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Similar to chloroplast loci, mitochondrial markers are frequently used for genotyping, phylogenetic studies, and population genetics, as they are easily amplified due to their multiple copies per cell. In a recent study, it was revealed that the chloroplast offers little variation for this purpose in central European populations of beech. Thus, it was the aim of this study to elucidate, if mitochondrial sequences might offer an alternative, or whether they are similarly conserved in central Europe. For this purpose, a circular mitochondrial genome sequence from the more than 300-year-old beech reference individual Bhaga from the German National Park Kellerwald-Edersee was assembled using long and short reads and compared to an individual from the Jamy Nature Reserve in Poland and a recently published mitochondrial genome from eastern Germany. The mitochondrial genome of Bhaga was 504,730 bp, while the mitochondrial genomes of the other two individuals were 15 bases shorter, due to seven indel locations, with four having more bases in Bhaga and three locations having one base less in Bhaga. In addition, 19 SNP locations were found, none of which were inside genes. In these SNP locations, 17 bases were different in Bhaga, as compared to the other two genomes, while 2 SNP locations had the same base in Bhaga and the Polish individual. While these figures are slightly higher than for the chloroplast genome, the comparison confirms the low degree of genetic divergence in organelle DNA of beech in central Europe, suggesting the colonisation from a common gene pool after the Weichsel Glaciation. The mitochondrial genome might have limited use for population studies in central Europe, but once mitochondrial genomes from glacial refugia become available, it might be suitable to pinpoint the origin of migration for the re-colonising beech population.
A large number of chemicals are constantly introduced to surface water from anthropogenic and natural sources. Although substantial efforts have been made to identify these chemicals (e.g potentially anthropogenic contaminants) in surface waters using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), a large number of LC-HRMS chemical signals often with high peak intensity are left unidentified. In addition to synthetic chemicals and transformation products, these signals may also represent plant secondary metabolites (PSMs) released from vegetation through various pathways such as leaching, surface run-off and rain sewers or input of litter from vegetation. While this may be considered as a confounding factor in screening of water contaminants, it could also contribute to the cumulative toxic risk of water contamination. However, it is hardly known to what extent these metabolites contribute to the chemical mixture of surface waters. Thus, reducing the number of unknowns in water samples by identifying also PSMs in significant concentrations in surface waters will help to improve monitoring and assessment of water quality potentially impacted by complex mixtures of natural and synthetic compounds. Therefore, the main focus of the present study was to identify the occurrence of PSMs in river waters and explore the link between the presence of vegetation along rivers and detection of their corresponding PSMs in river
water.
In order to achieve the goals of the present thesis, two chemical screening approaches, namely, non-target and target screening using LC-HRMS were implemented. (1) Non-target analysis involving a novel approach has been applied to associate unknown peaks of high intensity in LC-HRMS to PSMs from surrounding vegetation by focusing on peaks overlapping between river water and aqueous plant extracts (Annex A1). (2) LC–HRMS target screening in river waters were performed for about 160 PSMs, which were selected from a large phytotoxin database (Annex A2 and A3) considering their expected abundance in the vegetation, their potential mobility, persistence and toxicity in the water cycle and commercial availability of standards.
In non-target screening (Annex A1), a high number of overlapping peaks has been found in between aqueous plant extracts and water from adjacent location, suggesting a significant impact of vegetation on chemical mixtures detectable in river waters. The chemical structures were assigned for 12 pairs of peaks while several pairs of peaks
whose MS/MS spectra matched but no structure suggestion were made by the implemented software tools for retrieving possible chemical structure. Nevertheless, the pairs of peaks with matching spectra represented the same chemical structure. The identified compound belonged to different compound classes such as coumarins, flavonoids besides others. For the identified PSMs individual concentration up to 5 µg/L were measured. The concentration and the number of detected PSMs per sample were correlated with the rain event and vegetation coverage.
Target screening unraveled the occurrence of 33 out of 160 target compounds in river waters (Annex A2 and A3). The identified compounds belonged to different classes such as alkaloids, coumarins, flavonoids, and other compounds. Individual compound concentrations were up to several thousand ng/L with the toxic alkaloids narciclasine and
lycorine recording highest maximum concentrations. The neurotoxic alkaloid coniine from poison hemlock was detected at concentrations up to 0.4 µg/L while simple coumarins
esculetin and fraxidin occurred at concentrations above 1 µg/L. The occurrence of some PSMs in river water were correlated to the specific vegetation growing along the rivers while the others were linked to a wide range of vegetation. As an example, narciclasine and lycorine was emitted by the dominant plant species from Amaryllidaceae family (e.g. Galanthus nivalis (snow drop), Leucojum vernum and Anemone nemorosa) while intermedine and echimidine were from Symphytum officinale. The ubiquitous occurrence of simple coumarins fraxidin, scopoletin and aesculetin could be linked to their presence in a wide range of vegetation.
Due to lack of aquatic toxicity data for the identified PSMs (in both target and non-target) and extremely scarce exposure data, no reliable risk assessment was possible.
Alternatively, risk estimation was performed using the threshold for toxicological concern (TTC) concept developed for drinking water contaminants. Many of the identified PSMs
exceeded the TTC value (0.1 µg/L) thus caution should be taken when using such surface waters for drinking water abstraction or recreational use.
This thesis provides an overview of the occurrence of PSMs in river water impacted by the massive presence of vegetation. Concentration for many of the identified PSMs are well within the range of those of synthetic environmental contaminants. Thus, this study adds to a series of recent results suggesting that possibly toxic PSMs occur in relevant concentrations in European surface waters and should be considered in monitoring and risk assessment of water resources. Aquatic toxicity data for PSMs are extensively lacking but are required to include these compounds in the assessment of risks to aquatic organisms and for eliminating risks to human health during drinking water production.
Impact of biological education and gender on students' connection to nature and relational values
(2020)
The new concept of relational values (RVs) is gaining more and more attention in environmental research, but empirical analyses are still rare. However, this type of research is necessary because the RVs have an influence on environmental behavior. To evaluate the impact of biological education on attributing higher importance to RVs and connectedness to nature, we compared the connection to nature scores (using the inclusion of nature scale (INS) and connectedness to nature scale (CNS)) and RV scores of biologically interested high school students (n = 417) with first year (n = 593) and advanced biology (n = 223) students. While high school students showed significant lower connection to nature scores than university students, there was no significant difference in RVs between the test groups. These results suggest that there is a lack of factors in the university study of biology that can change RVs. The gender comparison of RVs and connection to nature showed a significant higher RV score for females while INS and CNS did not show a gender difference. Thus, the study makes an important contribution to the research, as it was able to prove that gender has an influence on a person's RVs but not on their connection to nature.
In der vorliegenden Arbeit wurde das Zinkfinger-µ-Protein HVO_2753 des halophilen Archaeons Haloferax volcanii hinsichtlich seiner biologischen Funktion und seiner Struktur charakterisiert.
Zinkfinger-µ-Proteine wurden bisher nur sehr wenig untersucht, während ihnen jedoch in den letzten Jahren steigendes Interesse entgegengebracht wird. Im Genom von H. volcanii sind mehr als 40 solcher Zinkfinger-µ-Proteine codiert. Von diesen besitzt mit HVO_2753 lediglich eines nicht nur zwei, sondern vier der charakteristischen C(P)XCG-Muster, was für die Anwesenheit von zwei Zinkfinger-Motiven spricht. Während Homologe von HVO_2753 in vielen Euryachaeota vorkommen und manche davon als Zink-Ribbon RNA-Bindeproteine annotiert sind, ist über ihre Funktion jedoch nichts bekannt. Zur Charakterisierung des Proteins wurde zunächst eine in frame-Deletionsmutante seines Gens erstellt und diese einer phänotypischen Charakterisierung unterzogen. Die Mutante wies, verglichen mit dem Wildtyp, keine Unterschiede im Wachstum in Komplexmedium oder in synthetischem Medium mit Glukose als Kohlenstoffquelle auf. Ein schweres Defizit konnte jedoch sowohl bei der Adhäsion und Biofilmbildung als auch der Schwärmfähigkeit der Deletionsmutante festgestellt werden. Während die Schwärmfähigkeit des Wildtyps durch plasmidische Expression von HVO_2753 in der Deletionsmutante teilweise wiederhergestellt werden konnte, war eine solche Komplementation bei der Biofilmbildung nicht möglich. Die Analyse der Relevanz ausgewählter Aminosäuren, wie beispielsweise das jeweils erste Cystein in jedem C(P)XCG-Muster zeigte, dass die Substitution jeder einzelnen der getesteten Aminosäuren einen Funktionsverlust des Proteins nach sich zieht. Die Untersuchung des HVO_2753-Transkripts mittels Northern Blot-Analyse bestätigte erste Hinweise aus vorangegangenen dRNA- und RNA-Seq-Studien, die eine Co-Transkription von HVO_2753 mit dem Nachbargen HVO_2752, das für den Translations-Elongationsfaktor aEF-1 beta codiert, aufzeigten. Daraufhin erfolgte eine Untersuchung des Ribosomenprofils, bei der keine Unterschiede zwischen der Deletionsmutante und der Überexpressionsmutante von HVO_2753 festgestellt werden konnten.
Eine Variante von HVO_2753 mit N-terminalem Hexahistidin-Tag wurde homolog überproduziert und aufgereinigt. Die Überproduktion und Aufreinigung wurden im Zuge dieser Arbeit weiter, speziell für HVO_2753, optimiert. So konnten große Mengen von HVO_2753n überproduziert und bei nativen Salzbedingungen mittels Nickel-Affinitätschromatographie und anschließender Größenausschlusschromatographie aufgereinigt werden. Eine massenspektrometrische Analyse bestätigte sowohl das Molekulargewicht als auch die Abwesenheit posttranslationaler Modifikationen. Die Untersuchung der Menge an gebundenem Zink im Protein erfolgte beim Zink-Assay mit Hilfe des hochsensitiven und hochspezifischen Fluorophors ZnAF-2F. Dabei konnte gezeigt werden, dass überraschenderweise lediglich ein Zink-Ion in HVO_2753 gebunden vorliegt.
Zur weiteren Funktionsaufklärung erfolgte eine Interaktionspartnersuche. Hierfür wurde HVO_2753 überproduziert, ein in vivo-Crosslink und anschließend eine native Aufreinung durchgeführt. Die massenspektrometrische Analyse ausgewählter Fraktionen nach der Größenausschlusschromatographie ergaben eine Vielzahl an möglichen Bindepartnern. Besonders häufig wurde hier die GalE family Epimerase/Dehydratase gefunden. Eine weitere Methode zur Suche nach Interaktionspartnern richtete sich auf RNAs. Hier konnten mittels eines eigens entwickelten Protokolls neben RNAs des Translationsapparates auch mehrfach die tRNA(Glu) gefunden werden.
Zusätzlich sollte die Transkriptomanalyse mittels RNA-Sequenzierung Unterschiede zwischen Wildtyp, Deletionsmutante und Komplementationsmutante aufzeigen. Hier wurden weitreichende Auswirkungen der Deletion von HVO_2753 gefunden. Zahlreiche Gene in mehreren Operons zur Motilität und Chemotaxis lagen in der Deletionsmutante stark herunterreguliert vor, während die Gene einiger Metallionen-Transporter und der Eisen(III)-Siderophor-Biosynthese hochreguliert vorlagen. In der Komplementationsmutante konnten nur von den letzteren Genen Transkriptlevel vergleichbar mit denen des Wildtyps wiedergefunden werden.
In dieser Arbeit konnte gezeigt werden, dass das kleine Zinkfinger-Protein HVO_2753 eine essenzielle Rolle in der positiven Regulation der Motilität, Chemotaxis und der Adhäsion bzw. Biofilmbildung spielt. Gleichzeitig übt HVO_2753 eine negative Regulation auf den Metallionen-Transport und die Biosynthese des Eisen(III)-Siderophors aus.
Climate change imposes severe stress on European forests, with forest degradation already visible in several parts of Europe. Thus adaptation of forestry applications in Mediterranean areas and central Europe is necessary. Proactive forestry management may include the planting of Mediter- ranean oak species in oak-bearing Central European regions. Five replicate common gardens of Greek and Italian provenances of Quercus ilex, Q. pubescens and Q. frainetto seedlings (210 each per plantation) were established in Central Italy, NE Greece (two) and Southern Germany (two, including Q. robur) to assess their performance under different climate conditions. Climate and soil data of the plantation sites are given and seedling establishment was monitored for survival and morphological parameters. After 3 years (2019) survival rates were satisfactory in the German and Italian sites, whereas the Greek sites exerted extremely harsh conditions for the seedlings, including extreme frost and drought events. In Germany, seedlings suffered extreme heat and drought periods in 2018 and 2019 but responded well. Provenances were ranked for each country for their performance after plan- tation. In Greece and Italy, Q. pubescens was the best performing species. In Germany, Q. pubescens and Q. robur performed best. We suggest that Greek or Italian provenances of Q. pubescens may be effectively used for future forestation purposes in Central Europe. For the establishment of Quercus plantations in Northern Greece, irrigation appears to be a crucial factor in seedling establishment.
Microplastics (MPs) are ubiquitous and persistent pollutants, and have been detected in a wide variety of media, from soils to aquatic systems. MPs, consisting primarily of polyethylene, polypropylene, and polyacrylamide polymers, have recently been found in 12% of samples of honey collected in Ecuador. Recently, MPs have also been identified in honey bees collected from apiaries in Copenhagen, Denmark, as well as nearby semiurban and rural areas. Given these documented exposures, assessment of their effects is critical for understanding the risks of MP exposure to honey bees. Exposure to polystyrene (PS)-MPs decreased diversity of the honey bee gut microbiota, followed by changes in gene expression related to oxidative damage, detoxification, and immunity. As a result, the aim of this perspective was to investigate whether wide-spread prevalence of MPs might have unintended negative effects on health and fitness of honey bees, as well as to draw the scientific community’s attention to the possible risks of MPs to the fitness of honey bees. Several research questions must be answered before MPs can be considered a potential threat to bees.
Photorhabdus and Xenorhabdus are Gram-negative, entomopathogenic bacteria, living in endosymbiosis with the soil-dwelling nematode of the genera Steinernema and Heterorhabditis. The life cycle of these nematodes consists of non-feeding infective juvenile (IJ) stage, which actively searches for insects in the soil. After penetrating the insect prey, Photorhabdus and Xenorhabdus bacteria are released from the nematode gut. The bacteria proliferate and produce toxins to kill the insect. Photorhabdus and Xenorhabdus support nematode development throughout the life cycle and to get rid of food competitors by providing a wide variety of specialized metabolites (SMs). However, little is known about which SMs function as so called “food signals” to trigger the development process.
The IJs develop into adult, self-fertilizing hermaphrodites in a process called recovery, while feeding on cadaver and bacterial biomass. Heterorhabditis and Steinernema proceed to breed until nutrients are exhausted. Next generation IJs (NG-IJs) develop and leave the cadaver to search for another insect prey.
Photorhabdus and Xenorhabdus can be cultivated in defined medium under laboratory conditions. By placing IJs on a plate containing their respective bacterial symbiont, the complete life cycle of the nematodes can be observed in vitro. The in vitro nematode bioassay was used as a tool to investigate the development of the nematode.
The aim of this study was to find the food signals responsible for nematode development. Different Photorhabdus deletion strains unable to produce one or several SMs were co-cultivated with nematodes in the nematode bioassay. Subsequently, two aspects of the life cycle were investigated: recovery and NG-IJ development.
As isopropyl stilbene (IPS) is postulated to function as a food signal to support nematode recovery, it was used as a starting point for investigations. This study was focused on the biosynthetic pathway of IPS, including intermediates, side products and derivatives to investigate which one is in fact responsible for supporting nematode development.
The biosynthesis of IPS requires two precursors, phenylalanine and leucine (Figure 5). The first topic was focused on the phenylalanine derived pathway. Photorhabdus laumondii deletion mutants, defective in intermediate steps of this pathway, were created. The deletion of the genes coding for the phenylalanine ammonium lyase (stlA), converting phenylalanine into cinnamic acid (CA), the coenzyme A (CoA) ligase (stlB) and the operon coding for a ketosynthase and aromatase (stlCDE), were used. These strains were used for nematode bioassay including complementation of mutant phenotypes by feeding experiments. Recovery of nematodes grown on the deletion strains was always lower than recovery of nematodes grown on wild type bacteria. Feeding IPS to a deletion strain did not restore wild type level nematode recovery, thus IPS cannot be the food signal. Instead, the food signal must be another compound derived from this part of biosynthetic pathway. Lumiquinone and 2,5-dihydrostilbene are suggested to function as food signals and need to be investigated in future work.
The second part of this study was focused on the leucine derived pathway, which involved the Bkd complex forming the iso-branched part of IPS. A deletion of bkd was created and phenotypically analysed, subsequently performed with the nematode bioassay. Not only IPS but also other branched SMs, like photopyrones and phurealipids are synthetised by the Bkd complex. Deletions strains defective in producing photopyrones and phurealipids were also performed in nematode bioassays to investigate effects of these SMs individually. Branched SMs did not have an impact on nematode development, but nematodes grown on the ΔbkdABC strain showed a reduced nematode recovery and almost diminished NG-IJs development. As the Bkd complex also produces branched chain fatty acids (BCFAs), feeding experiments were performed with lipid extracts of wild type and mutant strain. All lipid extracts improved recovery, but only wild type lipids could complement NG-IJ development. This strongly indicates that BCFAs play an important role in NG-IJ development, which needs to be proven with purified BCFA feeding. This is an interesting finding, which could improve nematode production for biocontrol agent usage.
The role of IPS derived to epoxy stilbene (EPS) for nematode development, was another focus in the nematode life cycle. Recently it was demonstrated that EPS does not support nematode development. However, EPS forms adducts with amino acids. In my thesis, novel adducts containing the amino acid phenylalanine or a tetrapeptide were characterized. Another adduct, most likely being an EPS dimer, was also characterized. The biological role of such adducts was discussed to be potentially important for insect weakening and the structure of the novel compounds need to be structure elucidated and tested for bioactivity.
Climatic niches describe the climatic conditions in which species can persist. Shifts in climatic niches have been observed to coincide with major climatic change, suggesting that species adapt to new conditions. We test the relationship between rates of climatic niche evolution and paleoclimatic conditions through time for 65 Old-World flycatcher species (Aves: Muscicapidae). We combine niche quantification for all species with dated phylogenies to infer past changes in the rates of niche evolution for temperature and precipitation niches. Paleoclimatic conditions were inferred independently using two datasets: a paleoelevation reconstruction and the mammal fossil record. We find changes in climatic niches through time, but no or weak support for a relationship between niche evolution rates and rates of paleoclimatic change for both temperature and precipitation niche and for both reconstruction methods. In contrast, the inferred relationship between climatic conditions and niche evolution rates depends on paleoclimatic reconstruction method: rates of temperature niche evolution are significantly negatively related to absolute temperatures inferred using the paleoelevation model but not those reconstructed from the fossil record. We suggest that paleoclimatic change might be a weak driver of climatic niche evolution in birds and highlight the need for greater integration of different paleoclimate reconstructions.
The factors that vary the aroma of Tuber magnatum fruiting bodies are poorly understood. The study determined the headspace aroma composition, sensory aroma profiles, maturity and bacterial communities from T. magnatum originating from Italy, Croatia, Hungary, and Serbia, and tested if truffle aroma is dependent on provenance and if fruiting body volatiles are explained by maturity and/or bacterial communities.
Headspace volatile profiles were determined using gas chromatography–mass spectrometry–olfactometry (GC-MS-O) and aroma of fruiting body extracts were sensorially assessed. Fruiting body maturity was estimated through spore melanisation. Bacterial community was determined using 16S rRNA amplicon sequencing.
Main odour active compounds were present in all truffles but varied in concentration. Aroma of truffle extracts were sensorially discriminated by sites. However, volatile profiles of individual fruiting bodies varied more within sites than across geographic area, while maturity level did not play a role. Bacterial communities varied highly and were partially explained by provenance. A few rare bacterial operational taxonomical units associated with a select few nonodour active volatile compounds.
Specificities of the aroma of T. magnatum truffles are more likely to be linked to individual properties than provenance. Some constituents of bacteria may provide biomarkers of provenance and be linked to nonodour active volatiles.
Although macroecology is a well-established field, much remains to be learned about the large-scale variation of fungal traits. We conducted a global analysis of mean fruit body size of 59 geographical regions worldwide, comprising 5340 fungal species exploring the response of fruit body size to latitude, resource availability and temperature. The results showed a hump-shaped relationship between mean fruit body size and distance to the equator. Areas with large fruit bodies were characterised by a high seasonality and an intermediate mean temperature. The responses of mutualistic species and saprotrophs were similar. These findings support the resource availability hypothesis, predicting large fruit bodies due to a seasonal resource surplus, and the thermoregulation hypothesis, according to which small fruit bodies offer a strategy to avoid heat and cold stress and therefore occur at temperature extremes. Fruit body size may thus be an adaptive trait driving the large-scale distribution of fungal species.