Rates and Patterns of Bryophyte Molecular Evolution

Home , Bryophyte, Jungermanniopsida, Lunularia, Marchantiopsida

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1850

Rates and patterns of bryophyte molecular evolution

ANNA-MALIN LINDE

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0740-4 UPPSALA urn:nbn:se:uu:diva-392462 2019 Dissertation presented at Uppsala University to be publicly examined in Zootissalen, EBC, Villavägen 9, Uppsala, Wednesday, 23 October 2019 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Jonathan Shaw (Duke University).

Abstract Linde, A.-M. 2019. Rates and patterns of bryophyte molecular evolution. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1850. 42 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0740-4.

Plants have been growing on land for at least 450 million years. The bryophytes comprising the three phyla liverworts, mosses and hornworts, are considered to be the closest extant relatives to the plants that colonized land. Bryophytes has been described as evolutionary “unchanging sphinxes of the past” regarding both morphological and genetic potential. This suggestion has some support in limited studies of molecular evolution within bryophytes, but has also been questioned based on e.g., studies of species diversification rates. To shed more light on this controversy, the overall aim of this thesis is to investigate rates and patterns of bryophyte molecular evolution. Our data suggest that the per nucleotide mutation rates in bryophytes are lower than those in angiosperms. Likewise, angiosperms are also more dynamic in terms of genome size, structural rearrangements, genome duplications and transposon activity. However, our data show that mutation rates of bryophytes are higher or at least on par with those of gymnosperms. Genome evolution in bryophytes is actually, in many aspects, similar to that of gymnosperms. Gymnosperms and bryophytes are both characterized by a low speciation rate, a low nucleotide mutation rate, low variation in chromosome numbers and relatively stable genome sizes. Studies have also suggested that macrosynteny is better conserved between conifer species compared with angiosperms, just as this study shows for bryophytes. Hybridization and introgression has been suggested to affect speciation and evolution. Recent genomic data shows that hybridization and introgression in angiosperms is more common then previously thought, but the question is less well studied in bryophytes. The present study gave some support to the occurrence of introgression between Marchantia polymorpha subspecies, but refute a previous hypothesis that M. polymorpha subsp. ruderalis is a new stabilized hybrid between M. polymorpha montivagans and polymorpha. An additional aspect of genome evolution and complexity is changes in gene regulatory networks. Gene regulatory networks generally appear more complex in angiosperms compared with bryophytes; also reflected in the circadian clock; with more gene components and more duplicated paralogous members, with possibly overlapping function, allowing a more robust and flexible system. Our studies of the plant circadian clock revealed that orthologs of most genes of the A. thaliana clock were present already in charophycean algae. Although gene numbers and complexity have generally increased during plant circadian clock evolution, our results suggest that gene loss has also been important in shaping the circadian clocks in the three bryophyte groups.

Anna-Malin Linde, Department of Ecology and Genetics, Plant Ecology and Evolution, Norbyvägen 18 D, Uppsala University, SE-752 36 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-0740-4 urn:nbn:se:uu:diva-392462 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-392462) List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Linde, A-M., Sawangproh, W., Cronberg, N., Szövényi, P., & Lagercrantz, U. Evolutionary history of the Marchantia polymorpha complex. Manuscript

II Linde, A-M., Eklund, D.M., Cronberg, N., Lagercrantz, U. Rates and patterns of molecular evolution in liverwort genomes, with focus of Marchantiopsida. Manuscript

III Linde, A-M., Eklund, D.M., Cronberg, N., Lagercrantz, U. Rates of structural changes in bryophyte genomes; exemplified by Marchan- tiopsida. Manuscript

IV Linde, A-M., Eklund, D.M., Kubota, A., Pederson, E, Holm, K., Gyllenstrand, N., Nishihama, R., Cronberg, N., Muranaka, T., Oya- ma, T., Kohchi, T., & Lagercrantz, U. (2017) Early evolution of the land plant circadian clock. New Phytologist, 216(2) 576-590

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 7 Bryophyte phylogeny and evolution ...... 8 The Marchantia polymorpha species complex ...... 8 Plant Genome Evolution ...... 10 Mutation and substitution rates ...... 11 Transposable elements ...... 11 Gene and genome duplications ...... 12 Genome size dynamics ...... 12 Hybridization, from a bryophyte perspective ...... 15 The Evolution of Gene Regulatory Networks ...... 16 The evolution of a specific functional network of genes: The circadian clock ...... 16 Aims of the thesis ...... 18 Materials and Methods ...... 19 Gene annotation of Marchantia sp. and Lunularia cruciata assemblies (Papers I, II, III) ...... 19 Genetic variation analysis of Marchantia polymorpha L (Paper I) ...... 19 Rate calculations and associations (Paper II) ...... 20 TE landscape, gene statistics and synteny (Paper III) ...... 20 Homolog search and functional analyses of clock genes in bryophyte and charophytes genomes (Paper IV) ...... 21 Results and Discussion ...... 22 The phylogenetic relationship between the three subspecies of Marchantia polymorpha (Paper I) ...... 22 Bryophytes do not have a uniquely low mutation rate (Paper II) ...... 23 Slow structural evolution of liverwort genomes (Paper III) ...... 26 Most components of the plant circadian clock are present in liverworts and in charophytes (Paper IV) ...... 28 Svensk sammanfattning ...... 30 Acknowledgement ...... 34 References ...... 36

Introduction

Based on spores found in the fossil record, plants have been living on land at least since Middle Ordovician, approximately 460 million years ago (Kenrick and Crane 1997). Land plants belong to the Embryophyta, whose members primarily live in terrestrial habitats and show a great variety of appearances. Now living embryophytes include hornworts, mosses, ferns, lycophytes, gymnosperms and angiosperms (flowering plants) (Figure 1). Together with some of the green algal lineages (Charophycean algae) they form the group streptophytes. All green algae and lands plants together comprise the clade Viridiplantae (“green plants”), which is the collective name of streptophytes and chlorophytes. Land plants form a monophyletic group which nests within the freshwater Charophycean algal clade, making it likely that land plant evolved from an ancestral freshwater or terrestrial alga (Bowman 2013; Harholt et al. 2016). It has been suggested that adaptations to land already evolved in early terrestrial charophytes (Delwiche and Cooper 2015; Harholt et al. 2016).

Figure 1. The phylogenetic relationship of plants, rooted with a charophytes algae. The phylogenetic order is adopted from Puttick et al. (2018). The branch lengths are not informative.

7 Bryophyte phylogeny and evolution The plants called “bryophytes” belong to three phyla, the liverworts (Marchantiopsida), mosses (Bryopsida) and hornworts (Antheroceropsida). Although these lineages represent a large fraction of the earth’s flora, exist on all continents and have significant ecological importance, they are often overlooked in the research of land plants. Traits they have in common include small size, comparatively small genomes, a predominant haploid gam- etophytic life phase and a reduced sporophytic phase (Shaw et al. 2011; Bowman et al. 2017; Rensing et al. 2008). The bryophytes are considered to be the closest extant relatives of the plants that first colonized land. Due to their phylogenetic position, they are crucial for understanding the evolutionary transition from freshwater algae to land plants and from structurally relatively simple early land plants to more complex forms. The phylogenetic relationships among the three bryophyte groups and their relationship to the vascular plants have long been debated. Seemingly well supported phylogenetic hypotheses resulting from different data and phylogenetic methods are often incongruent and this may be explained by heterogeneous rates of nucleotide evolution that is difficult to model (reviewed in Cox 2018). A well-supported phylogenetic hypothesis of their relationship, i.e. knowing if vascular plants have a bryophyte ancestor, is important for making correct evolutionary interpretations of traits specific to land plants such as their alternating life cycles and the development of stomata. (Sousa et al. 2019). Two recent studies (Puttick et al. 2018 and Sousa et al. 2019) both resolved bryophytes as a monophyletic group when including only slow-evolving sites. As summarized in Budke et al. (2018), 9000-13000 species are estimated to belong to the mosses, 5000-7500 species belong to the liverworts and 215 species to the smallest bryophyte phylum, the hornworts. Bryophytes has been described as evolutionary “unchanging sphinxes of the past” regarding both morphological and genetic potential (Crum 1972) and this hypothesis has been supported in a few molecular studies (Stenøien 2008; Villarreal et al. 2016; Chiang and Schaal 2000) although those studies only cover a limited number of mainly organellar genes. Higher diversification rates among mosses and liverworts than expected from this traditional view of slow molecular evolution has been reported (Renner et al. 2017; Laenen et al. 2014) which to the contrary indicate that the molecular evolution might not be as slow as suggested.

The Marchantia polymorpha species complex Marchantia polymorpha L. is a dioicous liverwort with a morphological variability that has led the taxonomists to describe more than fifty species, varieties, forms, subforms and subspecies during the last centuries. Linneaus

8 (Linneaus 1753) subdivided this species into three unnamed varieties. At present, three subspecies are recognized based on their different morphological characters, isozyme patterns and preferred habitats (roughly: in ruderal, wetland and montane habitats, respectively). The species geographical range is worldwide and it has been found both in cold and tropical regions. The subspecies have also been described as having a largely non-overlapping distribution due to niche differentiation, and therefore gene exchange between populations has been suggested to be restricted (Bischler-Causse and Boisselier-Dubayle 1991). Burgeff (1943) found restricted fertility between European material representing the three taxa and recognized them at species level using the names proposed by Nees (1838): M. polymorpha L. (being the largely rural morphotype), M. aquatica (Nees) Burgeff (representing the wetland morphotype) and M. alpestris (Nees) Burgeff (representing the montane morphotype). Later, Bischler-Causse and Boisselier-Dubayle (1991) found that the Lin- nean type specimen actually had the morphotype typical for wetland habitats (not the common ruderal morphotype), meaning that the name M. polymorpha applied at subspecific level must be used for the wetland morphotype. They reclassified them as the three subspecies commonly recognized today: M. polymorpha subsp. ruderalis, M. polymorpha subsp. polymorpha and M. polymorpha subsp. Montivagans (Figure 2), based on the detection of one putative hybrid using electrophoretic techniques, taken as evidence for occurrence of gene exchange in nature. Burgeff (1943) also postulated that one of the species (now subsp. ruderalis) is a stabilized hybrid involving the other two species (now subsp. montivagans and subsp. polymorpha). This postulate was based on the appearance of the subsp. ruderalis as a morphological intermediate between the other two and from the outcome of his crossing experiments. This hypothesis was promoted by the influential liverwort specialist Schuster (1983), who even proposed that the hybridization event had occurred in historic man-influenced habitats in which the two parental species were supposedly brought into first contact.

9 susubsp. polymorphapolymorpolymorphorphaha sussubsp.ubsp.ubspbsp.p rurudruderalisudederade alisalilis subssubsussubsp.ubbsspsp.sp.p mommontivagansonontivagtivagvavagaaggaganansn FigureFiigurere 1: Photosotoss of M.M polymorphapopolyolymorphaymymorphammomorporpphha sspspp.ppp. polymorphapolylyymorphaorphaa (left),(le(left)(lef(lleft)efeeft)), M.M polymorphappopolololylyymymoorpharp a spspp.pppp. ruderuderalisralis (middledlee) anandd M. ppolympolymorphaymorymorphmorrphahaa sspspp.ppp.pp. mmomontivagansonontntivivivagvagvagaganss (r(right).right).r ght)ght).ht).). NoNNoteoteoto thetthhe didifdifferencesffeerrenencennccescese oofffth ththehee tththickness of the blackblacbl midrimidribib off thallithalth llili (firstfifirstfirrsstt rowrrow),ro , theth size ofof thallithalthaththalliallli (seco(se(secondcoondond rrorow),owww),) aanandndd mmomormorphologyorphology of innermiinnermostnermosermost venventralentraltralralall sc sscaleses wwithithh 12112525 X magnificationmagnificatioagnificatioagnificatia n ((thirdtthhiiirdrd rorow).ow).

Figure 2. Images of M. polymorpha subsp. polymorpha (left), M. polymorpha subsp. ruderalis (middle) and M. polymorpha subsp. montivagans (right). Note the difference in the size of thalli (first row), the thickness of the black midrib (second row, arrow) and morphology of innermost ventral scales with 125x magnification (third row, arrow).

Plant Genome Evolution Evolution cannot proceed in the absence of genetic variation and there are many potential sources of such variation. Mechanisms that have been described as important sources of genetic variation and that are covered in this thesis are point mutations, gene duplications (reviewed in Panchy et al. (2016)), the activity of transposable elements (causing a range of different effects on the genomes including mutations and duplications; reviewed in Lisch (2013)) and introgression (reviewed in Suarez-Gonzalez et al. (2018)).

10 Mutation and substitution rates One type of genetic change contributing to the variation in genomes is point mutations. When a mutation is spread through the population it is called a substitution and the substitution rates can be estimated by comparing homol- ogous DNA sequences between two species. The substitutions can be synonymous (dS; do not lead to changes in protein sequence and are assumed to be selectively neutral) or non-synonymous (dN; leading to changes in protein sequence and potentially also change the phenotype of the organisms which may change its fitness). Due to its assumed neutrality, the synonymous substitution rate approximately equals the mutation rate. Often the ratio of non- synonymous to synonymous substitution rate ratio (dN/dS; also called omega) is used to represent the strength and mode of natural selection (Jeffares et al. 2015). When omega is larger than one it indicates that substitutions in the protein-coding gene were enriched for those that altered the amino acids, suggesting diversifying (positive) selection. By contrast, a value of omega that is smaller than one can be interpreted as purifying (negative) selection. When an amino acid change is neutral, the rate of fixation will be the same as that of a synonymous mutation and omega equals one. Most new non- synonymous mutations reduce the fitness of their carriers, being deleterious and will be selected against, so that the majority will be removed from the population (purifying selection) (Graur 2008). Several factors have been associated with differences in evolutionary rates between genes within the same plant genome. Among the most important are the level of gene expression (Gaut et al. 2011; Slotte et al. 2011) and how broadly the genes is expressed (Slotte et al. 2011), and both generally show strong negative correlation with the rate of evolution. Other factors that has been shown to contribute to variation in evolutionary rate among plant genes are functional category, physical location of the gene, recombination rate, GC content, gene lengths, mutation rate, chromatin environment, gene compactness and gene duplication status/gene family size (Guo et al. 2017; De La Torre et al. 2015; L. Yang and Gaut 2011). Factors that have been associated with differences in evolutionary rates between lineages in plants includes species richness, genome size, life history traits, environmental factors (energy, temperature, latitude, UV), generation time and type of reproduction (sexual/asexual) and mating system if the reproduction is sexual (Bromham et al. 2015; Lanfear et al. 2013).

Transposable elements Since the discovery of transposable elements (TEs) in maize in the 1940s by McClintock (1950) TEs have been found to be ubiquitously present in all eukaryotes. TEs are genetic elements that have the capacity to modify their position within the genome and some also have the ability to generate new

11 copies of themselves. They are divided into two classes, retrotransposons (class I) and DNA transposons (class II) based on their transposition mechanism. Class I elements transpose with a “copy and paste” mechanism, having an RNA intermediate while class II elements transpose by a “cut and paste” mechanism (Bennetzen and Wang 2014). Class I TEs are subdivided into elements with Long Terminal Repeats (LTR), also called LTR retrotransposons and elements without LTRs known as non-LTR retrotransposons such as LINEs and SINEs. The two main superfamilies of LTR retrotransposons are called Gypsy and Copia which are both common in plants (Suoniemi et al. 1998; Voytas et al. 1992). The TE activity is almost always deleterious or neutral for the plant but in the evolutionary timescale occa- sional selectively advantageous TE insertion does occur. Lisch (2013) summarized multiple evidence for mechanisms in which TEs have actually contributed to plant evolution: gene inactivation (knock out of gene function is the simplest and most common type of TE-induced phenotypic change), change of gene expression when inserted into repressors or enhancers, chromosomal rearrangements, exaptation of coding sequences of TEs by the host and epigenetic effects. The epigenetic silencing of TEs involves small RNAs, DNA methylation and various histone modifications and epigenetic changes can also be introduced in nearby genes, affecting their expression (Sigman and Slotkin 2016; Wang et al. 2013; Wheeler 2013).

Gene and genome duplications One other driving force of eukaryotic evolution is gene and genome duplications, where gene duplications are the primary source of new genes (Ohno 1970; Zhang 2003). Gene duplications can occur through different mechanisms such as whole genome duplication (WGD), segmental duplication, tandem duplication and transposon-induced duplication (Panchy et al. 2016) all of which might have different impacts on duplicate gene function and its evolutionary fate. Most duplicated genes will, however be purged from the genome. Panchy et al. (2016) estimated that in plants, on average 65% of the genes have a duplicate copy and the most common source of the duplication is WGD.

Genome size dynamics Each group of land plants is characterized by a distinctive genome size pro- file (Leitch and Leitch 2013) and the genome sizes are much more variable than the number of protein-coding genes. Genome size shows no correlation with organism complexity, an observation early referred to as the C-value paradox (Thomas 1971). The large variation in genome sizes is explained mainly by duplications and TE activity, especially the latter (Lee and Kim 2014). Such mechanisms, leading to expansion of genomes, are counteracted

12 by epigenetic suppressing mechanisms to control the TE activity and by genome-removal mechanisms that include illegitimate or unequal recombination between LTRs and other types of deletions (Tenaillon, Hollister, and Gaut 2010). Two alternative mechanisms can explain the evolution of very small genomes of some plants; either resulting from a rapid reduction of genome size due to effective removal of DNA, or from a low rate of genome size increase from an ancestral small genome. As discussed later, the importance of these two mechanisms differ between plant lineages. Because there are only a handful of genomes of flowering plants, ferns and vertebrates that exceed 100 Gb, and none above 150 Gb, Hidalgo et al. (2017) speculates that 150 Gb might be a biological upper limit for genome size. Despite the importance of transposable element amplification and poly- ploidy in generating evolutionary novelties, species with enormous genomes are the exception and most plant lineages are skewed towards small or mod- erate genome sizes suggesting that large genomes are at selective disad- vantage. The most common phenotypic modifications associated with genome size variations include nucleus and cellular sizes, growth rate and metabolic rate (Lefébure et al. 2017). For example, a large genome and a subse- quently prolonged replication time may result in longer generation times and inability to grow quickly enough to attain an annual life strategy.

Angiosperms Angiosperms is the plant lineage with the largest variation in genome sizes (Pellicer et al. 2018), and also the group with the largest species richness and phenotypic variation (Simonin and Roddy 2018). The estimated number of angiosperm species is around 352 000 (http://www.theplantlist.org/). Nucle- ar genomes vary enormously in overall DNA content, from the smallest genomes such as the genome of Genlisea tuberosa (61Mb/1C) to the largest genomes such as the genome of Paris japonica (149000Mb/1C) (Pellicer et al. 2018). The variation in genome sizes is explained partly by differences in ploidy, WGD events and other types of duplications but most of the differences are due a high count of a few transposable elements amplified in bursts of transposition (reviewed in Bennetzen and Wang 2014). High rates of genome size evolution (but not the actual size of the genome) correlate with high rates of speciation in angiosperms, consistent with previous predictions that genome size variability is linked to the success of flowering plants (Puttick et al. 2015).

Gymnosperms In contrast to angiosperms, gymnosperms have been described as slow- evolving plants (De La Torre et al. 2017) and several contrasting features have been mentioned in order to explain this, including contrasting reproduc- tive biology, generation time, genome sizes and population sizes (Lanfear et al. 2013; Buschiazzo et al. 2012). While gymnosperm genomes are, on aver-

13 age, larger than any other land plant group, they still remain one of the least variable groups concerning genome sizes. In addition, the genome size pro- file across species is not strongly skewed towards small DNA amounts, as in the other plant lineages (Leitch and Leitch 2013). There are around 1000 species of gymnosperms (http://www.theplantlist.org) and the conifers are the best studied. Conifer genes tend to accumulate long introns but no evidence has shown that this would reduce the level of gene expression (Liu and El-Kassaby 2019). The large genomes can be explained by large amounts of TEs but in contrast to large angiosperm genomes TEs are of low copy number in the gymnosperms. The large genome size is believed to be due to a constant accumulation of TEs together with slow a removal of DNA (De la Torre et al. 2014).

Ferns There are around 12 000 species of ferns (including horsetails; Nagalingum 2016) and in contrast to angiosperms and gymnosperms, there is a significant positive correlation between genome size and chromosome number (Clark et al. 2016). Clark et al. (2016) estimated the average (gametic) chromosome number to n = 60.5 based on the available chromosome counts for 2639 fern species. The fern Ophioglossum reticulatum has the highest chromosome number of all plants known so far (2n=1440). The high chromosome numbers of ferns have resulted from, at least partly, repeated cycles of WGDs. Ferns also have a higher rate of speciation events caused by poly- ploidy compared with other plants. Despite high average number of chromosomes, the variation in chromosome sizes is much smaller compared with angiosperms (Clark et al. (2016).

Lycophytes Lycophytes is the second and smaller group of vascular seedless plants (be- side ferns) with its approximately 1000 species members (Nagalingum 2016) Lycophytes are subdivided into three lineages, of which Selaginella is the sole genus of Selaginellales and the most studied. Selaginella is special in that it is the only clade of vascular plants that lack an ancient shared WGD event and the estimated genome sizes are generally small (1C=81.2-182.4 Mb). In contrast to other extremely small genomes of vascular plants, Baniaga et al. (2016) showed in a phylogenetic comparative analysis that the small genome sizes of Selaginella very likely is a consequence of low rates of genome size expansion rather than recent reductions in genome size. They used a comparison between S. moellendorffi and a flowering plant (Utricu- laria gibba) having an extremely small genome to further exemplify this: The Utricularia gibba genome contains not only smaller amount of noncod- ing DNA, but also the presence of solo LTRs which indicate large scale genome size reduction through TE deletions (as also found in A. thaliana; Wang et al. 2013). Furthermore, there is also a difference in the distribution

14 of TEs within genomes. Selaginella TEs are evenly distributed in the genome, whereas extremely small angiosperm genomes tend to have TEs con- centrated to certain regions (Baniaga et al. 2016).

Bryophytes In contrast to liverworts and hornworts, WGDs has been described in the evolutionary history of mosses (Lang et al. 2018). Gametophytes of most liverworts usually have 8-10 chromosomes and are unisexual. The bisexual gametophytes of some monoecious liverworts have 16 or more chromosomes (Fritsch 1991) and are therefore considered to be polyploid descendants of haploid dioecious ancestors (Haig 2016). The mosses have a larger variation in chromosome numbers, ranging from 4 to 72 (Fritsch 1991). The chromosome number of the model moss Physcomitrella patens is 27 (Rensing et al. 2008). Hornworts mostly have 5-6 chromosomes (Villarreal et al. 2010). Bryophytes are, in general, characterized by small genomes. The mean (median) genome size of bryophytes has been estimated to 244 Mb (205 Mb), 1844 Mb (751 Mb) and 504 Mb (433 Mb) for hornworts, liverworts and mosses, respectively (Pellicer et al. 2018).

Hybridization, from a bryophyte perspective Most genomic studies of hybridization and introgression in plants have so far been conducted on organisms with a diploid dominant generation. In bryophytes, the diploid sporophyte generation is the primary hybrid combining the parental genomes (comparable of the F1 generation in a vascular plant) and the thousands of spores formed in the sporophyte after meiosis are recombinants (comparable with the F2 generation) of the parental genomes in varying proportions. If viable, they might germinate to form a new haploid gametophyte generation and are immediately exposed to selection in the haploid phase. Also, because the sexual reproduction of bryophytes is dependent on water (so that the mobile gametes from the antheridia can swim to fertilize egg-cells from archegonia), the two species must grow close to each other in order for hybridization to occur. There are only rare cases of recombinants of hybrid origin documented in natural bryophyte populations, and it has been suggested that homoploid hybridization of bryophytes is rare, because hybrid sporophytes in most documented cases have failed to produce viable spores (reviewed in Natcheva and Cronberg (2004)). Although speciation resulting from hybridization is rare, it is more likely to result in introgression, i.e., the transfer of genetic material between species through hybridization and repeated backcrossing. To truly identify introgression, it has to be distinguished from shared ancestral variation (also called incomplete lineage sorting, ILS). Both introgression and ILS may result in incongruence between gene trees. Large population sizes and speciation events closely in time will increase the frequency of incongruent gene

15 trees arising from ILS and make the speciation and hybridization history more difficult to reveal. Introgression may increase standing genetic variation and adaption if introgressed alleles have positive fitness consequences in the recipient and thus maintained by natural selection (Suarez-Gonzalez et al. 2018).

The Evolution of Gene Regulatory Networks Species can have highly similar genome sequences and gene content but still quite different gene expression patterns. Such divergence in gene expression and regulation may be important in evolution and in giving rise to phenotypic variation between species (Voordeckers et al. 2015). Genes do not work independently, and throughout evolution, not only the genes themselves but also the regulatory networks they might participate in will change, expand and adapt, including novel genes and gene functions. Changes in transcription factors are important for the evolution of regulatory networks. Duplication of a gene allows one of the two copies to retain the ancestral function while the other is released from negative selective pressure and can mutate and evolve a different function (Ohno 1970; Zhang 2003; Voordeckers et al. 2015). It might switch to regulate another target or con- tinue to regulate the same target genes but respond to different signals or bind to different cofactors. Many transcription factors are known to arise by gene duplication and a number of them have acquired a new function. The majority of novel genes in eukaryote genomes are actually the results of duplications (Teichmann and Babu 2004). Two frequent fates of duplicated genes are subfunctionalization (division of labour) where each paralog evolve to regulate a subset of the target genes of the single ancestral transcription factor (this may give a more precise and specific regulation of target genes/fine-tuning of expression) and neofunctionalization where one of the paralogs acquires a novel function (Panchy et al. 2016). Duplication of target genes and subsequent diversification also contribute to the evolution of transcriptional networks. Transposable elements are suggested as an important source of DNA binding domains for transcription factors and also to provide new regulatory sequences (Voordeckers et al. 2015).

The evolution of a specific functional network of genes: The circadian clock For optimal fitness a plant needs to fine-tune its biological processes in response to day and night lengths and to seasonal changes. For this reason, circadian clocks have evolved, giving rise to circadian rhythms. The circadian clock is a self-sustaining oscillator and the approximately 24-hour rhythm

16 results from transcriptional and translational feedback loops (Harmer 2009b). The clock is on daily basis interacting with the environment and the major environmental cues, called zeitgebers, are light and temperature (McClung 2006). The clock also has to cope with unpredictable variations in sunlight and temperature and have evolved towards a more complex, flexible and robust architecture (Pfeuty et al. 2012). Circadian clocks are present in most organisms, from cyanobacteria to land plants and animals, even though the key components differ (McClung 2013). The plant circadian clock is well studied in the angiosperm model plant A. thaliana and the central components are shown in Figure 3. By comparing circadian clock genes in green algae and angiosperm it is clear that additional genes have been recruited during evolution giving successively more complex clocks with multiple feedback loops and partly duplicated structure (Matsuo and Ishiura 2010; Bouget et al. 2014; Harmer 2009). The first studies of the clock of bryophytes were conducted in the moss Physcomitrella patens (Holm et al. 2010; Okada et al. 2009) and the results suggested a less complex clock lacking homologs to several of the core clock genes in Ara- bidopsis thaliana. (Figure 3). Thus, the circadian clock seems to be a good study system to learn more about the evolution of gene regulatory networks.

Figure 3. An overview of the most important core clock and clock-related genes identified in Arabidopsis thaliana, the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens. The figure is inspired by Shim et al. (2017). The symbols indicate shared ancestry and the colors are for making the overview clearer. Time of expression indicated by time of day is only relevant for A. thaliana. The complete names of the genes, here abbreviated, can be found in paper IV.

17 Aims of the thesis

The overall aim of this thesis is to investigate different aspects of molecular evolution of bryophytes. The specific aims in each paper are:

Reconstruct the phylogeny of the three subspecies of Marchantia polymorpha, to analyze the degree of differentiation and hybridization between the three subspecies genomes and to test the hypothesis that subsp. ruderalis originated as a hybrid between the other two subspecies. (Paper I)

Investigate the molecular evolutionary rates of bryophytes with a special focus on nucleotide substitutions and liverworts within Marchantiopsida, and compare those estimates to other plants. (Paper II)

Study genome evolution of liverworts, focusing on large-scale changes such as gene duplication, transposable elements, and genome rearrangements. (Paper III)

Study the evolution of a specific functional network of genes: the circadian clock and to investigate its transition from a simple clock in algae to a complex one in angiosperms. (Paper IV)

18 Materials and Methods

Gene annotation of Marchantia sp. and Lunularia cruciata assemblies (Papers I, II, III) Genomic and transcriptomic sequences was extracted, sequenced and as- sembled as described in Papers I and II. Genome annotation was done using Maker version 3.01.2-beta (Holt and Yandell 2011) in two runs, and described in more detailed in Paper I. The aim of the first run was to obtain a first set of genes to be used to train the ab initio gene annotation tools Au- gustus (Stanke and Morgenstern 2005) and GeneMark-ES (Lomsadze et al. 2005). The transcriptome assemblies, Swissprot and the published proteome of M. polymorpha ruderalis were given as support. In the second run of Maker, the pipeline integrates the results of the gene prediction tools, with the protein- and transcript support. The transfer of gene models from the references of each subspecies to the additional samples of the same taxa was also done using Maker but with changed settings adjusted for this purpose.

Genetic variation analysis of Marchantia polymorpha L (Paper I) In total five sample IDs of subsp. ruderalis, three sample IDs of subsp. polymorpha and three sample IDs of subsp. montivagans was sequenced. One individual of subsp. polymorpha and one of subsp. montivagans were sequenced with PacBio long read sequencing (Roberts et al. 2013) and were together with the publicly available subsp. ruderalis genome (Bowman et al. 2017) considered as references for its respective taxa. Three different data sets were prepared and aligned as described in Paper I; genomic fragments (GF), coding sequences and organellar genomes. Phy- logenic trees were reconstructed using different phylogenetic methods. In- trogression statistics (i.e., Pattersons D statistic, Martins f statistic and Bd fraction) and pairwise nucleotide diversity between the subspecies were cal- culated using the R package Popgenome.

19 Rate calculations and associations (Paper II) Estimates of dS, dN and their ratio were obtained using codeML (Yang 2007) for the two different data sets used as input. The first data set is based on a set of 42 conserved genes identified in angiosperms and gymnosperms (De La Torre et al. 2017). Bryophyte orthologs to the A. thaliana genes were identified with a reciprocal blast approach in 10 moss species, 10 liverwort species and 3 hornwort species (listed in Paper II). By using the same genes the obtained results are more confidently comparable as it reduces the introduced biases (for example comparing highly conserved genes with less conserved one). The branch model (model=1) was used to allow variation between branches and to obtain one estimate of each of dS, dN and dN/dS per gene per species. Pairwise comparisons (runmode = -2) was also done to obtain one averaged value of dS, dN and dN/dS per gene and to compare the rates in pairs of species belonging to Jungermanniopsida and pairs of species belonging Marchantiopsida. Using the classification made by (Dierssen 2001) for each of the included bryophyte species were divided into three groups based on the humidity of its preferred habitat (as a proxy for desiccation tolerance) and its life span (as a proxy for generation time) for testing the hypothesis that those factors in- fluence the substitution rates. To get a genome-wide value of silent site substitution rate and dN/dS, alignments of more than 8000 single-copy orthologs in eight Marchanti- opsida species were used as input for codeML and with the option model=0, one averaged value of dN/dS per gene was obtained. The output from pairwise comparisons (runmode= -2) was used for calculating the synonymous substitution rate using the formula μ=dS/2T, where T is the pairwise divergence time. Expression data and gene statistics for M. polymorpha ruderalis were publicly available (Bowman et al. 2017) and correlated with the differences in rate observed between genes.

TE landscape, gene statistics and synteny (Paper III) The repeat content of M. polymorpha ruderalis, M. polymorpha polymorpha, M. paleacea, M. inflexa and L. cruciata was de novo annotated using Re- peatModeler (Arian et al. 2013) and this resulting species-specific repeat- library was used with RepeatMasker (Smot et al. 2013) to characterize the TE landscape. LTRharvest (Ellinghaus et al. 2008) and LTRdigest was used to find full-length and potentially functional LTRs in the genome assemblies. Gene statistics such as gene length and mean average intron length were extracted from the gene annotation files for a few liverworts together with representative species from other plant lineages. Duplicated genes were identified using the criteria in (Panchy et al. 2016) and tandem duplicated

20 genes were identified with the tool duplicate gene classifier in the MCscanX toolkit (Wang et al. 2012). The synteny of L. cruciata and M. polymorpha ruderalis was assessed by extracting their single-copy orthologs and by identify the position of each pair in each of the two assemblies. To get a percentage of collinear gene pairs the tool MCscanX_h in the MCscanX toolkit (Wang et al. 2012) was used.

Homolog search and functional analyses of clock genes in bryophyte and charophytes genomes (Paper IV) Homologs to circadian clock- and clock related genes known in Arabidopsis thaliana were identified in Marchantia polymorpha subsp. ruderalis, An- thoceros agrestis and Klebsormidim nitens using a best reciprocal blast approach together with phylogenetic reconstruction. Time series experiments were conducted and the variation of transcript levels for the putative clock genes were detected with qRT-PCR; plants of Marchantia polymorpha subsp. ruderalis, Anthoceros agrestis were first entrained in neutral day (12:12h; light: dark cycles) and then transferred to constant lightness, constant darkness or kept in neutral day before sampling and RNA extraction. Luciferase reporter lines were obtained by cloning of the promoters of the identified clock-gene homologs in Marchantia polymorpha subsp. ruderalis to the luciferase (LUC) gene and by adding its sub- strate D-Luciferin the spatial LUC expression could be imaged.

21 Results and Discussion

The phylogenetic relationship between the three subspecies of Marchantia polymorpha (Paper I) M. polymorpha is a complex of three subspecies, which together show a world-wide distribution. One of the subspecies subsp. ruderalis, has been adopted as one of the most important plant model plants, and, as such, acts as a liverwort representative and an object for functional studies with lower complexity. As such, it is important to disentangle its intraspecific relationship Our genome-wide phylogenetic analysis gave a clear separation of the three subspecies (subsp. ruderalis, subsp. polymorpha and subsp. montivagans) with high support, where the results of both a concatenation approach and a coalescent species tree approach was a topology with subsp. montivagans branching of first with subsp. ruderalis and subsp. polymorpha as sister group (Paper I). However, a unique branching orders of the subspecies could not be reconstructed using individual genomic fragments or genes. Using these data, we obtained a high frequency of supported trees for all three possible topologies. This suggests similar divergence times of the three subspecies and a high degree of incomplete lineage sorting (ILS), possibly accompanied by hybridization and introgression. The hypothesis that subsp. ruderalis is a new stabilized homoploid species created by hybridization of the other two subspecies was not supported by the data as its genomic sequence was not intermediate between the other two. This hypothesis has been questioned before by (Boisselier‐Dubayle et al. 1995), however, that study was based on a limited data set and therefore this study contribute to the discussion by testing this hypothesis at the level of whole genome. Limited introgression was indicated in two out of the 12 individuals test- ed, i.e. in one subsp. montivagans, and in one subsp. polymorpha individual. Interestingly both were sampled at locations where both their respective parental species occurred in sympatry. These data suggest that hybridization between the subspecies might be frequent, but in most cases foreign DNA fragments are rapidly shortened through backcrossing and recombination and are therefore difficult to detect. Clearly, further studies including more individuals are needed to clarify this issue.

Limited genetic exchange after hybridization might be explained by the haploid-dominant life cycle of bryophytes: Fertilization involving transfer of a male gamete from the haploid male to the haploid female initiates the more or less short-lived diploid sporophyte phase. The restoration of the dominant haploid phase takes place through numerous meiotic events in the spore cap- sule of the sporophyte prior to spore production. The diploid sporophyte is the “true” hybrid (comparable with the F2 generation in angiosperms) and a sporophyte can produce at least 300 000 spores in M. polymorpha with different recombination of the parental genomes. In this way, a single hybridization event may result in an enormous number of recombinant haplotypes which are immediately exposed to selection if the spores are able to germinate. Dependent on the degree of inter-genomic incompatibility, germination and survival rates may be higher for mildly admixed spore relative to strongly admixed spores, potentially resulting in an instant form of introgression involving few genes. To our surprise, we found that one pseudo-chromosome in subsp. montivagans showed more than twice the amount of genetic divergence to both subsp. polymorpha and ruderalis, as compared to other chromosomes. It also displayed more chromosomal rearrangements and a higher proportion of transposons. Two alternative explanations are that i) hybridization with a, for us so far unknown, Marchantia species has occurred introducing DNA material in this chromosome making it more divergent from subsp. ruderalis and subsp. polymorpha, or that ii) rearrangements have led to restricted gene flow between subsp. montivagans and the other two subspecies specifically for this chromosome. The current taxonomic rank of M. polymorpha ruderalis, M. polymorpha polymorpha and M. polymorpha montivagans is as subspecies. This has been questioned and discussed before by Schuster (1992), Damsholt (2002) and (Kijak et al. 2018), arguing that the taxa are morphologically and genetically differentiated and have largely non-overlapping distribution areas. Our study may be used as support a treatment at species level, although the taxonomi- cal ranking is influenced by the species concept applied.

Bryophytes do not have a uniquely low mutation rate (Paper II) Nucleotide substitution rate (i.e. the number of nucleotide sequence changes per unit of time) is an important aspect of molecular evolution. Substitution rates vary between species but also within the genome of one species and even within the same gene. In this paper, the silent site substitution rate of bryophytes was estimated and compared with other plant groups. Assuming

23 that the synonymous substitutions are approximately neutral, differences in silent site substitution rates reflect differences in mutation rates. Results based on 42 conserved genes suggest that the mutation rate of bryophytes is lower compared with angiosperms but not as low as for gymnosperms (Figure 4A). Thus, the idea of bryophytes as evolutionary sphinxes of the past is not supported at the molecular level. The average values of the silent substitution rates for each bryophyte group (liverworts, hornworts and mosses) were not significant different from each other although data suggests a higher rate for hornworts. However, comparing the major liverwort classes Marchantiopsida and Jungermanniop- sida we did observe a significantly higher rate in Marchantiopsida (Figure 4B; and lower dN/dS as illustrated in Figure 5B). This result is supported by studies on single nuclear genes (ribosomal large subunit 26S) (Wheeler 2000; Boisselier-Dubayle et al. 2002) but is in contrast to data from plastid DNA, that showed a lower substitution rate in complex thalloids (Marchan- tiopsida; Villarreal et al. 2016). Although we did not specifically test for a phylogenetic signal, the silent site substitution rate appears to show such a signal, as related species tend to display similar rates.

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Figure 4. Synonymous substitution rate in units of substitutions/site/MY for different plant groups visualized as boxplots.

Factors suggested to affect mutation rates include generation time and DNA repair mechanisms. The generation time hypothesis states that species with shorter generation times copy their genomes more often and consequently

24 accumulate more replication errors per time unit resulting in higher mutation rates (Thomas et al. 2010; Li et al. 1996). Our data are consistent with this hypothesis. We observe a trend where short-lived bryophyte species have a higher mutation rate. Thus, the generation time hypothesis might explain differences in mutation rates between bryophytes, but as the diversity in life span and generation time among them is large it might not explain the intermediate rates, between gymnosperms and angiosperms, that we observed for bryophytes. Bryophytes occupy many diverse habitats, with a majority of species being able to survive periods of desiccation (Budke et al. 2018) and they have evolved mechanisms to recover from drought-induced damages (Glime 2017; Oliver et al. 2005). Although not specifically studied, it seems likely that bryophytes have developed strategies to cope with desiccation-induced damages also within their DNA. In line with this hypothesis, our data suggested that bryophyte species living in a wet habitat (as a proxy for low desiccation tolerance) have a higher mutation rate.

The selection pressure is not higher on haploid bryophytes than diploid angiosperms (Paper II) Because liverworts spend most of their life cycle in the haploid phase, where new mutations are exposed to selection and not masked by another functional copy of the gene, it is expected, according to the masking hypothesis, that selection should be more efficient on haploid dominant bryophytes compared with other plants. Because far more new mutations are deleterious than beneficial, dN/dS should be lower in bryophytes. Even though a lower dN/dS was observed for bryophytes as compared to gymnosperms, bryophyte dN/dS estimates were in the range of those from the diploid dominant angiosperms (Figure 5A). Possibly, other factors such as gene expression levels and specificity, which show a strong correlation with dN/dS dominate and preclude detection of an effect of masking. It has repeatedly been shown that the dN/dS for gymnosperms is higher compared to angiosperms. In empirical studies, large effective population size is often associated with low dN/dS, consistent with the idea that selection is more efficient in large populations, and that most mutations are deleterious Accordingly, the larger effective population sizes of conifers as compared to flowering plants has been put forward to explain the relatively higher dN/dS observed in gymnosperms (Buschiazzo et al. 2012; Lanfear et al. 2013). We used the number of reported bryophyte species observations (https://www.gbif.org) as a proxy for population size, but no significant correlation with dN/dS was observed in our data (Paper II).

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Figure 5. Ratio of non-synonymous to synonymous substitutions for different plant groups, visualized as boxplots.

Slow structural evolution of liverwort genomes (Paper III) Lunularia cruciata has a genome size almost the double of that of Marchantia polymorpha and we could show that most of this increase in size was due to bursts of the LTR retrotransposon superfamily Ty3/Gypsy. Still, compared to other species, activities of transposons in liverwort genomes are relatively low, and bursts are relatively rare (Figure 6A). No WGDs have been identified in liverworts, which contrasts to the abundance of such duplications in angiosperms. In line with this observation, liverworts contain fewer genes, with numbers similar to that of the charophyte algae Klebsormidium nitens (Figure 6B). Furthermore, the rate of structural changes both in terms of genome size and chromosomal rearrangements also seems low as compared to angiosperms (Figure 6C). As a conclusion, difference in the patterns of genome evolution between liverworts and angiosperms was seen, resulting in less complex liverwort genomes. These differences in terms of structural changes could be the result of the generally lower transposon activity in liverworts.

Figure 6.A) Repeat content of the genome assemblies included in this study; LC=L. cruciata, MI= Marchantia inflexa, MPA= Marchantia paleacea, MPP= M. polymorpha polymorpha, MPR=M. polymorpha ruderalis B) Phylogenetic relationship and duplication information. C) Decrease of collinearity with increased pairwise divergence time using different combinations of s (number of genes required to call collinear blocks) and m (maximum gaps allowed).

27 Most components of the plant circadian clock are present in liverworts and in charophytes (Paper IV) Angiosperm clocks, most studied in A. thaliana, are described as an intricate network of interlocked transcriptional feedback loops (McClung 2014). In contrast, clocks of green algae have been modeled as a loop of only two genes, suggesting an acquisition of more genes and interactions (loops) during plant evolution (Matsuo and Ishiura 2010; Bouget et al. 2014; Harmer 2009). Homologues to most of the clock genes and important clock-associated genes identified in A. thaliana were found in the liverwort Marhantia polymorpha and in the hornwort Anthoceros agrestis, as well as in the charophytes algae Klebsormidium nitens (Paper IV). This suggests that the circadian clockwork of land plants may have arisen earlier than previously assumed, perhaps already in charophyte algae. Such a scenario fits with the hypothesis that physiological adaptations to land had already evolved in early terrestrial charophytes (Harholt et al. 2016; Delwiche and Cooper 2015). It was previous suggested that the less complex clock in P. patens lacking the core clock genes GI, TOC1 and ZTL homologs might represent a less complex, ancestral state (Holm et al. 2010). However, our data clearly show that the lack of these three components is the result of gene loss in bryophyta. The three mentioned genes were not found in any moss except the basal moss species Takakia lepidozioides but was on the other hand present in liverworts and charophytes (Figure 7). Additional examples of the loss of core clock gene were identified: TOC1 was absent also in hornworts, and CCA1 orthologs were absent in all liverworts. These results suggest that although gene numbers and network complexity have generally increased during plant circadian clock evolution gene loss has also being important in shaping the circadian clocks of the three bryophyte groups. Novel components identified in circadian clocks of charophytes and their descendants are the evening complex genes ELF3, ELF4, LUX, as well as GI and genes of the ZTL family, which all are crucial for angiosperm clock function (Harmer 2009a; C. McClung 2019).

Figure 7. An overview of the most important plant circadian clock and clock-related genes identified, now also including Klebsormidium nitens, Marchantia polymorpha and Anthoceros agrestis. The figure is inspired by Shim et al. (2017). The symbols indicate shared ancestry and the colors are used to make the overview clearer. The figure only attempt to show the time point of expression (morning, midday, evening and night) for the genes in Arabidopsis thaliana.

We also performed functional characterization of putative core clock genes in Marchantia polymorpha and Anthoceros agrestis to investigate their function within the circadian clock. Circadian rhythms were detected for most of the identified clock gene homologs in M. polymorpha and A. agrestis, and mutant analysis supports a role for MpPRR, MpRVE and MpTOC1 in M. polymorpha circadian clock. Analysis of circadian rhythms of putative clock genes in M. polymorpha further suggests that these rhythms are weak compared with those in angiosperms. The lack of homologs to a crucial component of angiosperm clocks (CCA1) in liverworts suggests that their circadian clocks contain fewer interactions (transcriptional feedback loops). This observation fits with the idea that a less complex clock with fewer transcriptional feedback loops and less control of degradation of its component results in a more rapidly dampened timer (Brown et al. 2012).

29 Svensk sammanfattning

Mossor har blivit kallade oföränderliga evolutionära sfinxer, som knappt har förändrats morfologiskt (till det yttre) under miljontals av år. Det har speku- lerats i huruvida denna (i alla fall till synes) långsamma evolution på utsidan även sammanfaller med en långsam evolution av deras arvsmassa. Denna teori har fått både stöd och mothugg. Syftet med denna avhandling är att studera olika aspekter av mossors evolution och bidra till diskussionen med mer data. Mossorna har historiskt sett hamnat i skymundan av de mer varierade och artrika kärlväxterna, främst blomväxterna, trots att de har en nyckelposition i många ekosystem. Man kan även se dem som evolutionära länkar mellan de första landväxterna som erövrade land för 450-500 miljoner år sedan och de nutida kärlväxterna. De första landväxterna tros likna dagens levermossor. Mossor är egentligen ett samlingsnamn för artgrupperna (divisionerna) bladmossor, levermossor och nålfruktsmossor, och utmärker sig främst genom att ha en livscykel med en dominerande haploid generation, där varje cell bara har en uppsättning kromosomer. Till skillnad från kärlväxterna har de inte heller djupgående rötter och är därmed mer utlämnade till den ome- delbara omgivningen – de kan enbart dra nytta av den fukt och de närings- ämnen som passerar deras växtplats. Till skillnad från kärlväxterna sprider de sig inte med frön, utan med sporer eller genom vegetativ förökning genom fragmentering, avknoppning eller med groddkorn. Kärlväxterna delas i sin tur in i lummerväxter, ormbunksväxter och fröväxter. Till fröväxterna hör blomväxter (angiospermer) och barrväxter (gymnospermer).

De tre underarterna av Marchantia polymorpha och deras släktskap I den första artikeln undersöks släktförhållandet mellan tre underarter av lungmossa: trädgårdslungmossa (Marchantia polymorpha ruderalis), fjäl- lungmossa (M. polymorpha montivagans) och vattenlungmossa (M. polymorpha polymorpha). Med denna studie vill vi testa hypotesen att trädgårds- lungmossa är en stabil hybridart som bildats genom naturlig korsning av de två andra underarterna. Vi ville också studera hur vanligt det är att dessa underarter hybridiserar samt huruvida de ska klassificeras som olika arter eller inte. Resultaten visar att de utgör tre separata taxa och att trädgårds- lungmossan inte är en ny art som har bildats genom hybridisering av fjäl- lungmossa och vattenlungmossa. Det tycks ske viss hybridisering mellan underarterna, men det är bara väldigt små bitar av deras arvsmassa som går

30 att spåra till ett ursprung från en annan underart. Om de bättre klassificeras som separata arter beror på vilken definition av artbegreppet man använder.

Mutationshastighet och selektionstryck hos mossor I den andra artikeln har substitutionshastigheten hos mossor undersökts och jämförts med andra växter. Evolution kan bara ske i närvaro av förändringar i arvsmassan (DNA) som ger variation mellan organismer och det finns många potentiella källor till uppkomsten av dessa förändringar. Ett sådant exempel är punktmutationer (utbyte av enstaka baser i DNA-spiralen). När en punktmutation har spridit sig till hela populationen kallas det för substitution och substitutionshastigheten kan beräknas genom att räkna de skillnader i DNA sekvenser som ses mellan arter. Substitutioner kan vara synonyma (= inga förändringar i proteinsekvensen; dS) eller icke-synonyma (= leder till förändringar i proteinsekvensen och potentiellt även i organismens fenotyp vilket kan påverka dess evolutionära framgång; dN). Det finns alltså två aspekter av evolutionär hastighet – dels kan färre substitutioner ha skett eftersom mutationshastigheten är lägre och dels kan färre substitutioner ha skett eftersom selektionen kraftfullt motverkar förändringar i proteinerna och nya mutationer därför försvinner fortare ur populationen. Genom att dela dS (som huvudsakligen reflekterar mutationshastigheten) med den evolutionära tid som utgör perioden då dessa förändringar skett får man ett estimat av mutationshastigheten per tidsenhet. Resultat visade att mossor generellt inte har lägre mutationshastighet än alla andra växter, de visar dock att den är lägre jämfört med blomväxter men inte lika låg som för barrväxter. Inte heller skiljer det sig mellan bladmossor och levermossor. Däremot verkar olika arter inom samma grupp av mossor vara mer lika varandra (samma mutationshastighet), vilket antyder en fylogenetisk signal. Dessutom hade mossor med kort generationstid en högre mutationshastighet (växter med kort livscykel anses genomgå fler celldelningar och därmed högre risk för att mutationer ska uppstå) medan mossor i torra habitat hade lägre mutationshastighet. Det senare kan tolkas så att selektionen har lett till bättre reparationsmekanismer för att återhämta sig från skador på arvsmassan orsakade av torka vilket skulle ge en lägre mutationshastighet. Vidare visade resultaten att selektionstrycket på mossor inte skiljer sig från blomväxterna. Växter har en livscykel som alternerar mellan en upp- sättning kromosomer (s.k. haploid generation) och dubbel uppsättning kromosomer (s.k diploid). Medan alla andra landväxter domineras av den diplo- ida generationen så dominerar mossors livscykel av den haploida generationen. Eftersom mossor lever större delen av sin livscykel i sin haploida fas då varje gen bara finns i en upplaga, så innebär det att det inte finns någon annan fungerande version av genen som kan maskera en eventuell effekt av en mutation. Enligt den så kallade maskningshypotesen förväntas haploider därför uppleva ett högre selektionstryck och eftersom de flesta mutationer som sker är skadliga en långsammare proteinevolution. Eftersom våra resul-

31 tat föreslår samma selektionstryck i mossor som blomväxter verkar andra mekanismer vara viktigare för regleringen av selektionstryck.

Strukturella förändringar i levermossornas arvsmassa I den tredje artikeln undersöks andra evolutionära aspekter än mutations- och substitutionshastigheter, nämligen förekomst av dupliceringar, transposoner och kromosomala rearrangemang, vilka är andra viktiga källor till variation i arvsmassan. Skillnader i dessa processer kan också bidra till att förklara var- för mossor har förblivit mindre komplexa jämfört med andra växter. Ibland när arvsmassan kopieras så råkar en gen eller en del av en gen kopieras två gånger och ibland blir till och med hela arvsmassan duplicerad. Det senare har skett upprepade gånger under blomväxternas historia. Organ- ismen behöver endast en kopia av en gen så överskottsgenerna kan användas på olika sätt och är en viktig källa till ny variation. Transposoner är en bit DNA som kan flytta sig själv i arvsmassan och är därmed ett så kallat mobilt genetiskt element (”hoppande gen”). Transposonerna kan även råka få med sig andra delar av arvsmassan när de flyttar vilket leder till omstrukturering- ar av kodande och icke-kodande områden av arvsmassan. Det är bara en liten del av arvsmassan som kodar för proteiner och även om antalet gener varie- rar mellan olika arter så är den variationen inte så stor jämfört med den enorma variation som ses om man tittar på den totala längden av arvsmassan (genomstorleken) mellan olika växtarter. Transposoner är den viktigaste orsaken till att arvmassan blir större och dupliceringar av växtens hela genom har också en betydande inverkan i vissa växtgrupper. Det finns dock cellulära processer som motverkar expansion av arvsmassan genom avlägs- nande av visst DNA samt kontrollsystem som minskar aktiviteten hos transposonerna. För att studera dessa aspekter har jag använt mig av arvsmassan från fem levermossor separerade från varandra med olika tidsavstånd, nämligen mån- lungmossa (Lunularia cruciata), Marchantia inflexa, Marchantia paleacea, vattenlungmossa samt trädgårdslungmossa. (När arterna anges enbart med vetenskapliga namn beror det på att arterna inte går att finna i Sverige och därför saknar svenskt namn). De förstnämnda fyra separerades från träd- gårdslungmossan för mellan cirka 220 och 7 miljoner år sedan (att jämföra med exempelvis människa och mus som separerade från varandra för cirka 90 miljoner år sedan). Våra analyser visar på tydliga skillnader i evolution av arvsmassan mellan levermossor och blomväxter, vilket vi anser bidra till en mindre komplex arvsmassa hos levermossor. Resultaten visar också att jäm- fört med övriga växter är både det totala antalet gener och duplicerade gener generellt lägre för de studerade levermossorna. Detta bekräftar att deras genom inte förändrats lika mycket under hundratusentals år som blomväxternas genom gjort. Hastigheten av strukturella förändringar av arvsmassan, både vad gäller storlekar och omlagringar verkar vara låg jämfört med blomväx- ter. Detta kan vara orsakat av lägre transposonaktivitet. Det gick, i våra ana-

32 lyser, att se spår av ökad aktivitet hos transposonerna, framför allt hos mån- lungmossan som har en arvsmassa som är dubbelt så stor som trädgårds- lungmossan och där den huvudsakliga orsaken till denna ökning är att transposoner har kopierat sig själva upprepade gånger.

Levermossors cirkadiska klocka har de flesta genkomponenter Ett regulatoriskt gennätverk är en uppsättning gener som interagerar och reglerar varandra (via deras RNA och proteinuttryck). Den cirkadiska klockan är ett exempel på ett sådant nätverk. Växter har precis som oss människor en inre klocka, som reglerar att funktionerna i cellerna sker vid rätt tidpunkt för organismen. Denna rytm kallas cirkadisk efter latinets ”circa diem” som betyder ”ungefär en dag”. Den inre klockan synkroniseras med signaler från omgivningen, så kallade ”zeitgebers”. Den viktigaste signalen utifrån är solljuset. Den cirkadiska klockan hos växter är mest studerad hos modellväx- ten backtrav (Arabidopsis thaliana). Medan backtrav har ett komplext nät- verk av sammanlänkade transkriptionella återkopplingsslingor så verkar de enklare grönalgerna klara sig med endast två gener. Klockan hos backtrav har flera duplicerade gener med delvis överlappande funktion i nätverket. Syftet med den fjärde artikeln var att undersöka övergången från en enkel klocka såsom i encelliga grönalger, till en komplex klocka såsom i kärlväx- ter. Därför inventerade vi vilka gener kopplade till den cirkadiska klockan som gick att hitta i levermossan samt även i svart nålfruktsmossa (Anthoce- ros agrestis) och karofytalgen Klebsormidium nitens. För att bekräfta att identifierade generna faktiskt har en roll i klockan även hos levermossor så testade vi om de hade en cirkadisk rytm. Homologer (= gener som har samma genetiska ursprung) till kärnklockgener som identifierats i backtrav hittades i mossorna men i färre kopior, vilket reflekterar den lägre graden duplicerade gener. Att de dessutom identifierades i Klebsormidium överens- stämmer med en nyare hypotes, att anpassningen till livet på land skedde tidigare än vad man tidigare har trott i karofytalgernas historia. Både genduplikation och förvärv av nya gener har varit viktiga i utvecklingen av den cirkadiska klockan från enkla alger till komplexa kärlväxter, men gen- förluster har också bidragit till att forma klockan hos mossor.

33 Acknowledgement

The work with this thesis was carried out at the Department of Ecology and Evolution, EBC, Uppsala University. To write this thesis has been a journey and want to express my gratitude to all of you that in various ways helped me along the way.

First of all, I would like to thank my supervisor Ulf Lagercrantz for giving me this opportunity, for invaluable support and always keeping your door open and having time for me whenever I had something to discuss.

Also many thanks to my co-supervisor Nils Cronberg for your support, your bryophyte expertise and your optimism, and to my other co-supervisor Mag- nus Eklund for your support and for teaching me how to become a more focused researcher.

I am also grateful to Karl Holm, my co-supervisor during my first years, for introducing me to the project and to the lab and to Niclas Gyllenstrand for your help.

My grateful thanks are also extended to all people our department, mentioned or not. Special thanks to Kerstin Jeppsson, for always being helpful both in and outside of the lab. I want to thank all former and current phD colleagues; Anna and Sofia- for welcoming me into our office; Anja- I am happy that you moved in to our office, and I have enjoyed travelling with you; Charlie and Fia– you walked this road just before me and patiently an- swered to my questions; Lili, Andres, Camille, Dmytro, Elodie, Froukje, Giulia, Kevin, Lina, Linus, Luis, Matt, Rosie, Tianlin and Xiadong.

Thanks to my co-author Weerachon Sawangproh for inspiring discussions over a coffee during your visit here.

Thanks to my friends that in different ways helped me to this. Tack Anne- Lie för alla tidiga frukostar och samtal före jobbet. Du är förutom en vän en förebild som kvinnlig forskare och jag tror att du har hjälpt mig mer än vad du vet om. Tack Cilla för alla luncher på blåsenhus när jag var en ny doktorand, för peppiga samtal under en period av mitt liv då jag lärde känna nya sidor av mig själv, och för din vänskap. Tack Ida för alla barn-

34 vagnspromenader och för att du och Anders såg till att jag och barnen fick mat under trötta kvällar under den intensiva sista perioden av avhandlingsskrivandet. Till mina vänner som jag har lärt känna via barnen, på öppna förskolan och i regnbågsgruppen, för att ni kompletterar mitt liv som doktorand med lekplatsdejter och golvhäng med våra små och ger mig annat att tänka på. Jag vill även tacka Maria, min bästis från tonåren för att jag fick låna ditt hus och dina sociala katter för en skrivarvecka (något mindre uppskattar jag de ”presenter” som katterna fångade och bar in till mig). Jag vill även nämna Ida, Jenny och Gerd, vi blev vänner då vi började plugga tillsammans och trots att vi inte har träffats på alldeles för länge så kommer ni alltid att förbli speciella för mig.

Tack till mina föräldrar för att ni alltid har trott på mig och för att ni är de bästa föräldrar man kan tänka sig. Jag vill tacka min farmor, du finns med i många av mina bästa barndomsminnen och jag kommer med kärlek minnas hur jag alltid var välkommen att komma förbi på håltimmar och hur du stekte de godaste pannkakorna.

Jag vill tacka min fru Susanna, för din uppmuntran, stöd och tålamod. Du har dragit ett stort lass hemma den senaste tiden, låtit mig fokusera på avhandlingsskrivandet och varit allmänt fantastisk. Till mina barn Agnes och Selma för att ni är de fantastiska människor ni är och för att jag får ha er i mitt liv.

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1850 Editor: The Dean of the Faculty of Science and Technology

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