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Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
The fungal genus Pestalotiopsis s.l. contains approximately 300 described species and is globally distributed. The monotypic genus Pestalotia is considered the closest relative of Pestalotiopsis s.l. This study aims to investigate the diversity and systematics within Pestalotiopsis s.l. and its relation to Pestalotia. Therefore, an integrative approach is used considering molecular phylogeny methods as well as examination of morphological characters.
Recently, Pestalotiopsis s.l. was split into three genera with the addition of the newly erected Neopestalotiopsis and Pseudopestalotiopsis. The species of these genera are usually saprotrophic, phytoparasitic, or endophytic, and have been isolated from soil, air, and many kinds of anorganic material. The asexual fruiting bodies appear on infected plant material as black acervuli that release conidia. The conidia are important to examine for morphological taxon recognition. The number of conidial cells is the feature that distinguishes Pestalotiopsis s.l. spp. with five celled conidia, from Pestalotia pezizoides with six celled conidia. However, the significance of morphological characters is controversially discussed among mycologists. In recent years, 55 new species were described based on minor genetic distances and marginal or no morphological differences. Thus, the value of certain morphological characters and genetic markers need to be reconsidered.
In this study, 102 herbarium specimens of 26 described species, with an emphasis on plant pathogenic species from North America, have been morphologically examined and documented through drawings and photographs. Morphological examination was complemented with a comprehensive molecular dataset obtained from 191 cultures representing the genera Neopestalotiopsis, Pestalotia, Pestalotiopsis, Pseudopestalotiopsis, and Truncatella. One novelty of this work is that, besides the well-established markers ITS, TEF1, and ß-tubulin, the protein-coding genes MCM7 and TSR1 were successfully sequenced and included in the analyses. Phylogenies using Maximum Likelihood and Bayesian inference methods of single loci and the combined dataset were calculated. By comparison of these phylogenies, MCM7 was identified as the most powerful one in terms of phylogenetic resolution and statistical support of nodes and is proposed as an additional barcoding marker in Pestalotiopsis s.l.
In Pestalotiopsis, species delimitation was tested using the Baysian Phylogenetics and Phylogeography (BP&P) program that tests an existing species scenario against Bayesian inference methods under a multispecies coalescent model. The program supported only ten species out of the predetermined 19 species scenario. Measurements of conidia for species detected by BP&P were explored using a TukeyHSD-Test in the program R to find means that are significantly different from each other. This test revealed that combinations of morphological characters are required to distinguish between the ten species found by BP&P.
Another purpose of this work was to clarify the status of Pestalotia with regard to Pestalotiopsis s.l. Therefore, fresh epitypic material of Pestalotia pezizoides, was collected, isolated, and cultivated. The molecular analysis of a combined dataset of the gene regions ITS and LSU for species of Amphisphaeriales nested P. pezizoides in the genus Seiridium. Thus, synonymy of Pestalotia with Seiridium is proposed here. This is supported by morphology of the conidia. Further, an epitype is proposed for the type species of Pestalotiopsis, P. maculans. On the other hand, the recently proposed epitype of P. adusta is rejected here as it conflicts with the taxonomic hypothesis obtained in this study and its introduction is inconsistent with the formal requirements for epitypification. A new topotypic specimen is proposed instead. Additionally, several nomenclatural changes become necessary in many species examined. These include three new combinations and six synonyms of species of Pestalotiopsis s.l.
The conclusion of this work is that morphological data have potential as a valuable, inexpensive and easy way to recognize species. However, it is not the best method for species discovery and delimitation bearing in mind that in microfungi and many other organisms, individual plasticity and analogous structures are inadequately investigated. By phylogenetic analyses of molecular sequence data, it is possible to compare a great amount of equivalent characters and to delimit species that are morphologically cryptic. This is especially important since species of Pestalotiopsis s.l. mostly lack sexual structures that are helpful for morphological species delimitation in other groups of fungi. Thus, the Genealogical Concordance Species Concept (GCSC) finds its application in many fungal taxa. Conflicts in the genealogy between phylogenetic trees of different markers are interpreted as recombination of the genetic material within a linage. Accordingly, the change from conflict to congruence in a set of different phylogenetic trees can be seen as the species limit. It can be expected that increased application of the GCSC will lead to further approximation of described species numbers to the real number of species, especially in complicated groups like asexual microfungi.
Seed dispersal is a key ecosystem function for plant regeneration, as it involves the movement of seeds away from the parental plants to particular habitats where they can germinate and transition to seedlings and ultimately adult plants. Seed dispersal is shaped by a diversity of abiotic and biotic factors, particularly by associations between plants and climate and between plants and other species. Due to the ongoing loss of biodiversity and changing global conditions, such interactions are prone to change and pose a severe threat to plant regeneration. One way to address this challenge is to study associations between plant traits and abiotic and biotic factors to understand the potential impacts of global change on plant regeneration. Plant communities have long been analyzed through the lens of vegetative traits, mainly ignoring how other traits interact and respond to the environment. For instance, while associations between vegetative traits (e.g., specific leaf area, leaf nitrogen content) and climate are well studied, there are few case studies of reproductive traits in relation to trait-environment associations in the context of global change.
Thus, the overarching aim of this dissertation is to explore how trait-environment associations, with a special focus on reproductive traits, can improve our understanding of the effect that global change may have on seed dispersal, and ultimately on plant regeneration. To this end, my research focuses on studying associations between plant traits and abiotic and biotic factors along an elevational gradient in both forests and deforested areas of tropical mountains. This dissertation addresses three principal research objectives.
First, I investigate the extent to which reproductive (seed and fruit traits) and vegetative traits (leaf traits) are related to abiotic and biotic factors for communities of fleshy-fruited plants in the Ecuadorian Andes. I used multivariate analyses to test associations between four (a)biotic factors and seven reproductive traits and five vegetative traits measured on 18 and 33 fleshy fruited plant species respectively. My analyses demonstrate that climate and soil conditions are strongly associated with the distribution of both reproductive and vegetative traits in tropical tree communities. The production of “costly” vs. “cheap” seeds, fruits and leaves, i.e., the production of few rewarding fruits and acquisitive leaves versus the production of many less-rewarding fruits and conservative leaves, is primarily limited by temperature, whereas the size of plant organs is more related to variation in precipitation and soil conditions. My findings suggest that associations between reproductive and vegetative traits and the abiotic environment follow similar principles in tropical tree communities.
Second, I assess how climate and microhabitat conditions affect the prevalence of endozoochorous plant species in the seed rain of tropical montane forests in southern Ecuador. I analyzed seed rain data for an entire year from 162 traps located across an elevational gradient spanning of 2000 m. I documented the microhabitat conditions (leaf area index and soil moisture next to each seed trap) at small spatial scale as well as the climatic conditions (mean annual temperature and rainfall in each plot) at large spatial scale. After a one-year of sampling, I counted 331,838 seeds of 323 species/morphospecies. My analyses demonstrate that the prevalence of endozoochorous plant species in the seed rain increases with temperature across elevations and with leaf area index within elevations. These results show that the prevalence of endozoochory is shaped by the interplay of both abiotic and biotic factors at large and small spatial scales.
Third, I examine the potential of seed rain to restore deforested tropical areas along an elevational gradient in southern Ecuador. For this chapter, I collected seed rain using 324 seed traps installed in 18 1-ha plots in forests (nine forest plots) and in pastures (nine deforested plots) along an elevational gradient of 2000 m. After a sampling period of three months, I collected a total of 123,039 seeds of 255 species/morphospecies from both forests and pastures along the elevational gradient. I did not find a consistent decrease in the amount and richness of seed rain between forests and pastures, but I detected a systematic change in the type of dispersed seeds, as heavier seeds and a higher proportion of endozoochorous species were found in forests compared to pastures at all elevations. This finding suggests that deforestation acts as a strong filter selecting seed traits that are vital for plant regeneration.
Understanding the role that trait-environment associations play in how plant communities regenerate today could serve as a basis for predicting changes in regeneration processes of plant communities under changing global conditions in the near future. Here, I show how informative the measurement of reproductive traits and trait environment associations are in facilitating the conservation of forest habitats and the restoration of deforested areas in the context of global change.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
In the light of emerging resistances against common drugs, new drug leads are required. In the past natural sources have been more yielding in this respect than synthetic strategies. Fungi synthesize many natural products with biological activities and pharmacological relevance. However, only a fraction of the estimated fungal diversity has been evaluated for biological activity, and much of the Fungi’s natural chemical diversity awaits discovery. Especially promising in this context are lichenized fungi. Lichens are well known for their particularly rich and characteristic secondary chemistry which allows them to withstand intense UV radiation, protects them against herbivory, and prevents them from being overgrown. The slow growth rates of lichens and difficulties and infeasibility of large scale cultivations in the laboratory render lichens inaccessible for applied purposes. These experimental challenges have led to a poor understanding of the molecular mechanisms underlying the biosynthesis of characteristic lichen secondary metabolites. The recent development of improved sequencing techniques has enabled new strategies to address multi-species assemblages directly through metagenome sequencing and survey their biosynthetic potential through genome mining. However, whole genome sequencing of entire lichen thalli to metagenomically assess the lichen-forming fungus without the need of cultivation has not been evaluated for lichens before. This approach will enable the reconstruction of fungal genomes from mixed DNA from lichen thalli and allow the exploration of biosynthetic gene content.
My thesis was conducted in two parts: a methodological evaluation of a metagenomic strategy to reconstruct genomes and gene sets of lichen-forming fungi, and the exploration of biosynthetic gene content with the help of comparative genomics and phylogenetics. For the first part, I evaluated the quality of metagenome-derived genome assemblies and gene sets by direct comparison to culture-derived reference assemblies and gene sets of the same species. I showed that metagenome-derived fungal assemblies are comparable to culture-derived references genomes and have a similar total genome size and fungal genome completeness. The quality of assemblies was affected strongly by the choice of assembler, but not by the method of taxonomic assignment or inference of non-mycobiont DNA sequences. The fungal gene space is well covered in metagenome-derived and culture-derived fungal gene sets and overlaps to 88-90 %. Finally, the metagenome-derived assemblies reliably recover gene families of secondary metabolism. This shows the suitability of metagenomically derived genomes for mining biosynthetic genes, and potentially also other gene families. Overall, the method validation showed a high similarity between metagenome- and culture-derived genome assemblies.
For the second part of my thesis, I explored the biosynthetic gene content in two different systems: Between two sister-species with different ecological requirements but similar chemical profile, and between two species which are metabolite-rich and economically relevant in the perfume industry. I compared the diversity of biosynthetic gene clusters between the species and in the broader context of other lichenized and non-lichenized fungi. Overall, the whole genome mining revealed a large number of uncharacterised secondary metabolite gene clusters in fifteen genomes of lichen-forming fungi compared to other fungal classes. Their number highly outweighs the number of known synthesized metabolites and highlights the hidden biosynthetic potential in lichen-forming fungi. Many biosynthetic gene clusters in the ecological distinct sister-species showed a high homology in accordance with the high synteny in gene content and order in both genomes. These clusters represent ideal candidates for secondary metabolites synthesized by both species, while the remaining clusters may encode for metabolites relevant for the different ecological requirements of both species. The metabolite-rich species used in the perfume industry showed a particularly high number of biosynthetic gene clusters. An in-depth characterization of architecture and gene content of homologous gene clusters together with hints from phylogenetic relatedness to functional characterized metabolites provides promising insights into the biosynthetic gene content of these lichen-forming fungi.
In conclusion, I showed that metagenome sequencing of natural lichen thalli is a feasible approach to reconstruct the fungal mycobiont genome of lichens and circumvent time-consuming and in some cases impossible cultivation of individuals. The genome mining for secondary metabolite gene clusters in lichen-forming fungi revealed a high biosynthetic potential for the discovery of new natural products. One of the focal species, Evernia prunastri, contained the highest ever reported number (80) of biosynthetic clusters in lichenized fungi. The comprehensive cluster characterizations through annotation, comparative mapping and phylogenetics provide first valuable hints for linking metabolites to genes in these lichen-forming fungi. My results pave the way for biotechnological strategies to unlock the vast richness of natural products from lichens for applied purposes.
Lichens are present in most land ecosystems, frequently occupying habitats where few other organisms are able to survive. Their contribution to the ecosystems in terms of biomass and ground cover increases with latitude and altitude, being, together with bryophytes, the most conspicuous component of alpine and polar landscapes. Whereas some polar lichens have reduced distributions and are restricted to high latitudes, most of them have very wide distributional ranges, which oven extend over several climatic regions. Many of them are common to Polar Regions of both hemispheres, a distributional pattern that has been denominated as bipolar, antitropical or amphitropical. Bipolar distributions are not exclusive to lichens, but common to many groups of organisms. The bipolar element in lichens is exceptional as it includes a large number of species, while in most other land organisms it includes genera or families but very seldom species.
In this dissertation I use the bipolar lichen Cetraria aculeata to give a first insight into the phylogeography of this biogeographic element in lichens. I discuss how and when the disjunct distribution of C. aculeata came to be, and try to partial out the roles that historical and ecological processes played in shaping its distribution.
Sampling was designed to cover a wide geographic extension. The main e"ort was made to collect in boreal, temperate and tropical mountain ranges in North and South America, as well to include Mediterranean populations in which specimens with deviant morphologies are observed.
I found that Cetraria aculeata forms a genetically congruent taxon. Although whether it should include C. muricata remains unsolved, I excluded all specimens identified as the latter from our analyses. Thee populations of both algal and fungal symbionts have a strong geographic structure. The study of the lichen fungus suggested that the species originated in the Eurasian continent and later expanded to acquire its current distribution during the Pleistocene. The results showed that all American populations originated from an ancestral population, more similar to the extant Arctic populations than to the Mediterranean ones.
The comparison between the structure of fungal and algal populations showed a high degree of coherence between them. However, the similarity in photobiont use between Arctic and Antarctic populations suggests that photobiont use responds not only to a history of codispersal in vegetative propagula, but it is also a result of a selective process related to climate. Since this climatic pattern of similarity is also found in the community of Alphaproteobacteria associated with C. aculeata, we concluded that lichens might be able to accommodate or to respond to different environmental conditions by selectively associating with different symbiotic partners.
Lastly, we found the Mediterranean populations of C. aculeata to be genetically differentiated in algal and fungal symbionts from the rest of the populations. While we found no grounds to believe that the overgrown morphs encountered in the region are due to the association with different algal lineages, I believe that a switch in photobiont use might be responsible for the pattern of genetic isolation encountered. Furthermore, I suggest that the Mediterranean and bipolar C. aculeata could be two different species, since both are ecologically, genetically and at least in part morphologically divergent.
The existence of all living organisms depends on their multidimensional adjustment to the conditions of the environment in which they live. Organisms must constantly deal with not only abiotic stress factors (such as water availability or extreme temperatures), but also with various biotic interactions (the competition between different organisms, both intraspecific and interspecies). When there is a consensus between an organism and the environment it means that this organism is well adjusted and increases its probability of survival.
Symbiotic organisms possess the ability to establish an intimate interaction with another species (symbiont) that provides benefits for survival. Organisms that are involved in obligate symbiosis may adapt to a new environment by switching to another symbiotic partner that is locally better adapted; or by reshuffling symbiont communities present in the holobiont. This ability potentially gives them the opportunity to flexibly react to changing environmental conditions.
In this thesis I studied the genetic diversity and geographic distribution of symbiont lineages in a lichen symbiosis to better understand environmental adaptation in symbiotic systems. Lichens are symbiotic associations of photobionts (one or several green-algal species or cyanobacteria), filamentous mycobionts (lichen-forming fungi) and co-inhabiting symbiotic microorganisms (lichen-associated bacteria, endolichenic fungi, and basidiomycete yeast). The coccoid green algae of the genus Trebouxia are the most common and the most studied lichen photobionts. However, the lack of formal Trebouxia taxonomy impedes our understanding of this photobiont diversity.
Different species of mycobionts may share the same photobionts and a single species of mycobiont may associate with multiple, genetically different photobionts. Interactions among symbionts are not random and are constrained by evolutionary and environmental processes. The ability to associate with specific symbiotic partner is considered as a lichen strategy to facilitate adaptation to the constantly changing environments.
The objectives of this thesis were to 1. Elucidate the intraspecific diversity of fungal and algal symbionts in the lichen Umbilicaria pustulata, given a range-wide (Europe-wide) sampling; 2. Evaluate species delimitation in trebouxioid photobionts based on molecular data, and 3. Quantify the climatic niches of photobiont lineages within U. pustulata, to establish whether the association with particular photobionts may modify the range and ecological niche of this lichen.
The main findings of this thesis are:
1. The genetic diversity within trebouxoid photobiont of U. pustulata is higher than within the mycobiont. The most variable photobiont loci are nrITS rDNA, psbJ-L, and COX2. RbcL is the least variable photobiont locus. The most variable mycobiont loci are MCM7 and TSR1. This study shows a lack of genetic variability in the mycobiont loci EF1, nrITS rDNA, RPB1, and RPB2.
2. U. pustulata shows a low level of selectivity and is associated with numerous (most likely six) putative algal species. All photobiont haplotypes found in U. pustulata are shared between other lichen-forming fungi species, showing different patterns of species-to-species and species-to-community interactions.
3. The geographic distribution of U. pustulata symbionts associations is strongly connected to changes in the climatic niches. The mycobiont-photobiont interactions change along latitudinal temperature gradients (cold-adapted hotspot) and in Mediterranean climate zones (warm-adapted hotspot). U. pustulata broadens its distribution range by switching between photobionts that posses specific environmental preferences.
Overall, this thesis contributes to the understanding of the symbiont diversity, fungal-algal association patterns and local adaptation linked to symbiont-mediated niche expansion in lichens. While identifying intraspecific diversity of both lichen symbionts is a key predisposition to understand symbiont interactions, population dynamics or co-evolution, my comparative study of the sequence-based molecular markers is relevant to reveal cryptic diversity in other lichen-forming fungi and their photobionts.
The determination of species boundaries in lichen symbionts is essential for the study of selectivity and specificity, co-distribution, and co-evolution. Whereas the phylogenetic relationships of Trebouxiophyceae are poorly understood, the application of a novel multifaceted approach based on phylogenetic relationships, coalescence methods and morphological traits presented in this thesis is a promising tool to address species boundaries within this heterogeneous genus.
This thesis provides evidence for symbiont-mediated niche expansion in lichens and highlights the preferential photobiont association from a niche-modeling perspective. My results shed light on symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation in the lichen symbiosis. The spatial genetic pattern found in U. pustulata symbionts supports the concept of ecological fitting and is consistent with patterns found in other lichen studies. Results presented here relate also to findings in different symbiotic systems, like reef-building corals, where different latitudinal patterns and symbiont switching has been reported as an adaptive response to severe bleaching events. Furthermore, this study is timely in light of global warming, because the identification of interaction hotspots among symbionts helps to understand how lichens or other symbiotic organisms adjust to the ongoing climate change. This knowledge will, in turn, facilitate the proper conservation of the most vulnerable lichen populations. My doctoral thesis provides a conceptual framework for analyzing symbiont diversity, interaction patterns, and symbiont-mediated niche expansion that could be applied to other types of lichen species as well as other organisms involved in facultative or obligate symbiosis.
Genetic and genomic tools have provided researchers with the opportunity to address fundamental questions regarding the reintroduction of species into their historical range with greater precision than ever before. Reintroduction has been employed as a conservation method to return locally extinct species to their native range for decades. However, it remains unknown how genetic factors may impact population establishment and persistence at the population and metapopulation level in the short- and long-term. Genetic methods are capable of producing datasets from many individuals, even when only low quality DNA can be collected. These methods offer an avenue to investigate unanswered questions in reintroduction biology, which is vital to provide evidence based management strategies for future projects. The Eurasian lynx (Lynx lynx) and European wildcat (Felis silvestris) are elusive carnivores native to Eurasia and have been the subject of multiple reintroduction attempts into their native range. During the 19th and 20th century, the Eurasian lynx was extirpated from West and Central Europe due to increasing habitat fragmentation and persecution. Similarly, the European wildcat was the subject of human persecution, residing in a few refugia in West and Central Europe. After legal protection in the 1950s, subsequent reintroduction projects of both species began in the 1970s and 1980s and continue to the present. Despite this large focus on species conservation, little attention has been given to the consequences these reintroductions have on the genetic composition of the reintroduced populations and if the populations have a chance of persisting in the long term. These species have not yet benefited from the large range of genetic and genomic techniques currently available to non-model organisms, leaving many fundamental aspects of their reintroduction poorly understood. In my dissertation, I investigate demography, population structure, genetic diversity and inbreeding at the population and metapopulation level in both species. In the introduction, which lays the foundation for the subsequent chapters of this PHD, I provide background on reintroduction, its role in conservation and the genetic consequences on populations, especially populations of apex and mesocarnivores. In Publication I, I investigated the reemergence of the European wildcat in a low mountain region in Germany using fine-scale spatial analysis. I found that the reintroduced population has persisted and merged with an expanding natural population. The reintroduced population showed no genetic differentiation from the natural population suggesting there is a good chance this population has retained sufficient genetic diversity despite reintroduction. In Publication II, I tracked population development and genetic diversity over 15 years in a reintroduced lynx population to determine the genetic ramifications on a temporal scale. I found slow genetic erosion after a period of outbreeding, which fits in line with other reintroduced taxa sharing similar demographic histories. I also found the number of genetic founders to be a fraction of the total released individuals, indicating that reintroduced populations of elusive carnivores may have fewer founder individuals than previously thought. In Publication III, I sampled all surviving lynx reintroductions in West and Central Europe as well as 11 natural populations to compare levels of genetic diversity and inbreeding across the species distribution. I found that all reintroduced populations have lower genetic variability and higher inbreeding than natural populations, which urgently requires further translocations to mitigate possible negative consequences. These translocations could stem from other reintroduced populations or from surrounding natural populations. The results contribute to a growing body of evidence indicating that inbreeding is likely to be more prevalent in wild populations than previously understood. Finally, in the discussion I explore how genetic methods can be applied to post-reintroduction monitoring of felid species to illuminate questions relating to genetic composition after release. The methods employed in these studies and in future work will be highly dependent on the research questions posed. Additionally, I investigate the drivers of the observed genetic patterns including founder size, source population, environmental factors, and population growth. I found that genetic diversity loss patterns across these two felid species are not clearly defined, however, management actions can be taken to mitigate the negative effects of reintroductions. These management actions include further translocation, introducing a sufficient number of released individuals and situating reintroductions adjacent to natural populations. All of these actions can minimize genetic drift and inbreeding, two factors which negatively impact small populations. This thesis further supports mounting evidence that genetic considerations should be assessed before releasing individuals, which allows for incorporation of scientific evidence into the planning process thereby increasing the overall success of reintroduction projects. Ultimately, the resources developed during this dissertation provide a solid baseline and foundation for future work regarding the consequences of reintroductions. This is especially important as an increasing number of species are at risk of extinction and reintroductions of both the European wildcat and Eurasian lynx, as well as many others, are planned in the coming years.
Droughts impair plant growth, limit global net primary production and are predicted to increase in the course of climate change. Knowledge of the plant drought response on a molecular level can facilitate the selection of drought resistant genotypes and genetic engineering and thereby can help to implement strategies, such as assisted migration projects or crop improvement, in order to preserve natural and agricultural vegetation against droughts.
Studies on gene expression under drought stress were conducted in three species each of the genera Quercus and Panicum, to shed light on the molecular drought response in these species and identify drought responsive genes as a basis for technical applications.
In the genus Quercus, gene expression studies were conducted in the three major European forest trees Q. ilex, Q. pubescens and Q. robur, for which a distributional shift caused by climate change is predicted for the 21st century. RNA-Seq experiments were conducted in the three Quercus species for the first time, ortholog groups were assigned and unregulated genes, as well as drought responsive genes, were identified (Madritsch et al. 2019). For a set of the unregulated genes, a stable expression over the course of long-term drought periods was evaluated in order to enable an application as reference genes for normalizing qRT-PCR experiments (Kotrade 2019a). The reference genes were used in subsequent experiments to generate gene expression profiles over the course of a two-year drought experiment with consecutive drought periods for a set of twelve drought responsive genes and revealed a highly variable gene regulation under long-term drought stress in the Quercus species (Kotrade et al. 2019b).
In the genus Panicum, the gene expression in response to drought was examined in the two wild crop species, P. laetum and P. turgidum, and in the less drought tolerant species P. bisulcatum via RNA-Seq experiments (Kotrade et al. 2020 (in revision). The transcriptomes of the species were sequenced for the first time, ortholog groups were assigned and the gene regulation was compared across the species. The common grounds of the drought response in Panicum were determined by identifying similarities across the species, while the identification of differences between the species led to genes that might contribute to the higher drought tolerance of P. laetum and P. turgidum
A comparison across the two genera showed large differences in the gene regulation upon drought. This might be largely explained by different experimental setups that resulted in different drought conditions in the genera, such as drought intensity, drought duration and velocity of drought development.
The sequence information and the drought responsive genes identified in the Quercus and Panicum species can be used to develop marker assays for marker-assisted selection. The genes that putatively contribute to the higher drought tolerance of the two wild crop Panicum species should be considered as candidate targets in genetic engineering studies. Marker-assisted selection and genetic engineering can be applied, for example, in assisted migration projects to support natural vegetation in the course of climate change or to breed more drought tolerant crop strains to mitigate crop failure rates caused by droughts.
The transition from the marine to the terrestrial realm is one of the most fascinating issues in evolutionary biology for it required the appearance, in different organisms, of several novel adaptations to deal with the demands of the new realm. Adaptations include, for instance, modifications in different metabolic pathways, development of body structures to facilitate movement and respiration, or tolerance to new conditions of stress. The transition to the land also gives an extraordinary opportunity to study whether evolution used similar changes at the genomic level to produce parallel adaptations in different taxa. Mollusks are among taxa that were successful in the conquest of the land. For instance, several lineages of the molluscan clade Panpulmonata (Gastropoda, Heterobranchia) invaded the intertidal, freshwater and land zones from the marine realm. In my dissertation, using tools from bioinformatics, phylogenetics, and molecular evolution, I used panpulmonates as a suitable model group to study the independent invasions into the terrestrial realm and the adaptive signatures in genes that may have favored the realm transitions. My work includes two peer-reviewed published papers and one manuscript under review. In Publication 1 (Romero et al., 2016a), I used mitochondrial and nuclear molecular markers to resolve the phylogeny of the Ellobiidae, a family that possesses intertidal and terrestrial species. The phylogeny provided an improved resolution of the relationships within inner clades and a framework to study the tempo and mode of the land transitions. I showed that the terrestrialization events occurred independently, in different lineages (Carychiinae, Pythiinae) and in different geological periods (Mesozoic, Cenozoic). In addition, the diversification in this group may not have been affected by past geological or climate changes as the Cretaceous-Paleogene (K-Pg) event or the sea-level decrease during the Oligocene. In Publication 2 (Romero et al., 2016b), I generated new mitochondrial genomes from terrestrial species and compared them with other panpulmonates. I used the branch-site test of positive selection and detected significant nonsynonymous changes in the terrestrial lineages from Ellobioidea and Stylommatophora. Two genes appeared under positive selection: cob (Cytochrome b) and nad5 (NADH dehydrogenase 5). Surprisingly, I found that the same amino acid positions in the proteins encoded by these genes were also under positive selection in several vertebrate lineages that transitioned between different habitats (whales, bats and subterranean rodents). This result suggested an adaptation pattern that required parallel genetic modifications to cope with novel metabolic demands in the new realms. In Manuscript 1 (Romero et al., under review), I de novo assembled transcriptomes from several panpulmonate specimens resulting in thousands of genes that were clustered in 702 orthologous groups. Again, I applied the branch-site test of positive selection in the terrestrial lineages from Ellobioidea and Stylommatophora and in the freshwater lineages from Hygrophila and Acochlidia. Different sets of genes appeared under positive selection in land and freshwater snails, supporting independent adaptation events. I identified adaptive signatures in genes involved in gas-exchange surface development and energy metabolism in land snails, and genes involved in the response to abiotic stress factors (radiation, desiccation, xenobiotics) in freshwater snails. My work provided evidence that supported multiple land invasions within Panpulmonata and provided new insights towards understanding the genomic basis of the adaptation during sea-to-land transitions. The results of my work are the first reports on the adaptive signatures at the codon level in genes that may have facilitated metabolic and developmental changes during the terrestrialization in the phylum Mollusca. Moreover, they contribute to the current debate on the conquest of land from the marine habitat, a discussion that has been only based in vertebrate taxa. Future comparative genome-wide analyses would increase the number of genes that may have played a key role during the realm transitions.