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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 selfincompatibility) (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. Mat-gene constitution of heterothallic N. crassa
1987).
APN2
mat A-3
(HMG)
mat A-2
mat A-1
(alpha-box)
(eat-2)
SLA2
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 382amino 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 7transmembrane (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 fruitingbody 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 selffertile 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 acomponent was flanked by SLA2 and APN2, and mat A is located at least 50220 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 Cterminal 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 deletionstrains 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. Lillgumman 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
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