A kiss is a lovely trick designed by Nature, to stop speech when words become superfluous
- Ingrid Bergman
Illustrations on front cover reprinted from Mycological Research, 108 (10), García, D., Stchigel, A.M., Cano, J., Guarro, J., Hawksworth, D.L., A synopsis and re-circumscription of Neurospora (Syn. Gelasinospora) based on ultrastructural and 28S rDNA sequence data, p. 1119-1142, October 2004, with permission from Elsevier.
Photography on back cover by Peter Halvarsson.
List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I Nygren, K., Strandberg, R., Wallberg, A., Nabholz, B., Gustafsson, T., García, D., Cano, J., Guarro, J., and Johannesson, H. (2011) A comprehensive phylogeny of Neurospora reveals a link between reproductive mode and molecular evolution in fungi. Molecular Phylogenetics and Evolution, 59:649–663.
II Gioti, A., Mushegian, A.A., Strandberg, R., Stajich, J.E., and Johannesson, H. Unidirectional evolutionary transitions in fungal mating systems and the role of transposable elements. Manuscript.
III Strandberg, R*., Nygren, K*., Gioti, A., Karlsson, M., and Johannesson, H. Deciphering the relationship between mating system and pheromone receptor gene evolution in species of Neurospora. Manuscript.
IV Strandberg, R., Nygren, K., Menkis, A., James, T.Y., Wik, L., Stajich, J.E., and Johannesson, H. (2010) Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the filamentous ascomycete genus Neurospora. Fungal Genetics and Biology, 47:869-878.
V Strandberg, R., Tzelepis, G., Johannesson, H., and Karlsson, M. Co-existence and expression profiles of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa. Manuscript.
*These authors contributed equally to this work.
Papers number I and IV were reprinted with permission from the publisher (Elsevier).
Contents
Introduction...... 9 Neurospora – a model system ...... 10 Sexual identity and the search of a compatible mate...... 12 Chemotropic interactions for attracting a mate...... 13 Additional roles for pheromones and pheromone receptors ...... 14 Alternative splicing of pre-1 ...... 15 Mating systems in Neurospora...... 16 Heterothallism...... 16 Homothallism...... 17 Pseudohomothallism ...... 18 Ancestral state and unidirectional transitions of mating systems ...... 19 Evolution of reproductive genes in fungi ...... 20 Expected limited gene flow of reproductive genes...... 20 Decay of reproductive genes in homothallic Neurospora ...... 21 Research aims ...... 22 Specific research aims:...... 22 Summaries of papers...... 24 Paper I – A comprehensive phylogeny of Neurospora reveals a link between reproductive mode and molecular evolution in fungi...... 24 Paper II – Unidirectional evolutionary transitions in fungal mating systems and the role of transposable elements...... 25 Paper III – Deciphering the relationship between mating system and pheromone receptor gene evolution in species of Neurospora...... 26 Paper IV – Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the ascomycete genus Neurospora...... 27 Paper V – Co-existence and expression profiles of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa.... 29 Concluding remarks and future perspectives...... 30 Sammanfattning på svenska ...... 32 Acknowledgements...... 34 References...... 37
Abbreviations
act Actin AS Alternative splicing ccg-4 Clock-controlled-gene-4 DNA Deoxyribonucleic acid FGSC Fungal Genetics Stock Center JGI Joint Genome Institute kb Kilobases mat Mating type MCMC Markov Chain Monte Carlo mfa-1 Mating factor expressed in mat a strains ML Maximum likelihood mRNA Messenger RNA pre Pheromone receptor qPCR Quantitative PCR PCR Polymerase chain reaction RNA Ribonucleic acid RT Reverse transcription TE Transposable element
Introduction
Sexual reproduction is necessary for the maintenance and regeneration of life in the majority of eukaryotic organisms, ranging from unicellular yeasts to humans. The fungal kingdom encompasses a great diversity of species with a wide range of habitats, morphologies and life cycles. There is a plethora of different pathways for reproduction in fungi, and the evolutionary dynamics behind these reproductive systems are intriguing. Hence, by engaging fungi in biological research, they become excellent model systems to study sex determination, mate recognition and mating-type evolution. To be able to form hypotheses and study the evolution of reproductive systems experimentally, a good system should contain a diverse set of species exhibiting different reproductive strategies, be easy to culture, have a short generation time, and a well-studied genetic basis. A suitable study system, fulfilling all just mentioned requirements, is the ascomycete filamentous fungal genus Neurospora.
In this thesis work, using Neurospora as model system, I have applied a candidate gene approach to study the evolution of genes involved in reproduction, as well as the evolution and evolutionary dynamics of the different reproductive systems. In the introductory section, proceeding the actual research papers, I will introduce the concepts important for understanding the evolution of reproductive systems in Neurospora, i.e., how sexual identity is determined, the chemotropic interactions which orchestrate mating behaviors, a description of the different reproductive systems, as well as covering the expectations and trajectories of reproductive genes from an evolutionary perspective.
Taken together, I hope this thesis work will contribute key pieces of the puzzle required to understand the evolution of the sexual reproductive system, including gene evolution, and further strengthen Neurospora as a model for research in evolutionary biology.
9 Neurospora – a model system
Neurospora has a rather long history side-by-side with modern human activity. In the mid-1800s Neurospora was intensively studied, since as a contaminant it invaded French bakeries, caused trouble to housewives and became known as the “red bread mould” (Davis, 2000). During the first half of the 1900s, Neurospora emerged as a model eukaryotic organism for genetic studies, since it possesses numerous advantageous properties; it is haploid during most of its life cycle, easy to cultivate, susceptible to mutagenesis, and on top of all, non pathogenic (Davis and Perkins, 2002). The first scientific description of Neurospora was made by Shear and Dodge (1927). They described four species possessing dark ascospores (sexual spores) with nerve like ornamentations, hence the name Neurospora (see cover illustration). In 1958 Beadle and Tatum were awarded the Noble Prize for their ‘one gene, one enzyme’ hypothesis, demonstrated with X-ray experiments in Neurospora.
Neurospora belong to the family Sordariaceae, containing about ten genera, including Sordaria and Gelasinospora. In this thesis work, I have included species from Sordaria, Gelasinospora and Neurospora, and for simplicity following the suggestion by Garcia et al. (2004), merged members of the two latter into Neurospora, since they are not reciprocal monophyletic groups.
Neurospora is a cosmopolitan genus; one of the first organisms to colonize and grow on fire-scorched plant debris, mostly found in tropical and subtropical regions (Perkins and Turner, 1988; Perkins et al., 2001), but can also be found in temperate regions, for example western North America (Jacobson et al., 2004). Neurospora grows on vegetation killed by fire, because fire produces a sterile environment rich in nutrients and the heat necessary for the sexual spores to germinate. An extensive, global collection of Neurospora cultures is available at the Fungal Genetics Stock Center (FGSC; University of Missouri, Kansas City, USA).
In recent years, Neurospora has truly emerged as an excellent model for studies in evolutionary biology (Dettman et al., 2003; Ellison et al., 2011; Menkis et al., 2008; Turner et al., 2011). It is particularly useful for studies on the evolution of reproductive traits and behavior, since the genus has
10 three different mating strategies: heterothallism (self-incompatibility), homothallism (self-compatibility), and pseudohomothallism (partial self- incompatibility) (description in later section) (Karlsson et al., 2008; Nygren et al., 2011; Wik et al., 2008). The most studied species of Neurospora is without a doubt the heterothallic N. crassa, while homothallic species of Neurospora have gained less attention. However, the versatility of reproductive systems within the genus has drawn the attention to more than Neurospora spp., making it possible to make hypotheses on the evolution of reproductive systems and genes involved in reproduction.
More than a decade ago, modern biology entered the era of whole genome sequencing. Neurospora research has also advanced rapidly into the genomic field. Ascomycete fungi are convenient to sequence since the genomes are relatively small, at least compared to other complex eukaryotes, and Neurospora is especially good as it has low repeat content. The 40 megabase genome of N. crassa consists of seven chromosomes (linkage groups) and was sequenced by the Broad Institute (Galagan et al., 2003). The genome was later predicted to encode approximately 10 000 genes (Borkovich et al., 2004). All of these predicted genes have been knocked out systematically in a huge scientific community effort. Additional Neurospora genomes have since been sequenced (N. tetrasperma, and N. discreta) by JGI (Joint Genome Institute; US Department of Energy (Grigoriev et al., 2012)). All three Neurospora genomes are publicly available. Together, the available Neurospora genomes provide a powerful resource for genomic comparative biology, exploring the evolutionary dynamics of these species.
11 Sexual identity and the search of a compatible mate
In eukaryotes, great diversity exists when considering the systems determining sexual identity. The different sexes in animals and plants are often determined by sex chromosomes. In fungi, there are no sexes in the ‘classical’ sense, i.e., defined by an individual being either female or male, since an individual can produce both female and male components (Coppin et al., 1997). Sexual identity in ascomycete fungi is determined by the mating-type locus, which is normally a limited chromosomal region (Fraser and Heitman, 2004, 2005). The different “alleles” at the mating-type locus are two highly dissimilar sequences, suggested to be unrelated by descent although located at the same place on the mating-type chromosome. These allelic sequences are denoted as different idiomorphs (Glass et al., 1988; Metzenberg and Glass, 1990).
Neurospora species are hermaphroditic, i.e., produce both ‘male’ (donor) and ‘female’ (receptor) structures from the same mycelia. In this section, for simplicity, I will begin to focus on heterothallic (or self-incompatible) Neurospora. Heterothallic strains of Neurospora carry either of two distinct idiomorphs, a and A, at the mating-type locus (Glass et al., 1988; Metzenberg and Glass, 1990) (Figure 1), thus, mating type in heterothallic Neurospora is strictly biallelic (Coppin et al., 1997). Unlike in yeast, Neurospora strains do not have a silent copy of the opposite mating type, and therefore do not have a mechanism of mating-type switching (Perkins, A. Mat1987)-gene constitution. of heterothallic N. crassa
APN2 mat A-3 mat A-2 mat A-1 (eat-2) SLA2 (HMG) (alpha-box)
mat a-1 (HMG) Figure 1. The mating-type (mat) gene constitution in heterothallic Neurospora crassa and adjacent genes. The mating type genes are known as the master regulators of sexual reproduction and encode transcription factors that regulate downstream targets (modified from Butler, 2007). HMG – High Mobility Group.
12 The mating-type genes encode transcription factors that are known as the master regulators of sexual reproduction (Kronstad and Staben, 1997). The a idiomorph is 3.2 kb and encodes a single ORF (mat a-1), resulting in a 382- amino acid with a HMG domain and DNA-binding activity (Staben and Yanofsky, 1990). A mini-ORF (mat a-2) has also been reported, although it is often overlooked (Pöggeler and Kuck, 2000) and from here on, I will only focus on mat a-1. The A idiomorph is 5.3 kb and contains three ORFs; mat A-1 confes mating identity and incompatibility (Glass et al., 1990), mat A-2 and mat A-3 seem to influence the efficiency of mat A-1 (Ferreira et al., 1998).
The mating-type locus does not solely control mating, it also controls vegetative incompatibility between A and a strains, i.e., inhibition of vegetative growth if hyphae of opposite mating types fuse (Beadle and Coonradt, 1944).
Strains of different mating types are attracted by strains of the opposite mating type. This attraction was tested experimentally in N. crassa by Bistis (1983), who showed that this was mediated by a pheromone receptor system. The pheromones and their cognate receptors in Neurospora seem to be under direct control of the mating-type genes (Debuchy, 1999; Pöggeler, 2000; Pöggeler and Kuck, 2001), as well as under the control of the circadian clock (Bobrowicz et al., 2002).
Chemotropic interactions for attracting a mate Pheromones are diffusible chemical signals used for communication between individuals of the same species; one individual sends out pheromones that cause a biological response in another individual of the same species (Karlson and Luscher, 1959). In fungi, the occurrence of pheromone receptor systems have been reported and investigated in numerous studies (Jones Jr. and Bennett, 2011). The best-described fungal system is Saccharomyces cerevisiae, baker’s yeast, where the pheromone signal transduction pathways have been characterized in molecular detail, from the initial chemoattraction and pheromone/receptor contact, to the subsequent activation of genes resulting in cells that are competent to mate.
In filamentous ascomycetes, those with the potential for both female and male structures in the same mycelia, pheromones are suggested to primarily guide mate attraction. As mentioned above, heterothallic Neurospora also find a suitable mating partner by a pheromone/receptor system, i.e., chemoattraction (Bistis, 1981). The pheromone precursor genes, mfa-1 and ccg-4, code for very short peptides that interact with their cognate
13 pheromone receptors pre-1 and pre-2, respectively (Kim and Borkovich, 2004; Pöggeler and Kuck, 2001). The pre-genes encode for 7- transmembrane (7-TM) G-protein coupled receptors, which are embedded in the cell membrane, with an extracellular (EC) and a cytosolic tail. The intracellular parts of the receptor (three loop regions and the EC tail) physically interact with a heterotrimeric G-protein complex that mediates regulatory signals to downstream targets of the expression cascade (Casselton, 1997). The G-protein complex consists of three subunits; Gα, Gβ, and Gγ, and after the exchange of GDP to GTP, the G-protein complex is disassociated and the transduction cascade is activated.
The chemoattraction event initiates the sexual cycle which leads to plasmogamy, i.e., the fusion of the ‘female’ trichogyne (receptive hyphae), emanating from the unfertilized fruiting body (protoperithecium), with the ‘male’ propagules. Plasmogamy is followed by karyogamy and meiosis, which takes place in the mature fruiting body (perithecium), and eventually results in eight haploid spores (four of each mating type) formed in the ascus. The spores are forcefully released from the ascus and these will germinate upon heat activation, propagate and start searching for a mate of opposite mating-type, eventually the starting the sexual cycle again (ref).
Both pheromones and their cognate receptors have been knocked out in N. crassa, to investigate what effects these genes have on the phenotype. Deletion of pre-1 in N. crassa results in female sterility of the A mating type, since the trichogynes are unable to grow towards and fuse with spermatia (Kim and Borkovich 2004). In addition, deletion of either ccg-4 or mfa-1 results in male infertility in the corresponding mating type, since spermatia can no longer attract female trichogynes (Kim and Borkovich 2006).
Additional roles for pheromones and pheromone receptors It is not known if additional functions of the pheromone receptor system exist in Neurospora. In other systems, numerous studies indicate additional roles for pheromones and their receptors; for example genes and transcripts of both types of pheromone precursor and receptor genes have been reported in the homothallic Sordaria macrospora (Pöggeler and Kuck, 2001), and double-deletion mutant strains of pheromone precursors and receptors in this species suggest that pheromone/receptor systems are pivotal for fruiting- body development and ascosporogenesis (Mayrhofer et al., 2006). In double knockouts of the receptor genes in the homothallic Aspergillus nidulans, the ability to form fruiting bodies and ascospores was completely eliminated (Seo et al., 2004). In homothallic Giberella zeae, genes from both
14 pheromone/receptor pairs have been identified, but only one pair seems to be involved in sexual reproduction (Lee et al., 2008). Furthermore, pheromones have been suggested to be involved in induction of meiosis in Schizosaccharomyces pombe (Chikashige et al., 1997), and stimulate filamentous growth in Ustilago maydis (Spellig et al., 1994). Finally, internuclear recognition has been suggested as a possible role for pheromones in Schizophyllum commune and Podospora anserina (Debuchy, 1999). Taken together, studies on the pheromone/receptor system from a wide range of taxa have indicated that these genes have functions in addition to simply mating.
Alternative splicing of pre-1 Alternative functions of genes can be mediated by alternative splicing, a mechanism that enables a single gene to give rise to multiple, differentially spliced versions of a protein. The process of alternative splicing increases the complexity of the genome without changing it, as well as enabling the fine-tuning of gene expression. Different splice mechanisms exist in most organisms (McGuire et al., 2008). In fungi the retained intron mechanism is most commonly adopted. To date, two splice variants of the pre-1 gene have been reported in N. crassa (Karlsson et al., 2008; Kim and Borkovich, 2004; Pöggeler and Kuck, 2001).
Not much is known about the expression of the different variants, but initial studies have been undertaken (Paper V). If the different splice variants were found to exist in different stages of the life cycle, or in species with different reproductive systems, this would contribute to our understanding of the regulation of the pheromone receptor pathway. This previously uninvestigated topic may even give clues to the additional roles of pheromones and pheromone receptors.
15 Mating systems in Neurospora
As briefly touched upon in the introduction, there are three different mating systems in Neurospora: heterothallism (self-incompatibility), homothallism (self-compatibility) and pseudohomothallism (partial self-incompatibility). The initiation of the sexual cycle is the step that predominately distinguishes sexual reproduction in heterothallic versus homothallic species (Coppin et al., 1997): heterothallic species require a partner for mating, whereas homothallic species are able to self-mate. Pseudohomothallic species are partially self-compatible, since they occasionally outcross. The different mating systems are schematically depicted in Figure 2.
Heterothallism The term heterothallism was first introduced by Blakeslee (1904), who found that sexual reproduction in the common bread mould Rhizopus stolonifer was possible between partners indistinguishable by morphology, but which were of different mating types.
As stated previously, heterothallic taxa of Neurospora have two distinct mating types, A and a, with completely dissimilar sequences (idiomorphs) at the mating-type (mat) locus (Glass et al., 1988; Metzenberg and Glass, 1990). For sexual reproduction to occur, strains of the two opposite mating types must meet (Figure 2). Heterothallic Neurospora are shown in analyses of population structure to be mostly outcrossing, although they are sexually compatible with 50 % of their siblings (Ellison et al., 2011; Powell et al., 2001).
In addition to sexual reproduction, many heterothallic and pseudohomothallic Neurospora are known to reproduce asexually, through the production of asexual spores (i.e., micro- and macroconidia) or by fragmentation of the vegetative hyphae. This way of reproduction is believed to occur when environmental (nutritional) conditions are advantageous. The different types of conidia are products of different developmental pathways, and are thought to fill different functions. The macroconidia can serve both as male fertilizing units during the sexual cycle, but also as asexual propagules. They are described as vivid and copious, and it is this colorful
16 (orange to salmon-pink) phenomenon which leads to the name ‘orange bloom’ (Perkins and Turner, 1988). The microconidia, on the other hand, are expected to function primarily as mating propagules (Pandit and Maheshwari, 1996), albeit when environmental conditions are poor, these fungi invest in sexual reproduction. Raju (1992) reported that female reproductive structures, protoperithecia, are formed upon nitrogen starvation. One of the suggested reasons to reproduce sexually is in order to produce sexual ascospores, which are more rigid and preserved for very long periods compared to the asexual spores. Almost all of the described taxa of homothallic Neurospora have lost this ability, only adopting the sexual route of propagation.
Homothallism Homothallic Neurospora are not believed to be capable of mating in the classical sense (Howe and Page, 1963; Nygren et al., 2011; Perkins, 1987). Homothallic Neurospora are self-compatible, or strictly self-reproducing, i.e., the species can complete the sexual cycle and go through all steps of meiosis without finding a mate since they possess all the genetic information necessary for sexual reproduction in one haploid genome (Casselton, 2002; Coppin et al., 1997) (Figure 2). Genetically speaking, this intra-haploid mating equals asexual reproduction (Nauta and Hoekstra, 1992). No evidence for alternative reproductive strategies has been observed for the homothallic taxa. In support of homothallic species being strictly self-reproducing is the lack of structures important for outcrossing, such as trichogynes (female receptive hyphae), micro- and macroconidia (male fertilizing units) (Howe and Page, 1963; Perkins, 1987). However, the support for homothallic Neurospora being strictly self-reproducing has only been investigated in a few species, and therefore it is impossible to conclude if this is true for all homothallic species.
Homothallic Neurospora can be divided into three different groups based on the organization of mat genes; 1) those that contain only mating-type sequences similar to the mat A idiomorph (Glass and Smith, 1994), 2) those that contain mating-type sequences similar to both the mat a and A idiomorphs, and 3) those with mat a-1, mat A-1, and mat A-2, but missing mat A-3 (Beatty 1994). The sexual cycle is achieved without seeking a mate and provides long-lived ascospores, which can counterbalance the absence of vegetative spores (conidia). N. africana is probably the most studied homothallic species of Neurospora (Glass and Smith, 1994). In this doctoral thesis work, some homothallic species, including N. africana, have been studied extensively (Paper II).
17 Pseudohomothallism The third described mating system in the genus Neurospora is pseudohomothallism. This is characterized by isolates harboring nuclei of both mating types in ascospores (A+a) and vegetative cells, resulting in self- fertile heterokaryons (Raju and Perkins, 1994) (Figure 2). Self-fertilization is the primary way of sexual reproduction, but the species N. tetrasperma has been reported to occasionally produce homokaryotic individuals that outcross in nature (Menkis et al., 2009; Powell et al., 2001). Therefore, in this thesis work, N. tetrasperma has been grouped together with the heterothallic taxa (Paper III and Paper IV).
Homothallism
A(a)
Heterothallism
A a Pseudohomothallism
A + a
A a
Figure 2. A conceptual view of the different mechanism for sexual reproduction between the three mating systems (heterothallism, homothallism and pseudohomothallism) in the genus Neurospora. The stars represent different individuals and the mating types (indicated by letters in circles) needed for sexual reproduction. Heterothallism is suggested to be the ancestral state of the genus (Paper I and II), and for species with this mating system, two isolates of different mating types must meet for completion of sexual reproduction. Homothallic isolates can complete the sexual cycle by themselves, without meeting a partner, since they have all mating components in their genomes. Pseudohomothallic isolates have both components in the same cell, but in different nuclei. Occasionally the two nuclei separate during spore morphogenesis, which creates a condition of functional heterothallism. The switches from heterothallism to homothallism in Neurospora appear to be unidirectional (Paper II).
18 Ancestral state and unidirectional transitions of mating systems Several examples from Ascomycete genera indicate that homothallic species have arisen from heterothallic ancestors (O'Donnell et al., 2004; Yun et al., 1999). Although in many other genera, it is likely that multiple independent transitions from heterothallism to homothallism and vice versa have occurred (Lee et al., 2010). In summary, it is well known that transitions of mating systems have occurred frequently, i.e., multiple times in evolutionary time, but the direction of the switch is often not known.
The ancestral mode in Neurospora appears to be heterothallism (Paper II). The evolution of different reproductive systems is probably correlated with the wide range of natural environments. Recent evolutionary and structural research suggest that the ancestral state in Neurospora was most likely heterothallism (Paper I and Paper II). In addition, two mechanisms for mating type switching (heterothallism to homothallism) have recently been explored (Paper II), i.e., translocation and unequal crossover. Two novel retrotransposable elements (npanLTR and nsubGypsy) are suggested to be drivers for these independent and unidirectional transitions (Paper II).
The repeated occurrence of homothallism within numerous genera and the predominance of homothallism in filamentous ascomycetes one might suggest that this mating system has selective advantage. Theoretical models predict that heterothallic systems are the ancestral (Nauta and Hoekstra, 1992). However a few reports have suggested that homothallism is the ancestral state (Williams et al., 1981).
From a theoretical point of view it is important to determine the ancestral state of the genera, since changing from outcrossing to self-fertility might lead to an evolutionary dead-end, that is the extinction of species in the long term (Paper II) (Takebayashi and Morrell, 2001).
19 Evolution of reproductive genes in fungi
The evolution of sexual reproduction has intrigued evolutionary biologists for hundreds of years. For example, both Linnaeus and Darwin devoted their lives to understanding different aspects of reproductive biology. In this thesis, the focus has been on mating type genes and pheromone receptor genes, from an evolutionary perspective, in order to understand the evolution of reproductive systems.
Reproductive proteins per se have been reported to evolve more rapidly than other genes (Swanson and Vacquier, 2002; Turner and Hoekstra, 2008). This pattern is found in organisms ranging from unicellular diatoms with no or little pre-mating barriers (Armbrust and Galindo, 2001) to mammals with more complex mating behaviors (Swanson et al., 2001). Rapid evolution of a gene could be a consequence of either 1) adaptive evolution promoted by natural selection of amino acid divergence, or 2) a lack of functional constraint, i.e., absence of purifying selection. The rapid evolution of reproductive genes could potentially be important for reproductive isolation and eventually speciation events.
In a previous study of the evolution of the pheromone receptor genes in heterothallic and pseudohomothallic Neurospora (Karlsson et al., 2008), the authors argued that purifying selection is the major force shaping genes, although they noted that the cytosolic C-terminal domains of both genes evolve rapidly. This divergence might be driven by both stochastic and directional processes.
In my thesis work I find evolutionary patterns agreeing, as well as disagreeing, with the expectations of reproductive genes.
Expected limited gene flow of reproductive genes Genes involved in reproduction are expected to show a limited gene flow between species, due to hybrid incompatibility (Baack and Rieseberg, 2007; Tao et al., 2003; Turelli and Begun, 1997). When we constructed phylogenies for the mating-type and pheromone-receptor genes using a collection of heterothallic Neurospora, we found that the so-called species
20 tree (based on four microsatellite-flanking regions) was in disagreement with the trees built from reproductive genes (Paper IV). We argued that the discrepancies between gene and species trees were caused by introgressional events. Introgression is the transfer of genetic material via hybridization from one species to another, and the subsequent backcrossing between the hybridized individual with an individual from the parental species.
Decay of reproductive genes in homothallic Neurospora One may expect that a switch in reproductive behavior can change the evolutionary trajectory of a reproductive gene. Previously, Wik et al. (2008) showed that mat-genes evolve rapidly in Neurospora, in accordance with the expectations of reproductive genes. The authors argued that this rapid divergence was a result of adaptive evolution in the heterothallic taxa, and that this was caused by a lack of selective constraints among homothallic taxa. In addition, this study showed that the mating type genes in homothallic Neurospora have disrupted reading frames causing pre-mature stop codons or frame-shifts, in gross contrast to heterothallic species where the mating type genes are highly conserved.
One might speculate that once a heterothallic Neurospora isolate switches its reproductive mode to homothallism, its selective pressure to maintain functional mat-genes disappears, resulting in pre-mature stop codons and frame-shift mutations, which consequently disrupt the open reading frames (ORF). Although for other homothallic fungal genera of ascomycetes, mating-type gene degeneration, are not found (Lee et al., 2003; Pöggeler et al., 2006). We studied the evolutionary trajectory of the pheromone receptor genes, that are supposed to be regulated down-stream of the mat-genes, and included species of both heterothallic and homothallic Neurospora. The results from our molecular evolution analyses suggest that pre-genes are functional (Paper III).
21 Research aims
The general aim of this thesis was to study the evolution of reproductive systems and traits in the ascomycete fungal genus Neurospora. More specifically; I explored the evolutionary forces shaping the genes involved in sexual reproduction, especially the mating-type and pheromone receptor genes. To do this, I have combined laboratory and computational work to gather data and perform molecular evolutionary analyses. The specific aims for each research paper are presented below.
Specific research aims:
Paper I • Build a robust phylogeny for the genus Neurospora in order to further strengthen the genus as a model in evolutionary biology. • Infer how many switches of reproductive mode have occurred in the evolutionary history of the genus. • Investigate genomic consequences of reproductive modes, by determining substitution rates between homothallic and heterothallic clades.
Paper II • By using the Neurospora phylogeny together with mating-type locus architecture among species, further address the question of the ancestry of fungal mating systems in order to understand the great variety of reproductive modes. • Investigate the directionality of mating-system transitions. • Elucidate the mechanism/s of the polyphyletic origins of homothallism in Neurospora. • Examine the role of transposable elements in Neurospora and their potential importance in driving the switches in reproductive modes.
22 Paper III • Based on the Neurospora phylogeny, investigate whether the evolutionary trajectory of the pheromone receptor genes in Neurospora differs between heterothallic and homothallic taxa, and among the homothallic lineages/clades representing independent switches from heterothallism to homothallism in the evolutionary history of the genus. • Study the gene expression of pheromone-receptors and mating-type genes in homothallic species of Neurospora during different life cycle stages.
Paper IV • Compare gene trees of the mating-type and pheromone receptor genes with the species tree in heterothallic Neurospora, and infer signatures of evolutionary processes, e.g. gene flow, of reproductive genes in natural populations.
Paper V • Confirm the co-existence of different pre-1 splice variants in tissue of heterothallic N. crassa. • Investigate if the two splice variants of pre-1 have different expression profiles during the life cycle of N. crassa.
23 Summaries of papers
Paper I – A comprehensive phylogeny of Neurospora reveals a link between reproductive mode and molecular evolution in fungi In this study we constructed a comprehensive phylogeny of the genus Neurospora using sequence information from seven nuclear loci and 43 taxa. The genus contains taxa with three different reproductive modes, i.e., heterothallism, homothallism, and pseudohomothallism. With this phylogeny we made theoretical predictions for which reproductive mode was the ancestral, as well as inferred how many times a switch in reproductive mode may have occurred in the evolutionary history of the genus. This robust phylogeny will further strengthen Neurospora as a model in evolutionary biology.
In order to construct the phylogenetic tree we analyzed the sequence alignments using both a maximum likelihood (ML) and a Bayesian approach. ML analyses were perfomed with the program RAxML (Stamatakis, 2006) and the Bayesian approach used MrBayes (Huelsenbeck and Ronquist, 2001). To reconstruct the evolutionary history of the reproductive modes, we applied the program BayesTraits (Pagel et al., 2004). We also used the codeml and basml programs, implemented in PAML package version 4.3 (Yang, 1997, 2007) to investigate correlation between molecular substitution rates and reproductive mode.
When a heterothallic ancestor was assumed, the resulting phylogeny revealed at least six switches from heterothallism to homothallism, with high support values for the different clades with different mating types. In addition, the phylogeny suggested two independent origins of pseudohomothallism. Although our results from the phylogeny-based ancestral state reconstruction analysis suggested a homothallic ancestor for the genus Neurospora, we argue for the heterothallic ancestor for several reasons. First, it seems to be ‘easy’ to change from heterothallism to homothallism, for example by occasional recombination resulting in a taxon having both mating-types in the same genome. Secondly, it was previously demonstrated that the mat genes are genetically degenerate in homothallic
24 taxa of Neurospora compared to the mat genes in the heterothallic taxa representing the terminal clade. Finally, many homothallic taxa appear to have lost both female and male complex reproductive structures. We argue that both functional mat genes and reproductive structures in an evolutionary perspective would be easier to loose than to gain.
Furthermore, we conclude that reproductive mode is an important factor driving genome evolution in Neurospora. Branches delineating homothallic taxa have a higher level of non-synonymous/synonymous (non-silent/silent) substitutions, implying a reduced power of purifying selection in these taxa. After further analyses, where we could not detect any signs of positive selection, and therefore concluded that homothallic clades show signs of less efficient purifying selection. This is in agreement with theoretical predictions, i.e., species exhibiting very low effective recombination rates have small effective population sizes, which in turn cause a lower selection efficiency compared to their outcrossing relatives (Charlesworth and Wright, 2001). To our knowledge, this is the first study that shows this pattern in fungi.
Finally, we found higher nucleotide substitution rates in heterothallic, conidia-producing taxa, than in homothallic, non-conidia producing taxa. This may be explained by a higher rate of mitotic divisions in outcrossing taxa. We further speculate that the loss of the asexual pathway in many homothallic taxa could have evolved to lower the rate of mutation accumulation.
Paper II – Unidirectional evolutionary transitions in fungal mating systems and the role of transposable elements Evolutionary transitions in fungal mating systems are well documented. In the genus Neurospora, switches in mating systems have been reported to occur multiple times in the history of the genus (Paper I). In this study, we study the mat locus structure in heterothallic and homothallic taxa of Neurospora across the phylogenetic tree. We show that the ancestor of Neurospora was heterothallic, and that transitions to homothallism are mechanistically feasible. We propose two mechanisms (translocation and unequal crossover) including the mat locus, to explain the transitions.
We used draft assemblies for genomes of the four homothallic species N. africana, N. pannonica, N. sublineolata, and N. terricola, to determine the structures of the mat locus. In addition, we performed traditional Sanger
25 sequencing to confirm linkage of genes in regions of the assemblies that are fragmented. N. africana possesses only mat A components, and they are organized similarly to mat A strains of N. crassa; mat A-1, mat A-2, and mat A-3 are juxtapositioned and flanked by the genes SLA2 and APN2. N. pannonica and N. terricola have both mat A- and mat a-genes positioned in close proximity. Finally, in N. sublineolata we found that the mat a- component was flanked by SLA2 and APN2, and mat A is located at least 50- 220 Kb away from mat a, but most likely located on another chromosome.
The four species are thought to have originated from independent transitions. By combining the phylogenetic framework of Neurospora (Paper I) and the newly acquired information about structural organization of the mating-type genes including neighboring genes (Paper II), we could conclude that the transitions in mating system are unidirectional, i.e., heterothallism to homothallism.
Additional analyses suggest that repetitive elements have shaped the mat locus architecture. By scanning the genomes we found two novel transposable elements in Neurospora, and named them npanLTR and nsubGypsy. These elements are predicted to code for retrotransposons. In conclusion, we propose that the transitions to self-fertile life styles in fungi mediated by transposable elements are mechanistically feasible.
Paper III – Deciphering the relationship between mating system and pheromone receptor gene evolution in species of Neurospora Here we present a study of the molecular evolution of the pheromone receptor genes (pre-1 and pre-2) of a total of 30 heterothallic and homothallic taxa of the model genus Neurospora. Our general aim was to make use of the phylogenetic framework presented by Nygren et al. (2011) (Paper I) to investigate whether the evolutionary trajectory of the pheromone receptor genes in Neurospora differs between heterothallic and homothallic taxa, and between the homothallic lineages/clades indicated previously to represent independent switches from heterothallism to homothallism in the evolutionary history of the genus. For this study we applied phylogenetics, molecular evolution (using PAML), RCA (Reverse Complementation Analyses) and real-time quantitative PCR.
For pre-1, molecular evolution analyses suggest that there is variation in dN/dS among the branches of the phylogeny, but we found no support for either a mating system or a switch-independent evolution for this gene. For
26 pre-2, we found statistical support for a mating-system dependent evolution of the gene. A local model of dN/dS (non-synonymous/synonymous substitutions), assuming a mating-system dependent evolution, was the simplest model providing a significantly good fit for the pre-2 data, although the differences in dN/dS between branches delineating heterothallic and homothallic clades were small. The result from the molecular evolution analysis suggests that the pre genes are functional in homothallic taxa of Neurospora, even though individual homothallic taxa were found to have frameshift mutations causing premature stop codons, which may indicate a loss of function for these particular taxa.
Both RCA and dN/dS studies show that most of the variation, for both homothallic and heterothallic taxa, in pre-1 is located to the cytosolic C- terminal tail. This tail is interacting with the G-protein complex and mediates downstream cascades. Noteworthy, none of the codons that showed signs of positive selection were common between homothallic and heterothallic species of Neurospora. This could influences properties of the protein, and one might speculate that the different species have evolved slightly different regulation of the genes. Our results from the expression study of both mat- and pre-genes do not support a general pattern for regulation for neither mat- nor pre-genes. Based on these results, we hypothesize that pre genes are important and functional during sexual development in the majority of homothallic taxa. This conclusion is in contrast to a previous molecular evolution study of mat-genes in Neurospora, were the authors found degeneration of mat genes in homothallic taxa (Wik et al., 2008).
Paper IV – Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the ascomycete genus Neurospora In this phylogenetic study, we derived the genealogies of genes important for sexual identity, i.e. mating type (mat) and pheromone-receptor (pre) genes, among heterothallic and pseudohomothallic taxa of Neurospora. The resulting genealogies were compared with the species phylogeny derived from non-coding sequences published by Dettman et al. (2003).
A total of 35 strains belonging to ten phylogenetic taxa and four strains of the pseudohomothallic N. tetrasperma were used in this study. The four mating types (mat a-1, mat A-1, mat A-2, and mat A-3) were sequenced. We used previously published data from microsatellite flanking regions to infer
27 the species tree. The phylogenetic analyses were done with PAUP* 4.0b10 (Swofford, 2003) using ML default heuristic settings and the best-fit model of sequence evolution as estimated from the Akaike information criteria in ModelTest 3.06 (Posada and Crandall, 1998). The node supports were obtained from ML bootstrap analyses using PHYML 2.4.4 (Guindon and Gascuel, 2003) and Bayesian Markov Chain Monte Carlo (MCMC) analyses using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001). For both support analyses the best model of sequence evolution was used.
We found two major conflicting topologies between the mat genealogies and the species phylogeny, and one conflicting topology between the mat a and mat A gene trees; 1) in the species tree N. crassa subgroup A, B, and C (NcA, NcB, and NcC), form a monophyletic group, but in both mat-gene trees NcC form a monophyletic group together with N. intermedia, 2) the placement of N. tetrasperma and N. metzenbergii differ between gene and species tree, and 3) the placement of N. sitophila and N. hispaniola, differ between the mat gene trees. All three conflicts were supported by both node support analyses and likelihood tests on the relative fit of datasets to alternative phylogenetic topologies.
When comparing pre-genealogies and species tree, we identified three conflicts; 1) in the pre-1 genealogy the N. intermedia subgroups A and B, do not cluster together as in the species tree, 2) in the pre-2 genealogy NcA, NcB, and NcC, do not form a monophyletic group as in the species tree, and 3) N. perkinsii and N. intermedia subgroup A form ‘within species subgroups’ in the pre-2 genealogy.
Taken together, this study indicates that reproductive genes are more permeable to introgression than other genes, which is in contrast to theoretical expectations (Baack and Rieseberg, 2007; Tao et al., 2003; Turelli and Begun, 1997). Wingfield et al. (2011) also reported a conflict between the MAT idiomorphs of species in the Gibberella fujikuroi complex and the recognized species tree, and between species introgression of the mating-type genes were also found by Paoletti et al. (2006). The authors of both these studies speculated that through the cross species introgression of reproductive genes, such as MAT, sexuality can be restored where once it had been lost. The evolutionary consequences of the mat gene introgression in our study is yet unknown. In contrary to my findings, other studies, including a study on the homothallic F. graminearum complex show congruencies between gene and species tree (O'Donnell et al., 2004).
28 Paper V – Co-existence and expression profiles of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa The alternative splicing of gene transcripts permits translation of different protein variants from the same gene. In heterothallic Neurospora crassa, strains of different mating type use a pheromone receptor system to find a compatible mating partner. Previously, only a single splice variant was reported from a single tissue (Karlsson et al., 2008; Kim and Borkovich, 2004; Pöggeler and Kuck, 2001). In this study, we show that two splice variants of the pheromone receptor gene (pre-1) co-exist in both vegetative and reproductive tissues of N. crassa. The two splice variants are spliced by intron retention of intron i3, which is predicted to result in a premature stop codon and loss of 322 amino acids from the C-terminal cytosolic region of PRE-1. In addition, we use quantitative PCR and showed that expression of the retained intron splice variant is on average 10-fold lower than the expression of the spliced intron variant. Both splice variants are induced by mycelial age, with higher transcript numbers after 14 days in culture compared with both one or seven days. Our data indicate that sexual reproduction and growth media composition did not influence the expression of either splice variant.
In a previous evolutionary analysis of interspecific sequence variation of the pre-1 gene in heterothallic Neurospora, the authors showed that the most variable region was the cytosolic tail (Karlsson et al., 2008). A cytosolic tail which exhibits a high proportion of codons that either evolve under relaxed selective constraints or under positive selection, correlates well with the third exon of pre-1 (Karlsson et al., 2008).
It has also been shown previously that alternatively spliced exons exhibit higher rates of non-synonymous substitutions than constitutively spliced exons. This is due to weaker selective constraints, which in turn can contribute to functional divergence (Chen et al. 2006). Therefore, we argue that alternative splicing of the third intron of pre-1 may be the mechanism behind the relaxed selective constraints or positive selection associated with the C-terminal cytoplasmic part of PRE-1 (splice variant I). One might speculate that this in turn can result in functional divergence of this region of pre-1. The functional divergence would be even more interesting to study further, by also including homothallic species of Neurospora (see Paper III).
29 Concluding remarks and future perspectives
I think fungi are amazing study organisms for evolutionary biology. The diverse fungal kingdom is interesting to study because one can find a plethora of different mechanisms to control sexual development, and different stories are found in different genera as well as species. This diversity may result from adaptations to different environments.
In this thesis, several pivotal questions were addressed in order to explore the dynamics of reproductive systems in Neurospora. The backbone of this thesis is the robust Neurospora phylogeny (Paper I), were the multiple transitions in reproductive life style are defined phylogenetically. The Neurospora phylogeny together with the findings of mat locus structure in homothallic Neurospora (Paper II), conclude that the ancestor of Neurospora was most likely heterothallic, implicating polyphyletic origins of homothallism in Neurospora. The multiple shifts in reproductive systems in this genus, further indicates that the transitions are mechanistically feasible. The idea that the polyphyletic origin of homothallism in Neurospora is facilitated by transposable elements is also intriguing, and might be influential for future research.
During this thesis work, the phylogeny of Neurospora has also been used to test if gene evolution, i.e., pheromone receptor genes in hetero- and homothallic Neurospora, is dependent on mating systems and/or even the switches themselves (Paper III). This study also included expression analyses of both pre- and mat-genes, and in conclusion, we also see different patterns in different taxa.
The power of phylogenetic analysis has been proven time after time to be very valuable for the evolutionary questions we have addressed. The introgression pattern we find when comparing mating-type and species phylogeny of Neurospora is an example of the usefulness of phylogenetic analyses (Paper IV). It would be interesting to further investigate if the genomic region outside the mating type will show the same pattern or if it is specific to the genes themselves.
30 The alternative splicing of pre-1 is exiting, since it could explain the patterns we see (Paper V). It would also be interesting to study if the two different splice-variants of pre-1 also exist in homothallic taxa of Neurospora.
Of course, much is left to be done in order for a more complete understanding of the evolution of reproductive systems in Neurospora. First, phenotypic studies of mating-type and/or pheromone-receptor deletion- strains in different homothallic species of Neurospora would be extremely valuable. For example, the mating-type genes exist in homothallic Neurospora, although they seem to evolve with low selective constraints, which could indicate that they are superfluous (Wik et al., 2008), but mutagenesis would provide the ultimate proof for this. Second, experimental evolution studies would provide fitness-data to support predictions for analyses of sequence data, for example whether homothallic Neurospora experience a fitness decline after many generations, which would support our analyses of molecular evolution of an accumulation of deleterious mutations in these species. Third, protein analyses of candidate genes are needed for the full picture of the phenotypic effects of reproductive behavior, for example it would provide ultimate evidence on the existence and function of the two splice variants of pre-1.
This work of this thesis will hopefully inspire other researchers in evolutionary genetics.
31 Sammanfattning på svenska
Denna avhandling undersöker evolutionen av reproduktiva system i svampsläktet Neurospora. Släktet är utmärkt för denna typ av studier tack vare att det har representanter för tre olika parnings-system: heterothallism (utkorsning), homothallism (självbefruktning) och pseudohomothallism (en kombination av utkorsning och självbefruktning). Neurospora är en kosmopolit och hittas i tropiska, subtropiska och tempererade områden. En global samling av isolat finns tillgänglig genom Fungal Genetics Stock Center (FGSC; University of Missouri, USA). Genetiken bakom Neurospora är välstuderad och hel-genom från tre olika arter finns tillgängliga.
I svampriket finns inga kön i klassisk bemärkelse. En svamp kan producera både han- och honstrukturer från samma mycel, och dess identitet definieras genom vilken parningstyp individen har. Neurospora crassa och N. intermedia är två exempel på heterothalliska arter. Utkorsande Neurospora kan antingen vara av parningstyp a eller A. För blotta ögat skiljer sig dock inte a- och A-svamparna sig åt. Det som avgör om två svampar av samma art är kompatibla är istället en genetisk sekvens som styr deras parningstyp (mat a och mat A, respektive). För att kunna fortplanta sig sexuellt måste individen hitta en individ med annan parningstyp. Svamparna attraheras av varandra genom ett feromon/receptor-system.
Andra arter av Neurospora kan fullborda den sexuella cykeln på egen hand, och är därmed självkompatibla (homothalliska), eftersom de har alla genetiska komponenter (mat a och mat A) som behövs för fullbordandet av den sexuella cykeln i samma genom. Det finns dock undantag, exempelvis homothalliska N. africana som endast har mat A-komponenter men som ändå är självkompatibel. Det tredje reproduktiva systemet, pseudohomothallism, karaktäriseras av att varje sexuell individ bär på både mat a- och mat A-komponeneter lokaliserade i samma cell. Ibland separeras de två komponenterna under celldelningen, vilket möjliggör fortplantning via utkorsning. Den mest studerade pseudohomothalliska Neurospora arten är N. tetrasperma.
I min doktorsavhandling har jag studerat hur släktskapet mellan olika Neurospora-arter genom att konstruera en robust fylogeni baserad på sju olika genetiska markörer. Denna fylogeni visar att under Neurosporas
32 evolutionära historia har flera övergångar skett mellan olika reproduktiva system (Artikel I). I och med den efterföljande studien, där vi fokuserar på mat-lokusets struktur i fyra arter av homothalliska Neurospora, har vi dragit slutsatsen att det ursprungliga systemet för Neurospora var heterothallism (Artikel II). Tittar vi på fylogenin och antar en heterothallisk anfader, ser vi att övergångarna har skett oberoende av varandra, minst sex gånger till homothallism och två gånger till pseudohomothallism. Under studien av mat-lokusets struktur upptäckte vi att övergångarna möjligen kan ha drivits av transposoner, det vill säga genetiska element som kan förflytta sig på eller mellan olika kromosomer i genomet.
Vi har i efterföljande studier använt oss av fylogenin över Neurospora för att undersöka om evolutionen av reproduktiva gener, i det här fallet feromonreceptorerna, är beroende av vilket parningssystem som svamparna har (Artikel III). Vi har även testat om evolutionen är specifik för varje övergång mellan systemen. Dessa studier har vi kompletterat genom att titta på genuttryck av både mat- och pre-gener. Metodologiskt är kombinationen av molekylär evolution och genuttrycksstudier något av ett nytt tillvägagångssätt. Vår förhoppning var att med de olika metoderna i kombination, peka på en tydlig trend vad gäller evolutionen av pre-gener i Neurospora.
I den fjärde studien undersöker vi om kan hitta fylogenetiska signaler som kan ge ledtrådar till vilka evolutionära processer som influerat den heterothalliska gruppen som innefattar N. crassa ser ut. Vi jämför släktträdet (baserat på sekvenser från regioner som flankerar fyra mikrosatelliter) med respektive genträd för parningstyp-generna (mat a och mat A) och feromonreceptorgenerna (pre-1 och pre-2). När vi jämför genträd med släktträdet ser vi att de inte har samma förgrening. De mest intressanta mönstren föreslår vi stamma ur introgression, en process där genetiskt material sprider sig genom hybridisering mellan två arter, och sedan återkorsning mellan hybriden och föräldraarten. Sammantaget visar vår studie att reproduktiva gener i Neurospora kan vara mer benägna att flytta runt mellan arter än vad teorin föreslår.
Vi har även studerat genuttryck av två olika splice-varianter av pre-1, det vill säga en av feromonreceptorgenerna i Neurospora crassa. Det är första gången som en ingående studie av dessa splicevarianters uttryck genomförts. Vi studerar om uttrycket skiljer sig mellan olika tillväxtmedier och under olika åldrar och utvecklingsstadier.
33 Acknowledgements
This thesis work was performed at the Department of Ecology and Genetics (Sub-department of Evolutionary Biology), Uppsala University, Sweden. First and foremost I would like to thank my brilliant supervisor Hanna Johannesson, who accepted me as a PhD student in her research group. The time spent in your group has been a great experience, and I have learned so much. Your endless support in all ways has been extraordinary. I think few PhD students are as lucky as I have been. I also wish to express my gratitude to my two assistant supervisors: I owe a great deal of thanks to Magnus Karlsson, who guided me through the beauty of qPCR experiments! Without you my thesis would not have been as good! Professor Hans Ellegren, who has provided an excellent research environment and cheering in the corridor.
I have had the privilege to be a member of the super fun Fungus group. Lots of people have passed through since I started, and together we have visited both the mushroom forest as well as Cambridge University. Kristiina Nygren! You are my idol! When I am running up-and-down being confused, you have always been calm and given me good advice. We have had great fun together, working on manuscripts and crossing the Atlantic. I will not forget the picnic basket! Tim James and Audrius Menkis, you were always helpful and taught me how to deal with lab and computer difficulties! Eric Bastiaans, your time in the fungus group was fun, thanks for visiting me in Göttingen. Nicklas Samils, being a supportive colleague and cheerful rock’n’roll icon. Good luck at SLU! Anastasia Gioti, thanks for always being supportive and questioning, I wish you all the best in your future research career. Soon you will have your own fungus group! Pádraic Corcoran and Yu Sun – thanks for always being helpful! Thanks Sasha Mushegian for loving pancake heaven! Ioana Brännström for good times in the fungus lab!
Besides of the fungus group, the vivid atmosphere at the department would not have been the same without Urban’s Drosophila swarm, Mattias’ human population genetics group, Tanja’s Capsella-research, Anders’ ancient DNA, Jochen’s crow crowd, Hans’ bird evolution and Simone’s zebra fish. All people, former and present, at the Department of Evolutionary Biology. Especially, Gunilla Kärf, Malin Johansson and Jessica Magnusson, for helping me with both small and big problems, and keeping the lab in good shape!
34 When I started as a PhD student Ülo Väli and Karin Berggren Bremdal kept me company in the office. (Det var roligt att prata svenska med dig Ülo! Jag hoppas du trivs i Estland!) Karin, you were always the gal who gave me good advice. Emma Svensson, thanks for all good chats. I think you are the coolest person! José Padial, you were not the early bird, and I still remember when you comforted me when my computer crashed. Rebecca Dean, we only shared office for a few weeks, but they were fun weeks! My new office mates, Lucie Gattepaille and Carina Schlebusch, thank you for being supportive the last months when I was writing up my thesis!
Vielen Dank zu Stephan Seiler in Göttingen, Germany. It was great fun to work in your lab for four cold winter months. Thanks to all lab members who kept me busy with bowling and Sambesi dinners: Sabine, Immo, Danni, Corinna, Yvonne and Sonja.
My greatest thoughts go to the Department of Forest Mycology and Pathology, SLU, especially Magnus (thanks again for being a great supervisor), Janne, Åke and Petra. Biking back and forth to SLU has always been rewarding! You all made me feel very welcome.
I would also like to thank my friends! Who during all of my PhD years kept my mind busy on things other than genetics. Maria Wilbe keeping me in good shape, your Pump it-class rocks! It was fun that although our research fields were different, we could still find a way to travel together. Jen Meadows for super excellent proof-reading of my thesis and YES, there will be dancing at my dissertation party! Wear your best dress! Elisabeth Sundström eating lunch with you and talking research and non-trivial things have always been a good distraction. Jeanette Axelsson, thanks for lunch breaks and giving me good advice. Thanks for all the coffee breaks Cecilia Wärdig! You are the best coffe and cake girl ever! I missed you when you moved to SLU. Peter Halvarsson, you crazy bird man, thanks for saving me! I will never forget the great escape. Thank you Eva Daskalaki for always being supportive and a great friend! Jenny Sågetorp, I remember when we went for coffee breaks downtown. I wish I could visit you again in beautiful Gbg! Daniel Svensson-Rothes, thank you for being my toastmaster and helping me organize my dissertation party!
Shiying Wu and Kasia Zaremba! You girls are great! My teaching time with you was excellent! Robert Fast, thanks for being a fun course organizer!
During my PhD, I also got the privilege to be a part of the IBG. Thanks to Torgny Persson. It was so nice to work together with you. Thanks to Ingela Frost for giving me interesting assignments.
Big thanks to the Neurospora Research Community and the FGSC! It has
35 been fun to be a part of this cool community of researchers. Special thanks to the Uppsala Graduate School in Biomedical Research (UGSBR). If I hadn’t been accepted to your program I would probably never have started PhD studies. Thanks to the EBC Grad School!
A number of funding bodies have been very generous and helped me to buy expensive lab consumables, travel around the world and present my research. The Royal Swedish Academy of Sciences, the Lars Hierta Memorial Foundation, Kungliga Fysiografiska Sällskapet i Lund, different Uppsala University funds (Liljewalchs, Sederholms, Gertrud Thelins), Norrland’s Nation, Helge Ax:son Johnson Foundation, the Royal Swedish Academy of Agriculture and Forestry, and Vidfelts foundation (The Swedish Forest Society).
Finally, I have loving and supportive family in Lapland! Mina snälla föräldrar Christina och Ingemar, som alltid uppmuntrat och stöttat mig. Lill- gumman har saknat er! Roligt att ni kommer på festen! Mina supercoola brothers, Anders and Erik, jag önskar vi kunde träffas oftare. Tack mormor och moster Sita!
Tack Joakim för att du gjort de sista stressiga månaderna under avhandlingsskrivandet så mycket roligare. Du är helt underbar på alla sätt! Nu blir det sovmorgnar och långa frukostar! ♥
36 References
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