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Regulation of gene expression by the control of transcription is essential for any cell to adapt to the environment and survive. Transcription regulators, i.e. sequence-specific DNA binding proteins that regulate gene expression, are central elements within the gene networks of most organisms. Transcription regulators are grouped into distinct families based on structural features that determine, to a large extent, the DNA sequence(s) that they can recognise and bind. Less is known, however, about how the DNA binding preferences can diversify within transcription regulator families during evolutionary timescales, and how such diversification can affect the biology of the organism.
In this dissertation I study the SREBP (sterol regulatory element binding protein) family of transcriptional regulators in yeasts, and in Candida albicans in particular, as an experimental system to address these questions. The SREBPs are conserved from fungi to humans and represent a subgroup of basic helix-loop-helix DNA binding proteins. Early chromatin immunoprecipitation experiments with SREBPs from humans and yeasts showed that these proteins bound in vivo to the canonical DNA sequence, termed E-box, most basic helix-loop-helix proteins bind to. By contrast, most recent analysis carried out with less-studied fungal SREBPs revealed a non-canonical DNA motif to be the most overrepresented sequence in the bound regions.
This study aims to establish the intrinsic DNA binding preferences of key branches of this family and to determine how the divergence in DNA binding affinities originated. To this end, I combined phylogenetic and ancestral reconstruction with extensive biochemical characterisation of key SREBP proteins. The results indicated that while the most-studied SREBPs (in mammals) indeed show preference for the E-box, a second branch of the family preferentially binds the non-E-box, and a third one is able to bind both sequences with similar affinity. The preference for one or the other DNA sequence is an intrinsic property of each protein because their purified DNA binding domain was sufficient to recapitulate their in vivo binding preference. The ancestor that gave rise to these two different types of SREBPs (the branch that binds E-box and the one that binds non-E-box DNA) appears to be a protein with a broader DNA binding capability that had a slight preference for the non-canonical motif. Thus, the results imply these two branches originated by either enhancing the original ancestral preference for non-E-box or tilting it towards the E-box DNA and flipping the preference for this sequence.
The main function associated with members of the SREBP family in most eukaryotes is the control of lipid biosynthesis. I have further studied the function of these proteins in the lineage that encompasses the human associated yeast C. albicans. Strikingly, the three SREBPs present in the fungus’ genome contribute to the colonisation of the mammalian gut by regulating cellular processes unrelated to lipid metabolism. Here I describe that two of the three C. albicans SREBPs form a regulatory cascade that regulates morphology and cell wall modifications under anaerobic conditions, whereas the third SREBP has been shown to be involved in the regulation of glycolysis genes.
Therefore, I posit that the described diversification in DNA binding specificity in these proteins and the concomitant expansion of targets of regulation were key in enabling this fungal lineage to associate with animals.
New experimental methods have drastically accelerated the pace and quantity at which biological data is generated. High-throughput DNA sequencing is one of the pivotal new technologies. It offers a number of novel applications in various fields of biology, including ecology, evolution, and genomics. However, together with those opportunities many new challenges arise. Specialized algorithms and software are required to cope with the amount of data, often requiring substantial training in bioinformatic methods. Another way to make those data accessible to non-bioinformaticians is the development of programs with intuitive user interfaces.
In my thesis I developed analyses and programs to tackle current problems with high-throughput data in biology. In the field of ecology this covers the establishment of the bioinformatic workflow for pollen DNA meta-barcoding. Furthermore, I developed an application that facilitates the analysis of ecological communities in the context of their traits. Information from multiple public databases have been aggregated and can now be mapped automatically to existing community tables for interactive inspection. In evolution the new data are used to reconstruct phylogenetic trees from multiple genes. I developed the tool bcgTree to automate this process for bacteria. Many plant genomes have been sequenced in current years. Sequencing reads of those projects also contain data from the chloroplasts. The tool chloroExtractor supports the targeted extraction and analysis of the chloroplast genome. To compare the structure of multiple genomes specialized software is required for calculation and visualization of the relationships. I developed AliTV to address this. In contrast to existing programs for this task it allows interactive adjustments of produced graphics. Thus, facilitating the discovery of biologically relevant information. Another application I developed helps to analyze transcriptomes even if no reference genome is present. This is achieved by aggregating the different pieces of information, like functional annotation and expression level, for each transcript in a web platform. Scientists can then search, filter, subset, and visualize the transcriptome.
Together the methods and tools expedite insights into biological systems that were not possible before.
Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males.
Understanding the emergence of species' ranges is one of the most fundamental challenges in ecology. Early on, geographical barriers were identified as obvious natural constraints to the spread of species. However, many range borders occur along gradually changing landscapes, where no sharp barriers are obvious. Mechanistic explanations for this seeming contradiction incorporate environmental gradients that either affect the spatio-temporal variability of conditions or the increasing fragmentation of habitat. Additionally, biological mechanisms like Allee effects (i.e. decreased growth rates at low population sizes or densities), condition-dependent dispersal, and biological interactions with other species have been shown to severely affect the location of range margins. The role of dispersal has been in the focus of many studies dealing with range border formation. Dispersal is known to be highly plastic and evolvable, even over short ecological time-scales. However, only few studies concentrated on the impact of evolving dispersal on range dynamics. This thesis aims at filling this gap. I study the influence of evolving dispersal rates on the persistence of spatially structured populations in environmental gradients and its consequences for the establishment of range borders. More specially I investigate scenarios of range formation in equilibrium, periods of range expansion, and range shifts under global climate change ...
The human genome has been sequenced since 2001. Most proteins have been characterized now and with everyday more bioinformatical predictions are experimentally verified. A project is underway to sequence thousand humans. But still, little is known about the evolution of the human proteome itself. Domains and their combinations are analysed in detail but not all of the human domain architectures at once. Like no one before, we have large datasets of high quality human protein-protein-protein interactions and complexes available which allow us to characterize the human proteome with unmatched accuracy. Advanced clustering algorithms and computing power enable us to gain new information about protein interactions without touching a pipette. In this work, the human proteome is analysed at three different levels. First, the origin of the different types of proteins was analysed based on their domain architectures. The second part focuses on the protein-protein interactions. Finally, in the third part, proteins are clustered based on their interactions and non-interactions. Most proteins are built of domains and their function is the sum of their domain functions. Proteins that share the same domain architecture, the linear order of domains are homologues and should have originated from one common ancestral protein. This ancestor was calculated for roughly 750 000 proteins from 1313 species. The relations between the species are based on the NCBI Taxonomy and additional molecular data. The resulting data set of 5817 domains and 32868 domain architectures was used to estimate the origin of these proteins based on their architectures. It could be observed, that new domain architectures are only in a small fraction composed of domains arisen at the same taxon. It was also found that domain architectures increase in length and complexity in the course of evolution and that different organisms like worm, and human share nearly the same amount of proteins but differ in their number of distinct domain architectures. The second part of this thesis focuses on protein-protein interactions. This chapter addresses the question how new evolved proteins form connections within the existing network. The network built of protein-protein interactions was shown to be scale free. Scale free networks, like the internet, consist of few hubs with many connections and many nodes with few connections. They are thought to arise by two mechanisms. First, newly emerged proteins interact with proteins of the network. Second, according to the theory of preferential attachment, new proteins have a higher chance to interact with already interaction rich proteins. The Human Protein Reference Database provides an on in-vivo interaction data based network for human. With the data obtained from chapter one, proteins were marked with their taxon of origin based on their domain architectures. The interaction ratio of proteins of the same taxa compared to all interactions was calculated and higher values than the random model showed for nearly every taxa. On the other hand, there was no enrichment of proteins originated at the taxon of cellular organisms for the node degree found. The node degree is the number of links for this node. According to the theorie of preferential attachment the oldest nodes should have the most interactions and newly arisen proteins should be preferably attached to them not together. Both could not be shown in this analysis, preferential attachment could therefore not be the only explanation for the forming of the human protein interaction network. Finally in part three, proteins and all their interactions in the network are analysed. Protein networks can be divided into smaller highly interacting parts carrying out specific functions. This can be done with high statistical significance but still, it does not reflect the biological significance. Proteins were clustered based on their interactions and non-interactions with other proteins. A version with eleven clusters showed high gene ontology based ratings and clusters related to specific cell parts. One cluster consists of proteins having very few interactions together but many to proteins of two other clusters. This first cluster is significantly enriched with transport proteins and the two others are enriched with extracellular and cytoplasm/membrane located proteins. The algorithm seems therefore well suited to reflect the biological importance behind functional modules. Although we are still far from understanding the origin of species, this work has significantly contributed to a better understanding of evolution at the protein level and has, in particular, shown the relation of protein domains and protein architectures and their preferences for binding partners within interaction networks.
This thesis extends the classical theoretical work of Macevicz and Oster (1976, expanded by Oster and Wilson, 1978) on adaptive life history strategies in social insects. It focuses on the evolution of dynamic behavioural patterns (reproduction and activity) as a consequence of optimal allocation of energy and time resources. Mathematical modelling is based on detailed empirical observations in the model species Lasioglossum malachurum (Halictidae; Hymenoptera). The main topics are field observations, optimisation models for eusocial life histories, temporal variation in life history decisions, and annual colony cycles of eusocial insects.
In the various groups of social bees, different systems of communication about food sources occur. These communication systems are different solutions to a common problem of social insects: efficiently allocating the necessary number of workers first to the task of foraging and second to the most profitable food sources. The solution chosen by each species depends on the particular ecological circumstances as well as the evolutionary history of that species. For example, the outstanding difference between the bumble bee and the honey bee system is that honey bees can communicate the location of profitable food sources to nestmates, which bumble bees cannot. To identify possible selection pressures that could explain this difference, I have quantified the benefits of communicating location in honey bees. I show that these strongly depend on the habitat, and that communicating location might not benefit bees in temperate habitats. This could be due to the differing spatial distributions of resources in different habitats, in particular between temperate and tropical regions. These distributions may be the reason why the mostly temperate-living bumble bees have never evolved a communication system that allows them to transfer information on location of food sources, whereas most tropical social bees (all honey bees and many stingless bees) are able to recruit nestmates to specific points in their foraging range. Nevertheless, I show that in bumble bees the allocation of workers to foraging is also regulated by communication. Successful foragers distribute in the nest a pheromone which alerts other bees to the presence of food. This pheromone stems from a tergite gland, the function of which had not been identified previously. Usage of a pheromone in the nest to alert other individuals to forage has not been described in other social insects, and might constitute a new mode of communicating about food sources. The signal might be modulated depending on the quality of the food source. Bees in the nest sample the nectar that has been brought into the nest. Their decision whether to go out and forage depends not only on the pheromone signal, but also on the quality of the nectar they have sampled. In this way, foraging activity of a bumble bee colony is adjusted to foraging conditions, which means most bees are allocated to foraging only if high-quality food sources are available. In addition, foraging activity is adjusted to the amount of food already stored. In a colony with full honeypots, no new bees are allocated to foraging. These results help us understand how the allocation of workers to the task of food collection is regulated according to external and internal nest conditions in bumble bees.
Safer without Sex?
(1999)
Highly eusocial insect societies, such as all known ants, are typically characterized by a reproductive division of labor between queens, who are inseminated and reproduce, and virgin workers, who engage in foraging, nest maintenance and brood care. In most species workers have little reproductive options left: They usually produce haploid males by arrhenotokous parthenogenesis, both in the queenright and queenless condition. In the phylogenetically primitive subfamily Ponerinae reproductive caste dimorphism is much less pronounced: Ovarian morphology is rather similar in queens and workers, which additionally retain a spermatheca. In many ponerine species workers mate and may have completely replaced the queen caste. This similarity in reproductive potential provides for the evolution of diverse reproductive systems. In addition, it increases the opportunity for reproductive conflicts among nestmates substantially. Only in a handful of ant species, including Platythyrea punctata, workers are also able to rear diploid female offspring from unfertilized eggs by thelytokous parthenogenesis. The small ponerine ant P. punctata (Smith) is the only New World member of the genus reaching as far north as the southern USA, with its center of distribution in Central America and the West Indies. P. punctata occurs in a range of forest habitats including subtropical hardwood forests as well as tropical rain forests. In addition to queens, gamergates and thelytokous workers co-occur in the same species. This remarkable complexity of reproductive strategies makes P. punctata unique within ants and provides an ideal model system for the investigation of reproductive conflicts within the female caste. Colonies are usually found in rotten branches on the forest floor but may also be present in higher strata. Colonies contained on average 60 workers, with a maximum colony size of 148 workers. Queens were present in only ten percent of the colonies collected from Florida, but completely absent both from the populations studied in Barbados and Puerto Rico. Males were generally rare. In addition, morphological intermediates between workers and queens (so-called intercastes) were found in 16 colonies collected in Florida. Their thorax morphology varied from an almost worker-like to an almost queen-like thorax structure. Queen and intercaste size, however, did not differ from those of workers. Although workers taken from colonies directly after collection from the field engaged in aggressive interactions, nestmate discrimination ceased in the laboratory suggesting that recognition cues used are derived from the environment. Only one of six queens dissected was found to be inseminated but not fertile. Instead, in most queenless colonies, a single uninseminated worker monopolized reproduction by means of thelytokous parthenogenesis. A single mated, reproductive worker (gamergate) was found dominating reproduction in the presence of an inseminated alate queen only in one of the Florida colonies. The regulation of reproduction was closely examined in ten experimental groups of virgin laboratory-reared workers, in which one worker typically dominated reproduction by thelytoky despite the presence of several individuals with elongated, developing ovaries. In each group only one worker was observed to oviposit. Conflict over reproduction was intense consisting of ritualized physical aggression between some nestmates including antennal boxing, biting, dragging, leap and immobilization behaviors. The average frequency of interactions was low. Aggressive interactions allowed to construct non-linear matrices of social rank. On average, only five workers were responsible for 90 percent of total agonistic interactions. In 80 percent of the groups the rate of agonistic interactions increased after the experimental removal of the reproductive worker. While antennal boxing and biting were the most frequent forms of agonistic behaviors both before and after the removal, biting and dragging increased significantly after the removal indicating that agonistic interactions increased in intensity. Once a worker obtains a high social status it is maintained without the need for physical aggression. The replacement of reproductives by another worker did however not closely correlate with the new reproductive's prior social status. Age, however, had a profound influence on the individual rate of agonistic interactions that workers initiated. Especially younger adults (up to two month of age) and callows were responsible for the increase in observed aggression after the supersedure of the old reproductive. These individuals have a higher chance to become reproductive since older, foraging workers may not be able to develop their ovaries. Aggressions among older workers ceased with increasing age. Workers that already started to develop their ovaries should pose the greatest threat to any reproductive individual. Indeed, dissection of all experimental group revealed that aggression was significantly more often directed towards both individuals with undeveloped and developing ovaries as compared to workers that had degenerated ovaries. In all experimental groups reproductive dominance was achieved by callows or younger workers not older than four month. Age is a better predictor of reproductive dominance than social status as inferred from physical interactions. Since no overt conflict between genetical identical individuals is expected, in P. punctata the function of agonistic interactions in all-worker colonies, given the predominance of thelytokous parthenogenesis, remains unclear. Physical aggression could alternatively function to facilitate a smooth division of non-reproductive labor thereby increasing overall colony efficiency. Asexuality is often thought to constitute an evolutionary dead end as compared with sexual reproduction because genetic recombination is limited or nonexistent in parthenogenetic populations. Microsatellite markers were developed to investigate the consequences of thelytokous reproduction on the genetic structure of four natural populations of P. punctata. In the analysis of 314 workers taken from 51 colonies, low intraspecific levels of variation at all loci, expressed both as the number of alleles detected and heterozygosities observed, was detected. Surprisingly, there was almost no differentiation within populations. Populations rather had a clonal structure, with all individuals from all colonies usually sharing the same genotype. This low level of genotypic diversity reflects the predominance of thelytoky under natural conditions in four populations of P. punctata. In addition, the specificity of ten dinucleotide microsatellite loci developed for P. punctata was investigated in 29 ant species comprising four different subfamilies by cross-species amplification. Positive amplification was only obtained in a limited number of species indicating that sequences flanking the hypervariable region are often not sufficiently conserved to allow amplification, even within the same genus. The karyotype of P. punctata (2n = 84) is one of the highest chromosome numbers reported in ants so far. A first investigation did not show any indication of polyploidy, a phenomenon which has been reported to be associated with the occurrence of parthenogenesis. Thelytokous parthenogenesis does not appear to be a very common phenomenon in the Hymenoptera. It is patchily distributed and restricted to taxa at the distant tips of phylogenies. Within the Formicidae, thelytoky has been demonstrated only in four phylogenetically very distant species, including P. punctata. Despite its advantages, severe costs and constraints may have restricted its rapid evolution and persistence over time. The mechanisms of thelytokous parthenogenesis and its ecological correlates are reviewed for the known cases in the Hymenoptera. Investigating the occurrence of sexual reproduction in asexual lineages indicates that thelytokous parthenogenesis may not be irreversible. In P. punctata the occasional production of sexuals in some of the colonies may provide opportunity for outbreeding and genetic recombination. Thelytoky can thus function as a conditional reproductive strategy. Thelytoky in P. punctata possibly evolved as an adaptation to the risk of colony orphanage or the foundation of new colonies by fission. The current adaptive value of physical aggression and the production of sexuals in clonal populations, where relatedness asymmetries are virtually absent, however is less clear. Quite contrary, thelytoky could thereby serve as the stepping stone for the subsequent loss of the queen caste in P. punctata. Although P. punctata clearly fulfills all three conditions of eusociality, the evolution of thelytoky is interpreted as a first step in a secondary reverse social evolution towards a social system more primitive than eusociality.