Open Access

Increasing environmental changes primarily due to anthropogenic impacts, are affecting organisms all over the planet. In general, scientists distinguish between three different ways in which organisms can respond to environmental changes in their habitat: extinction, dispersal and adaptation. An example of organisms which are highly adaptable and can easily cope with new and changing environments are invasive species which are able to colonize new habitats with only few individuals. To successfully survive in their new environment, invasive species adapt fast to novel abiotic and biotic parameters, such as different temperature regimes. Phenotypic plasticity which enables organisms to quickly modify their phenotype to new environmental conditions, explains the success in adaptation of invasive species. While underlying mechanisms of phenotypic plasticity are not fully understood, one possible “motor” of phenotypic plasticity is epigenetics. Especially DNA methylation could explain the fast changes of the organism’s phenotype due to plasticity when experiencing changing environments, as invasive species do. DNA methylation could even contribute to the adaptation of invasive species via phenotypic plasticity, especially with clonally reproducing species. Methods such as common garden experiments with clonally reproducing species are a useful tool to differentiate between phenotypic plasticity and genetic adaptation because the confusing effects of genetic variation are lowered in clonally reproducing species. Our overall goal was to evaluate the genetic adaptive potential of New Zealand mud snail (Potamopyrgus antipodarum) populations from Europe since they went through an extreme bottleneck after colonizing Europe only 180-360 generations ago. Seemingly, two different clonal lineages colonized Europe because two 16 s rRNA and cytochrome b haplotypes were found across different European countries, haplotypes t and z. The NZMS is a highly successful invasive species that is nowadays nearly globally distributed. The shells of the NZMS show a habitat-dependent high variability and are a fitness-relevant trait. The high variability in shell morphology is due to both genetic variation and phenotypic plasticity. To disentangle genetic from environmental effects on the shell morphology NZMS, we conducted a common garden experiment. We kept asexually reproducing females from eleven European populations in climate cabinets with three different temperatures to produce offspring. We compared shell size and shape across three generations using the geometric morphometrics approach. Furthermore, we estimated reaction norms, maternal effects, broad-sense heritability, the coefficient of genetic variation (CVA) and evolvability (IA) in shell size and shape across different temperature conditions. Additionally, we investigated the reproductive rate of the parental generation. Results showed that the shell morphology of the parental generation differed across populations. In contrast, the shell morphology of offspring generations became more similar. The reaction norms of the F1 generation were rather variable across the three temperatures. However, we were able to observe a haplotype-dependent pattern across the reaction norms suggesting a restricted genetic differentiation among NZMS in Europe. We detected high heritability values in size indicating a high genetic influence. Heritability values for shape were lower than in size. Generally, heritability varied slightly depending on temperature. Size seemed to have a higher evolvability than shape. However, the values of all our calculations were very low which indicates that the European NZMS populations are genetically diminished. The reproductive rate of the parental generation was rather haplotype than temperature dependent. In summary, we were able to display that the NZMS is capable to plastically adapt its shell morphology to different temperatures showing significant differences between the two haplotypes. Nevertheless, the low evolvability values indicate that little genetic variation has formed since the arrival of the NZMS in Europe and therefore, European NZMS seem to have a reduced ability to react to selection. These results implied that phenotypic plasticity is important for the adaptation to different environmental conditions in the NZMS and maybe other molluscan species. Since classical experimental approaches can only describe the resulting phenotypes, we also intended to shed more light on the mechanistic side of environmentally induced phenotypic modifications using DNA methylation analysis. Although molluscs represent one of the most diverse taxa within the metazoan and are found in many different habitats, our knowledge of the DNA methylation in molluscs is scarce. Therefore, we aimed at deepening and summarizing our understanding about DNA methylation in molluscs. Publicly available molluscan genomic and transcriptomic data of all eight mollusc classes was downloaded to search for DNA methyltransferases (DNMTs 1-3) responsible for DNA methylation. Additionally, we estimated the normalized CpG dinucleotide content (CpG o/e) indicating the presence/absence and the frequency of DNA methylation in the genome. The CpG o/e ratio refers to the level of DNA methylation in the genome. Based on the sensitivity of methylated cytosines to mutate into thymine residues, species having a high germline methylation in genomic regions over evolutionary time, also have a lower CpG content, which is called CpG depletion. In contrary, species with a limited germline methylation in genomic regions over evolutionary time, show a higher CpG content and lack CpG depletion. The presence or absence of CpG depletion can be calculated with the CpG o/e ratio. Ultimately, the goal of our analyses was to gain insight into the evolution of methylation in molluscs. We detected DNMTs in all eight mollusc classes and in most of the species. It is therefore plausible that the last common ancestor of molluscs has already had the enzymatic machinery which is needed for DNA methylation. However, various species did not possess the complete DNMT toolkit indicating evolutionary modification in DNA methylation. In general, we found a wide distribution of the bimodal CpG o/e pattern in six mollusc classes, resulting from CpG depletion. The genes in these groups seem to be divided into genes with a high degree of methylation and genes with a lower degree of methylation. This implies that DNA methylation seems to be rather common in molluscs. Species of Solenogastres and Monoplacophora were not or only sparsely methylated. It seems that those mollusc groups have undergone a reduction in DNA methylation. We hope that our investigations will demonstrate the lacking knowledge in epigenetics of molluscs and encourage scientist to execute and continue genetic studies on molluscs.

Amid the current global biodiversity crisis, being able to accurately monitor the changing state of biodiversity is essential for successful conservation actions and policy. Despite the pressing need for reliable and cost-effective monitoring methods, collecting such data remains extremely difficult for elusive species, such as temperate zone bats. Although bats are important indicators of environmental changes, monitoring bat populations is challenging because they are nocturnal, volant, small, and highly sensitive to human activities and disturbance. Thus far, population trends of temperate zone bats have been mainly based on visual surveys, including winter hibernation counts at underground sites. However, as bats may not always be roosting in visible locations within the hibernacula, it is currently unknown how these estimates relate to actual population sizes. Infrared light barriers combined with camera traps are a novel method to monitor bats at underground sites. When installed at the entrance of hibernacula, infrared light barriers have the potential to estimate site-level population sizes more accurately than visual surveys, by counting all bats flying in and out of the site. Moreover, camera traps, consisting of a digital camera and white flash, can be used for species-level identification. However, for this new method to be applicable as a large-scale bat monitoring technique, it is important to characterize it with regard to three main criteria: is the method minimally invasive, is it accurate, and is it scalable in terms of spatial and temporal resolution? Therefore, the purpose of this thesis was to investigate the invasiveness and accuracy of this novel bat monitoring method, and to develop standardized and automated data analysis pipelines, both for the light barrier and camera trap data, to support the deployment of this method at scale. In Publication I, we used light barrier data, infrared video recordings and acoustic data from an experimental field study to investigate whether the white flash of the camera trap has any measurable short- or long-term effect on bat activity and behavior. The flash of the camera trap was turned on and off every week at each site, which allowed us to compare the activity and behavior of bats between flash-on and flash-off nights. We found that despite the high sensitivity of bats to disturbance, they did not change their nightly activity patterns, flight direction, echolocation behavior, or long-term site use in response to the white flash of the camera trap. Based on these results, we concluded that camera traps using a white flash are a minimally invasive method for monitoring bat populations at hibernacula, providing high quality images that allows species-level identification. In Publication II, we used infrared video surveillance to quantify the accuracy of infrared light barriers, and we described a standardized methodology to estimate population sizes and trends of hibernating bat assemblages using light barrier data. We showed that light barrier accuracy varies based on the model and location of the installation relative to the entrance, with the best combination achieving nearly perfect accuracy over the spring emergence phase. When compared to light barrier-based estimates, we found that visual counts markedly underestimated population sizes, recovering less than 10% of the bats at the most complex hibernacula. Moreover, light barrier-based population trends showed regional patterns of growth and decline that were not detectable using the visual count data. Overall, we established that the light barrier data can be used to estimate the population size and trends of hibernating bat assemblages with unprecedented accuracy and in a standardized way. In Publication III, we described a deep learning-based tool, BatNet, that can accurately and efficiently identify bat species from camera trap images. The baseline model was trained to identify 13 European bat species or species complexes using camera trap images collected at 32 hibernation sites (i.e., trained sites). We showed that the baseline model performance was very high across all 13 bat species on trained sites, as well as on untrained sites when the camera angle and distance from the entrance were comparable to the training images. At untrained sites with more atypical camera placements, we demonstrated the ability to retrain the baseline model and achieve an accuracy comparable to the trained sites. Additionally, we showed that the model can learn to identify a new species, while maintaining high classification accuracy for all original species. Finally, we established that BatNet can be used to accurately describe ecological metrics from camera trap images (i.e., species diversity, relative abundance, and species-specific phenology) that are relevant for bat conservation. We conclude that infrared light barriers and camera traps offer a minimally invasive and accurate method to monitor site-level bat population trends and species-specific phenological estimates at underground sites. Such remote data collection approaches are particularly relevant for monitoring large, complex hibernation sites, where traditional visual surveys are not feasible or account only for a small fraction of the actual population. Combining this automated monitoring method with a deep learning-based species identification tool, BatNet, allows us quickly and accurately analyze millions of camera trap images resulting from large-scale, long-term camera trap studies. As a result, we can gain unprecedented insights into the behavior and population dynamics of these enigmatic species, drastically improving our ability to support data-driven bat conservation.

In the light of the accelerating pace of environmental change, it is imperative to understand how populations and species can adapt to altered environmental conditions. This is a crucial step in predicting current and future population persistence and limits thereof. Genetic adaption and phenotypic plasticity are two main mechanisms that can mediate the process of adaptation and are of particular importance for non-dispersing species. While phenotypic plasticity may enable individuals to cope with short term environmental changes, genetic adaptation will often be required for populations to survive in situ over longer time spans. However, a rapid genetic response is expected particularly in species with fast life histories or large population sizes, leaving species with slow life histories potentially at higher extinction risk. The Bechstein’s bat (Myotis bechsteinii) is a mammal of 10 g weight that - despite its small size - is characterized by a slow life history, with low reproductive output and long lifespan, and is already considered to be of high conservation concern. Past work demonstrated body size to be a highly fitness-relevant trait in Bechstein’s bats. Body size is further known to be a pivotal trait shaping the pace of life histories in numerous species. Simultaneously, many studies reported noteworthy changes in body size as a response to shifting environments across different taxa. This suggested a potential for high plasticity in this trait in Bechstein’s bats as well; however, changes in body size could have vital impacts on demographic rates. Therefore, this dissertation investigated the following questions: firstly, what shapes the fundamental development of body size in M. bechsteinii, and, specifically, is there an impact of weather conditions on body size? If so, in what form and magnitude? Secondly, how does body size subsequently influence the pace of life in females? What is the cost of a faster or slower pace of life, and how does fitness compare across individuals with slow and fast life histories? And finally, to what extent can changes in body size be attributed to either phenotypic plasticity or genetic adaptation? What is the evolutionary potential of body size in the populations? And, consequently, what implications can we draw regarding population persistence of these colonies? To answer these questions, we analyzed a long-term dataset of over two decades collected from four wild Bechstein’s bat colonies. We used individual-based data on survival, reproduction and body size, built multi-generational pedigrees, and combined everything with meteorological data. In Manuscript 1 we found that, in contrast to the declining body size observed in many species, body size in Bechstein’s bats increased significantly over the last decades. We demonstrated that ambient temperature was linked to the development of body size and identified a sensitive time period in the prenatal growth phase, in which body size was most susceptible to the impact of temperature. We established that warmer summers resulted in larger bats, but that these large bats had higher mortality risks throughout their lives. Manuscript 2 then revealed the influence of body size on the pace of life in Bechstein’s bats and demonstrated high plasticity in intraspecific life history strategies. Large females were characterized by a faster pace of life and shorter lifespans, but surprisingly, lifetime reproductive success remained remarkably stable across individuals with different body sizes. The acceleration of their pace of life means that larger females compensated for their reduced longevity by an earlier reproduction and higher fecundity to reach similar overall fitness. Ultimately, differences in body size resulted in changes in population growth rate via the impact of size on generation times. Results of Manuscript 3 were then able to clarify the extent to which changes in body size were founded on either phenotypic plasticity or genetic adaptation. We demonstrated a particularly low heritability in hot summers, indicating that variance in body size was mostly driven by phenotypic plasticity, with few genetic constraints. During cold summers, behavioural adaptations by reproducing bats seem to be able to mitigate negative effects of cold temperatures. These behaviours, such as social aggregation or preference for warm roosts, are, however, essentially irrelevant in hot environments. In addition, a low evolvability of forearm length points to a low capacity to respond to selection pressures associated with the trait. We can conclude that body size in M. bechsteinii has increased over the last two decades as a response to global warming and is only slightly constrained by its genetic underpinnings. We can further demonstrate a direct link between body size and the pace of life histories in the Bechstein’s bat populations and how changes in body size impact demographic rates via this linkage. In the context of climate change and hotter summers, our findings consequently suggest that body size will likely increase further if warm summers continue to become more frequent. Whether this plastic response of body size proves to be adaptive in the long term, however, remains to be seen. While, up to this point, switching to a faster life history has been successful in compensating fitness losses, this strategy requires sufficient habitat quality and is likely risky in times when extreme weather events are becoming more frequent, as predicted by most climate change scenarios.

Species are the basic units of evolution and biodiversity, and the process of speciation has been one of the most important questions in biology. The evolution of species with common descent is considered to be mainly driven by natural and sexual selection. The material basis and mechanical cause of organismic evolution were recognized during the formation of the modern synthesis of the evolutionary theory in the early 20th century, providing the framework for speciation studies. During this period, the biological species concept was developed in the frame of population genetics, putting emphasis on the reproductive isolation between populations. The phylogenetic species concept developed in the 1980s, on the other hand, does not make any particular assumption about evolutionary or speciation processes. It defines species via their unique combination of character states which are compatible with phylogenetic practices. However, the aforementioned two species concepts are difficult to apply in alpha-taxonomy, where newly discovered species are largely described by the morphological (typological) species concept for practical reasons. Nevertheless, the description of morphological species provides the basis for further assessments of species delimitation via other species concepts and approaches. One of the tools for assisting the identification and discovery of animal species is DNA barcoding, which uses a standard region of mitochondrial DNA sequence as a universal DNA barcode. However, its assumption of intraspecific genetic distances being smaller than interspecific genetic distances does not always hold. Species-level poly-/paraphyly is prevalent due to the discrepancy between the phylogenies of mitochondrial DNA and species. This suggests that the application of DNA barcodes must be combined with an integrative taxonomic approach. Beside the application as a tool for assisting species identification, the information from mitochondrial DNA sequences opens up a window for looking into the complex history of species. Sexual selection is a potential mechanism driving the evolution of species. It favors traits that increase mating probability and mating success. It can result from intrasexual competition, female preference or sexual conflict. However, previous comparative studies using the degree of sexual dimorphism as a proxy for the strength of sexual selection have yielded inconsistent results as to the relationship between sexual selection and species richness. A possible cause of the inferred low association are factors other than sexual selection, which can also lead to the evolution of sexual dimorphism, such as selection for increased female fecundity. In order to assess the effect of sexual selection on speciation, the lability and evolvability of traits need to be studied that are clearly under sexual selection. The aim of this thesis is to improve the knowledge about dwarf spider (Erigoninae, Linyphiidae) diversity and taxonomy, and to assess the evolutionary patterns of dimorphic traits that are under sexual selection. I focused on the abundant and diverse male prosomal modifications in dwarf spiders that are linked to the transfer of secretions from the male to the female during courtship and mating (gustatory courtship). This approach explores the process of speciation and the role of sexual selection on species diversification. I described new erigonine species and revised the classification of known species based on phylogenetic analyses. I also applied X-ray micro-computed tomography (micro-CT) to investigate the distribution and evolutionary pattern of the gustatory glands to tease apart the evolution of prosomal shape and glandular equipment. This cumulative thesis consists of three publications: Publication 1: This publication aimed at contributing to the knowledge of erigonine diversity. The genus Shaanxinus previously contained only two species from China. I collected dwarf spiders from multiple locations in Taiwan from above-ground vegetations with a seldom applied collecting method. Inspection of the collected material resulted in the discovery of 13 Shaanxinus species. An additional species from Vietnam was described from a museum collection. I provided a revision of the genus Shaanxinus. A phylogenetic analysis using morphological characters was conducted for determining the possible generic synamomorphies. I also reconstructed the glandular distribution associated with male prosomal modifications, as well as the detailed structure of a male secondary sexual organ (pedipalp) by micro-CT. Furthermore, I conducted phylogenetic analyses based on sequences from two mitochondrial and one nuclear loci, and assessed the efficacy of different criteria in species identification using DNA barcoding. Distinction of morphologically similar species have been assisted by molecular data. The species level poly-/paraphyly found in mitochondrial DNA sequences caused the low efficacy of many distance- and tree-based species identification methods, while the nearest neighbor method showed high identification success. The non-monophyly is likely caused by instances of interspecific hybridization and recent parapatric speciation. The genus Shaanxinus thus lend itself as an ideal group for congeneric phylogeographic studies addressing the interactions between closely related species. Published in: Lin, S.-W., Lopardo, L., Haase, M. & Uhl, G. 2019. Taxonomic revision of the dwarf spider genus Shaanxinus Tanasevitch, 2006 (Araneae, Linyphiidae, Erigoninae), with new species from Taiwan and Vietnam. Organism Diversity & Evolution, 19, 211-276. Publication 2: Sexually dimorphic prosomal modifications that are related to gustatory courtship occur in many dwarf spider species. These features evolved in the context of sexual selection, which has a potential effect on species diversification. In contrast to many erigonine genera which present little variability in male prosomal traits, the genus Oedothorax presents higher diversity in male prosomal structures among species not only in the position and shapes of the modifications, but also in the degree of modification, ranging from absent to highly elaborated. This genus thus lends itself as a suitable target group for studying the effect of gustatory-courtship-related traits on species diversification. I conducted a revision of the 82 species previously belonging to this genus. Based on the result of a phylogenetic analysis, this genus was re-delimited with 10 species as Oedothorax sensu stricto, while taxonomic decisions were made for other species including synonymization with species from other genera and transferring species to other existing and newly defined genera. 25 species were deemed as “Oedothorax” incertae sedis. The reconstruction of character state evolution suggested multiple origins of specific prosomal modification types. Convergent evolution of these traits among different lineages suggests that sexual selection has played an important role in the species diversification of dwarf spiders. Published in: Lin, S.-W., Lopardo, L. & Uhl, G. 2021. Evolution of nuptial-gift-related male prosomal structures: taxonomic revision and cladistic analysis of the genus Oedothorax (Araneae: Linyphiidae: Erigoninae). Zoological Journal of the Linnean Society, XX, 1-168. Publication 3: Although sexually dimorphic traits have inspired the concept of sexual selection as the driving force of their evolution, they might also have evolved due to other ecological factors. These factors include the sexual signal adaptation to the environment as well as sexual differences in ecological relations and parental investment. In contrast, the gustatory courtship in dwarf spiders is associated with sexually dimorphic male prosomal modifications, which have clearly evolved in the context of sexual selection. Multiple origins of various external prosomal modifications have been shown in erigonine phylogeny, but the evolutionary pattern of the associated glands has not been investigated. Our phylogenetic analysis incorporated the characters related to the glandular distribution in the male prosoma as well as the external shapes yielded from X-ray micro-computed-tomography showed a single origin of gland among the investigated erigonine taxa. The internal anatomy revealed previously undetected trait lability in attachments of muscles to the cuticular structures, as well as the presence/absence and differences in glandular distribution even in species without external modification. Our finding further supports that erigonine male prosomal traits are under divergent selection, and corroborates the argument that erigonines are a suitable group for investigating the effect of sexual selection on speciation. Published in: Lin, S.-W., Lopardo, L. & Uhl, G. 2021. Diversification through gustatory courtship: an X‑ray micro‑computed tomography study on dwarf spiders. Frontiers in Zoology, 18: 51.   The results of this thesis corroborate the importance of applying phylogenetic methods and an integrative approach in the description of new species, as well as in revising taxa which might not be monophyletic. Overall, the studies contributed to a more comprehensive knowledge about erigonine species diversity, phylogeny and the possible diversifying effect of sexual selection on male traits associated with gustatory courtship.

Abstract The two important mechanisms influencing the response of phytoplankton communities to alterations of abiotic factors in their environment are difficult to distinguish: species sorting resulting from a change in interspecific competitive pressure, and phenotypic plasticity (here explicitly physiological plasticity i.e. species‐specific physiological adjustment). A shift in species composition as well as physiological adjustments in species can lead to changes in fatty acid composition that determine the food quality for zooplankton consumers. We used phytoplankton communities consisting of five species and exposed them to two different light intensities, two light conditions (constant and variable), and two levels of phosphorus supply. Changes in fatty acid and species composition were analyzed. We compared community pairs differing in one factor by calculating the Bray‐Curtis similarity index for the composition of both variables. Comparing the Bray‐Curtis similarity index of the species composition with the index of the fatty acid composition was used to estimate the effects of species sorting and physiological plasticity. Changes in nutrient supply influenced fatty acid responses based on species sorting and physiological plasticity the most. On one hand, the relevance of physiological plasticity was highest at cultivation in different nutrient supplies but the same light environment. Conversely with low nutrients species sorting appeared to dominate the response to changes in light, while at high nutrients physiological plasticity appeared to influence the response. Overall, under low phosphorus supply the communities showed a lower total fatty acid content per carbon and had increased proportions of saturated and monounsaturated fatty acids. Instead, communities in low light produced more of eicosapentaenoic acid. Our results suggest that the relevance of species sorting and physiological plasticity in shaping the community response highly depends on the environmental factors that influence the system. Nutrient supply had the largest effect, while light had more limited conditional effects. However, all of these factors are important in shaping the food quality of the phytoplankton community for higher trophic levels.