conservation-related questions in bats, using behavioural, ecological, molecular, physiological and demographic methods. This includes automatic monitoring of animals marked with RFID-tags as well as DNA genotyping and sequencing. As a result, long-term individualised field data and detailed genetic data are available for populations of four bat species living in different areas in Germany. So far, we could show that in female Myotis bechsteinii, long forearms were associated with lower survival in our study population in Franconia, for which we have a data set spanning 25 years. Moreover, we could show that rare catastrophic events during the hibernation period have a strong negative effect on population dynamics in different bat species. Our results suggest that long-lived hibernating bat species have the potential to plastically adjust to changing climatic conditions, but that this potential differs between species.

Working hypotheses and work plan: Building upon our previous findings, our next research goals are to assess to which degree the relative impact of genetic versus plastic responses differs among populations of the same species as well as between species and how this may effect population persistence. In the proposed project, we aim to test whether behavioural, demographic and morphological responses to varying weather conditions differ between syntopic bat species as well as among populations of the same bat species. Our ultimate goal is to understand to which degree the responses to environmental conditions vary within species (e.g., in replicated populations living in different regions of Germany) and to use these data in order to model the fate of the studied populations under different global change scenarios.
The relative importance of larger body size (in response to increasing temperature), behaviour (e.g., phenology) on fitness (survival, reproductive success) and ultimately population persistence evaluation will be done in cooperation with our local partners Daniel Romero Mujalli (Projekt P), J. Kreyling (A6) and our external partners: J. Gampe from the Max-Planck-Institute for Demographic Research in Rostock and M. Dammhahn (Univ. of Potsdam).

>>>> Maria Grimm, Jörg Bernhard & Katharina Riedel (2nd cohort) <<<<

Thesis topic: The lichen microbiome and its role in adaptation to climate change associated factors.

State of the art: Symbiotic communities such as lichens represent a common and very successful lifeform on earth. Lichens consist of a heterotrophic fungus (mycobiont) and an autotrophic photosynthetic partner (photobiont), generally green algae and/or cyanobacteria. Within the last decade, bacterial communities were identified as surprisingly abundant, stable, specific, and structurally integrated partners of the classical lichen symbiosis. Recently, the application of integrated culture-independent meta-omics approaches has added valuable knowledge on the lichen symbiosis, not only about the taxa involved in lichen interactions, but also about their contributed functions. There is now strong evidence that the diverse microbiota contributes multiple aspects to the symbiotic system, including health, growth, and fitness. A similar observation has recently been made for Drosophila melanogaster, where physiological responses to changes in environmental conditions have been shown to arise from the resident microbial community (Fly 12: 1). We thus believe, that the strategy of functional diversification supports longevity of lichens under harsh conditions and that the highly versatile microbial community enables the lichen holobiont to cope with environmental changes. 

Working hypotheses and work plan: This project contributes to RESPONSE by investigating whether composition and functionality of the microbiome of lichens, defined as complex symbiotic communities composed of fungal, algal and bacterial partners, plays a pivotal role in the successful response to environmental changes, i.e., global warming. We hypothesise, that changes in the composition and functionality of its microbiome enable the lichen holobiont to cope with environmental changes. The composition of the microbiome can be considered as ecological trait mediating a kind of ‘extended’ phenotypic plasticity. This project aims thus to decipher molecular mechanisms of the model lichen L. pulmonaria and especially of its microbiome during adaptation to varying climate conditions, e.g., temperature and moisture. Comprehensive state-of-the-art meta-proteomic analyses of the protein profiles of all lichen partners combined with phenotypic analyses of lichens sampled at different geographic sites (2^nd cohort PhD-topic) or, if feasible, kept under defined conditions in climate chambers, will be used to test this hypothesis. As a starting point, we will continue to improve protein extraction and mass spectrometric analysis techniques aiming at a higher sensitivity, sample throughput and effectiveness while measuring lichen metaproteomes. This will be followed by a comprehensive characterisation of the genetic and phenotypic plasticity as well as metaproteomic analyses of lichen samples, collected at different sites in Austria, Croatia, North Germany, Denmark and Sweden during different seasons (2^nd cohort). These sampling sites differ in altitude, rainfall, average temperature, summer-winter temperature range and the influence of alpine and marine environmental parameters. Moreover, we will determine lichen fitness factors such as CO₂-emission, chlorophyll content, probably stictic acid content (a secondary metabolite repelling small grazing herbivores) as well as growth, which can subsequently be used to validate our metaproteomic data by in-situ analyses of laboratory-cultured L. pulmonaria subjected to different temperature and moisture conditions or other environmental stressors (2^nd cohort). Global scale analyses, integration, and visualisation of local climate, phenotypic and omics-data by using in-house designed bioinformatics tools for the separate partners of the holobiont will lead to a more detailed description and deeper understanding of functional, metabolic and molecular interactions among the symbiotic partners and their genotypic and phenotypic plasticity with varying environmental conditions (3^rd cohort PhD topic). Finally, in order to be able to explicitly define the role of the microbiota in adjusting the lichen to a changing environment, reciprocal transplantation experiments will be carried out in the longer term. For this purpose, lichens from higher Alpine locations are to be transplanted to lower locations and vice versa. Environmental parameters are recorded before and after transplantation with data loggers and the transplants and endemic reference samples are characterised metaproteomically and compared (3^rd cohort).

>>>> Anteneh Bogale, Jörg Bernhard & Mia Bengtsson (3rd cohort) <<<<

Thesis topic: Establishing advanced techniques for metaproteome analysis to clarify the role of the L. pulmonarias microbiome.

State of the art: Lichens consist of a heterotrophic fungus (mycobiont) and an autotrophic photosynthetic partner (photobiont), generally green algae and/or cyanobacteria forming a symbiotic meta-organism. Additionally, lichen associated bacterial communities were identified as stable, specific, and structurally integrated partners of this symbiosis. Our culture-independent meta-omics approaches have created a comprehensive data collection (along geographical/climatic gradients) and have added valuable knowledge to the lichen symbiosis, not only about the taxa involved in lichen interactions, but also about their contributed functions (Front. Microbiol. 17:180). We found, that the microbiota seems to contribute multiple aspects to the symbiotic system, including health, growth, and fitness(ISME J. 9:412, J Proteome Res. 16:2160, Microbiome 5:82). Some of the data indicate, that the strategy of functional diversification may support longevity of lichens and survival under harsh conditions. The highly versatile microbial community may enable the lichen holobiont to adapt to environmental changes (such as global warming). Our project complements RESPONSE by investigating phylogenetic and functional changes of the lichen symbiotic community affected by different climate conditions. 

Working hypotheses and work plan: This project will apply new data analysis techniques to reveal molecular mechanisms of the model lichen L. pulmonaria during adaptation to environmental changes along climatic temperature and moisture gradients. Preliminary analyses, comparing samples of L. pulmonaria from a variety of European sites suggest the presence of a partly stable microbiome, however, also indicated significant differences between the proteomes of several lichen microbiomes in contrast to the rather stable fungaland algal protein profiles (see also J Proteome Res. 16:2160). We hypothesize, that especially changes in the composition and functionality of the microbiome enable the lichen holobiont to cope with environmental changes. We will extend our current knowledge: by (1) a deeper analysis of the already collected data from the geographic/climatic gradients, (2) applying more advanced data processing techniques such as multiple missing value imputations and multiple and corrected statistic testing to perform reliable statistical analysis (Masterthesis Jasmin Franke, 2020), (3) evaluating the generated hypothesis by specifically designed transplantation experiments in close collaboration with the Karl-Franzens-University Graz and (4) supporting the application of computer models predicting the properties of the sampling site from the found constituents of the microbiome.

>>>> David Würth, Pascal Eusemann & Martin Schnittler (1st cohort) <<<<

Thesis topic: Comparative individual performance in adult and offspring cohorts of Abies alba and Picea glauca.

State of the art: Within the life span of long-living trees the local climate may change to a degree that would not allow the very same individuals to establish again today. Therefore, the individuals performing best today may not necessarily be those that will perform best tomorrow, but once established, they still produce most of the offspring. This allows for two contrasting adaptation scenarios: (1) In undisturbed habitats with dense stands intraspecific competition is high. Plants with ‘modern’ genotypes well adapted to current conditions suffer from competition with established plants adapted to past conditions, decreasing the speed of adaptation. (2) In contrast, disturbed or novel habitats (e.g. northwards or upslope of current treelines) may provide unoccupied space for seedling recruitment. Selection in trees acts mainly during establishment. In these environments, selection for modern genotypes should therefore act faster than under stable conditions. This will be tested in white spruce (Picea glauca) which forms monospecific forest and treeline stands in the North American subarctic, a region of pronounced warming during the last decades. To study of the applicability of these processes over species and ecosystem boundaries, we will also study silver fir (Abies alba), which occurs in a mosaic of old-growth and frequently disturbed stands in Germany. In contrast to the temperature-limited Picea forests, Abies is limited by interspecific competition in these environments.

Working hypotheses and work plan: We will compare populations representing undisturbed (closed canopy sites) and disturbed conditions (disturbed or arctic treeline sites). Both adult trees and established seedlings (>30 cm tall) will be mapped, measured and genotyped. As a first step, the relationship between individual performance (annual increment, photosynthetic rate, fungal parasite load) and fitness (seed set and number of established offspring) will be investigated for the adult cohort. We will then measure performance of the offspring (yearly height growth, photosynthetic rate, fungal parasite load) to study whether those parents performing best also produce the most and best performing offspring. All plants will be genotyped and pedigrees reconstructed using microsatellite markers. This will allow calculation of heritability in traits related to fitness and estimation of genetic and plastic components of trait expression. Additionally, trait plasticity under natural conditions will be assessed using naturally occurring clones.

>>>> Melanie Zacharias, Manuela Bog & Martin Schnittler (2nd cohort) <<<<

Thesis topic: Phenotypic plasticity and local genetic adaptation in Picea glauca.

State of the art: Rapid warming (Clim Change 46: 159) has a clear impact on growth of Picea glauca, the main timber tree in Alaska. Trees that were well-adapted to local conditions as seedlings may turn out to be less well adapted towards the end of their life span, if local conditions have changed in the meantime (e.g., because of global warming), unless individuals are backed up by high phenotypic plasticity. Annual growth is a primary phenotypic trait of practical and commercial interest that can be related to the fitness of a tree (Evol Appl 9: 271) and it is also suggested that growth rate can be related to local adaptation (Glob Change Biol 19: 1645). Adaptation in turn is difficult to show with neutral markers such as microsatellites (Int J Mol Sci 12: 3966) and especially challenging in forest trees (Mol Ecol 17: 3599). Consequently, most recent studies focusing on the adaptive potential of forest trees try to identify fitness-related SNPs (Mol Ecol 27: 1428). An atlas of SNPs for Picea glauca which is anchored onto a gene catalogue (Mol Ecol Resour 13: 324) and a workflow to identify potentially adaptively relevant genes, is available (Appli Plant Sci 1: 1200179).

Working hypotheses and work plan: This project contributes to RESPONSE by studying plastic and genetic components of trait variation (annual growth, needle morphology) in core and edge populations of white spruce (Picea glauca) in Alaska, a region experiencing a rapid change towards a warmer and drier climate. During the first funding period, it became obvious^4,5 that annual growth exhibits high plasticity, caused by micro-site conditions and competition between trees². Annual growth data (assessed in partner project B2) are available for many subsequent years, yet shows high plasticity. However, such data are only easily available for trees above 5 cm dbh. Within the currently running PhD project (2^nd cohort), we therefore plan (1) to test micro-increment borers to record annual growth for younger trees, and (2) quantify needle morphology (length, width, stomatal density as a second target trait available for trees of all ages) by a semiautomatic analysis of stacked macro images. Using a reciprocal transplant experiment (Brooks Range vs. Interior) carried out in the first funding phase of project B2, we will compare the morphological needle traits between the two provenances under different environmental conditions and test if there are significant differences. This way we aim at assessing (1) phenotypic plasticity (one transplanted tree – two environments) and (2) local genetic adaptation (trees perform better at the site of origin, measured as survival rate and annual growth, in the experiment available also for young trees through yearly perimeter measurements). To test the initial hypothesis that best-performing individuals are not necessarily the fittest ones, we will now perform paternity analysis with offspring from seeds collected in 2017, since we could not reach statistical power with the low numbers of putative offspring found by parentage analysis. Fitness will be estimated via seedling performance (survival rate and annual growth). For annual growth - measured by coring (old trees), or via perimeter (offspring raised from seeds) - and needle morphology, this setting allows us to estimate heritability via parent-offspring regression. Using a selection of differently performing trees from (1) seedlings raised in common garden and (2) trees with low and high relatedness from the field, we will search for fitness-related SNPs (which in our case can be expected for genes that play a role in drought adaptation), since we assume summer drought to be the key factor for adaptation to changing climate. Making use of the climate gradient between the tree plots, the 3^rd cohort PhD project will assess the adaptive potential, using the identified SNPs to see to which extent local environmental conditions influence tree performance. In addition, by use of aerial photographs from different time periods we will examine if individuals in advancing treelines differ in SNP patterns from those in long established forests.

>>>> Kristina Kuprina, María Sánchez Luque, Manuela Bog & Martin Schnittler (3rd cohort) <<<<

Thesis topic: The role of microenvironment on plant performance: Influence of habitat adapted symbioses at the treeline in White Spruce.

State of the art: White spruce (Picea glauca) is the main northern and elevational treeline species in Alaska, forming often monospecific stands. A previous study shows that the genetic differentiation of populations as measured with neutral markers is low, which hints towards effective long-distance gene flow mediated by pollen. Further, it was found that most of the variance in growth performance of individual trees could not be explained by genetic constitution or mesoclimatic differences between plots, thus yet unknown microenvironmental factors appear to be crucial (Zacharias et al., in prep.). Symbiotic interactions represent one part of the microenvironment and it was documented that members of the Pinaceae family will not grow into adult trees without their co-dispersed ectomycorrhizal fungi (Plant Soil 454:3) which gives them a decisive role in the development of the trees. It was further shown that symbioses can lead to an advantage at the seedling stage of long-lived trees (J Ecol 108:908) and increase the fitness to abiotic stress which may play a big role in how plants react to climate change (PLoS ONE 6: e14823). In addition, variation in trait expression and overall tree performance seems to be very high, which makes sense since climate may change during the lifetime of a tree (PLoS Biology 8: e1000357). In trees, selection is mainly effective during establishment (Theor. Popul. Biol. 42: 172) and site conditions are crucial for recruitment success. In cooperation with the working group Prof. Wilmking (B2) which records growth performance and will try to trace back abiotic microenvironmental factors, we will focus on symbiotic interactions of white spruce.

Working hypotheses and work plan: Subject to the global health situation, individual trees in the established plots in Alaska will be examined for their symbiosis partners by DNA metabarcoding to assess the influence of microbial interaction partners on tree performance and to identify possible benefits of such interactions at extreme locations (treeline) compared to reference locations (forest stands). Already established SSR markers will be used to assign sampled fine roots to already known genotypes for the trees (Canad. J. Forest Res. 48:1577). Further comparative cultivation studies with inoculated and non-inoculated tree seedlings under controlled conditions are planned to test if the choice of mycorrhizal partners reflects local genetic adaptation or will be determined by the micro-environment. The project is carried out in close cooperation with project B2 (Prof. Wilmking). Within the frame of a structured PhD school, field work in exciting landscapes, with a highly motivated team and the opportunity to write joint publications will help you to avoid the pitfalls of a “lonely PhD” and make the best out of your skills.

>>>> Lena Muffler, Andrey Malyshev & Jürgen Kreyling (1st cohort) <<<<

Thesis topic: Phenotypic plasticity and its heritability in recruitment cohorts of F. sylvatica under various climates.

State of the art: Fagus sylvatica (European Beech) is the dominant natural forest tree in Central Europe and thrives under a wide range of climatic and environmental conditions. Yet, F. sylvatica is expected to suffer from climate change due to a low seed dispersal capacity and drought sensitivity. Phenotypic plasticity, however, within and among populations is high and can be expected to buffer against climate change in situ. Local adaptation to different climates has been demonstrated in common garden experiments. Limits of phenotypic plasticity and consequences of selection by local climate on genetic constitution, however, are unclear. Such knowledge will ultimately allow for improved projections of range shifts, and provide guidance for long-term sustainable forest management.

Working hypotheses and work plan: We will quantify phenotypic plasticity and genetic adaptation at the recruitment stage of F. sylvatica using individuals that germinated and established under local climates different from their origin. Failure to establish will indicate limits of plasticity (and adaptation). We will study the importance of phenotypic plasticity of mothers (quantified by tree ring analyses) and their offspring under various climates, allowing estimating heritabilities and additionally genetic variation using neutral markers (microsatellites). We will focus on establishment and juvenile growth as traits of crucial importance for fitness. Seeds from several mother trees, originating from different populations covering major climatic gradients over the species’ range, will be exposed to the full range of ambient conditions at all places of origin (full reciprocal transplantation) and most interestingly also to conditions well beyond the current range. In addition, the importance of selection through drought stress on establishment will be quantified for individuals with or without drought stress during establishment. Establishment and survival will further be tested in climate chambers for causal interpretation of field data.

>>>> Jonas Schmeddes, Andrey Malyshev & Jürgen Kreyling (2nd cohort) <<<<

Thesis topic: (1) Phenotypic plasticity in recruitment cohorts of F. sylvatica under various climates. (2) Phenotypic plasticity and genetic adaptation to climate in F. sylvatica.

State of the art: Dominant temperate-zone tree species such as F. sylvatica thrive under a wide range of climatic and environmental conditions (Ann Forest Sci 63: 355). Yet, F. sylvatica is expected to suffer from climate change due to a low seed dispersal capacity and its drought sensitivity (Glob Change Biol 12: 2163; Trees 21: 1). Phenotypic plasticity within and among populations, however, is high (Agr Forest Meteorol 180: 76; Silvae Genet 44: 343) and can be expected to buffer against climate change in situ. Furthermore, F. sylvatica shows high genetic diversity within populations, fairly low within-region differentiation but distinct genetic variation over larger scales across Europe (New Phytol 171: 199). Local adaptation to different climates has been demonstrated in common garden experiments. Limits of phenotypic plasticity and consequences of selection by local climate on genetic constitution, however, are unclear. Such knowledge will ultimately allow for improved projections of range shifts, and provide guidance for long-term sustainable forest management. This project adds to RESPONSE by investigating phenotypic plasticity and genetic adaptation within and among populations and their role for the continued success of a key forest tree

Working hypotheses and work plan: This project contributes to RESPONSE by studying potentials and limits of phenotypic plasticity and genetic adaptation in populations across the range of Fagus sylvatica, the dominant natural forest tree of central Europe. We quantify and differentiate among phenotypic plasticity and genetic adaptation of F. sylvatica by a series of common garden experiments (allowing for the quantification of genetic adaptation) along a climatic gradient (i.e. under different environmental conditions, allowing for the quantification of phenotypic plasticity) in the reciprocal transplantation experiment (1^st cohort). Failure of plants to establish will indicate limits of plasticity and adaptation. Studying phenotypic plasticity (1^st cohort) and genetic variance of mothers and their offspring under various climates, furthermore allows us to estimate heritabilities (2^nd cohort). Additionally, genetic variation using neutral markers (microsatellites), and for selected samples from different origins, SNPs and epigenetics will be analysed by cooperation partners (2^nd cohort). We will focus on growth and phenology as traits of crucial importance for fitness, stomata density and SLA as stress-related traits, and leaf area as morphological trait. The obtained estimates of plasticity, heritability, and genetic adaptation will be used to inform hybrid species distribution models (e.g.,Ecol Lett 19: 1468) in order to estimate species persistence under climate change (3^rd cohort). In addition to the reciprocal translocation experiment, the importance of selection through drought and frost stress on establishment are quantified for individuals with or without drought or frost stress during establishment (1^st cohort & 2^nd cohort).

>>>> Niels Preuk, Daniel Romero Mujalli & Jürgen Kreyling (3nd cohort) <<<<

Thesis topic: Species persistence and future distribution of Fagus sylvatica - the role of genetic adaptation, phenotypic plasticity and its heritability

State of the art: Dominant temperate-zone tree species such as Fagus sylvatica thrive under a wide range of climatic and environmental conditions (Ann. Forest Sci. 63: 355). Yet, F. sylvatica is expected to suffer from climate change due to a low seed dispersal capacity and drought sensitivity (Glob. Change Biol. 12: 2163; Trees 21: 1). Phenotypic plasticity within and among populations, however, is high (Agr. Forest. Meteorol. 180: 76; Silvae Genet. 44: 343) and can be expected to buffer against climate change in situ. Furthermore, F. sylvatica shows high genetic diversity within populations, fairly low within-region differentiation but distinct genetic variation over larger scales across Europe (New Phytol. 171: 199). Local adaptation to different climates has been demonstrated in common garden experiments. Limits of phenotypic plasticity and consequences of selection by local climate on genetic constitution for species persistence, however, are unclear. A better understanding of these factors and their incorporation into projections of species persistence and potential future range shifts are crucial to provide guidance for long-term sustainable forest management.

Working hypotheses and work plan: We will quantify the importance of phenotypic plasticity and genetic adaptation of F. sylvatica for projections of species persistence and range shifts. Existing data of phenotypic plasticity, heritability, and genetic adaptation will be used to constrain hybrid species distribution models (e.g. Ecol. Lett. 19: 1468) and to develop species distribution models taking within-species variability into account (e.g. Ecol. Evol 3: 437-449). This project focuses on modelling and profits from existing data. Additional field sampling in existing, long-term provenance trials is possible, but not mandatory.

>>>> Mathilde Borg Dahl, Thomas Hoppe & Martin Schnittler (1st cohort) <<<<

Thesis topic: Genetic diversity, reproductive options and gene flow in the genus Meriderma.

State of the art: All mechanisms to cope with changing environments, plastic responses, genetic adaptation and dispersal in search for new habitats infer costs. Which of these constitutes a suitable way for free living protists will be investigated for plasmodial slime molds (myxomycetes). These organisms inhabit soils with unicellular, haploid amoebae but form minute fruit bodies dispersing meiospores (Soil. Biol. Biochem. 43: 2237). Only well adapted genotypes can support plasmodia and are able to fruit (FEMS Microbiol. Ecol. 31: 103). We will investigate how environmental stability (time of contiguous snow cover over elevational transects) determines survival in nivicolous myxomycetes (Meriderma spp.). Only a long-lasting, stable snow cover allows amoebae to multiply at a constant temperature of 0-0.5°C and finally fruit. Beside sexual spore formation, culture studies and a model for habitat colonization (Mycol. Res. 112: 697) mount evidence for the existence of a second, apomictic life cycle resulting in fruit bodies with automictic spores (Mycosphere 4: 233).

Working hypotheses and work plan: Field work will be conducted in the northern Caucasus and in the German Alps (Fungal Divers 59: 109). Molecular work will include sequencing of marker genes (SSU, EF1a, PLoS One e22872, e35359). In addition, we will attempt barcoding using environmental PCR of soil samples and subsequent Illumina sequencing to assess genetic diversity in fruiting and non-fruiting populations. Amoebae will be isolated from soil or raised from spores to test reaction norms regarding temperature and freeze/thaw cycles. We will assess plastic/genetic components of trait variation by comparing trait expression (spore number, diameter, ornamentation and hydrophobicity) in fructifications within/between genotypes. Environmental sensitivity of selection will be estimated as change in phenotypic traits of fructifications and compared with length of contiguous winter snow cover as main environmental parameter (revealed by data loggers). Comparing genotypes in several mountain ranges we will test if local genetic adaptation or phenotypic plasticity and rapid dispersal is the best strategy for these organisms.

>>>> Jan Woyzichovski, Nikki Dagamac & Martin Schnittler (2nd cohort) <<<<

Thesis topic: Trait evolution in myxomycete fructifications in dependence on climatic conditions.

State of the art: Nivicolous myxomycetes are Amoebozoan protists (Leontyev et al.; under revision) and inhabit as amoebae the uppermost soil layer. They prey on microbial communities that live under the snow and fruit with spring snow melt to disperse airborne spores. Spore formation critically depends on autumn frost events and the duration of snow cover, although amoebal populations are found in lower elevations as well (Dahl et al.; under revision). Plasmodia form under the snow, segregate into fragments which fruit over an extended period of time with snow melt, converting their biomass within 24-48 hrs into colonies of typically 100-1,000 sporocarps. Morphologically discernible species rely on long-distance dispersal (Naturwissensch 96: 147-151), but among mountain ranges they show a pronounced genetic structure with often several reproductively isolated populations. According to Stoke’s law, terminal velocity and thus dispersal ability strongly depends on spore size; survival on the degree of melanisation (UV-shelter); and dispersal distance can be modified by spore ornaments, which make spores hydrophobic, enabling them to take off repeatedly from wet surfaces. Spore sizes vary by 2-4 µm within a species, and we expect a trade-off between the amount of transported resources and spore numbers/dispersal ability.

Working hypotheses and work plan: This project contributes to RESPONSE by studying dispersal in free-living populations of nivicolous myxomycetes that live in alpine environments, strongly affected by global warming. We investigate the influence of spore traits (size, melanisation, ornamentation) and their plastic/genetic components on dispersal in dependence on elevation and macroclimate (proxy for climate changes determining the stability of snow cover). Based on the data collected (barcoded specimens from different mountain ranges, snow cover data for 3 years), we now want to understand how environmental conditions drive the evolution of spore traits. Our hypotheses are: (1) spore size increases with elevation (spores need more resources but can rely on stronger winds in open, alpine habitats); (2) melanisation increases with elevation (higher UV radiation); (3) spore ornaments are less pronounced in drier environments (reducing costs). Within the recently started 2^nd cohort PhD-project, we are focusing on measurements of spore size as the most critical trait, developing a method to measure spore size and melanisation automatically from mounted slides (sample size ca. 20,000 spores). As an add-on, we will try to improve a method (developed during the 1^st cohort phase) for analysis of spore ornamentation via SEM. Since nivicolous myxomycetes cannot be cultivated, we will estimate heritability of spore size by measuring five sporocarps from different colonies (all members of one genotype) collected on different days. Repeatedly assessing spore size of several colonies that developed at different times under slightly different environmental conditions from the same (fragmenting) plasmodium will allow us to estimate the plastic component of variation. By comparison with other genotypes, this should allow for an estimate of broad-sense heritability. Genotypic identity will be assessed in a two-step approach, first determining the ribotype with the barcoding procedure developed in the first funding period, second applying extremely polymorphic intron markers and/or SNP genotyping. Dispersal abilities will be (1) determined by a direct experiment with compound fruiting species (providing 10⁹-10¹² spores, constructing a dispersal kernel for distances up to 500 m, M.Sc.-thesis work), and (2) by assessing the occurrence of certain genotypes in different European mountain ranges (200-3,000 km, long-distance dispersal, associated PhD student). For the 3^rd cohort PhD-project, we will use direct snow cover data (transect of the German Alps) and estimate potential snow cover from macroclimatic conditions (European mountain ranges) to model the distribution of (1) amoebal populations (including data from metabarcoding studies) and (2) fructifications, assuming different dispersal capacities (mitigated by changes in spore traits). This will be done in collaboration with project P.

>>>> Maho Inoue, ÁngelaLópez-Villalba & Martin Schnittler (3rd cohort) <<<<

Thesis topic: Local genetic adaptation through evolution of reproductive systems in myxomycetes

State of the art:Amoebae of nivicolous myxomycetes (slime molds, Amoebazoa) are common in alpine (Sci. Rep. 8: 11662) and many lowland (Fungal Ecol. 39: 80) soils, playing a crucial role in soil ecosystems (PLoS ONE e2527) as predators of bacteria. Dispersal occurs via airborne spores produced in fruiting bodies; gene flow between mountain ranges seems to be high but not unlimited (Protist 167: 234). This may lead to outbreeding depression, but many species seem to segregate by a yet unknown mechanism into reproductively isolated units (putative biospecies), which should allow for local genetic adaptation. This is the case for our model species Physarum albescens, with more than a dozen of such units, recognizable by their barcodes (Shchepin et al., in prep.) and occurring in most mountain ranges of the northern hemisphere (Dagamac et al. 2020, Fungal Ecol., in review). In addition, asexual propagation via spores may occur (Janik et al. 2020, Protist, in press). A hint on the underlying mechanisms give macrospores showing roughly the double volume of normal spores (Woyzichovski et al. 2020, submitted).

Working hypotheses and work plan:We first want to focus on the distribution of putative biospecies, using a combination of fruitbody sampling (barcoding plus genotyping by sequencing, GBS) and metabarcoding (to detect possible non-fruiting units that are out of their optimum range) to (i) assess their distribution and (ii) model ecological niches with mesoclimatic data. In addition, spore size will be quantitatively assessed via particle measurement (flow cytometry) as a proxy for dispersal potential. Subject to the global health situation, we want to extend the sampling on the entire northern hemisphere to relate gene flow between mountain ranges to differences in spore size. As a second line of evidence, we want to use GBS to test for asexual reproduction via spores (clones that cover distances that cannot be bridged by active/passive amoebal migration) to test the hypothesis that asexual reproduction is a favourable strategy to colonize marginal habitats. This could lead to a new response model for very dispersive organisms on changing environments: on one hand, long distance dispersal allows to run out changing environments, on the other hand, segregation into reproductively isolated units with limited occurrence allows local genetic adaptation.

>>>> Mario Trouillier, Marieke van der Maaten-Theunissen & Martin Wilmking (1st cohort) <<<<

Thesis topic: Role of phenotypic plasticity in range dynamics of Picea glauca under rapidly changing climatic conditions in Alaska.

State of the art: Treelines have been a classical ecological example to study the ability of species to colonise new habitats (Ecology 15: 80) or to retreat under unfavourable conditions (Ecology 86: 1687). Recent climate change is expected to lead to substantial treeline advance at leading edge populations, and dieback of trailing edge populations, though the role of increasing climate extremes is unresolved (PNAS 106: 19723). Limiting factors for tree growth in northern regions might shift from cold to drought (Nature 405: 668), possibly influenced by decreasing seasonality (Nat. Clim. Change 3: 581). Picea glauca is the main northern and elevational treeline species in Alaska, also occupying the dry edge of coniferous tree growth in Interior Alaska (Can. J. For. Res. 43: 331). Both range edges are undergoing massive environmental change (projected to intensify in the future) with associated range dynamics (Can. J. For. Res. 40: 1197), most likely not limited by dispersal (Ecology 86: 1687).

Working hypotheses and work plan: To increase the understanding on how P. glauca range edges can cope with rapidly changing mean and extreme climatic conditions in Alaska, the role of phenotypic plasticity and in situ adaptation on the advance and dieback of this tree species will be investigated. Therefore, the history of climatic stress (especially the effects of extreme events) and phenotypic variability in growth and wood anatomy of the mother trees will be quantified by tree-ring analyses (in cooperation with Dr. U. Sass-Klaassen; Wageningen University) and related to phenotypic variability in growth and wood anatomy of the offspring, identified via genetic analyses.

>>>> Timo Pampuch, Alba Anadon-Rosell & Martin Wilmking (2nd cohort) <<<<

Thesis topic: Role of phenotypic plasticity in range dynamics of Picea glauca under rapidly changing climatic conditions in Alaska.

State of the art: Treelines have been a classical ecological example to study the ability of species to colonise new habitats (Ecology 15: 80) or to retreat under unfavourable conditions (Ecology 86: 1687). Recent climate change is expected to lead to substantial treeline advance at leading edge populations, and die-back of trailing edge populations, though the role of increasing climate extremes is unresolved (PNAS 106: 19723). Limiting factors for tree growth in northern regions might shift from cold to drought (Nature 405: 668), possibly influenced by decreasing seasonality (Nat Clim Change 3: 581). Picea glauca is the main northern and elevational treeline species in Alaska, also occupying the dry edge of coniferous tree growth in Interior Alaska (Can J For Res 43: 331). Both range edges are undergoing massive environmental change (projected to intensify in the future) with associated range dynamics (Can J For Res 40: 1197), most likely not limited by dispersal (Ecology 86: 1687). Here we will investigate the role of phenotypic plasticity and in-situ adaptation on the advance and dieback of P. glauca range edges under rapidly changing mean and extreme climatic conditions in Alaska.

Working hypotheses and work plan: This project contributes to RESPONSE by studying the range dynamics of a major boreal treeline species, taking into account plastic and genetic responses to varying climate conditions. The project combines extensive field and monitoring efforts with common garden and climate manipulation experiments and is closely aligned with project A5. Building on results of the first funding period, our next research goals are to assess to which extent the observed fitness-relevant responses are caused by genetic adaptation and/or phenotypic plasticity, and to quantify the heritability of such responsive traits (2^nd cohort PhD-topic). We then will test whether the relative impact of genetic versus plastic responses depends on adaptation to local conditions of our target species. Results will inform hybrid species distribution models in scenario runs of potential range dynamics (3^rd cohort PhD-topic). Our previous results underline the importance of individualised data sets for the study of local adaptation and fitness consequences of responses to environmental changes in our long-lived target species. We now have growth records of over 1,000 genotyped trees at our sites and will 1) intensify the analysis of wood anatomical properties and 2) combine the existing field data from Alaska with new experimental tests performed along stress gradients with local populations (in our labs in Greifswald). These experimental treatments will be supplemented with continued monitoring efforts of the reciprocal transplant experiments and six established field sites (see also A5). Ultimately, functional anatomical traits will be analysed on all samples (field data, experimental treatment and transplant experiment) to test to which extent local adaptation is a prerequisite for range expansion at the leading edge and the resistance to range contraction at the trailing edge.

>>>> Saroj Basnet, Mario Trouillier & Martin Wilmking (3rd cohort) <<<<

Thesis topic: Treeline dynamics of Picea glauca in Alaska – From plot scale mechanisms to species distribution models

State of the art: Treelines have been a classical ecological example to study the ability of species to colonize new habitats (Ecology 15: 80) or to retreat under unfavourable conditions (Ecology 86: 1687). Recent climate change is expected to lead to substantial treeline advance at leading edge populations (Glob Change 23: 5509), and die-back of trailing edge populations, though the role of increasing climate extremes and local adaptation is unresolved. Picea glauca is the main northern and elevational treeline species in Alaska, also occupying the dry edge of coniferous tree growth in Interior Alaska (Can. J. For. Res. 43: 331). Both range edges are undergoing massive environmental change with associated range dynamics (Ecology Letters, 14, 373). But how well are local populations adapted to expected climatic extremes and gradual shifts in climate? Here we will combine field and modelling studies to investigate the role of phenotypic plasticity and in-situ adaptation on the advance and dieback of P. glauca range edges under rapidly changing mean and extreme climatic conditions in Alaska.

Working hypotheses and work plan:This project will take advantage of a large existing field and genetic data set of white spruce from several study sites across Alaska (over 1000 trees) and the species distribution range (over 10000 trees). Additional field work will target specific data needs, and the student will develop and “fine-tune” his/her project with the help of the supervisor after acceptance. Ideally, the student will use field-based data on growth performance, seedling success and phenotypic plasticity of white spruce to inform local and regional species distribution models. You will become part of a dynamic, international working group, and your project can develop close cooperation with project A5 (supervised by Prof. Schnittler). And, last but not least, awesome landscapes and exciting field work is waiting for you, as is the small but vibrant Baltic sea coast town of Greifswald.

>>>> Franziska Spitzner, Andy Sombke & Steffen Harzsch (1st cohort) <<<<

Thesis topic: Effects of abiotic stress on neurogenesis in the developing brain of crustacean larvae.

State of the art: Climate change already has a major impact on marine ecosystems including plankton communities (Prog. Oceanograph. 81: 207). In particular semi-enclosed seas (e.g. the Baltic Sea) will be increasingly affected by rising surface temperatures and decreasing salinity due to a higher river runoff (J. Exp. Mar. Biol. Ecol. 400: 52). Decapod crustaceans may be particularly sensitive to such changes, owing to their complex life cycle including a pelagic larval and a benthic juvenile-adult stage. Here, larval survival critically depends on the ability to respond appropriately to chemical, mechanosensory and visual cues to control tidal transport and onshore recruitment (Invert. Reprod. Dev. 49: 175). Larval settlement is of utmost importance for colonisation and population survival, relying on e.g. odour identification indicating preferred habitats and the presence of conspecifics (Mar. Freshwater Behav. Physiol. 39: 269). Hence, the ability to sense environmental cues is an ecologically important trait intimately associated with fitness. However, larval growth and bioenergetics in decapod larvae are strongly affected by thermal and osmotic stress (Invert. Reprod. Dev. 33: 159). While concomitant effects on growth patterns are well understood, knowledge on the effects of abiotic stress on the development of specific, energy-demanding organs such as the nervous system and hence on behavioural performance is lacking. Therefore, we will here explore the effect of abiotic stress on the development of the larval olfactory system and olfactory-guided behaviour.

Working hypotheses and work plan: Current climate change may expose crustacean planktonic larvae to increasing abiotic stress (see above). We hypothesise that (1) thermal and salinity stress will negatively affect neurogenesis and thereby larval behavioural performance, and that (2) negative effects will differ depending on the respective species’ ability to tolerate challenging environmental conditions. To this end, we will study the effects on osmotic and thermal stress on the development of the olfactory system in larvae of six European decapod crustaceans, which are known to differ in their stress tolerance (Aquat. Biol. 21: 249). Investigations will include (1) in vivo incorporation of an s-phase specific mitosis marker to quantify neurogenesis; (2) immunohistochemistry against neuropeptides and confocal laser-scan microscopy to analyse the formation of neuronal networks within the central olfactory pathway, (3) scanning and transmission electron microscopy to analyse the ontogeny of the chemosensory organs; (4) bioessays to analyse larval responses and sensitivity ranges to environmental stimuli. A major part of the breeding experiments will be carried out at the “Biologische Anstalt Helgoland”, a marine biological station on the island of Helgoland/North Sea in cooperation with Dr. Gabriela Torres and Dr. Luis Gimenez (Bangor, Wales).

>>>> ZoranŠargač, Jakob Krieger & Steffen Harzsch (2nd cohort) <<<<

Thesis topic: Development of crab larvae Carcinus maenas from different European populations reared under combined thermal and osmotic stress

State of the art: Decapod crustaceans are key species in many marine habitats, and crab species are renowned for their massive impact on coastal ecosystems (Environ Sci Pollut Res21: 9129). Crabs display a complex life cycle that include pelagic larvae in addition to the benthic juvenile-adult stages (Invertebr Reprod Dev49: 175). Crab larvae of the Zoea type, as the main dispersing stage, are essential for range expansion as well as for population persistence and connectivity (Biol Rev125: 3465) and represent the life history stage that is most sensitive to fluctuations of environmental parameters. Global ocean change is already affecting abiotic factors such as temperature, salinity and pCO₂ (Nat Clim Change6: 83). In particular, semi-enclosed seas such as the Baltic Sea and North Sea will be increasingly affected by rising surface temperatures and decreasing salinities due to a higher river run-off (Global Change Biol21: 117). Therefore, research on the impacts of global ocean change on marine organisms focusses on possible synergistic and antagonistic effects of multiple environmental drivers (Global Change Biol24: 2239, Ecol Lett 21: 568). For a given population, one way of reacting to environmental changes is shifting its range. In the north European seas, climate models predict that various taxa will extend their range northwards, and these models coincide with current observations (Nat Clim Change 6: 83, Evol Applicat 7: 104). Species which are not able to track their preferred environment in space may adapt in situ to avoid extinction when the rate of environmental change is high, and these new selective pressures may lead to the evolution of new genetic adaptations and increased phenotypic plasticity (Evol Applic7: 104). The larvae of decapod crustaceans are well-established models in ecological developmental biology, a discipline that seeks to examine how organisms develop in ‘real-world’ environments including insights into the organism’s evolutionary potential to adapt to the changing physical and biotic environmental conditions created by anthropogenic climate change. Because survival of early life stages of crustaceans will determine patterns of recruitment and population/species persistence, research on future species distribution in crustaceans addresses the capacity of larvae to tolerate threats such as thermal and osmotic stress (Sci Rep6: 32263, Mar Biol 150:1275, Aquat Biol 12: 249).

Working hypotheses and work plan: This project contributes to RESPONSE by quantifying the combined effects of abiotic drivers (temperature and salinity as proxy for expected ocean change conditions) on various aspects of larval quality as a key feature for a crustacean’s potential for dispersal in a multi-population common garden approach.  For marine ecosystems, multi-population comparisons using common-garden approaches (Heredity116: 249) are more and more seen as instrumental to understand local adaptation and phenotypic plasticity in order to predict pending ecosystem changes (Sci Rep6: 32263, Mar Biol 150: 1275). Therefore, we will assess if C. maenas larvae from mothers originating from different populations across the native range display different levels of resistance to environmental drivers (2^nd cohort PhD topic) to contribute insights that help us predicting range expansions of C. maenas in the future ocean (3^rd cohort PhD topic). The European coast, with a temperature gradient from Spain to Norway and a salinity gradient from the Atlantic/North Sea across Skagerak and Kattegat into the Baltic Sea, provide an ideal natural playground to analyse phenotypic plasticity vs. genetic adaptations to changing environmental parameters at the level of local populations of a single species (Evol Applic 7: 104). Quantifying larval reaction norms is important because temperature controls the dispersal potential through changes in the length of the dispersal phase and through effects on larval fitness. Exposure to low salinity can lead to either increased mortality or reduced growth in dispersive larval stages (Aquatic Biol12: 249-260). We will analyse larvae from females obtained from different local populations of C. maenas along a temperature and a salinity gradient: Norway vs. Portugal, Wales vs. Helgoland vs. Baltic. We will rear larvae of selected populations (e.g., Norway – Portugal for temperature gradient; Helgoland – Baltic for salinity gradient) under the previously tested (phase 1) regime of combined abiotic thermal and osmotic stress. As in the 1^st cohort PhD project, we will measure larval survival, developmental duration, dry weight, elemental composition. We will also measure a previously identified morphological key trait, the amount of lipid inclusions in resorptive cells (R cells) of the digestive epithelium in the midgut gland as the central metabolic organ. As additional traits, we will perform immunolocalization of NaK-ATPase (NKA) in the larval transport epithelia. For larvae obtained from all populations (extract larval rRNA on site), we will perform molecular analyses by qPCR to measure NKA, NaClC-transporter, and heat shock proteins using established primers for these enzymes in adult C. maenas. We will also quantify the NKA protein expression using Western Blot analysis. From all populations, we will obtain samples for population genetic studies that will be conducted by our project partners. Species distribution predictions of C. maenas so far have been based on thermogeography, heat tolerance and potential for thermal acclimation of adult animals (J Exp Biol217: 1129). Using modelling approaches, in collaboration with project P, we will use information on larval reaction norms in combination with available data on thermogeography of adults to contribute to predictions about the range expansion of this species in the future ocean.

>>>> Jan Phillipp Geißel, Jakob Krieger & Steffen Harzsch (3rd cohort) <<<<

collaboration partners: Dr. G. Torres & Dr. L. Giménez, Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung

Thesis topic: Global ocean change drives range expansion of marine species: exploring northern distribution limits

State of the art: Coastal and shelf seas are characterized by a high biodiversity and unique ecosystems and are fundamentally important for sustaining and enhancing human welfare and industry. It is forecasted that in the next 50 – 100 years, between 50 and 70% of the population will be living in coastal areas which creates competing demands from different sectors such as tourism, fisheries and aquaculture, on scarce coastal and shelf sea resources. Furthermore, anthropogenic stressors related to climate change already have a major impact on coastal ecosystems by affecting environmental drivers such as water temperature, salinity, pH and oxygen content. Crab species are key organisms in many marine habitats and are renowned for their massive impact in coastal ecosystems where they act as strong predators but also serve as an important link in the food web because of their complex life cycle. In many crustaceans, dispersing pelagic larvae are essential for population persistence and dispersal, and larval stages of crabs are known to be more vulnerable to fluctuations in environmental key parameters in the water column than the adult animals. Therefore, one area of research on population persistence and range expansion in crustaceans addresses the capacity of crustacean larvae to tolerate the combined effects of environmental drivers. Within the framework of RESPONSE, we have previously quantified the combined effects of abiotic stress on various aspects of larval quality in the European shore crab Carcinus maenas, an iconic member of intertidal ecosystems of many European coasts. These experimental studies were carried out at the Alfred-Wegener-Institute (AWI) at the island of Helgoland using animals from the local population of Carcinus maenas. Because multi-population comparisons are instrumental to species responses to climate change, we are also analysing if larvae of this species taken from mothers from different populations across most of its native range (from S. Spain to S. Norway) display different levels of resistance to environmental stress (temperature and salinity).

Working hypotheses and work plan: Our previous studies have suggested, C. maenas has a strong potential to expand its native range northwards along the Norwegian coast, as a consequence of global warming. The project advertised here seeks to predict the future northern range expansion of this species and will be carried out in collaboration with Dr. Gabriela Torres (AWI Helgoland) and Dr. Luis Giménez (AWI Helgoland and School of Ocean Sciences, Bangor University, Wales). In a common garden experiment, larvae from Norwegian crab populations will be reared under a previously tested regime of thermal stress at the AWI Helgoland. To that end, we will compare the performance of larvae from edge populations at the northern distribution limit with those from the core of the Norwegian distribution range. Building on the expertise from the previous project, we will measure key parameters of larval performance such as survival, developmental duration, respiration and feeding rates, biomass, and gene expression of target genes. In a second step, we will establish predictions for the northern range expansion of this species using phenological models.  

>>>> Elisabeth Reim, Isabell Karl & Klaus Fischer (1st cohort) <<<<

Thesis topic: Factors affecting dispersal ability in a butterfly.

State of the art: Current global change has caused range shifts in a plethora of species, and butterflies have become important model organisms for such range dynamics. For populations trying to colonise new habitats their potential to persist or even thrive in edge populations is of crucial importance. Edge populations are usually not a random subset of the genotypes found in central populations but are biased towards genotypes showing enhanced dispersal ability and shorter life cycles. The consequences of the concomitant increased energy allocation to such genetically-based ‘range-shift’ syndromes are hitherto poorly understood. Especially in flying ectotherms, which disperse by highly energy-demanding flight, these syndromes are expected to generate trade-offs with other energy-demanding functions, e.g. the immune response, which may feedback on the ability to persist. Investigating potential interactions with disease resistance is particularly interesting as increased heat stress resistance may be traded off against immune function, and because the distribution and prevalence of infectious diseases are predicted to change with global warming. Here we will investigate factors affecting dispersal ability in replicated populations of the currently northward expanding butterfly Lycaena tityrus, paying special attention to range-shift syndromes and associated trade-offs. Thus, we will investigate differences in and constraints on dispersal ability at the environmental and genetic level.

Working hypotheses and work plan: The replicated populations will originate from recently established edge (Estonia, Russia) and core (Germany) populations of L. tityrus. Using well-established protocols we will investigate whether proxies of dispersal ability are affected by genetic (population comparisons) and environmental (temperature, resource availability) factors, and whether they are traded off against other energy demanding functions (immune response, reproduction). Dispersal ability will be measured at the morphological (e.g. wing loading, aspect ratio), physiological (fat reserves) and behavioural (dispersal) level under laboratory and semi-natural conditions. We will test the following hypotheses: (1) dispersal ability is enhanced in edge populations; (2) core and edge populations show heritable variation in dispersal ability (parent-offspring regression); (3) dispersal ability is associated with intrinsic (morphology, physiology) and external (abiotic conditions, resource availability) factors; (4) immune response and reproduction are traded off against genetically based range-shift syndromes, with trade-offs being more pronounced in edge populations (in collaboration with Prof. R. Stoks, Univ. Leuven); (5) newly established edge populations show a reduced genetic diversity at neutral loci (microsatellites) but not at loci important for local adaptation (e.g. PGI, HSP).

>>>> Anaïs Degut, Michaël Beaulieu & Peter Michalik (2nd cohort) <<<<

Thesis topic: Genetic and environmental effects on the morphology, physiology and behaviour of a European butterfly.

State of the art: In holometabolous insects, adult characteristics, among which morphological traits related to flight capacity, are largely determined during their early stages of life (eggs, larvae; Funct Ecol 21: 38). For instance, adult caddis flies (Odontocerum albicorne), resulting from larvae with increased investment into silk defences, show lighter thoraces and smaller wings than control individuals(Proc R Soc B 266: 1049). Similarly, adult Drosophila simulans flies show smaller wings when growing under hot conditions(Evolution57: 2773), thereby suggesting that developmental thermal conditions may determine the ability of adult insects to fly and disperse. Such changes in flight ability are also likely to generate trade-offs with other energy-demanding functions (e.g., physiology, thermal tolerance). .

Working hypotheses and work plan: This project adds to the overall aims of RESPONSE by investigating the role of morphological, physiological and behavioural characteristics in the ability to disperse in the butterfly Pieris napi. We will investigate plastic and genetic variation in morphology (wing characteristics, pigmentation, body mass, thorax musculature), physiology (oxidative status, immune response, lipid composition, cuticle desiccation resistance, pigmentation) and behaviour (flight characteristics) in replicated populations of the butterfly P. napi exposed to different thermal regimes during development. We will also examine how environmental conditions affect the transmission of such mechanisms across generations. We will use replicated populations of P. napi first in Germany (1^st year) and then across a latitudinal gradient across Europe from Italy to Germany (i.e. from warm to cold environments; 2^nd year) to examine inter-relationships between morphology, physiology and behaviour and to examine their heritability under variable environmental conditions. We have chosen P. napi for this project as it is widely distributed across Europe, is easy to breed in captivity, and because of previous experience with this species in the project A4. In the second phase of RESPONSE, we will test the following hypotheses: (1) morphological, physiological and behavioural characteristics are inter-related; (2) the effects of developmental thermal conditions on the morphology, the physiology and the behaviour of butterflies vary with the latitudinal origin of butterflies; (3) flight ability determined by developmental thermal conditions is negatively related to the thermal tolerance of adults; (4) heritability is not significantly affected by thermal conditions (parent-offspring regressions will be used to estimate heritability). Based on the data collected during the 1^st and 2^nd cohort of RESPONSE, and the population genetic analysis performed during the 3^rd cohort, we will model the future distribution of this species using available climate predictions.

>>>> Dustin Kulanek, Peter Michalik & Klaus Fischer (3rd cohort) <<<<

Thesis topic: Drivers of rapid range expansion in an European butterfly: beyond climate and dispersal potential

State of the art: For investigating responses to anthropogenic climate change and specifically range dynamics, butterflies seem to comprise an eminently suitable taxon (Parmesan et al. 1999). This is because poleward range shifts, as a possible response to climate change, have been repeatedly documented, owing to particularly solid data bases in terms of species records and a high sensitivity to thermal conditions (Konvicka et al. 2003; Settele et al. 2008). Additionally, important ecological, evolutionary, and behavioural traits are particularly well understood in butterflies, including knowledge on the temperature-dependence of phenology as well as physiological and behavioural adaptations to climatic conditions (Dennis 1993; Klockmann et al. 2016). Results obtained from this model taxon will also be relevant to other climate-sensitive organisms with similar population structures such as grasshoppers, beetles, rodents, and frogs (Parmesan et al. 1999).

This study explores the factors driving rapid range expansions, exploiting the spectacular case of the southern small white Pieris mannii. This butterfly was confined to the Mediterranean and was first found north of the Alps in 2008. Since then it gradually extended its range and is now even sighted in northern Germany. Recent work on this species indicated that the recent range expansion is unlikely to be directly caused by anthropogenic climate change. Rather, expanding as compared with historical populations show a more generalist lifestyle, namely a reduced host-plant specialization, and an overall increased fecundity. Currently, it is unclear whether these genetic changes occurred before the range expansion or during the course of it.

Working hypotheses and work plan: We will here usepopulation genetic analyses to determine (1) the geographic origin of newly established edge populations, (2) the degree of genetic differentiation between edge and source populations, and (3) the potential role of genetic modifications (e.g. admixture) as a potential driver of range shifts, and (4) landscape features affecting gene flow and thus dispersal. We will apply double digest restriction-site associated DNA sequencing (ddRADseq) to call SNPs using a de novo pipeline. Using the resulting SNP panel we will (1) explore the population genetic structure (STRUCTURE), (2) investigate the impact of landscape features on population structure (distance-based redundancy analyses, dbRDA), (3) model spatial patterns of genetic diversity and gene flow (estimated effective migration surface; EEMS), (4) analyse isolation by distance, resistance and environment based on distance and resistance surfaces derived from MAXENT distribution modelling, and (5) search for outlier loci under selection (BAYPASS, FDIST2). The combination of using (i) a species with a particularly well-documented and outstandingly rapid range expansion and (ii) modern population and landscape genetic methods is expected to generate novel insights into the factors underlying a species’ ability to establish in new habitats.

We will test the following hypotheses:

1. We hypothesize that the populations established in Germany originate from southern France (subspecies alpigena). We further predict an admixture event between alpigena and the Italian subspecies rossii, which has promoted the emergence of more dispersive phenotypes and an enhanced ability to settle within the new range.
2. We expect intraspecific genetic differentiation between edge and core populations to be high, due to short-distance dispersal in combination with allele surfing. Thus, the frequency of specific alleles will be high at the leading edge, while overall genetic diversity will be low.
3. Landscape features, namely mountain regions, which strongly impact dispersal routes.
4. Newly established edge populations show local adaptation as indicated by outlier loci under selection.

Finally, we may compare our findings with those obtained from the Green-veined white Pieris napi, which is a species with a circumboreal distribution and has not shown any recent range expansions. A much less pronounced population genetic structure and a weaker impact of geographic features is expected here.

>>>> Michael Klockmann & Gabriele Uhl (1st cohort) <<<<

Thesis topic: Environmental and genetic effects on dispersal behaviour in a range expanding spider.

State of the art: Global climate change often results in poleward range expansions. A new and promising model species for rapid range expansion is the orb-weaving spider Argiope bruennichi that moved from the Mediterranean region into continental climates and up to Scandinavia and Finland within less than 100 years. Consequently, its current distribution covers very different climates and environments. The rapid northward expansion of A. bruennichi was probably facilitated by admixture of formerly isolated lineages through global warming, resulting in an introgression of Asian alleles into the central European genepool (Mol. Ecol. 22: 2232). Taking advantage of the available population genetic and phylogeographic data as well as a fully sequenced genome and a solid understanding of the biology of the species, we will investigate adaptive responses in dispersal behaviour within and between populations along a latitudinal gradient. Dispersal behaviour is of crucial importance for rapid range expansion as well as for establishing populations.

Working hypotheses and work plan: We will investigate dispersal strategies of populations from the northern range limit (Sweden, Finland), the original range (Mediterranean), and genetically distinct island populations from the Azores to determine causes and consequences of variability in dispersal behaviour. We hypothesise that (1) populations differ in the propensity to disperse with northern range populations showing the strongest and island populations the weakest propensity, (2) behavioural plasticity is strongest in northern populations since the shorter period for growth requires immediate responses to local conditions. In breeding experiments under varied conditions, we will determine the degree of genetic and phenotypic variation. We will further test the hypotheses that (3) dispersal probability is affected by resource availability, population density (response to chemical cues), the future risk of inbreeding, and sex, that (4) dispersing phenotypes are of better condition and have higher competitive ability than philopatric phenotypes and that (5) maternal effects partly determine dispersal behaviour through selective provisioning of eggs. The project is conducted in close collaboration with Prof. J. Schneider (Univ. Hamburg, Germany) and Prof. D. Bonte (Univ. Gent, Belgium).

>>>> Monica Sheffer & Gabriele Uhl (2nd cohort) <<<<

Thesis topic: Environmental and genetic effects on fitness relevant traits in a range expanding spider.

State of the art: Global climate change results in poleward range expansions in many species. Our model for rapid range expansion is the orb-weaving spider Argiope bruennichi that moved from the Mediterranean region into continental climates and up to Scandinavia and Finland in less than 100 years. Consequently, its current distribution covers different climates and environments. The rapid northward expansion of A. bruennichi was probably facilitated by admixture of formerly isolated lineages through global warming, resulting in an introgression of Asian alleles into the central European gene pool (Mol Ecol 22: 2232). Based on the available population genetic and phylogeographic data¹, and on our solid understanding of the biology of the species, we study the morphological, physiological, behavioural and genetic responses to variation in environmental conditions in A. bruennichi. To this end, we compared populations from the range expanding front of the distribution (Baltic countries) with those of the central area in Southern France, and - in the 1^st cohort PhD project - complemented the picture by adding island populations from the Azores.

Working hypotheses and work plan:  This project contributes to RESPONSE by using the orb-weaving spider Argiope bruennichi, a species that has undergone a large recent range expansion. This project explores the relative importance of phenotypic plasticity, genetic adaptation, and admixture for colonising new habitats. As a next step, to quantify phenotypic plasticity and genetic adaptation, we will expand our investigation on the variation between populations from the northern range limit (Baltic countries) and the centre of distribution (France) to physiological traits (e.g., cold resistance and metabolic signatures) and survival in a reciprocal transplant experiment in which spiderlings overwinter in the respective winter conditions. We hypothesise that colonisation success is connected to temperature tolerance: spiderlings from the edge will show higher plasticity while being overall more cold-tolerant and showing faster metabolic reaction than spiderlings from the core region. To assess the role of genetic admixture in range expansion, we will scrutinise populations from north-west Poland to south-west Germany with double digest Restriction-site Associated DNA (ddRAD) sequencing. The width of the cline within the transect of potentially hybridising populations will reveal the intensity of selection acting on genotypes. We will further scrutinise core and edge populations for signatures of selection with the help of an annotated genome that is currently being processed. We will use this suite of data to perform targeted experiments focussing on physiological, behavioural and morphological traits to understand the factors that facilitated rapid range expansion.

News:

• PhD student Monica Sheffer hosted two interns in summer 2019 to work on projects related to metabolomic responses to different winter conditions in Argiope bruennichi spiderlings.
• Jasper Murphy and Kate Miller, bachelors students from UC Berkeley, stayed from May 26th to August 17th, 2019.
• Monica applied for and received funding for Jasper's internship through the RISE DAAD Germany Program. His project focused on the detection and abundance of cryoprotectant molecules in A. bruennichi spiderlings using GC-MS. Kate applied for her funding through the Berkeley-Greifswald Research Fellowship program, run by our International Office here at the University of Greifswald and the Institute for European Studies in Berkeley. Kate's project aimed to test the homeoviscous adaptation hypothesis in A. bruennichi by investigating the fatty acid composition of spiderlings, also using GC-MS.
• Both projects were in collaboration with the working group of Prof. Dr. Michael Lalk in the Biochemistry department at our University

>>>> Carolina Ortiz Movliav & Gabriele Uhl (3rd cohort) <<<<

Thesis topic: Environmental and genetic effects on fitness-relevant traits in a range expanding spider.

State of the art:Global climate change often results in poleward range expansions. A new and promising model species for rapid range expansion is the orb-weaving spider Argiope bruennichi, which moved from the Mediterranean region into continental climates as far as Scandinavia and Finland in less than 100 years. Consequently, its current distribution spans different climates and environments. The rapid northward expansion of A. bruennichi was probably facilitated by admixture of formerly isolated lineages through global warming, resulting in an introgression of Asian alleles into the central European genepool (Mol. Ecol. 22: 2232). To understand the range expansion dynamics, and subsequent adaptation of these spiders in novel environments, we use common garden experiments to compare populations of A. bruennichi from the range expanding front of the distribution (Baltic countries) with those of the original range in the Mediterranean.

We have developed state-of-the-art genomic resources for the species (bioRxiv: doi.org/10.1101/2020.05.21.103564), as well as detailed population genomic data, which we use to detect adaptation at the sequence level. Common garden physiological experiments at the phenotypic level point to both genetic adaptation and plasticity as important factors contributing to cold tolerance and overwintering success. Gene expression studies and metabolomic data will provide the connecting link between genomic and phenotypic evidence for adaptation. Our work contributes to the overall aim of the RTG by pinpointing the mechanisms driving success in species that colonise, and subsequently adapt to, new habitats.

Working hypotheses and work plan: We will investigate variation between populations from the northern range limit (Estonia) and the core of the range (Southern France). In this project, we aim to assess the degree of phenotypic plasticity and genetic adaptation underlying the variability in traits related to temperature tolerance, specifically in adaptation to colder winters. The methodology will include field collection of spiders, with overwintering of offspring in a common garden design followed by physiological measurements, including gene and metabolite expression. We will work closely with Henrik Krehenwinkel (Uni Trier) and an interdisciplinary collaboration network at our university to generate and analyse the expression data. Analysis will be aided by the use of our high quality genome assembly. We will use these fitness-relevant data to parameterize various models with the aim of predicting population persistence and distribution of A. bruennichi in the future.

>>>> Lisa Lehnen, Sébastien Puechmaille & Gerald Kerth (1st cohort) <<<<

Thesis topic: Spatio-temporal variation in dispersal in R. hipposideros and implications for fitness and genetic diversity.

State of the art: The European vertebrate fauna is highly dynamic due to both historical climatic variation and anthropogenic pressures. For some species, such as the Palearctic bat species Rhinolophus hipposideros, regressions and advances are quite well documented. In Western Europe, this species underwent a drastic population decline in the mid 20th century, with a pronounced southward receding of its distribution edge. Since several decades, however, re-colonization of former habitat via dispersal has been observed for colonies in central Germany, which are among the northernmost ones on the European continent. A species' dispersal capacity and thus, its ability to track environmental changes by range shift are expected to be a major determinant of extinction risk (Science 313: 789). Elucidating dispersal ability and mechanisms in the lesser horseshoe bat and identifying parameters determining these is necessary to test the explanatory power of current models which predict a northward progression of this species during the next decades (Global Change Biol. 16: 561). We investigate colony formation via dispersal in expanding R. hipposideros colonies in central Germany, study trade-offs / associations between dispersal and life history traits, and elucidate factors influencing dispersal, paying special attention to social and environmental factors.

Working hypotheses and work plan: Droppings will be collected from selected colonies in central Germany in three consecutive years. Bat DNA from this material will be genotyped at 9 microsatellite loci to estimate realized dispersal and further demographic parameters such as population size, reproduction rate, and vitals (survival / mortality). These will reveal a great deal about the lesser horseshoe bat's potential for life strategy adaptation to habitat availability – an important prerequisite for taking advantage of global warming rendering higher latitudes climatically suitable. Furthermore, ecological and environmental factors of interest are tested for their influence on R. hipposideros dispersal and genetic structure with a landscape resistance modeling approach. Specifically, we will investigate (1) How new maternity colonies are formed, (2) If expanding colonies in central Germany exhibit an adaptation in their life strategy to benefit from habitat availability, resulting in increased reproduction and dispersal rates, (3) Which ecological and environmental factors facilitate or impede dispersal in the lesser horseshoe bat. The study is carried out in collaboration with local conservationists in central Germany (fmthuer.de) and Dr. E. Petit, INRA (Rennes, France).

>>>> Thomas Näf, Jaap van Schaik & Gerald Kerth (2nd cohort) <<<<

Thesis topic:  Range expansion and dispersal in the lesser horseshoe bat Rhinolophus hipposideros.

State of the art: The ability of a species to track climatic changes by range shift is expected to be a major determinant of extinction risk (Science 313: 789). For some species, as the Palearctic bat R. hipposideros, range regressions and advances are quite well documented and represent unique opportunities to study how and why range limits move. Such dynamic range boundaries can be observed at the Northern edge of the current distribution of R. hipposideros, regions in which climate change models predict a northward progression of this species during the next decades (Glob Change Biol 16: 561). However, these predictions do not take into account the species’ dispersal capacity and trade-offs between life history traits that evolve in expanding populations (Ecol Lett 13: 1210). Life history traits are constrained by a whole network of trade-offs related to the species biology and evolutionary history as well as its environment. When colonising a new area, dispersal rates of the populations located at the expansion front are expected to be higher than in demographically stable populations located at the core (Ecol Evol Res 4: 1119; Ecol Entom 32: 437). After settlement, dispersal propensity is supposed to rapidly decrease in order to allocate more energy to reproduction, resulting in an increase in population size, until the carrying capacity of the environment drives the population to a trade-off between dispersive, competitive and reproductive abilities similar to the one observed in stable populations (Am Nat 164: 378; Ecol Lett 13: 1210). Additionally, these range fluctuations are continuously reshaping species distribution leading to populations of contrasting genetic diversity. As genetic diversity represents the raw material upon which selection can act, allowing species to respond to changing environments, it is crucial to better understand factors shaping genetic diversity in leading-edge populations.

Working hypotheses and work plan: This project adds to the overall aims of RESPONSE by investigating factors influencing dispersal and the links between dispersal, population dynamics and genetic diversity in a long-lived mammal that currently extends its range in Central-Europe. Building upon the findings described above, our next research goals are to assess to which extent the dispersal tendency (movement between breeding colonies) of individuals is heritable, following individual movements between colonies over years and through pedigree analyses, and to predict range expansion in the species. The results from the first funding phase reported above underline the importance of individualised data sets for studying fitness consequences of dispersal. The existing field and genetic data collected for 3-4 years will thus be combined with new data from the same colonies in Germany where substantial dispersal activity is observed. We will not add data from the French colonies as these are not expanding and we have enough data to infer all life-history traits at the colony level. The interconnected colonies at the northern edge of the species’ range will be surveyed according to the same protocols developed in the 2^nd cohort PhD project. Briefly, droppings will be collected from all colonies and genotyped using 8 microsatellite loci plus one sex-linked marker, allowing for individual identification¹. One sample per individual will be further genotyped for a set of 200 SNPs to facilitate pedigree reconstruction over multiple generations (Methods Ecol Evol 9: 1959). Non-invasive genotyping over consecutive years in the colony networks will allow for estimating demographic parameters (i.e. population size, population growth, vital and dispersal rates). An individual will qualify as a disperser if it is born in a specific colony but later found in another colony. Multigenerational family pedigrees will be combined with capture-mark recapture models under a capture-recapture animal model, allowing the estimation of life history trait heritability, including dispersal (J Evol Biol 23: 2176; Ecol Evol 7: 7334). In the 3^rd cohort PhD-project, we aim to compare life-history traits within the German meta-population: colonies at the edge of the expansion front vs. colonies at the core (colonies surrounded by other colonies). The results obtained will be used to parametrise a hybrid correlative/mechanistic model to predict range expansion in the species (Methods Ecol Evol 5: 388).

>>>> Antonia Hammer, Jaap van Schaik & Gerald Kerth (3rd cohort) <<<<

Thesis topic: Combining population genetic data and modelling to explore the influence of environmental and social factors on range expansion and colony formation in R. hipposideros.

State of the art: The ability of a species to track climatic changes by range shift is expected to be a major determinant of extinction risk (Science 313: 789). For some species, as the Palearctic bat R. hipposideros, range regressions and advances are quite well documented and represent unique opportunities to study how and why range limits move. Such dynamic range boundaries can be observed at the Northern edge of the current distribution of R. hipposideros, regions in which climate change models predict a northward progression of this species during the next decades (Glob Change Biol 16: 561). However, these predictions do not take into account the species’ dispersal capacity and trade-offs between life history traits and sociality that evolve in expanding populations (Ecol Lett 13: 1210). For example, (obligate) coloniality may fundamentally constrain the effect of individual dispersal decisions. To date, how dispersal leads to novel colony formation, and thereby range expansion at the edge of a species’ distribution, remains unknown for bats. The differing ways in which dispersal can lead to the foundation of new colonies and range expansion will be reflected in the genetic structure and diversity of the newly established population. As genetic diversity represents the raw material upon which selection can act, allowing species to respond to changing environments, it is crucial to better understand factors shaping genetic diversity in leading-edge populations.
During the first two project phases of RESPONSE, our international team obtained over 17,000 multilocus genotypes from 20 colonies (2015-2020) of an expanding meta-population of R. hipposideros located in Thuringia. Life history traits were estimated from an integrated population model that was developed to make full use of the information contained in non-invasively collected multilocus genotypes. By using population size estimates, sex ratios and genetic diversity data in a combined model we estimated survival, fecundity and dispersal in our target colonies. Results indicated a higher rate of dispersal (ca. 2-5 %) in German populations than in those of a stable population in France (movement between colonies; < 0.1 %), fitting our expectations of higher dispersal rate in the expanding population. In the second phase, individual genotyping is being supplemented with higher resolution SNP data to yield individual identity, in order to track dispersal events and gain a better understanding of the genetic composition of newly established colonies.

Working hypotheses and work plan: Building upon the findings described above, non-invasive genotyping over consecutive years in the colony networks will be used to estimate demographic parameters (i.e. population size, population growth, vital [births, deaths] and dispersal [emigrations, immigrations] rates), most notably in newly establishing colonies. These parameters will be used to parametrize models to predict colony formation and range expansion in R.hipposideros and compare it to other bat species for which similar data are available. Modelling will be done in cooperation with our local partner Barbara Bauer (Projekt P).

Daniel Romero Mujalli, Barbara Bauer, Jürgen Kreyling & Alexander Wacker

Research question: The evaluation of species persistence is the overarching aim of all projects in RESPONSE. This project will provide a general framework for the individual projects and aims at integrating them, thereby providing added value beyond single target species.

State of the art: Classical Species Distribution Models (SDMs) transpose observed species distributions within current environmental space onto predicted future environmental conditions. They therefore ignore the dynamic processes that determine range boundaries, and do not consider how plastic responses and genetic adaptation might moderate species’ environmental tolerances, which may well affect species persistence (Ecol Lett 17: 1351). ‘Mechanistic population models’, in contrast, focus on phenotypic traits affecting population persistence and underlying biological processes (J Evol Biol 22: 124) but lack the level of geographical and environmental details that SDMs provide (J Evol Biol 27:866-875). Hybrid SDMs and process-based population persistence models span the divide between purely correlative and mechanistic models (Meth Ecol Evol 5: 273). Process-based mechanistic models typically require more parameters but less calibration data compared to hybrid SDMs, because of the explicit representation of processes and mechanisms driving the eco-evolutionary dynamics (Reg Env Change doi: 10.1007/s10113-018-1406-7). In hybrid SDMs, physiological tolerances can define the fundamental theoretical limits, as opposed to observed, realised limits, and it is possible to consider plasticity of the relevant traits and how such traits would evolve under selection to modify expected range boundaries. Furthermore, dispersal dynamics can be included in hybrid models, which consider fluctuation of range shifts to changing climate conditions (Ecography 35: 458). This advanced approach to project species persistence, however, is strongly hampered by a lack of the necessary empirical data to feed such models (PLoS Biol 8: e1000357; Science 341: 504; Ecol Lett 19: 1468).

Working hypotheses and work plan: Current population persistence models (PLoS Biol 8: e1000357) or hybrid species distribution models (Ecol Lett 19: 1468) require data on genetic variance, phenotypic plasticity, and either environmental sensitivity of selection (i.e. population persistence models) or dispersal capacity and species distribution (i.e. hybrid species distribution models such as the generic modelling framework AdaptR). These parameters have been or will be quantified within the different individual projects in RESPONSE. While the mathematical population persistence models will not require additional modelling skills and can be applied by the respective doctoral researchers of the 3^rd cohort in RESPONSE, the advanced hybrid models such as AdaptR (Ecol Lett 19: 1468) do require modelling expertise.Though the inclusion of adaptation is the most distinctive feature of AdaptR, the inclusion of dispersal dynamics are also a significant improvement on most SDM applications because they control fluctuation of range shifts to changing climate conditions (Ecography 35: 458). As a result, the adaptive and dynamic elements correspond well to those individual projects within RESPONSE that focus on plastic and genetic in-situ responses but also have at least basic information on dispersal kernels. Thus, AdaptR provides a mechanism for predicting how these traits will influence the spatio-temporal patterns of species persistence. Habitat suitability is initially determined on the basis of species observed records and spatial environmental layers using an SDM algorithm such as Maxent (Ecol Model 190: 231). AdaptR predicts the distribution (presence/absence) of a species at each generation over time, given its distribution in the previous generation, relevant environmental changes and specified species tolerance traits (Global Ecol Biogeograph 24: 1192). Species can persist locally if environmental conditions are suitable, and, depending on the magnitude of exposure to selection, tolerance will respond to selection defined by the phenotypic variance, plasticity and heritability of that trait. Over time, the initial value of the trait can increase or decrease depending on the intensity of selection, and shift its distribution in response to changes in user-defined environmental scenarios. Where necessary, exposure to extremes will be statistically downscaled from climate models using NicheMapper (Methods Ecol Evol 5: 273). Most individual RESPONSE projects will be able to either measure the relevant parameters directly or obtain them from the literature. However, in cases where certain parameters are uncertain, sensitivity analyses provide a basis for identifying their impact on possible outcomes (Nature 470: 479). In order to allow for the comparison of the relative importance of plasticity, adaptation, and movement for species persistence in the face of environmental change, we plan to use such integrative modelling of the empirical results obtained in the individual projects. To do this, we plan for a postdoc position parallel to the 3^rd cohort in RESPONSE. This postdoctoral researcher will have a strong background in the modelling of species persistence. A close cooperation with the developers of AdaptR, A. Bush and K. Mokany, has already been agreed upon. This will allow the postdoctoral researcher to closely interact with these leading experts in the field of hybrid models. At least one of them will come to Greifswald for extended stays to support the postdoctoral researcher (see Modules 4.2 and 8.3). The postdoctoral researcher will also have the resources for external lab exchanges.

Envisaged topics: Beyond modelling persistence of the single target species in RESPONSE in cooperation with the respective doctoral researchers, the postdoc will work particularly on the understanding when and where genetic adaptation might aid species persistence (given a certain capacity for dispersal). Exploring the major differences among the species, such as contrasting life-cycles (k vs. r strategy), as well as the consequences of land-use change for dispersal and hence range shifts, are promising directions of research. We believe these higher-level implications of such studies are likely to generate high impact for the postdoc, as well as improve the visibility of individual RESPONSE projects.