Soils and plant microbial communities are intricately linked to ecosystem functioning as they play important roles in nutrients dynamics as decomposers and feedback to plant communities as mutualists and pathogens. Numerous soil physicochemical factors as well as the land use management are shaping the composition and dynamics of microbial community. In addition, global warming and climate change are the most prominent of all environmental factors that influence all kinds of the living organisms including microbes associated to the plant soil systems. A better understanding of the environmental drivers shaping these microbial communities especially under future climate will help to understand and predict the expected changes of ecosystems functions and accordingly of the services they provide. In addition, such knowledge will help to detect potential ways on how soil microorganisms can be harnessed to help mitigating the negative consequences of climate change.The Global Change Experimental Facility (GCEF) is settled in the field research station of the Helmholtz Centre for Environmental Research (UFZ) in Bad Lauchstädt, Saxony-Anhalt, Germany (51_22’60 N, 11_50’60 E, 118 m a.s.l.). This facility has been designed to investigate the consequences of a predicted future climate scenario expected in 50-70 years in Central Germany on ecosystem processes under different land-use regimes applied on large field plots in comparison to similar sets of plots under the ambient climate. We performed our study using this research facility, with the aim to analyze the impact of future climate conditions, soil physicochemical factors, and/or land use type and intensity on microbial communities in different habitats (rhizosphere soil, plant endosphere, and plant residues) in grassland and cropland ecosystems. To assess the microbial communities, we used the highly sensitive and powerful highthroughput next generation sequencing, Illumina Miseq.This thesis constitutes the first assessment of microbial communities in the GCEF experimental facility. The samples were collected in 2015 for manuscript 4, while for manuscripts 1, 2, 3, 5, 6, the samples were collected in 2018-2019. Manuscript 1: (Sansupa, Wahdan, Hossen et al., 2021; Applied Science 2021, 11, 688) “Can we use functional annotation of prokaryotic taxa (FAPROTAX) to assign the ecological functions of investigated the potential use of FAPROTAX for bacterial functional annotation in non-aquatic ecosystems, specifically in soil. For this study, we used microbial datasets of soil systems including rhizosphere soil of Trifolium pratense from the extensively used meadow plots in the GCEF. We hypothesized that FAPROTAX can be used in terrestrial ecosystems. Our survey revealed that FAPROTAX tool can be used for screening or grouping of 16S derived bacterial data from terrestrial ecosystems and its performance could be enhanced through improving the taxonomic and functional reference databases. Manuscript 2: (Wahdan et al., 2021; Frontiers in Microbiology 12:629169) “Targeting the active rhizosphere microbiome of Trifolium pratense in grassland evidences a stronger-than-expected belowground biodiversity-ecosystem functioning link”. In this study, we used the bromodeoxyuridine (BrdU) immunocapture technique combined with pair-end Illumina sequencing to differentiate between total and active microbiomes (including both bacteria and fungi) in the rhizosphere of T. pratense. In the same rhizosphere soil samples, we also measured the activities of three microbial extracellular hydrolytic enzymes, (ß-glucosidase, N-acetylglucosaminidase, and acid phosphatase), which play central roles in the C, N, and P acquisition. We investigated the proportion of active and total rhizosphere microbiomes, and their responses to the manipulated future climate in the GCEF. In addition, we identified the possible links between total and active microbiomes and the soil ecosystem function (extracellular enzyme production). Our results revealed that the active microbes of the rhizosphere represented 42.8 and 32.1% of the total bacterial and fungal operational taxonomic units (OTUs), respectively. Active and total microbial fractions were taxonomically and functionally diverse and displayed different responses to variations of soil physicochemical factors. We also showed that the richness of overall and specific functional groups of active microbes in rhizosphere soil significantly correlated with the measured enzyme activities, while total microbial richness did not. Manuscript 3: (Wahdan et al., 2021; Microbiology Open 10:e1217) “Deciphering Trifolium pratense L. (red clover) holobiont reveals a resistant microbial community assembly to future climate changes predicted for the next 50–70 years”. We investigated the microbial communities of bacteria and fungi associated with four plant parts of T. pratense (the rhizosphere and the endopheres of the roots, whole shoot system (leaves and stems), and of the flower) and evaluated their potential ecological and metabolic functions in response to future climate conditions. This study was performed on the GCEF extensively managed grassland plots. Our analyses indicated that plant tissue/compartments differentiation enables the formation of a unique ecological niches that harbor specific microbial communities. Except for the fungal communities of the aboveground compartments, T. pratense microbiome diversity and community composition showed a resistance against the future climate changes. We also analyzed the predicted bacterial metabolic functional genes of red clover. Thereby, we detected microbial genes involved in plant growth processes, such as biofertilisation (nitrogen fixation, phosphorus solubilisation, and siderophore biosynthesis) and biostimulation (phytohormone and auxin production), which were not influenced by the future climate. Manuscript 4: (Wahdan et al., 2021; Environmental Microbiology) “Organic agricultural practice enhances arbuscular mycorrhizal symbiosis in correspondence to soil warming and altered precipitation patterns”. This study was performed on the conventional and organic farming plots under both ambient and future climate conditions. We evaluated the effect of climate (ambient vs. future), agricultural practice (conventional vs. organic farming) and their interaction on Arbuscular Mycorrhizal Fungi (AMF) community composition and richness inside wheat roots. In addition, we evaluated the relationship between molecular richness of indigenous root AMF and wheat yield parameters. Future climate altered the total AMF community composition and a sub-community of Glomeraceae. Further, application of different agricultural practices altered both total AMF and Glomeraceae community, whereby organic farming appeared to enhance total AMF and Diversisporaceae richness. Under the future climate scenario, organic farming enhanced total AMF and Gigasporaceae richness in comparison with conventional farming. Our results revealed a positive correlation between AMF richness and wheat nutrient contents not only in organic farming system but also under conventionally managed fields. Manuscript 5: (Wahdan et al., 2020; Microorganisms 8, 908) “Future climate significantly alters fungal plant pathogen dynamics during the early phase of wheat litter decomposition”. This study was performed on the conventional farming plots. We investigated the structure and ecological functions of fungal communities colonizing wheat during the early phase of decomposition (0, 30, and 60 days) under current and future climate conditions. We found that plant pathogenic fungi dominated (~87% of the total sequences) within the wheat residue mycobiome. Destructive wheat fungal pathogens such as Fusarium graminearum, Fusarium tricinctum, and Zymoseptoria tritci were detected under ambient and future climates. Additionally, the future climate brought new pathogens to the system. Manuscript 6: (Wahdan et al., 2021; Microbial Ecology 10.1007/s00248-021-01840-6) “Life in the wheat litter: effects of future climate on microbiome and function during the early phase of decomposition”. This study was performed on the conventional farming plots. We assessed the effects of climate change on microbial richness, community compositions, interactions and their functions (production of extracellular enzymes) in decomposing residues of wheat. In addition, we investigated the effects of climate change on litter residues physicochemical factors as well as on mass loss during the early phase of decomposition. Future climate significantly accelerated litter mass loss as compared with ambient one. Our results indicated that future climate significantly increased fungal richness and altered fungal communities over time, while bacterial communities were more resistant in wheat residues. Fungi corresponded to different physicochemical elements of litter under ambient (C, Ca2+ and pH) and future (C/N, N, P, K+, Ca2+ and pH) climate conditions. Also, a highly correlative interactions between richness of bacteria and fungi were detected under future climate. Activities of microbial β-glucosidase and N-acetylglucosaminidase in wheat straw were significantly higher under future climate. Such high enzymatic activities were coupled with a significant positive correlation between microbial (both bacteria and fungi) richness and community compositions with these two enzymatic activities only under future climate.:CONTENTS BIBLIOGRAPHIC DESCRIPTION……………………………………………….......III ZUSAMMENFASSUNG………………………………………………………...........V SUMMARY……………………………………………………………………………..X GENERAL INTRODUCTION…………………………………………………………………...............1 I-1 Ecosystem functions carried out by soil and plant microbiomes…………………..2 I-2 Biodiversity and functional diversity and maintenance of ecosystem functions……………..3 I-3 Total vs. active microbial diversity for assessing ecosystem functions……………4 I-4 Factors influencing soil and plant microbiota…………………………………..……6 I-4.1 Elements of climate changes……………………………………………................7 I-4.2 Climate changes influence microbes in an interacting, complex manner………8 I-4.3 Environmental factors controlling the response of microorganisms to climate changes………………………………………………………………………………….....10 I-5 Interplay between climate and land use intensity in agroecosystems……………11 I-6 Study site, and overall objectives………………………………………………....…12 I-7 Methods used for the taxonomic and functional characterization of the microbiomes……...15 I-8 Presentation of aims and hypotheses of the publications/manuscripts in different chapters.................................................................................................................16 I-9References.........................................................................................................20 CHAPTER 1 Can we use functional annotation of prokaryotic taxa (FAPROTAX) to assign the ecological functions of soil bacteria? .....................................................................29 Publication…………………………………………………………………………...........31 Supplementary materials…………………………………………………………….......42 CHAPTER 2 Targeting the active rhizosphere microbiome of Trifolium pratense in grassland evidences a stronger-than-expected belowground biodiversity-ecosystem functioning link………………..........................................................................…49 Publication………………………………………………………………………………51 Supplementary materials……………………………………………………………..67 CHAPTER 3 Deciphering Trifolium pratense L. holobiont reveals a microbiome resilient to future climate changes……………………………………………….…………………………..89 Publication………………………………………………………………………………….91 Supplementary materials……………………………………………………………….111 CHAPTER 4 Organic agricultural practice enhances arbuscular mycorrhizal symbiosis in correspondence to soil warming and altered precipitation patterns………………125 Publication……………………………………………………………………………….127 Supplementary materials………………………………………………………….......140 CHAPTER 5 Future climate significantly alters fungal plant pathogen dynamics during the early phase of wheat litter decomposition…...................………………….……………..156 Publication………………………………………………...…………….….…………...158 Supplementary materials………………………………………………….…....……..175 CHAPTER 6 Life in the wheat litter: effects of future climate on microbiome and function during the early phase of decomposition…………………………………….....……....…….181 Publication…………………………………..…………………………………….....…...183 Supplementary materials………………………………………………………………..199 GENERAL DISCUSSION…………………………………………………………….......210 D-I Approaches and main findings of the result chapters………………………..…211 D-2 Conclusion and implications of the study findings…………………………...…215 D-3 Technical limitation of the study……………………………………………......…217 D-4 Future prospects of the study field ...……………………………………………217 D-5 References…………………………………………………………………………..219 DATA AVAILABILITY……………………………………………………………………...223 ACKNOWLEDGEMENTS……………………………………………………………......224 CURRICULUM VITAE……………………………………………………………….....…225 LIST OF PUBLICATIONS………………………………………………………….........226 CONFERENCE PROCEEDINGS…………………………………………………….....227 STATUTORY DECLARATION………………………………………………................228 VERIFICATION OF AUTHOR PARTS……………………………………………........229

Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life. Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions. So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants. Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex. Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex. Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme. In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 82 / Phosphorylierungen von Aminosäuren ist eine der verbreitetsten post-translationalen Modifikationen für zelluläre Signalübertragungswege und zur Regulation des Metabolismus auf Proteom-Ebene. Mit der reversiblen Protein-Phosphorylierung eng verbunden ist die unabhängige Modulation der Aktivität von Enzymen ungeachtet der Genom- und Epigenom-basierten Genexpression. Die evolutionäre Zunahme der zellularen Komplexität äußert sich in zunehmend komplexeren Regulations-Netzwerken in mehrzelligen eukaryotischen Organismen basierend auf dem Transfer von Phosphatgruppen vorzugsweise auf die Aminosäuren Serin, Threonin und Tyrosin. Die Natur hat evolutionär als Baustein der inter- und intrazellulären Kommunikation Phosphat gewählt, welches auch ein integraler Bestandteil der Nukleinsäuren ist und somit als das „Molekül der Wahl“ für das Leben bezeichnet werden darf. Die Lebenswissenschaften sind gegenwärtig daran interessiert das Netzwerk der Proteinphosphorylierung aufzuklären, welches durch zwei antagonistisch wirkende Enzymklassen, die Proteinkinasen und Proteinphosphatasen charakterisiert ist. Dabei profitieren wir gegenwärtig von den Fortschritten der „Proteomics-Ära“ auf dem Gebiet der massenspektrometrischen Proteinidentifizierung. Ausdruck dessen ist eine Vielzahl von Proteom-Studien, die jedoch meist nur eine einfache Inventarisierung der unter mehr oder weniger gut definierten zellulären Bedingungen existierenden Proteine in ihrer Phosphat-modifizierten oder unphosphorylierten Form darstellen. Die beteiligten Enzyme werden dabei kaum berücksichtigt. Insbesondere gilt dies für extra-cytoplasmatische Ereignisse. Bisher gelang es nur in wenigen Fällen eine Korrelation der physiologischen Rolle dieser Proteinmodifikation, z.B. durch den Vergleich der Phospho-Proteome unter zwei unterschiedlichen physiologischen Bedingungen, herzustellen. Eine andere Strategie, die auch Gegenstand dieser Arbeit ist, sieht ein Screening von Mutanten vor, die durch Deletionen von Genen, die für Proteinkinasen bzw. –phosphatasen kodieren, gekennzeichnet sind. Dieser Ansatz profitiert von der Existenz und leichten bioinformatischen Vorhersagbarkeit charakteristischer Kinase- bzw. Phosphatase- Sequenzmotive. In Kombination mit dem Hauptziel der Arbeit – Licht ins Dunkel der Proteinphosphorylierung im mitochondrialen Kompartiment zu bringen – wurde die Hefe Saccharomyces cerevisiae als Modellsystem gewählt, insbesondere vor dem Hintergrund ihres fermentativen Metabolismus. Als Beleg der prinzipiellen Funktionalität des vorgeschlagenen Ansatzes konnten zwei Kinasen (Pkp1p, Pkp2p) und zwei Phosphatasen (Ppp1p, Ppp2p) als Schlüsselkomponenten der Regulation des Pyruvatdehydrogenase (PDH) Komplexes identifiziert und charakterisiert werden. Darüber hinaus konnte sowohl das Zielprotein der Phosphorylierung, Pda1p, die α-Untereinheit des Komplexes, als auch die modifizierte Aminosäure (Serin 313) experimentell bestätigt werden. Ferner konnte der Atmungsdefekt von Stämmen mit einer nicht-funktionellen Abc1p-Kinase mit dem Phosphorylierungszustand der Untereinheit Coq3p des Ubiquinon-Biosynthese Komplexes und dem Ausfall der Ubiquinonsynthese korreliert werden. Eine alternative Herangehensweise, die Überexpression einer Kinase, führte zur Identifizierung möglicher Zielproteine von Sat4p. Vergleichende Analysen des 2D-gelelektrophoretisch separierten mitochondrialen Genoms mit dem des Wildtyps legen die Vermutung nahe, dass Sat4p eine direkte oder indirekte Rolle bei der Regulation der „Aconitase-Typ“ Eisen-Schwefel (Fe/S) Proteine besitzt. Der darüber hinaus beobachtete Effekt einer Abnahme von Liponsäure-tragenden mitochondrialen Enzymen, ist wahrscheinlich sekundärer Natur und kann durch die Zugehörigkeit der Liponsäure-Synthase zur oben erwähnten Gruppe der „Aconitase-Typ“ -Fe/S-Proteine erklärt werden. Im Verlauf der Arbeit wurde deutlich, dass das regulatorische Netzwerk der Proteinphosphorylierung der Hefe eher den Kriterien einer evolutionären Adaptation an eine spezifische ökologische Nische – der temporären Verfügbarkeit zuckerreicher Substanzen – entsprechen. Das schränkt die Übertragbarkeit der gewonnen Einsichten in die Regulation des mitochondrialen Metabolismus auf höhere Eukaryonten ein. Es zeigt jedoch, dass Hefe in erster Linie ein exzellentes Modellsystem für die prinzipiellen molekulare Mechanismen ist, die sie mit den höheren Eukaryonten teilt.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 82

The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals. Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis. In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.

The great crested newt (Triturus cristatus) is an amphibian species of European conservation concern that has suffered severe declines, primarily due to habitat loss and fragmentation. This pond-breeding amphibian lives in spatially structured populations (SSPs) where dispersal strongly influences population dynamics, genetics and thereby the long-term persistence of the whole SSP. This dissertation investigates the effects of habitat quality on demography and how such environmental as well as individual factors influence different stages of the dispersal process and consequently the population structure of great crested newts. The evaluation of a commonly used habitat suitability index (HSI) model showed no relationship between habitat quality and individual survival probability or body condition but a positive association with reproduction probability and abundance, making it a useful tool to identify habitats of higher priority for species conservation. A comprehensive analysis of dispersal and population structure combining extensive demographic and genetic data highlights the importance of habitat quality for driving context-dependent dispersal and therefore demography and genetic structure in a patchy population of great crested newts. Finally, the monitoring of 18 newly created ponds revealed that ponds were rapidly colonized, mostly over short distances, and that newts captured in new ponds were younger and tended to be larger than those in established ponds (phenotype-dependent dispersal), indicating that colonization is predominantly the result of natal dispersal by large individuals. Implications for conservation management are being discussed including corresponding recommendations.:Zusammenfassung..........1 Summary..........7 Introduction..........12 Chapter I (Linking habitat suitability to demography in a pond-breeding amphibian)..........24 Chapter II (The relationship between habitat suitability, population size and body condition in a pond-breeding amphibian)..........38 Chapter III (Context-dependent dispersal determines relatedness and genetic structure in a patchy amphibian population)..........50 Chapter IV (Pond construction for amphibian conservation: phenotypic traits influence the colonization process)..........99 Acknowledgements..........127 Author contributions..........128

Während der Gewebeentwicklung beeinflusst die Anpassung der mechanischen Spannung bei Zell-zu-Zell-Kontakten das Gewebewachstum, die Musterbildung und die Morphogenese. Die Erzeugung und Kontrolle der mechanischen Spannung hängt von Komponenten des Zytoske- letts wie dem Aktomyosin und den Mikrotubuli-Netzwerken ab. Die Bildung von Komparti- mentgrenzen ist ein wichtiger Entwicklungsprozess, der auf der Anpassung mechanischer Spannungen beruht. Kompartimentgrenzen sind Abstammungsbeschränkungen, die Zellen mit unterschiedlichen Funktionen und Identitäten innerhalb von Geweben trennen. Zellverbindun- gen entlang der Kompartimentgrenzen sind häufig durch eine Anreicherung von filamentösen (F-) Aktin sowie nicht-muskulären Myosin II (Myosin II) Motorprotein und erhöhter mechani- scher Spannung gekennzeichnet. Die Mechanismen, durch die F-Aktin und Myosin II an die- sen Verbindungsstellen angereichert werden, sind jedoch kaum verstanden. Hier zeigen wir, dass an der sich bildenden anteroposterioren Kompartimentgrenze der Puppenepidermis von Drosophila melanogaster F-Aktin und Myosin II vorübergehend angereichert werden. Die An- reicherung von F-Aktin scheint nicht von mechanischer Spannung abzuhängen. Die Fluores- zenzerholung nach Photobleichversuchen (Fluorescence recovery after photobleaching, FRAP) weist eher darauf hin, dass Myosin II vorzugsweise an Zellübergängen entlang der Komparti- mentgrenze stabilisiert wird. Darüber hinaus zeigen wir unter Verwendung einer photokonver- tierbaren Form von Myosin II, dass Myosin II vorzugsweise aus einem zytosolischen Pool an Zellverbindungen entlang der Kompartimentgrenze rekrutiert wird. Um die Rolle des Mikro- tubuli-Netzwerks bei der Bildung von Kompartimentgrenzen zu testen, haben wir außerdem dessen Organisation in der Puppenepidermis charakterisiert. Wir zeigen, dass sich Mikrotubuli und das Mikrotubuli-Minus-Ende-bindende Protein Patronin in einem Streifen anteriorer Zellen entlang der Kompartimentgrenze ansammeln. Interessanterweise haben die Zellen in diesem Streifen, im Vergleich zu anderen Zellen in der Epidermis, eine unterschiedliche Form. Zusammengefasst enthüllen unsere Daten Unterschiede in der Organisation der Mikrotubuli, die mit Kompartimentgrenzen verbunden sind, und zeigen, dass die Anreicherung von Myosin II entlang der Kompartimentgrenze der Puppen-Abdominalepidermis sowohl eine bevorzugte Stabilisierung als auch eine Rekrutierung beinhaltet.

Studies of comparative morphology clearly distinguish the shoulder morphology of Homo from that of the other hominoids. While the shoulder morphology of non-human hominoids is thought to signal adaptations to arboreal locomotion, human shoulder morphology is understood to have lost this adaptation during hominin evolution. Ideas how non-human hominoid shoulder morphology is advantageous in an arboreal context suggest that the specific shoulder morphological traits enhance the arm-raising mechanism. However, this idea has not been biomechanically tested. This thesis constitutes the first analysis of the biomechanical consequences of two distinct shoulder morphologies within Hominoidea by comparing the glenohumeral muscle capabilities of Gorilla to Homo. The biomechanical capabilities are evaluated by constructing a computational musculoskeletal model of a gorilla thorax, shoulder girdle and upper arm, which is used to predict relevant biomechanical metrics such as muscle moments and moment arms. Muscle moments and moment arms are predicted for two important mechanisms, arm-raising and arm-lowering. The predictions are compared to those of an already existing human musculoskeletal model in order to evaluate differences in arm-raising and arm-lowering capability based on the two distinct thorax and shoulder girdle morphologies. The results of the biomechanical analyses show that the arm-lowering mechanism is enhanced in Gorilla compared to Homo, instead of the arm-raising mechanism. The enhanced arm-lowering mechanism is evident by greater moment capacities of two important arm-lowering muscles, pectoralis major and teres major. The greater moments are the result of greater muscle force capacities and greater moment arms, due to the beneficial musculoskeletal geometry of Gorilla. The results highlight that a more distal muscle insertion along the humerus has the greatest enhancing effect on the arm-lowering moment arms of teres major and pectoralis major. Furthermore, thorax and shoulder girdle morphological traits that are well known to distinguish non-human apes from humans were found to contribute to the enhancement of the arm-lowering mechanism. The more cranially oriented glenoid, obliquely oriented scapular spine and cranial scapula position on the thorax enabled certain muscles to act as arm-lowering muscles in Gorilla, contrary to the arm-raising action capability that is predicted for Homo. The enhanced arm-lowering capability is likely advantageous for the arboreal locomotion of apes. During hoisting behaviours that are known to occur during suspension and vertical climbing, arm-lowering is used to lift the heavy body of the apes upward. The results of this thesis in conjunction with earlier EMG studies suggest those muscles which are highly activated during these hoisting behaviours also have enhanced arm-lowering capacities in Gorilla and potentially other non-human hominoids compared to Homo. As such, the results highlight shoulder morphological traits that are biomechanically important for the arboreal locomotor behaviour of apes. By this, the thesis demonstrates a link between the conformation of shoulder morphological traits and their biomechanical capability, which will aid future functional interpretations of extant and extinct species.:Acknowledgements Bibliographische Darstellung Summary Zusammenfassung Chapter 1: Exploring the functional morphology of the Gorilla shoulder through musculoskeletal modelling Chapter 2: Comparison of the arm-lowering performance between Gorilla and Homo through musculoskeletal modeling Conclusion Appendix A: Supplementary Information for Chapter 1 Appendix B: Supplementary Information for Chapter 2 Appendix C: Curriculum Vitae Appendix D: Author Contributions

The sensation of mechanical signals is vital for all animals. For this task Drosophila larvae are equipped with chordotonal organs. These are specialized mechanosensory organs which are composed of multicellular subunits. In this study I show how metabotropic signaling by the adhesion GCPR CIRL interacts with part of the ionotropic pathways during mechanosensation in sensory neurons of the pentascolopidial chordotonal organ (lch5). CIRL modulates cAMP levels in sensory neurons and thereby shapes the receptor potential response to mechanical stimuli. Here, CIRL forms a functional interaction with the TRP channel NOMPC in which nompC is epistatic to Cirl. Furthermore, the evidence presented suggest the presence of another target of CIRL and the involvement of a further signaling pathway besides cAMP modulation. In the second part of the study, I describe a method to express the anion-selective channelrhodopsin GtACR1 in individual of the five neurons of the lch5. For this I used the MARCM approach which generates genetic mosaics during the development of the neurons of interest. Thereby a specific subset of cells deriving from a common precursor expresses the desired protein GtACR1.

Body-size variability results from a variety of extrinsic and intrinsic factors (environmental and biological influences) underpinned by phylogeny. In ostracodes it is assumed that body size is predominantly controlled by ecological conditions, but investigations have mostly focused on local or regional study areas. In this study, we investigate the geographical size variability (length, height, and width) of Holocene and Recent valves of the salinity-tolerant ostracode species Cyprideis torosa within a large geographical area (31◦–51◦ latitude, and 12◦–96◦ longitude). It is shown that distant local size clusters of Cyprideis torosa are framed within two large-scale geographical patterns. One pattern describes the separation of two different size classes (i.e., morphotypes) at around ∼42◦ N. The co-occurrence of both size morphotypes in the same habitats excludes an environmental control on the distribution of the morphotypes but rather could point to the existence of two differentiated lineages. Generally, correlations between valve size and environmental parameters (salinity, geographical positions) strongly depend on the taxonomic resolution. While latitude explains the overall size variability of C. torosa sensu lato (i.e., undifferentiated for morphotypes), salinity-size correlations are restricted to the morphotype scale. Another large-scale pattern represents a continuous increase in valve size of C. torosa with latitude according to the macroecological pattern referred as Bergmann trend. Existing explanations for Bergmann trends insufficiently clarify the size cline of C. torosa which might be because these models are restricted to intraspecific levels. The observed size-latitude relationship of C. torosa may, therefore, result from interspecific divergence (i.e., size ordered spatially may result from interspecific divergence sorting) while environmental influence is of minor importance. Our results imply that geographical body-size patterns of ostracodes are not straightforward and are probably not caused by universal mechanisms. Consideration of phylogenetic relationships of ostracodes is therefore necessary before attempting to identify the role of environmental controls on body size variability.

The rapid spread of antibiotic resistance is a major global health concern, jeopardizing the successful treatment of bacterial infections. Natural environments are potential hotspots for the emergence and spread of antibiotic resistance genes (ARGs). Among these potential hotspots, aquatic ecosystems are of particular concern, as they receive wastewater containing antibiotic-resistant bacteria and ARGs originating from both human and animal sources. Several key questions remain to be addressed. What is the fate of ARGs in receiving water bodies? What are implications of environmental ARGs for human health? How does wastewater discharge impacts aquatic microbial communities with regard to the overall ecosystem well-being? The objective of this work was to investigate the impact of wastewater, seasonal variations, and the riverine compartment on the prevalence of selected ARGs and the composition of natural microbial communities in a near-pristine river, and to specifically assess the effect of antibiotics on riverine microbial communities. Quantitative real-time PCR was used to monitor the abundance of three indicator ARGs (sul1 and sul2, conferring resistance against sulfonamide antibiotics, and intI1, a marker for anthropogenic pollution) upstream and downstream from a wastewater treatment plant (WWTP). Furthermore, the impact of WWTP effluent on the riverine microbial community was examined through 16S rRNA amplicon sequencing. Wastewater was the main source of all three target genes and significantly altered the microbial community in the river. The surface water compartment served as a dissemination route for ARGs, with increased prevalence even 13 km downstream of the WWTP, particularly during the summer season when the proportion of wastewater in the river was high. Notably, riverbed biofilms served as a local reservoir for ARGs only at the discharge point, with little abundance of target genes further downstream. The sulfonamide antibiotic sulfamethoxazole (SMX) was persistent in both near-pristine and wastewater-impacted river water when introduced at a concentration of 12.5 µg/L, but had neglectable effects on the microbial community diversity. Interestingly, concentrations as high as 100 µg/L SMX induced a short-term increase in microbial activity in both surface water and biofilm compartment, as revealed by bulk and nanoscale measurements. Altogether, this work underscores the fundamental role of wastewater treatment in combating the environmental dissemination of antibiotic resistance.:Summary 1 Zusammenfassung 5 1 Introduction 9 1.1 Rundown of the global antibiotic resistance crisis 9 1.1.1 History of antibiotics 9 1.1.2 Emergence of antibiotic resistance 9 1.1.3 Integrons as vehicles for antibiotic resistance 10 1.1.4 Risks related to environmental antibiotic resistance 12 1.2 Fate of antibiotic resistance genes in the aquatic environment 14 1.2.1 Genetic indicators for antibiotic resistance 14 1.2.2 River surface water compartment as dissemination route for antibiotic resistance 15 1.2.3 River biofilm compartment as reservoir for antibiotic resistance 17 1.3 Impact of antibiotics on aquatic microbial communities 18 1.4 Fate and effect of sulfamethoxazole in surface waters 20 2 Scope of the thesis 22 3 Main findings and scientific implications 24 3.1 Fate of antibiotic resistance genes after wastewater discharge into a near-pristine river 24 3.1.1 Wastewater is the primary source for aquatic antibiotic resistance 24 3.1.2 Drought increases the antibiotic resistance prevalence in surface waters 25 3.1.3 Riverbed biofilms serve as local reservoirs for antibiotic resistance genes 26 3.2 Anthropogenic pollution is the key driver for microbial community alteration 26 3.3 Sulfamethoxazole increases the microbial activity of aquatic microbial communities 27 4 Conclusions and future perspective 29 5 References 31 6 Publications 43 6.1 Publication 1 43 6.2 Publication 2 56 6.3 Publication 3 69   7 Appendix 94 7.1 Declaration of independent work 94 7.2 List of publications and conference contributions 95 7.2.1 Publications 95 7.2.2 Conference contributions 96 7.3 Contribution of Co-authors 97 7.4 Curriculum vitae 101 7.5 Acknowledgements 104 7.6 Supplementary Material 105 7.6.1 Supplementary Material for Publication 1 105 7.6.2 Supplementary Material for Publication 2 118 7.6.3 Supplementary Material for Publication 3 125

This thesis examines the biodiversity and evolutionary ecology of Lumnitzera mangroves in the Indonesian Archipelago. Using a combination of genomics and metabolomics, the study arrived at several key findings: (1) Investigation into 14 populations of L. littorea and 21 populations of L. racemosa revealed low genetic variation, with significant barriers like Sulawesi and the Java Sea influencing genetic differentiation. (2) Specifically for L. littorea, Wallace's line was identified as a key biogeographical separator, marking divergent evolutionary pathways and separating phylogroups, whereas L. racemosa showed a mixing population in the Wallacea region. (3) Uniquely, the research discovered a diversity in sulfated constituents, including previously unknown compounds like Lumnitzeralactone. (4) Further emphasizing the mangroves' medicinal importance, antibacterial potential was uncovered in the species. (5) The study innovatively linked phylogenetic data with chemical analyses, offering a comprehensive view of mangrove evolution and ecology. (6) Finally, the findings highlighted the need for specific conservation strategies, considering the genetic differentiation within populations, to protect the mangroves' ecological significance and medicinal value across Indonesia.:Preface 4 Summary 5 Zusammenfassung 10 1. Introduction 15 1.1. Characteristics, significance, and threat of mangroves 15 1.1.1. Characteristics of mangroves 15 1.1.2. Significance of Indonesian mangroves 16 1.1.3. Threats to mangroves 18 1.2. Evolutionary processes and diversity of mangroves 19 1.2.1. Evolution of mangroves 19 1.2.2. Sea surface currents in Indonesia shape genetic structure 20 1.2.3. The relevance of Wallace’s line to mangrove evolution 22 1.2.4. Isolation by geographical distance 24 1.2.5. Genetic diversity and population structure 25 1.3. Diversity of bioactive compounds of mangrove genus Lumnitzera 27 1.3.1. Sulfur-containing metabolites 27 1.3.2. Phylogenetics 28 1.3.3. Anti-infective potential 29 1.4. Study species 30 1.5. Aim of the thesis 33 2. Material and Methods 37 2.1. Sampling and sample design 37 2.2. Laboratory procedures and genetic analysis 38 2.2.1. DNA isolation 38 2.2.2. ddRADseq sequencing, and bioinformatics 38 2.2.3. Genetic diversity, population structure and differentiation 40 2.2.4. Identifying barriers and areas of connectivity 41 2.2.5. Isolation by distance and sea surface current connectivity 42 2.2.6. Polymerase Chain Reaction (PCR), and phylogenetic analyses 43 2.3. Laboratory procedures and phytochemical analysis 44 2.3.1. Root sample extraction 44 2.3.2. TLC, Low-resolution ESI-MS spectra, HPLC, and NMR 45 2.3.3. UHPLC-ESI-QqTOF-MS and MS/MS 46 2.3.4. RP-UHPLC-ESI-LIT-Orbitrap-MS 47 2.3.5. Extraction and isolation of compounds 47 2.3.6. Anti-infective bioassays 51 3. Results 52 3.1. Genetic diversity and population structure 52 3.1.1. Genetic diversity 52 3.1.2. Population structure and genetic differentiation 54 3.1.3. Effective migration 58 3.1.4. Isolation by distance and sea surface current connectivity 59 3.2. Unusual-sulfated constituent and anti-infective properties 61 3.2.1. Phytochemical screening 61 3.2.2. Phylogenetic tree of Lumnitzera 69 3.2.3. Evaluation of anti-infective properties 70 4. Discussion 74 4.1. Population genomics of Lumnitzera mangroves in Indonesia 74 4.1.1. The genetic diversity paradox in mangroves 74 4.1.2. Phylogroups and the Sunda-Wallacea biogeographical pattern 76 4.1.3. Limited mixture among phylogroups and populations by sea surface currents 78 4.1.4. Restricted gene flow by geographical distance 80 4.1.5. Evolutionary ecology of Lumnitzera inferred by genetics and chemodiversity 81 4.2. Bioactive compounds and anti-infective potential of Lumnitzera 82 4.2.1. Diversity of bioactive compounds 82 4.2.2. Sulfated and nonsulfated ellagic acid supported by phylogenetic pattern 83 4.2.3. Anti-infective properties and their restriction to particular locations 86 5. Conclusion and future perspective 89 6. References 94 7. Appendix 111 Curriculum vitae 119 Declaration of independent work 122 Acknowledgments 123 Author contributions statement 125 Authors’s Addendum / Diese Arbeit untersucht die biologische Vielfalt und evolutionäre Ökologie der Lumnitzera-Mangroven im indonesischen Archipel. Unter Verwendung einer Kombination aus Genomik und Metabolomik gelangte die Studie zu mehreren wichtigen Ergebnissen: (1) Die Untersuchung von 14 Populationen von L. littorea und 21 Populationen von L. racemosa ergab eine geringe genetische Variation, wobei signifikante Barrieren wie Sulawesi und die Javasee die genetische Differenzierung beeinflussen. (2) Speziell für L. littorea wurde die Wallace-Linie als wichtige biogeografische Trennlinie identifiziert, die divergierende Evolutionspfade markiert und Phylogruppen trennt, während L. racemosa eine Mischpopulation in der Wallacea-Region aufweist. (3) Einzigartig war die Entdeckung einer Vielfalt an sulfatierten Bestandteilen, darunter bisher unbekannte Verbindungen wie Lumnitzeralacton. (4) Ein weiterer Beleg für die medizinische Bedeutung der Mangroven ist das antibakterielle Potenzial, das in der Art entdeckt wurde. (5) Die Studie verknüpfte auf innovative Weise phylogenetische Daten mit chemischen Analysen und bot so einen umfassenden Einblick in die Evolution und Ökologie der Mangroven. (6) Schließlich verdeutlichten die Ergebnisse die Notwendigkeit spezifischer Erhaltungsstrategien, die die genetische Differenzierung innerhalb der Populationen berücksichtigen, um die ökologische Bedeutung und den medizinischen Wert der Mangroven in ganz Indonesien zu schützen.:Preface 4 Summary 5 Zusammenfassung 10 1. Introduction 15 1.1. Characteristics, significance, and threat of mangroves 15 1.1.1. Characteristics of mangroves 15 1.1.2. Significance of Indonesian mangroves 16 1.1.3. Threats to mangroves 18 1.2. Evolutionary processes and diversity of mangroves 19 1.2.1. Evolution of mangroves 19 1.2.2. Sea surface currents in Indonesia shape genetic structure 20 1.2.3. The relevance of Wallace’s line to mangrove evolution 22 1.2.4. Isolation by geographical distance 24 1.2.5. Genetic diversity and population structure 25 1.3. Diversity of bioactive compounds of mangrove genus Lumnitzera 27 1.3.1. Sulfur-containing metabolites 27 1.3.2. Phylogenetics 28 1.3.3. Anti-infective potential 29 1.4. Study species 30 1.5. Aim of the thesis 33 2. Material and Methods 37 2.1. Sampling and sample design 37 2.2. Laboratory procedures and genetic analysis 38 2.2.1. DNA isolation 38 2.2.2. ddRADseq sequencing, and bioinformatics 38 2.2.3. Genetic diversity, population structure and differentiation 40 2.2.4. Identifying barriers and areas of connectivity 41 2.2.5. Isolation by distance and sea surface current connectivity 42 2.2.6. Polymerase Chain Reaction (PCR), and phylogenetic analyses 43 2.3. Laboratory procedures and phytochemical analysis 44 2.3.1. Root sample extraction 44 2.3.2. TLC, Low-resolution ESI-MS spectra, HPLC, and NMR 45 2.3.3. UHPLC-ESI-QqTOF-MS and MS/MS 46 2.3.4. RP-UHPLC-ESI-LIT-Orbitrap-MS 47 2.3.5. Extraction and isolation of compounds 47 2.3.6. Anti-infective bioassays 51 3. Results 52 3.1. Genetic diversity and population structure 52 3.1.1. Genetic diversity 52 3.1.2. Population structure and genetic differentiation 54 3.1.3. Effective migration 58 3.1.4. Isolation by distance and sea surface current connectivity 59 3.2. Unusual-sulfated constituent and anti-infective properties 61 3.2.1. Phytochemical screening 61 3.2.2. Phylogenetic tree of Lumnitzera 69 3.2.3. Evaluation of anti-infective properties 70 4. Discussion 74 4.1. Population genomics of Lumnitzera mangroves in Indonesia 74 4.1.1. The genetic diversity paradox in mangroves 74 4.1.2. Phylogroups and the Sunda-Wallacea biogeographical pattern 76 4.1.3. Limited mixture among phylogroups and populations by sea surface currents 78 4.1.4. Restricted gene flow by geographical distance 80 4.1.5. Evolutionary ecology of Lumnitzera inferred by genetics and chemodiversity 81 4.2. Bioactive compounds and anti-infective potential of Lumnitzera 82 4.2.1. Diversity of bioactive compounds 82 4.2.2. Sulfated and nonsulfated ellagic acid supported by phylogenetic pattern 83 4.2.3. Anti-infective properties and their restriction to particular locations 86 5. Conclusion and future perspective 89 6. References 94 7. Appendix 111 Curriculum vitae 119 Declaration of independent work 122 Acknowledgments 123 Author contributions statement 125 Authors’s Addendum