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Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
The continuous conversion of natural wildlife habitats into agricultural areas, as well as the fragmentation of the last wildlife refuges, is increasing the interface between people and wildlife. When wildlife negatively impacts on people and vice versa, we speak about human-wildlife conflicts (HWCs). This definition includes losses on both sides and takes into consideration the rooting of most of these conflicts between different groups of interest, such as advocates for nature conservation and economic groups. The centres of highest biodiversity are located in developing countries, which are also characterized by poverty. In African and Asian countries, people living in the vicinity of national parks and other conservation areas mostly receive only little support through the government or conservation organisations. Especially for those people who are dependent on agriculture, damage to fields and harvests can have catastrophic consequences. If the species causing damage is protected by national or even international law, the farmer is not allowed to use lethal methods, but has to approach the authority in charge. If this agency, however, cannot offer appropriate support, resentment, anger or even hate develops, and the support for wildlife conservation activities declines. For this reason, HWCs were declared as one of the most important conservation topics today, being particularly relevant for large and threatened species such as the African and Asian elephant, hippopotamus and the greater one-horned rhino, as well as for large predators. Up to today, no general assessment scheme has been recommended for damage caused by protected wildlife species.
In my study, HWCs in Asia and Africa are compared, focussing on all herbivorous species identified which damaged crops. For the French NGO Awely, des animaux et des hommes, I developed a detailed assessment scheme suitable for all terrestrial ecosystems, and any type of HWCs and any species (Chapter 2). This HWC assessment scheme was used in four different study areas located in two African countries (South Luangwa/Zambia (SL), Tarangire/Tanzania (TA)) and two Asian countries (Bardia/Nepal (BA) and Manas/India (MA)). This scheme ran for six consecutive years (2009 to 2014) for Zambia, Nepal and India and two years (2010 to 2011) for Tanzania. To carry out the assessments, I trained local HWC officers (Awely Red Caps) to assess HWCs by field observations (measurement of damage, identification of species through signs of presence, landscape attributes etc.) and interviews with aggrieved parties (socio economic data). Results of this assessment are presented in Chapters 2-4.
To determine whether elephants prefer or avoid specific crop species, two field experiments were carried out, one in SL and one in BA (Chapter 5 and 6). For this, two test plots were set up and damage by elephants (and other herbivores) were quantified.
Within this doctoral thesis, 3306 damage events of 7408 aggrieved parties were analysed. In three out of the four study areas (SL, BA, MA), elephants caused the highest number of damage events compared to all other wildlife species, however, in TA, most fields were damaged by zebra. Furthermore, the greater one-horned rhino, hippopotamus, wild boar, bushpig, deer and antelope, as well as primates, caused damage to fields and harvests. Damage to houses and other property were nearly exclusively caused by elephants.
With this doctoral thesis I was able to show that season, crop availability, type and the phenological stage of the crop played an important role for crop damaging behavior of herbivores (Chapter 2). Elephants especially damaged rice, maize and wheat and preferred all crop types in a mature stage of growth. In contrast, rhinos preferred wheat to rice and similar to antelope and deer, they preferred crops at earlier stages of growth, before ripening. Crop damage by wildlife species varied strongly in size; most damages fell below 40% of the total harvest per farmer, but in several cases (3 to 8% depending on the study area), harvests were completely destroyed. Interestingly, during times of low nutritional availability in the natural habitat (dry season), crop damages in all four study areas were significantly less than during other seasons.
In all four study areas, crop protection strategies, such as active guarding in the fields, chasing wildlife with noise or fire torches or erecting barriers, were used. In some cases protection strategies were combined. Analysis of data revealed that traditional protection strategies did not reduce the costs of damage (Chapter 3). In some cases, costs of damage, on protected fields were even higher than for unprotected fields. Only in MA did strategic and cohesive guarding significantly reduce crop damage by wildlife species.
Besides damage in the fields, elephants also caused damage to properties in the villages. In search for stored staple crops, they damaged houses, grain stores and kitchens. Such damage was analysed in three study areas (SL, BA, MA) (Chapter 4). Although property damage occurred less frequently compared to crop damage in the fields, the mean cost of this damage was found to be double in BA/MA and four times higher in SL, compared to the costs of crop damage in the fields. It is further remarkable that property damage significantly increased towards the dry season, when the harvest was brought into the villages.
The findings of this study underpin the assumption that wildlife herbivores, especially elephants, are lured to fields and crops because the highly nutritional food (crop) being readily available. Traditional crop protection is cost and labour intensive and does not reduce the costs of damage. For this reason, crop types, which are thought to be not consumed by elephants were systematically tested on their attractiveness in field experiments in SL and BA (Chapter 5 and 6). In SL, lemon grass, ginger and garlic were proven to be less attractive to African elephants than maize and in BA, basil, turmeric, chamomile, coriander, mint, citronella and lemon grass were found to be less attractive to Asian elephants than rice.
The results of this doctoral thesis are relevant for the management of wildlife conservation as they can lead to new approaches to the mitigation of HWCs in African and Asian countries. Finally, specific needs for more scientific research in this field have been identified.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
Cardiovascular disease is the leading cause of death worldwide. Aging is among the greatest risk factors for cardiovascular disease. Cardiovascular disease comprises several diseases, for example myocardial infarction, elevated blood pressure and stroke. Many processes are known to promote or worsen cardiovascular disease and in the present study, cellular senescence and inflammatory activation were of special interest, as they have a strong association to aging and can be seen as hallmarks of cellular aging.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. In recent years, numerous regulatory functions were shown for these transcripts and lncRNAs were shown to directly interact with DNA, RNA and proteins. The long noncoding RNA H19 was among the first described noncoding RNAs and was initially shown to act as a tumor suppressor. More recently, several studies showed oncogenic roles for H19. In regards to the cardiovascular system, H19 was not analyzed before.
We show that H19 is the most profoundly downregulated lncRNA in endothelial cells of aged mice compared to young littermates. Microarray analysis of human primary endothelial cells upon pharmacological H19 depletion revealed an involvement of H19 in cell cycle regulation. Loss of H19 in human endothelial cells in vitro led to reduced proliferation and to increased senescence. H19 depletion was shown to counteract proliferation before, but none of the described mechanisms applied to endothelial cells. We show that the reduction in proliferative capacity and the pro-senescent function of H19 is most probably mediated by an upregulation of p16ink4A and p21 upon H19 depletion.
When we compared the angiogenic capacity of aortic endothelial cells from young and aged mice in an aortic ring assay, rings from aged mice showed a reduced cumulative sprout length. Interestingly, pharmacological inhibition of H19 in aortic rings of young animals, where H19 is highly expressed, was sufficient to reduce the cumulative sprout length to levels we observed from aged animals. Furthermore, overexpression of human H19 in aortic rings of aged mice, where H19 is poorly expressed, rescued the impaired angiogenic capacity of aged endothelial cells.
We generated inducible endothelial-specific H19 knockout mice (H19iEC-KO) and subjected these animals to hind limb ischemia surgery followed by perfusion analysis in the hind limbs by laser-doppler velocimetry and histological analysis. Perfusion in the operated hind limb was increased in H19iEC-KO compared to Ctrl littermates, which was in contrast to a reduction in capillary density in the operated hind limbs of H19iEC-KO animals compared to Ctrl littermates and to our previous results. Analysis of arteriogenesis revealed an increase in collateral growth upon EC-specific H19 depletion in the ischemic hind limbs, which explains the increase in perfusion despite the reduction in capillary density. Further characterization of the animals revealed an increase in leukocyte infiltration into the tissue in the ischemic hind limbs upon endothelial-specific H19 depletion, indicating a potential role of H19 in inflammatory tissue activation.
Reanalysis of the microarray data from human primary endothelial cells upon H19 depletion revealed an association of H19 with inflammatory signaling and more specifically with IL-6/JAK2/STAT3 signaling. Analysis of cell surface adhesion molecule expression revealed an upregulation of ICAM-1 and VCAM-1 on mRNA level and an increase of the abundance of the two proteins on the cell surface of human primary endothelial cells. Consequently, adhesion of isolated human monocytes to human primary endothelial cells was increased upon H19 depletion in vitro. Interestingly, TNF-α mediated inflammatory activation of primary human endothelial cells repressed H19 expression. H19 did not function via previously described mechanisms. We excluded a competitive endogenous RNA (ceRNA) function for H19 in endothelial cells and showed that miR-675, which is processed from H19, does not play a role in the endothelium. Furthermore, H19 did not regulate previously described genes or pathways.
Analysis of transcription factor activity upon H19 depletion and overexpression revealed a differential activity of STAT3. STAT3 phosphorylation at TYR705 and thus activation was increased upon H19 depletion. Inhibition of STAT3 activation using a small compound inhibitor abolished the effects of H19 depletion on mRNA expression of p21, ICAM-1 and VCAM-1 and on proliferation, indicating that the effects of H19 are at least partially mediated via STAT3. STAT3 was shown to have positive effects on the cardiovascular system before, most likely due to upregulation of VEGF in a STAT3-dependent manner. We were not able to confirm previously described mechanisms for STAT3 in the present study and propose a new mechanism of action for the H19-dependent regulation of STAT3. Taken together, these results identify the long noncoding RNA H19 as a pivotal regulator of endothelial cell function. Figure 38 summarizes the described functions of H19 in endothelial cells.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
Savannas provide essential ecosystem services for human well-being in West Africa. Thus, ecosystem change not only directly affects biodiversity but also human livelihoods. Human land use considerably shaped these savanna ecosystems for millennia, particularly agriculture, livestock grazing, logging and the collection of non-timber forest products (NTFPs). NTFPs are wild plant products and comprise all organic matter from herbaceous plants, shrubs, and trees (excluding timber). Current increasing land use pressure through fast demographic changes is widely esteemed as a severe threat for savanna biodiversity and the socio-economy of rural communities. In consideration of the pivotal role of NTFP species for biodiversity and livelihoods, it is important to evaluate the effect of increasing land use change on savanna vegetation and on its provisioning service for human well-being. Thus, the major aim of this thesis is to investigate the impacts of land use intensification on vegetation composition, diversity and function and its consequences for provisioning ecosystem services (NTFPs) and human well-being in a West African savanna.
The research for this study was conducted in the North Sudanian vegetation zone of south-eastern Burkina Faso, where population growth exceeds the nationwide trend. Generally, Burkina Faso belongs to the worldwide poorest countries, where nearly one quarter of the population suffers from malnutrition (FAO 2014). The integration of NTFPs and particularly wild food species into rural household economies is, thus, an important measure in the national combat against poverty and food insecurity (FAO 2014). Against this background, I focus on vegetation changes, the economic importance of NTFPs as well as the decrease and substitution of wild food species in this study.
Vegetation resurveys of different vegetation types since the early 1990s showed that land use change led to more pronounced changes in the herbaceous than in the woody vegetation layer. Most woody vegetation types stayed stable in species composition and richness, even though some highly useful tree species (Vitellaria paradoxa, Parkia biglobosa) declined in some woody vegetation types. In contrast, in most herbaceous vegetation types species richness increased and species composition considerably changed. This change might be explained by a general ruderalisation process through a pronounced increase of wide-ranging herbaceous species. However, in spite of a general species increase in the herbaceous layer, a decrease of preferred herbaceous fodder species was found. Thus, the decline of useful species in both layers is alarming. Herbaceous vegetation types also showed more pronounced changes in plant functional trait characteristics in comparison to woody vegetation types. However, an increase of smaller plant species and species with a high diaspore terminal velocity (VTerm) was found in both vegetation layers. Since these two trait responses are generally related to grazing and browsing, the strong increase of livestock herds is likely to be responsible for the detected vegetation changes.
In addition to the vegetation study, interviews showed that all useful food species were widely considered to decline. The two economically most important tree species, the shea tree (Vitellaria paradoxa) and the locust bean tree (Parkia biglobosa) that contribute with 70% to wild food income, were considered among the most declining species of all cited wild food species. On this matter, local perceptions of species decline and results from field observations are in accordance. However, a wide range of cited substitutes indicated a great knowledge on alternative plant species in the area. Most wild food species are, however, substituted by other highly valued wild food species. Although our results suggest that rural communities are able to cope with the decrease or absence of wild food species, growing decline of one species would concurrently increase the pressure on other native food species. Therefore, the need to counteract the decrease of highly useful wild food species should be of high priority in management measures. In general, I showed that NTFPs are an essential component in rural households, since it contributed with 45 % to total household income. Significant differences in NTFP dependency between the two investigated villages and across the three main ethnic groups were detected, reflecting different traditional uses and harvesting practices. In general, it was shown that poorer households depend more on NTFP income than wealthier households. Against the background of this study, management strategies for agroforestry systems and poverty alleviation should consider local differences, and ethnicity-dependent NTFP-use patterns.
Overall, the combination of field studies on temporal and functional vegetation change with socio-economic and ethno-botanic interviews increases the knowledge on qualitative and quantitative vegetation changes and on the consequences for rural populations. This thesis gives a thorough insight into decreasing trends of economically valued plant species and thus gives evidence on the consequences of vegetation changes for ecosystem services of West African savanna ecosystems. Further, different NTFP-dependencies and use preferences according to socio-economic and cultural variables, such as ethnicity, present a valuable basis for specific decision-making and should be considered in management plans.
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
Die neuronalen Mechanismen, welche den meisten kognitiven Prozessen zu Grunde liegen, bestehen aus dem Zusammenspiel verschiedener Neuronen-Typen und deren spezifischen Funktionsmechanismen, sowohl in lokalen, als auch in globalen neuronalen Netzwerken. Eine funktionelle Interaktion mit diesen Netzwerken ist unumgänglich um das „kognitive“ Gehirn zu studieren, da neuronale Gruppen in einer hierarchischen, nicht linearen Weise miteinander interagieren, und dabei charakteristische raum-zeitliche Muster aufweisen. In dieser Arbeit untersuchten wir die Struktur und Funktion eines wichtigen Merkmals kortikaler Prozesse: Die neuronale gamma-Band Oszillation.