Finding the closest relatives of Podospora anserina Resolving the phylogeny in a part of the Lasiosphaeriaceae fungal family Ylva Jondelius Degree project in biology, Bachelor of science, 2018 Examensarbete i biologi 15 hp till kandidatexamen, 2018 Biology Education Centre and Department of Organismal Biology, Uppsala University Supervisors: Hanna Johannesson, Aaron Vogan and Sandra Lorena Ament Velásquez Cover illustration credit: Ylva Jondelius, 2018 Abbreviations BLAST: Basic Local Alignment Search Tool Blastn: Basic Local Alignment Search Tool nucleotide BS/bs: Bootstrap βT: β-tubulin. Protein coding gene. bp: basepairs CBS: Central Bureau of Fungal Cultures dnH2O: De-nucleased-water EBC: Evolutionary Biology Center, Uppsala University EF1α: Elongation factor-1 complex. Protein coding gene. GTR + Γ: General Time Reversible + Gamma distribution. Mathematical substitution model allowing for variable base frequencies and a symmetrical substitution matrix with gamma distributed rate variation among sites. HKY+F+I+G4: Hasegawa-Kishino-Yano + empirical base Frequencies + proportion of Invariable sites + discrete Gamma distribution model rate category 4. Mathematical substitution model. ITS: The Internal Transcribed Spacer 1 & 2. Ribosomal gene. LSU: Large-sub-unit rRNA. Ribosomal gene. MAFFT: Multiple Alignment using Fast Fourier Transform MCM7: Mini- chromosome maintenance protein. Protein coding gene. NCBI: National Center for Biotechnology Information PCR: Polymerase Chain Reaction RAxML: Randomized Axelerated Maximum Likelihood. Software. RBootstraps/rbs: Rapid bootstraps rDNA: ribosomal-DNA RPB2: DNA-directed RNA polymerase II. Protein coding gene. TN+F+G4: Tamura-Nei + empirical base Frequencies + discrete Gamma distribution model rate category 4. Mathematical substitution model. TN+F+I+G4: Tamura-Nei + empirical base Frequencies + proportion of Invariable sites + discrete Gamma distribution model rate category 4. Mathematical substitution model. TN+F+R3: Tamura-Nei + empirical base Frequencies + FreeRate model 3. Mathematical substitution model. TNe+R2: Tamura-Nei for equal base frequency + FreeRate model 2. Mathematical substitution model. TSR1: Ribosome Maturation Factor. Protein coding gene. UFBootstraps/UFbs: Ultra-fast bootstraps Abstract The phylogeny and diversity within the Lasiosphaeriaceae family of Ascomycete fungi is poorly known in many aspects. In this study a phylogenetic hypothesis for a subclade within Lasiosphaeriaceae was developed using nucleotide sequence data from the ribosomal LSU and ITS, and the nuclear protein coding βT, RPB2, MCM7, and TSR1 genes. The phylogenetic analyses based on maximum likelihood revealed non-monophyly in seven out of eight studied genera, among them Podospora with the model species P. anserina. Data on spore number and collection substrate type were optimized on the concatenated nucleotide maximum likelihood tree using an equal rate maximum likelihood model. Three independent switches from 8 to 4 spores and five unambiguous independent substrate switches from soil to dung were inferred. Key words: Fungi, Podospora anserina, Ascomycota, phylogeny, spores, substrate. Introduction The Fungal kingdom is estimated to comprise between 2.2-3.8 million species, of which currently only ~120 000 are described (Hawksworth & Lücking 2017). Some species are only superficially know whilst others have been studied in depth. One of the most studied fungal species is the ascomycete Podospora anserina, which has been used as model organism for the study of various biological processes such as senescence and genomic conflict (van der Gaag et al. 2000; Geydan et al. 2012). Despite the in-depth knowledge about P. anserina and its features, its closest relatives remain fairly unknown. Within the phylum Ascomycota, P. anserina belongs to the family Lasiosphaeriaceae. Miller & Huhndorf (2005) established a phylogenetic backbone of the family, and diagnosed some internal clades based on the morphology of the ascomal walls. Building on their work, Kruys et al. (2014) expanded the phylogeny using molecular markers for the ribosomal large subunit (LSU) and the protein coding gene β-tubulin (βT) resulting in further division of the Lasiosphaeriaceae family into five clades: I, II (including Sordariaceae), III, IV, and Chaetomiaceae (Fig. 1). Kruys et al. 2014 found support for three sub-groups within clade IV: they are here referred to as clades A, B and C. Clade A includes Podospora comata, clade B includes Cladorrhinum samala and clade C includes Cercophora grandiuscela. A complication of the classification within Lasiosphaeriaceae is that many genera are indicated to be non-monophyletic (Huhndorf 2004; Kruys et al. 2014; Miller & Huhndorf 2005). This problem is rampant within clade IV, which includes the genera Apisordaraia, Arnium, Cercophora, Cladorrhinum, Podospora, Triangularia, and Zopfiella (Chang et al. 2010; Cai et al. 2006). The limited set of molecular markers resulted in a relatively poorly supported hypothesis for the interrelationships of the species within Lasiosphaeriaceae (Kruys et al. 2014). I. Lasiosphaeriaceae II. Lasiosphaeriaceae incl. Sordariaceae Soridariales Ascomycota III. Lasiosphaeriaceae IV. Lasiosphaeriaceae Other Ascomycetes Fungi Chaetomiaceae Other fungus Fig 1. Illustration of clade IV of the Lasiosphaeriaceae family’s position in the fungal kingdom. Clade IV, the focus of this study circled in pink. In the code of nomenclature for algae, fungi, and plants (McNeill et al. 2012) it is stipulated that a type species must be designated for every genus that is recognized. The type species is the bearer of the generic name. Nomenclatural changes must follow if a type species is found to be nested within another genus, separate from the other species of its nominal genus if the aim is to maintain monophyletic genera. In the hypothesis put forward by Kruys et al. (2014) there are several examples where type species within Lasiosphaeriaceae clade IV are separate from some of their congeneric species: Podospora (type species: Podospora fimiseda (von Niessl 1883)), Triangularia (type species: Triangularia bambusae (Beyma 1933)), Cladorrhinum (type species: Cladorrhinum foecundissimum (Saccardo & Marchal 1885)), and Apisordaria (type species: Apisordaria verruculosa (Arx & Gams 1967)). If this separation of type species from their congeners is supported in more robust phylogenetic hypotheses, it will become necessary to implement a number of classificatory changes. It is not only the phylogenetic relationships that are poorly known for Lasiosphaeriaceae clade IV; there is little data regarding biological features such as mating systems and ecological traits for most of the species (Kruys et al. 2014; Lundqvist & Degelius 1972; Huhndorf et al. 2004). The development of a strongly corroborated phylogenetic hypothesis will enable consistent naming of currently known species within this group as well as those that may be discovered in the future, and facilitate analyses of character evolution e.g. spore morphology. One of the most interesting arguments for implementing a phylogenetic classification is that through establishing a strongly supported phylogeny for the clade it might be possible to predict biological traits in the unstudied species based on what is previously known about e.g. P. anserina and its relatedness to the species in question. In this study I aim to develop a robust phylogenetic hypothesis for the Lasiosphaeriaceae clade A by increasing taxonomic sampling and investigating more markers than previous studies. This hypothesis can be associated with selected biological traits, building a character evolution for the biological traits within the clade. Methods Species sampling All taxa used in this study and associated strain identification numbers are presented in Table 1. Live cultures of the strains sequenced in this study were acquired through central storage facilities, either from the Westerdijk Fungal Biodiversity Institute (CBS-KNAW Fungal culture collection, The Netherlands) or from the NITE Biological Resource Center (NBRC Culture Collection, Japan) for cultivation in lab. Clade A, from the phylogeny put forward by Kruys et al. (2014), containing amongst others P. comata, make up the species to be investigated in more detail, and for which this study aims to establish a phylogeny. The outgroup for this study was selected from representatives of clades B and C from the study by Kruys et al. (2014), from clade B Cladorrhinum bulbillosum, Cladorrhinum samala, Podospora fimiseda, and from clade C, Cercophora grandiscuela. Cultivation and DNA extraction The specimens were cultivated on a cellophane covered plate of either PSAM2.0 (van Diepeningen et al. 2008) or 3% Malt-extract medium. Mycelium, the source of DNA, was harvested after ~2-7 days when covering ≥ 50% of the plate as development rate varied depending on the strain and cultivation media. Caution was taken not to leave cultures growing too long as the mycelium starts degrading the cellophane and hinders the extraction of pure mycelia. In order to facilitate DNA extraction, mycelium was stored at -20°C for at least 24h, then placed in -80°C for at least 1h before being lyophilized for 12h prior to extraction. DNA was extracted using the ZR Fungal/Bacterial DNA Miniprep™ kit (Zymo Reasearch, Irvine, California, USA) following the instructions for dry samples with the exception of leaving the extraction in the Lysis solution for a minimum of 10 minutes. β-mercaptoethanol was added to the Fungal/Bacterial