The Evolution of Secondary Metabolism Regulation and Pathways in the Aspergillus Genus

Home , Aquamarina, Aspergillus calidoustus, Aspergillus fischerianus, Aspergillus lentulus, Aspergillus rambellii, Aspergillus ruber

THE EVOLUTION OF SECONDARY METABOLISM REGULATION AND PATHWAYS IN THE ASPERGILLUS GENUS

Abigail Lind

Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY in Biomedical Informatics August 11, 2017 Nashville, Tennessee

Approved: Antonis Rokas, Ph.D.

Tony Capra, Ph.D.

Patrick Abbot, Ph.D.

Louise Rollins-Smith, Ph.D.

Qi Liu, Ph.D.

ACKNOWLEDGEMENTS

Many people helped and encouraged me during my years working towards this dissertation. First, I want to thank my advisor, Antonis Rokas, for his support for the past five years. His consistent optimism encouraged me to overcome obstacles, and his scientific insight helped me place my work in a broader scientific context. My committee members, Patrick

Abbot, Tony Capra, Louise Rollins-Smith, and Qi Liu have also provided support and encouragement.

I have been lucky to work with great people in the Rokas lab who helped me develop ideas, suggested new approaches to problems, and provided constant support. In particular, I want to thank Jen Wisecaver for her mentorship, brilliant suggestions on how to visualize and present my work, and for always being available to talk about science. I also want to thank

Xiaofan Zhou for always providing a new perspective on solving a problem.

Much of my research at Vanderbilt was only possible with the help of great collaborators.

I have had the privilege of working with many great labs, and I want to thank Ana Calvo, Nancy

Keller, Gustavo Goldman, Fernando Rodrigues, and members of all of their labs for making the research in my dissertation possible.

I have been supported by a National Library of Medicine training grant T15LM007450 during my time at Vanderbilt, which has also given me the opportunity to travel to conferences and present my work. I am grateful to both the Department of Biomedical Informatics and the

National Library of Medicine for their support.

I am grateful to my family and friends for their support, love, and encouragement. My husband Nathan has helped me in more ways than I can possibly name during the last five years, but especially by encouraging me during times of self-doubt.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

LIST OF ABBREVIATIONS ...... ix

Chapter

I. INTRODUCTION ...... 1 Ecological roles of fungal secondary metabolites ...... 1 Secondary metabolic pathways in fungal genomes ...... 5 Regulation of fungal secondary metabolism...... 7 Chapter previews ...... 9

II. EXAMINING THE EVOLUTION OF THE REGULATORY CIRCUIT CONTROLLING SECONDARY METABOLISM AND DEVELOPMENT IN THE FUNGAL GENUS ASPERGILLUS ...... 12 Authors ...... 12 Introduction ...... 12 Methods...... 16 Genome sequences and orthogroup definitions for A. nidulans, A. fumigatus, A. oryzae, and A. niger ...... 16 Gene category definitions ...... 17 Strains and culture conditions ...... 17 RNA extraction ...... 18 RNA sequencing ...... 18 RNA-seq read alignment and differential gene expression ...... 19 Statistical analyses ...... 19 Results ...... 20 The majority of SM gene clusters in Aspergillus are species-specific ...... 20 Aspergillus SM genes are significantly less conserved than genes for primary metabolism .24 VeA regulates the same biological processes as well as the same fraction of the genome in both A. nidulans and A. fumigatus ...... 26 MtfA’s regulatory role is smaller in scope in A. fumigatus compared to A. nidulans ...... 29 Regulation of similar processes regardless of gene conservation ...... 30 Discussion ...... 32 Aspergillus secondary metabolic genes and gene clusters are largely species-specific ...33 The evolution of the circuit regulating secondary metabolism and development ...... 35

iii

III. REGULATION OF SECONDARY METABOLISM BY THE VELVET COMPLEX IS TEMPERATURE-RESPONSIVE IN ASPERGILLUS ...... 38 Authors ...... 38 Introduction ...... 38 Methods...... 41 Strains and culture conditions ...... 41 RNA isolation ...... 41 RNA sequencing ...... 42 Gene expression analysis ...... 42 Functional enrichment analysis...... 43 Gene cluster expression ...... 43 Data availability ...... 44 Results ...... 44 Temperature shift changes the expression of 10% of all genes and of more than half of the genes in SM gene clusters ...... 44 VeA regulates a much large number of genes at 37ºC than at 30ºC ...... 46 LaeA regulates similar numbers and types of genes at 30ºC and 37ºC ...... 48 VeA and LaeA have greater regulatory overlap at 37ºC than at 30ºC ...... 50 Many SM gene clusters are regulated by both VeA and LaeA at 37ºC, but only by LaeA at 30ºC ...... 53 Discussion ...... 55

IV. TRANSCRIPTIONAL REGULATORS OF ASEXUAL DEVELOPMENT CONTROL BOTH SPORE AND HYPHAL SECONDARY METABOLIC PATHWAYS IN ASPERGILLUS FUMIGATUS ...... 59 Authors ...... 59 Introduction ...... 59 Methods...... 61 Growth conditions and RNA isolation ...... 61 RNA sequencing ...... 61 Differential gene expression ...... 61 Functional enrichment analysis...... 62 Gene cluster expression ...... 62 Results ...... 63 Genome-wide transcriptional impact of BrlA, AbaA, and WetA ...... 63 Media-specific effects of asexual regulators...... 69 Shared and specific targets of BrlA, AbaA, and WetA ...... 70 Secondary metabolic gene clusters and their products are regulated by BrlA, AbaA, and WetA ...... 71 BrlA regulates secondary metabolites both associated with asexual tissues and associated with hyphal growth ...... 72 Discussion ...... 73

V. DRIVERS OF GENETIC DIVERSITY IN SECONDARY METABOLIC GENE CLUSTERS IN A FUNGAL POPULATION ...... 75 Authors ...... 75

Introduction ...... 75 Methods...... 78 Strains analyzed ...... 78 Single nucleotide variant (SNV) and indel discovery...... 79 De novo genome assembly and gene annotation ...... 80 Secondary metabolic gene cluster annotation and discovery ...... 80 Phylogenetic analysis ...... 81 Results ...... 82 Single-nucleotide and indel polymorphisms ...... 85 Gene content polymorphisms ...... 86 Whole gene cluster gain and loss polymorphisms ...... 90 Idiomorph polymorphisms ...... 95 Genomic location polymorphisms ...... 98 Discussion ...... 102

VI. SUMMARY ...... 107

REFERENCES ...... 112

APPENDIX

A. ROLE OF THE STUDENT ...... 133

B. STRAINS USED IN CHAPTER V ...... 134

C. GENE PHYLOGENIES FROM CHAPTER V ...... 138

LIST OF TABLES

Table Page 2-1. Differentially expressed genes in ΔveA vs. WT and ΔmtfA vs. WT comparisons for A. fumigatus and A. nidulans ...... 26 4-1. Differential gene expression in ΔbrlA, ΔabaA, ΔwetA and wild-type during growth on GMM and RPMI media...... 63 5-1. Types and rates of SM gene cluster variants in A. fumigatus strains ...... 84

LIST OF FIGURES

Figure Page

2-1. SM gene clusters in Aspergillus show minimal evolutionary conservation ...... 21 2-2. SM genes in A. fumigatus, A. nidulans, A. niger, and A. oryzae are less conserved than genes involved in primary metabolism ...... 24 2-3. GO term enrichment analysis of genes differentially expressed in ΔveA and ΔmtfA in A. fumigatus and A. nidulans ...... 27 2-4. Orthology of SM and development genes differentially expressed in ΔveA and ΔmtfA in A. fumigatus and A. nidulans ...... 30 2-5. Model of gene regulatory network evolution in Aspergillus ...... 35 3-1. Expression analysis of veA and laeA in A. fumigatus wild type and respective mutants at 30° and 37° by qRT-PCR...... 41 3-2. Comparison of enriched functional categories and overlapping differential gene expression ...... 45 3-3. Comparison of enriched functional categories and differential gene expression in ΔveA and ΔlaeA strains at 30º and 37º ...... 49 3-4. Differential expression of SM gene clusters in all conditions ...... 51 3-5. Expression and differential expression of the fumagillin and pseurotin clusters in wild-type and ΔveA at 37º and 30º ...... 52 3-6. Expression and differential expression of gene clusters showing a higher change in gene expression in ΔveA 37º than at 30º ...... 54 4-1. Differential gene expression in ΔbrlA, ΔabaA, ΔwetA and wild-type during growth on GMM and RPMI media...... 64 4-2. Heatmap of Euclidean distance between all RNA-seq samples ...... 66 4-3. Gene Ontology enrichment analysis for genes under-expressed in (A) ΔbrlA, (B) ΔabaA, and (C) ΔwetA strains as compared to wild-type...... 68 4-4. Overlap of genes under-expressed in A) ΔbrlA, (B) ΔabaA, and (C) ΔwetA as compared to wild-type during growth on GMM media versus growth on RPMI media...... 69 4-5 Shared and specific targets of BrlA, AbaA, and WetA. (A) Genes under-expressed in ΔbrlA, ΔabaA, and ΔwetA compared to wild-type on GMM media. (B) Genes under- expressed in ΔbrlA, ΔabaA, and ΔwetA compared to wild-type on RPMI media ...... 70

vii

4-6. Gene expression and fold changes of spore and hyphal associated secondary metabolic gene clusters in all strains and conditions. (A,B) Gene expression and log2 fold change of the endocrocin gen cluster, a spore-associated secondary metabolite. (C,D) Gene expression and log2 fold change of the fumigaclavine gene cluster, a spore-associated ergot alkaloid. (E, F) Gene expression and log2 fold change of the gliotoxin gene cluster, a metabolite produced throughout filamentous growth. (G,H) Gene expression and log2 fold change of the fumisoquin gene cluster, a metabolite produced throughout filamentous growth...... 72 5-1. SNP-based phylogeny of A. fumigatus strains...... 82 5-2. Differences in gene content in SM gene cluster 14 in A. fumigatus strains and closely related species ...... 86 5-3. Alignments showing deletion of genes in SM gene clusters ...... 88 5-4. Pseudogenization and gene loss in SM gene clusters...... 90 5-5. Novel SM gene clusters in A. fumigatus strains ...... 93 5-6. Six alleles of an idiomorphic SM gene cluster ...... 95 5-7. Two alleles of the idiomorphic SM gene cluster 10 present in one strain each ...... 96 5-8. Multiple genomic locations of a horizontally transferred SM gene cluster ...... 98 5-9. Genomic contexts of mobile SM gene cluster 5 ...... 101 5-10. Types and frequencies of all SM gene cluster variants in the A. fumigatus population .....102

viii

LIST OF ABBREVIATIONS

SM ...... Secondary metabolism

SMGC ...... Secondary metabolic gene cluster

VeA ...... Velvet A

MtfA ...... Master Transcription Factor A

LaeA ...... Loss of AflR Expression A

VosA ...... Viability of Spores A

VelB ...... Velvet-like B

BrlA...... Bristle

AbaA ...... Abacus

WetA ...... Wet

PKS ...... Polyketide synthase

NRPS...... Non-ribosomal peptide synthase

Anid...... Aspergillus nidulans

Afum ...... Aspergillus fumigatus

Aory ...... Aspergillus oryzae

Aniger ...... Aspergillus niger

AspGD ...... Aspergillus Genomes Database

Ogroup ...... Orthogroup

SNV...... Single-nucleotide variant

GMM...... General minimal media

RPMI ...... Roswell Park Memorial Institute

MFS...... General minimal media

MCL ...... Markov cluster algorithm

DHN ...... 1,8-dihydroxynaphthalene

BiNGO ...... Biological Networks Gene Ontology

CHAPTER I

INTRODUCTION

Filamentous fungi produce a diverse array of bioactive organic small molecules known as

secondary metabolites. Secondary metabolites are critical to the fungal lifestyle, with functions

in defense, virulence, and communication (O’Brien and Wright 2011). Secondary metabolites

also have large impacts on human health in a variety of ways. Many SMs function as toxins, as

in the case of the highly carcinogenic secondary metabolite aflatoxin produced by Aspergillus

flavus and Aspergillus parasiticus, which is found in contaminated food and causes acute

aflatoxicosis and liver cancers (J. W. Bennett and Klich 2003). Alternatively, some secondary metabolites have been repurposed as effective medications, including the widely prescribed cholesterol lowering statins, the iconic antibiotic penicillin, and the immunosuppressant cyclosporine (Keller, Turner, and Bennett 2005).

Ecological roles of fungal secondary metabolites

Secondary metabolites play diverse ecological roles in the lifestyle of filamentous fungi, including enhancing virulence, facilitating communication, and self-protection. Secondary metabolites are so named because they are generally not required for basic cellular functions, distinguishing them from primary metabolites that are (J. Bennett and Bentley 1989). However, the line separating primary and secondary metabolism is sometimes blurred by metabolites that play roles in both primary and secondary processes. Some siderophores, for example, can be produced intracellularly for basic iron metabolism; or, as in the opportunistic human pathogen

Aspergillus fumigatus, secreted to enhance virulence by scavenging iron during infection of the

animal lung (Schrettl et al. 2007). Nevertheless, most secondary metabolites share general

ecological, chemical, and genetic features that separate them from primary processes.

Many filamentous fungi are saprotrophs that grow in soil environments containing other

species of microbes, including fungi, bacteria and microbial eukaryotes. These environments are

competitive and crowded, and secondary metabolites can give a competitive edge to the fungus

as well as function to mediate signaling both within and between species (Macheleidt et al.

2016). The particular ecological role of a given secondary metabolite is highly dependent on

context. Differences in concentrations of an SM, for example, can determine whether it functions

as a broad spectrum antibiotic or functions as a signaling molecule to alter the behavior of other

microbes (J. Bennett and Bentley 1989; Yim, Wang, and Davies 2007). In addition, secondary

metabolites functioning as signaling molecules can be antagonistic or synergistic (Macheleidt et

al. 2016); for example, fusaric acid produced by Fusarium graminearum represses the

production of an antifungal compound in Pseudomonas fluorescens CHA0 (Notz et al. 2002),

while several secondary metabolites produced by Fusarium oxysporum function as

chemoattractants and promote hyphal colonization by Pseudomonas fluorescens WCS365 (de

Weert et al. 2004).

Secondary metabolites also play roles in virulence in pathogenic filamentous fungi, both on animal and plant hosts. Plant-fungal interactions are complex and ancient, likely dating to the emergence of land plants (Krings, Taylor, and Dotzler 2012; Hibbett and Matheny 2009), and

phylogenetic evidence suggests that plant pathogenic fungi are typically closely related to fungal

endophytes, which colonize plants without causing disease (Delaye, García-Guzmán, and Heil

2013; Spatafora and Bushley 2015). In contrast to their endophytic relatives, pathogenic fungi

produce secondary metabolites that function as toxins with various roles in pathogenesis.

Fusarium graminearum, a cereal pathogen, produces terpenes including the trichothecene deoxynivalenol that aid the fungus in colonizing barley and wheat (Kazan, Gardiner, and

Manners 2012). In some cases, the secondary metabolites produced by plant pathogenic fungi specify host range and host-specific virulence. A classic example of this phenomenon is T-toxin, a polyketide secondary metabolite produced by Cochliobolus heterostrophus race T which is hypervirulent on corn varieties carrying the mitochondrial Texas cytoplasmic male sterility gene

(Turgeon and Baker 2007). Secondary metabolites produced by plant pathogens are also targets of the plant immune system in the evolutionary arms race between plants and their pathogens; the rice blast pathogen Magnaorthe grisea contains a PKS-NRPS hybrid gene that is recognized by certain rice cultivars, rendering the fungus avirulent (Böhnert et al. 2004).

Filamentous fungi can also be animal pathogens, though are typically opportunistic rather than obligate animal pathogens. Members of the Aspergillus and Mucor genus of filamentous fungi, in particular Aspergillus fumigatus, can cause a variety of diseases in respiratory tracts of humans and other animals, ranging in severity from asthma and breathing related disorders to invasive fungal infections (Pfaller, Pappas, and Wingard 2006; Gibbons and Rokas 2012). These fungi cause infections beginning in the lung when their conidia are inhaled and germinate.

Conidia and germlings are generally cleared efficiently by the host immune system, and these infections are much more likely to affect immune compromised individuals than healthy individuals (Roilides, Katsifa, and Walsh 1998). However, some fungi have defenses against the immune system that render them more effective pathogens. For example, the conidia of

Aspergillus fumigatus contain a secondary metabolite derived melanin pigment required for virulence that prevents killing of conidia by phagocytes by interfering with acidification in

3 phagolysosomes and additionally inhibits apoptosis of macrophages (Heinekamp et al. 2012).

This pigment also plays a role in protecting conidia from UV radiation. Other secondary metabolites with known roles in animal virulence include siderophores, which are produced by

A. fumigatus to scavenge for iron in the iron-poor environment of the lung (Schrettl et al. 2007).

Other fungi produce siderophores during animal infection, such as the entomopathogenic fungus

Metarhizium robertsii (Giuliano Garisto Donzelli, Gibson, and Krasnoff 2015). In both fungi, siderophores are essential for virulence. Other secondary metabolites produced by animal pathogenic fungi have not been linked to in vivo biological mechanisms, though some are produced at higher levels during infection. While some of these metabolites are dispensable for virulence, the gliotoxin secondary metabolite in Aspergillus fumigatus enhances virulence during mouse infection and is produced at higher levels during infection (Sugui et al. 2008; Bignell et al. 2016).

There are many more known cases of secondary metabolite mediated specific host- pathogen interactions between plants and fungi than there are animals and fungi. One explanation for this is that most filamentous fungi, with the exception of insect-infecting fungi, are only rarely successful in infecting healthy individuals (Abad et al. 2010). The relatively recent increase in the immune compromised population through the spread of HIV/AIDS and through increased use of immunosuppressants correlates with the increased incidence of filamentous fungal infections (Latge 1999). While there are clear roles for some secondary metabolites in pathogenesis, it is likely that these metabolites have other ecological functions in the fungi that produce them.

Secondary metabolic pathways in fungal genomes

A unique genetic feature of secondary metabolism in filamentous fungi is that the biosynthetic genes that synthesize a given compound are found in contiguous gene clusters in the genome, which is atypical of biosynthetic pathways in other eukaryotes (Keller and Hohn 1997).

These clusters contain backbone synthesis genes, such as non-ribosomal peptide synthases,

polyketide synthases, dimethylallyl tryptophan synthases, and terpene synthases, as well as

additional biosynthetic genes that modify the chemical product of the backbone enzyme.

Backbone synthesis genes are often large, multi-domain and in the case of nonribosomal peptide

synthases, multi-modular biosynthetic enzymes. Additional biosynthetic enzymes found in SM

gene clusters include oxidoreductases, methyltransferases, acetylases, and esterases (Keller,

Turner, and Bennett 2005).

In addition to biosynthetic genes, these clusters contain transporters involved in either

secreting the metabolite or, in some cases, sequestering the metabolite in a vesicle-vacuole

fusion derived organelle known as a toxisome (Keller 2015). Toxisomes play roles in

biosynthesis and export of reactive or toxic metabolites. One example of this process occurs

during aflatoxin biosynthesis, in which the penultimate metabolic intermediate is transported into

a toxisome where it is converted to the final product and secreted via exocytosis (Chanda et al.

2009). Secondary metabolic gene clusters can also contain additional types of detoxifying genes

that protect the fungus from reactive metabolites; for example, the gliotoxin gene cluster contains

an oxidoreductase that reduces the reactivity of the metabolite (Dolan et al. 2015; Scharf et al.

2010). Finally, these clusters often contain a cluster-specific transcription factor that regulates

the expression of the genes in the cluster. The most common type of cluster-specific transcription

factors are from the Zn2Cys6 zinc finger transcription factor family, but Cys2His2 transcription factors can also be found in SM gene clusters (Chang and Ehrlich 2013; Tsuji et al. 2002).

Because of this unique organization, the position of gene clusters in fungal genomes can be computationally predicted from genome sequences. The most widely used algorithms for gene cluster prediction are antiSMASH and SMURF (Medema et al. 2011; Khaldi et al. 2010), which scan fungal genomes using profile hidden Markov models trained on experimentally characterized gene clusters. Additional computational methods for gene cluster prediction rely on the assumption that secondary metabolic gene clusters are co-expressed and use gene expression data to scan fungal genomes for co-expressed gene clusters (Umemura et al. 2013; Andersen et al. 2013). While experimentally characterized SM gene clusters predate the genomic era by decades (Keller and Hohn 1997), genome sequencing revealed scores of previously unknown

SM gene clusters and demonstrated that they are extremely common in fungal genomes

(Nierman et al. 2005; Brakhage and Schroeckh 2011).

Secondary metabolites are extraordinarily diverse among filamentous fungi, reflecting their often highly specialized ecological roles. Individual metabolites are often only produced by one or a handful of species, and the taxonomic distribution of these metabolites can be either narrow (i.e., restricted to a small number of closely related species) or discontinuous (i.e., produced by a small number of unrelated fungi). Phylogenetic analyses have demonstrated that the genes producing these metabolites also often follow that discontinuous distribution (Bushley

and Turgeon 2010). The discontinuous distribution of SM gene clusters is caused by gene and

gene cluster loss as well as horizontal gene transfer of entire SM gene clusters from bacteria to

fungi as well as between fungi (Bushley and Turgeon 2010; Slot and Rokas 2011; Wisecaver and

Rokas 2015). In some characterized cases, horizontal transfer of SM gene clusters occurs

between fungi and other microbes with overlapping ecologies, likely reflecting a shared benefit

of the metabolite (Greene et al. 2014).

Regulation of fungal secondary metabolites

Around 60% of experimentally characterized and computationally predicted SM gene

clusters contain a transcription factor, which in many cases regulates the transcription of the

remaining genes in the cluster (Brakhage 2013). Well-characterized examples of this

phenomenon are the aflR transcription factor in the aflatoxin gene cluster in Aspergillus flavus

and the zinc finger transcription factor gliZ in the gliotoxin gene cluster in Aspergillus fumigatus

(Chang et al. 1995; Bok, Chung, et al. 2006). In both cases, presence of the transcription factor is required for metabolite production, and overexpression of the transcription factor leads to an increase in production of the metabolite. Some cluster-specific SM gene cluster regulators are located outside of the cluster that they regulate. For example, the asperfuranone cluster in

Aspergillus nidulans is regulated by the ScpR transcription factor which is found in a different

SM gene cluster (Bergmann et al. 2010).

In addition to pathway-specific regulation, secondary metabolism production is coordinated by environmentally responsive master SM regulators (Brakhage 2013). Most

secondary metabolites are not produced constitutively throughout the filamentous fungal life

cycle, and instead are produced under specific environmental conditions or certain stages of

development (O. Bayram and Braus 2012). Abiotic environmental conditions that activate or

repress certain secondary metabolites include light availability, carbon source, nitrogen source,

ambient pH, redox status, presence of trace metals, among many others. The induction or

repression of SM gene cluster expression in response to environmental conditions is controlled

by master SM regulators that regulate multiple SM gene clusters. In contrast to the pathways

they regulate, these master regulators are generally well conserved across filamentous fungi. One

such example is PacC, a pH-responsive master regulator activated at alkaline pH and initiates the

transcription of the penicillin biosynthetic gene cluster and represses transcription of the

sterigmatocystin gene cluster in Aspergillus nidulans (Bignell et al. 2005; Espeso and Penalva

1996; Then Bergh and Brakhage 1998). One ecological explanation for PacC’s regulation of

penicillin is that β-lactam antibiotics such as penicillin are generally more toxic against bacteria at alkaline pH (Arst 1996; Brakhage 2013).

Some other master regulators of secondary metabolism are also general environmentally

responsive regulators with multiple roles in fungal biology. Master regulators of secondary

metabolism that activate or repress the expression of SM gene clusters in response to different

nutrient source also regulate primary metabolic pathways. For example, the master SM regulator

AreA is activated by changes in nitrogen source and regulates nitrogen utilization genes as well

as multiple SM gene clusters (Caddick and Arst 1998). The carbon catabolite repressor CreA is

also a master regulator of SM gene clusters, including penicillin in Aspergillus nidulans (Dowzer

and Kelly 1991). Other processes, including asexual or sexual development, can be bridged by

master SM regulators. The Velvet regulatory complex, comprised of the Velvet domain proteins

VeA and VelB and the putative methyltransferase LaeA, regulate both sexual development and

many SM pathways across filamentous fungi (Ö. Bayram et al. 2008; O. Bayram and Braus

2012). This regulatory complex is itself regulated by light availability. When the fungus is

exposed to light, the VeA/VelB dimer is inhibited from entering the nucleus, while under dark

conditions VeA and VelB are both localized in the nucleus where they form the heterotrimeric

Velvet complex with LaeA and interact with other regulatory proteins to control secondary

metabolism and start the sexual developmental cycle.

Chapter previews

In this dissertation, I address several central questions concerning the evolution of secondary metabolism and its regulation using species from the model filamentous fungal genus

Aspergillus. Several unique features of secondary metabolism raise questions with respect to how its regulation evolves and changes in response to different environmental signals and different stages of the fungal life cycle. Here, I address how divergent and often species-specific secondary metabolic pathways are regulated by conserved master regulators alongside more conserved processes such as development, and also examine the genetic drivers behind the species-specificity of secondary metabolic gene clusters.

First, while secondary metabolites are fast evolving and often species-specific, the transcriptional regulators that control these metabolites are typically well conserved across filamentous fungi. In Chapter II, I examine the ways these regulatory networks have evolved across species through transcriptional rewiring. I quantify the conservation and divergence of

SM gene clusters across the Aspergillus genus of fungi using four representative and well annotated genomes and sets of SM gene clusters. I next examine the genome-wide regulatory roles of two master regulators of SM and development, VeA and MtfA, in Aspergillus nidulans and Aspergillus fumigatus using RNA sequencing. This reveals if and to what extent transcriptional rewiring contributes to SM diversity, as well as how transcriptional rewiring impacts the conserved process of development.

In Chapter III, I address whether master SM regulators are able to regulate secondary metabolite production based on combinations of environmental signals. While most master regulators of secondary metabolism are described as responding to one environmental signal, it is likely that these regulators combinatorially control SM production to fine-tune the metabolic profile of a fungus to changing environments. This possibility is supported by work showing that multiple environmental cues and transcription factors can regulate production of the SM terrein in Aspergillus terreus (Gressler et al. 2015), that both the light-responsive regulator VeA and the nitrogen regulator AreA are required for wild-type levels of SM-producing gene transcription in

Fusarium oxysporum (López-Berges et al. 2014) and that small changes in glucose concentration impact SM production in A. nidulans by changing VeA’s subcellular location (Atoui et al. 2010).

In chapter III, I examine whether the light-sensitive Velvet complex can respond to additional environmental signals by determining whether the complex is also sensitive to temperature. This work demonstrates that secondary metabolic regulators can fine-tune their regulatory programs based on multiple environmental signals.

Next, I investigate how development impacts secondary metabolism in filamentous fungi.

The onset of asexual development and secondary metabolism production often occur at the same time in many species of filamentous fungi. Secondary metabolites that are produced during asexual development are typically localized in developmental tissues and can play roles in spore protection as well as virulence. In Chapter IV, I investigate how these processes are coordinated by determining the impact of central asexual regulatory pathways in Aspergillus fumigatus on secondary metabolism production. Early, intermediate, and late stages of asexual development are controlled by the three transcriptional regulators BrlA, AbaA, and WetA, respectively, in all

Aspergillus species. These regulators are linked in a circuit where BrlA activates AbaA, which

activates WetA. Using RNA-sequencing, I determine the genome wide regulatory roles of these

three transcription factors and the shared and regulator-specific targets of the circuit. I further dissect the individual contributions of each regulator in controlling spore-specific as well as non-

developmentally linked metabolites. This work demonstrates how the asexual developmental

program and secondary metabolism production are coordinated transcriptionally in filamentous

fungi.

In Chapter V, I determine the genetic drivers that generate diversity in secondary

metabolic gene clusters. In Chapter II, I demonstrate that secondary metabolic gene clusters are

divergent and rapidly evolving across different species of Aspergillus. In order to determine the

mechanisms that result in this rapid divergence, I examine the secondary metabolism clusters in

a large population-based sample of Aspergillus fumigatus strains. I utilize novel whole-genome

sequences of 8 strains of A. fumigatus as well as an additional 58 publicly available genome

sequences. By looking at population-level changes rather than species-level differences, this

analysis is able to catch diversity-generating mechanisms “in the act”. From these data, I propose

likely mechanisms by which secondary metabolic gene clusters are evolving.

CHAPTER II

EXAMINING THE EVOLUTION OF THE REGULATORY CIRCUIT CONTROLLING

SECONDARY METABOLISM AND DEVELOPMENT IN THE FUNGAL GENUS

ASPERGILLUS

Authors

Abigail L. Lind, Jennifer H. Wisecaver, Timothy D. Smith, Xuehuan Feng, Ana M. Calvo, and

Antonis Rokas

Introduction

Filamentous fungi produce diverse repertoires of small molecules known as secondary metabolites (SMs) (Keller, Turner, and Bennett 2005). SMs include widely used pharmaceuticals such the antibiotic penicillin (A. A. Brakhage 1998), the cholesterol-reducing drug lovastatin

(Kennedy et al. 1999), and the immunosuppressant cyclosporine (Weber et al. 1994), as well as potent mycotoxins such as aflatoxin (P K Chang et al. 1995) and fumonisin (Daren W Brown et al. 2007; Robert H Proctor et al. 2003). SMs play key ecological roles in territory establishment and defense, communication, and virulence (Yim, Wang, and Davies 2007; Rohlfs et al. 2007;

Vining 1990; Demain and Fang 2000; Rohlfs and Churchill 2011).

The genes involved in fungal SM pathways are often physically linked in the genome, forming contiguous SM gene clusters (Keller and Hohn 1997). These gene clusters are typically characterized by a backbone gene, such as those encoding nonribosomal peptide synthetases

(NRPSs), polyketide synthases (PKSs), hybrid NRPS-PKS enzymes, and prenyltransferases

(PTRs), whose protein products are responsible for synthesizing the proto-SM. Additional

genetic components of SM gene clusters include genes for one or more tailoring enzymes that chemically modify SM precursors, transporter genes responsible for exporting the final product, and transcription factors that drive expression of additional genes in the cluster. For example, the gene cluster responsible for the synthesis of the mycotoxin gliotoxin in the opportunistic human pathogen Aspergillus fumigatus contains 13 genes including a non-ribosomal peptide synthase

(gliP), multiple tailoring enzymes (gliI, gliJ, gliC, gliM, gliG, gliN, gliF), a transporter gene

(gliA), a transcription factor (gliZ), and a gliotoxin oxidase gene that protects the fungus from the harmful effects of gliotoxin (gliT) (Gardiner and Howlett 2005; Scharf et al. 2012).

Surveys of fungal genomes show that for any given fungus there are many more gene clusters than known SMs, suggesting that the currently characterized SMs might be only a small fraction of the SMs that a fungus can produce (Khaldi et al. 2010; Hoffmeister and Keller 2007).

For example, 33 of the 37 putative SM gene clusters in A. fumigatus have no characterized products, despite evidence from metabolomics surveys suggesting that the fungus produces many

SMs (Lin et al. 2013; Inglis et al. 2013; Frisvad et al. 2009). This is likely due to the fact that characterizing the function of SM gene clusters and matching them to specific SMs is a non- trivial task; most SM gene clusters contain many genes and are often only activated under specific ecological conditions such as the availability of different nutrients or the presence of other species (Axel a Brakhage 2013).

Filamentous fungi exhibit a huge amount of SM biochemical diversity. Individual SMs are often known to be produced by only one or a handful of species, and the SM chemotypic profiles of closely related fungi are typically non-overlapping (Keller, Turner, and Bennett 2005;

J. Bennett and Bentley 1989). For example, the meroterpenoid fumagillin, originally isolated

from Aspergillus fumigatus, has only been detected in Aspergillus fumigatus and some isolates of

Penicillium raistrickii (Christensen, Frisvad, and Tuthill 1999; McCowen, Callender, and Lawlis

1951). The gene cluster required for its production appears to be conserved in the A. fumigatus

close relative, Aspergillus fischerianus, though only intermediate compounds have been detected

from cultures of this and other closely related species (Lin et al. 2013; Asami et al. 2004;

Wiemann, Guo, et al. 2013). In some genera, including Aspergillus (Frisvad, Andersen, and

Thrane 2008), the extent of fungal SM distribution is so taxonomically narrow that SM

chemotypic profiles have been used as unequivocal species-level identifiers.

As might be expected given their key roles in fungal ecology, SM production – and as a consequence SM gene cluster transcriptional activity – is tightly controlled by a complex network of master SM regulators triggered by a wide variety of environmental cues such as temperature, light, pH, and nutrient availability (Axel a Brakhage 2013). Among the master SM regulators identified to date are members of the fungal-specific Velvet protein family, which regulate SM production in response to dark conditions in the model filamentous fungus

Aspergillus nidulans (Ö. Bayram et al. 2008; Kato, Brooks, and Calvo 2003; Stinnett et al. 2007;

Purschwitz et al. 2008). The founding member of the Velvet family, VeA, stimulates production of diverse types of SMs in various fungal genomes under dark conditions, and has been shown to regulate gliotoxin, fumagillin, fumitremorgin G, and fumigaclavine C gene cluster expression and metabolite production in A. fumigatus (Sourabh Dhingra et al. 2013). Recently, a VeA- dependent regulator of secondary metabolism, MtfA, was identified in A. nidulans, which–unlike

VeA–is localized in the nucleus regardless of light conditions (Ramamoorthy et al. 2013). MtfA regulates terrequinone, sterigmatocystin, and penicillin in A. nidulans; in A. fumigatus, MtfA is

necessary for normal protease activity, and virulence assays using the moth Galleria mellonella

suggest it plays a role in pathogenicity (Smith and Calvo 2014).

In addition to regulating SM, both of these regulators have been linked to the regulation

of asexual and sexual development. Timing of SM production with developmental changes is

well established in filamentous fungi, and the presence/absence of certain SMs has been linked

with developmental changes (Tsai et al. 1998; A. M. Calvo et al. 2002; A. M. Calvo 2008). It has

been suggested that regulators that coordinate SM and development allow filamentous fungi to

have more complex lifestyle and diverse natural products than their unicellular yeast relatives,

which lack veA as well as backbone synthesis genes necessary for SM production (Roze,

Chanda, and Linz 2011; Kroken et al. 2003; A. M. Calvo 2008).

Remarkably, both veA and mtfA appear to be broadly conserved in filamentous fungi with non-overlapping SM profiles (Ramamoorthy et al. 2013; O. Bayram and Braus 2012). We used four well-studied organisms from the fungal genus Aspergillus, a highly diverse genus and producer of some of the most iconic SMs, including gliotoxin and penicillin, to investigate the evolutionary variability in the distribution of SM gene clusters and its interaction with these two broadly conserved global transcriptional regulators that differ in their response to light, veA and mtfA. Our evolutionary analyses show that although both the SM gene clusters as well as their gene content are poorly conserved between Aspergillus species, explaining the narrow taxonomic distribution and distinctiveness of their SM profiles, the effects of the global transcriptional regulators on SM production in response to environmental cues are largely conserved across these same species. In contrast, examination of the role of veA and mtfA in development, a process that involves genes that are highly conserved between the two species and whose regulation is intimately linked to SM regulation, yields a very different pattern; whereas the role

of veA is conserved, mtfA regulates development in the homothallic A. nidulans but not in the

heterothallic A. fumigatus.

Methods

Genome sequences and orthogroup definitions for A. nidulans, A. fumigatus, A. oryzae, and A.

niger

All genome sequences and annotations for A. nidulans FGSC A4 s10-m02-r03, A. fumigatus AF293 s03-m04-r11, A. oryzae RIB40 s01-m08-r21 and A. niger CBS 513.88 s01- m06-r10 were taken from the Aspergillus Genomes Database (AspGD) (Arnaud et al. 2010).

Orthogroups for these four genomes were taken from AspGD’s orthology assignments for 16

Aspergillus species, which were generated using a Jaccard clustering approach (Crabtree et al.

2007). AspGD orthogroups contain groups of genes that are thought to have descended from the

Aspergillus common ancestor; genes from the same species that are part of a given orthogroup are defined as in-paralogs that have duplicated at some later point after the species diverged from the Aspergillus common ancestor. Species-specific genes, which were absent from AspGD orthogroups, were organized into species-specific orthogroups using the MCL algorithm in combination with all-versus-all protein BLAST search (Enright, Van Dongen, and Ouzounis

2002). Proteins with BLAST hits with 60% query and subject coverage, an e-value of less than

1e-5, and a percent identity of greater than 60% were subsequently clustered in MCL with an

inflation parameter of 2 and were considered species-specific orthogroups. Proteins that did not

pass the BLAST cutoffs were considered single-gene, species-specific orthogroups.

Gene category definitions

Genes involved in secondary metabolism were taken from a previous study that expertly

annotated secondary metabolic gene clusters in the four species under study (Inglis et al. 2013).

Manually curated gene cluster boundaries were used when available. Primary metabolism genes were annotated using a previously described enzyme classification pipeline which utilizes KEGG

Enzyme Commission annotations (J. H. Wisecaver, Slot, and Rokas 2014). Genes involved in development were determined from all genes in A. fumigatus and A. nidulans annotated to the

GO term “developmental process” (GO:0032502) in AmiGO (Carbon et al. 2009). This data was accessed on 2014-07-19.

Strains and culture conditions

The strains used in this study include A. fumigatus CEA10, TSD1.15(∆veA) and

TTDS4.1(ΔmtfA) (S Dhingra, Andes, and Calvo 2012; Smith and Calvo 2014) and A. nidulans

TRV50.2 (Ramamoorthy et al. 2012), TXFp2.1(∆veA) generated in this study, and TRVp∆mtfA

(Ramamoorthy et al. 2013). Many A. nidulans studies have used a veA partial deletion (H.-S.

Kim et al. 2002). For the present study, we generated a strain with a complete deletion of the veA coding region, TXFp2.1(∆veA). This strain was generated as follows. First, The veA deletion cassette was obtained by fusion PCR as previously described (Szewczyk et al. 2006). A 1.4 kb 5’

UTR and a 1 kb 3’ UTR veA flanking regions were PCR amplified from wild type FGSC4 genomic DNA with primers veA_comF and AnidveA_p2, and ANVeASTagP3 and

ANVeASTagP4 primers sets, respectively. The A. fumigatus pyrG (pyrGA.fum) selectable marker

was amplified with AnidveA_p5 and ANVeASTagP6 primers from plasmid p1439. The 5’ and

3’ UTR fragments were then PCR fused to pyrGA.fum to generate the veA replacement construct

using primers AnidveA_P7 and AnidveA_P8. The deletion cassette was transformed into A.

nidulans RJMP1.49 strain (Shaaban et al. 2010). The resulting transformants were then

transformed with the pSM3 plasmid containing the A. nidulans pyroA to generate prototrophs, obtaining the ∆veA strain. This strain was confirmed by DNA analysis (data not shown) and designated as TXFp2.1.

All strains were grown in liquid stationary cultures in Czapek-Dox medium (Difco) in the

dark. The experiments were carried out with two replicates. After 72 hours of incubation at 37oC

mycelia samples were harvested, immediately frozen in liquid nitrogen and lyophilized.

RNA extraction

Total RNA was isolated from lyophilized mycelia using the directzol RNA MiniPrep Kit

(Zymo) according to the manufacturer’s instructions. RNA then was quantified using a nanodrop

instrument. Expression patterns of veA and mtfA were verified in the A. fumigatus and A.

nidulans wild types as well as in the deletion mutants by qRT-PCR prior to RNA sequencing

(not shown), conforming the absence of transcripts in the deletion mutants.

RNA sequencing

RNA-Seq libraries were constructed and sequenced at Vanderbilt Technologies for

Advanced Genomics using the Illumina Tru-seq RNA sample prep kit as previously described

(Gibbons, Salichos, et al. 2012; Gibbons, Beauvais, et al. 2012; Sourabh Dhingra et al. 2013). In

brief, total RNA quality was assessed via Bioanalyzer (Agilent). Upon passing quality control,

poly-A RNA was purified from total RNA and the second strand cDNA was synthesized from

mRNA. cDNA ends were then blunt repaired and given an adenylated 3’ end. Next, barcoded

adapters were ligated to the adenylated ends and the libraries were PCR enriched, quantified,

pooled and sequenced an on Illumina HiSeq 2500 sequencer. Two biological replicates were

generated for each strain sequenced.

RNA-seq read alignment and differential gene expression

Raw RNA-seq reads were trimmed of low-quality reads and adapter sequences using

Trimmomatic using the suggested parameters for single-end read trimming (Bolger, Lohse, and

Usadel 2014). Trimmed reads were aligned to A. nidulans and A. fumigatus genomes using

Tophat2 using the reference gene annotation to guide alignment and without attempting to detect novel transcripts (parameter –no-novel-juncs) (D. Kim et al. 2013). Reads aligning to each gene were counted using HTSeq-count with the intersection-strict mode (Anders, Pyl, and Huber

2014). Differential expression between ΔveA and WT and ΔmtfA and WT strains of A. fumigatus and A. nidulans were determined using DESeq2 (M. I. Love, Huber, and Anders 2014). Genes were considered differentially expressed if their adjusted P-value was less than 0.1 and their log2

fold change was greater than 1 or less than -1.

Statistical analyses

GO term enrichment was determined for over- and under-expressed genes in all four

conditions tested (A. nidulans and A. fumigatus ΔveA vs. WT and ΔmtfA vs. WT) using the

Cytoscape plugin BiNGO (Shannon et al. 2003; Maere, Heymans, and Kuiper 2005). To allow

for a high-level view of the types of differentially expressed gene sets, the Aspergillus GOSlim

term subset developed by AspGD was used. The Benjamani-Hochberg multiple testing correction was applied, and terms were considered significantly enriched if the adjusted P-value was less than 0.05.

Fisher’s exact tests were performed using the R function fisher.test with a two-sided

alternative hypothesis (R Core Team 2014). P-values were adjusted for multiple comparisons using the R function p.adjust with the Benjamini-Hochberg multiple testing correction

(Benjamini and Hochberg 1995) Figures were created using the R plotting system ggplot2

(Wickham 2009) and circos (Krzywinski et al. 2009).

Results

The majority of SM gene clusters in Aspergillus are species-specific

Figure 2-1. SM gene clusters in Aspergillus show minimal evolutionary conservation. (A) Venn diagram showing homologous SM gene clusters between A. fumigatus, A. nidulans, A. niger, and A. oryzae. Two SM gene clusters were considered homologous if greater than 50% of their genes were orthologs. Numbers in parenthesis indicate the total number of SM gene clusters present in each species. The asterisk (*) is to clarify that that two SM gene clusters in A. oryzae are homologous to one gene

cluster in A. niger. (B) Circos plot showing all SM gene clusters in A. fumigatus, A.

nidulans, A. niger, and A. oryzae. The outer black track shows the relative SM gene

counts in each of the four species. SM clusters are indicated by the alternating light and

dark grey wedges of the inner track; wedge thickness is proportional to number of

clustered genes. The sterigmatocystin gene cluster in A. nidulans is colored red. Links

indicate SM clusters containing one or more genes assigned to the same orthogroup as

gene(s) in the sterigmatocystin gene cluster; link color indicates the number of shared

genes.

The genomes of A. fumigatus, A. nidulans, A. oryzae, and A. niger contain 317, 498, 725, and 584 secondary metabolic genes, respectively, which are organized in 37, 70, 75, and 78 corresponding secondary metabolic gene clusters (Inglis et al. 2013). We considered SM gene clusters to be conserved between species if greater than half of the genes in the larger gene cluster were orthologous to greater than half of the genes in the smaller gene cluster. Even with this very liberal definition of gene cluster conservation, we found that no SM gene clusters were conserved across all four species. Moreover, 91.9-96.1% of SM gene clusters were specific to each species, with only 7 SM gene clusters conserved between any species (Figure 2-1a). As none of these SM gene clusters have chemically characterized products, little can be inferred about the similarity or differences of the products of these conserved gene clusters.

While very few conserved SM gene clusters can be identified between these four species,

SM gene clusters do contain genes whose orthologs are parts of other, non-homologous, SM gene clusters. For example, the 25 genes in the sterigmatocystin gene cluster in A. nidulans, one of the largest SM gene clusters present in the genomes analyzed, have orthologs in 25 SM gene

clusters in the other three species as well as inparalogs in 8 other A. nidulans SM gene clusters

(Figure 2-1b). However, in all but one case, less than 20.0% (5 genes) of the sterigmatocystin

gene cluster is present in the other gene cluster. The only exception is the truncated aflatoxin

gene cluster of A. oryzae, which shares 11 orthologs with the ST gene cluster. Although the A.

oryzae aflatoxin gene cluster is non-functional (Machida et al. 2005; Kiyota et al. 2011; Gibbons,

Salichos, et al. 2012), the evolutionary conservation between the aflatoxin and sterigmatocystin gene clusters is reflected in the fact that sterigmatocystin is the penultimate precursor product of the aflatoxin biosynthetic pathway (D W Brown et al. 1996).

Aspergillus SM genes are significantly less conserved than genes for primary metabolism

Figure 2-2. SM genes in A. fumigatus, A. nidulans, A. niger, and A. oryzae are less

conserved than genes involved in primary metabolism. For each species, dark blue bars

indicate the percentage of primary metabolism orthogroups that is species-specific when

compared to all genes similarly annotated as participating in primary metabolism in the

other three species; light blue bars indicate the percentage of each species’ primary

metabolism orthogroups that is species-specific compared to all genes, irrespective of their annotation, in the other three species. Similarly, light orange bars indicate the percentage

of each species’ SM backbone synthesis orthogroups that is species-specific when

compared to all genes similarly annotated as SM backbone synthesis genes in the other

three species; dark orange bars indicate the percentage of each species’ SM backbone

synthesis orthogroups that is species-specific compared to all other genes. Light red bars

indicate the percentage of each species’ SM orthogroups that is species-specific when

compared to all genes similarly annotated as SM genes in the other three species; dark red

bars indicate the percentage of each species’ SM orthogroups that is species-specific

compared to all other genes. Asterisks indicate statistically significant differences based on

a P-value ≤ 0.01 (*), ≤ 0.001 (**), or ≤ 0.0001 (***) in a two-tailed Fisher’s exact test.

To determine the percentage of lineage-specific orthogroups, we determined the number of orthogroups annotated to a particular GOSlim term with at least one gene present in at least one other genome as well as the number of orthogroups with at least one gene annotated to the same functional category in at least one other genome. We found that SM orthogroups were significantly far less conserved than primary metabolic orthogroups in all four genomes examined (adjusted P < 1e-10 for all combinations). No more than 18% of primary metabolic

orthogroups were lineage-specific in any Aspergillus species; this low percentage was observed

both for comparisons between just primary metabolic genes as well as across all genes (Figure 2-

2). In contrast, SM orthogroups as well as orthogroups containing just SM backbone genes were

more likely to be lineage-specific (Figure 2-2). For example, in A. fumigatus, the smallest

genome in our analysis, 8.9% (117/1322) of primary metabolic orthogroups were lineage-

specific versus 19.0% (4/21) of SM backbone orthogroups and 26.0% (73/281) of SM

orthogroups. Strikingly, genes involved in secondary metabolism in at least 2 genomes were by

far the least conserved in our analysis. Between 53.7% (in A. fumigatus, with 37 SM gene

clusters) to 74.7% (in A. niger, with 77 SM gene clusters) of SM orthogroups had no SM

ortholog in any of the other species examined. SM backbone genes were conserved at a similar

rate when compared to all genes and all SM backbone genes, which reflects the accurate

prediction of polyketide synthase and non-ribosomal peptide synthase genes in the organisms

under study.

VeA regulates the same biological processes as well as the same fraction of the genome in both

A. nidulans and A. fumigatus

Table 2-1. Differentially expressed genes in ΔveA vs. WT and ΔmtfA vs. WT comparisons for A. fumigatus and A. nidulans

Total dif. Per. diff. Under- Over- Condition Species expresseda expresseda expresseda expresseda A. fumigatus 3,101 31.7% 1,555 1,546 ΔveA A. nidulans 2,836 26.5% 1,671 1,165 A. fumigatus 97 0.9% 63 34 ΔmtfA A. nidulans 968 9.0% 568 400 aNumber of differentially expressed genes relative to wild type

We next examined the function of the conserved secondary metabolic regulator VeA by

performing RNA sequencing (Lin et al. 2013; Gibbons, Salichos, et al. 2012; Gibbons, Beauvais,

et al. 2012) of ΔveA and wild-type (WT) A. fumigatus strains TSD1.15 (S Dhingra, Andes, and

Calvo 2012) and CEA10 and A. nidulans strains TXFp2.1 and TRV50.2 (Ramamoorthy et al.

2012) and analyzing the data to identify genes that are differentially regulated in ΔveA vs WT in the two species. Of the 9,783 transcribed genes in the A. fumigatus genome, 1,546 (15.8%) were over-expressed and 1,555 (15.9%) were under-expressed in the ΔveA vs WT analysis in A. fumigatus (Table 2-1). We observed very similar numbers of genes differentially regulated in the

A. nidulans ΔveA vs WT analysis; out of 10,709 genes in the A. nidulans genome, were 1,165

(10.9%) were over-expressed and 1,671 genes (15.6%) were under-expressed. In total, approximately 32% and 26% of protein coding genes were differentially regulated in ΔveA compared to WT in A. fumigatus and A. nidulans, respectively.

Figure 2-3. GO term enrichment analysis of genes differentially expressed in ΔveA

and ΔmtfA in A. fumigatus and A. nidulans. Gene ontology (GO) categories statistically

overrepresented in under-expressed (red) and over-expressed (blue) gene sets in ΔveA

and ΔmtfA relative to wild type. Arrows point to GO term ancestors. Horizontal bars

show the percentage of each gene set assigned to a particular GO term with black bars

indicating significant enrichment (Benjamini & Hochberg adjusted P-value ≤ 0.05 in a

hypergeometric test); white bars indicate no significant enrichment.

To characterize the broad functional categories of these differentially regulated genes, we

performed GO term enrichment analysis using the Aspergillus GOSlim term hierarchy

(Ashburner et al. 2000; Arnaud et al. 2010). Four GO terms, namely SECONDARY METABOLIC

PROCESS, CARBOHYDRATE METABOLIC PROCESS, OXIDOREDUCTASE ACTIVITY, and -

EXTRACELLULAR REGION, are significantly enriched in under-expressed genes in both A. nidulans

and A. fumigatus, showing that VeA is a positive regulator of similar processes in both species

(Figure 2-3). Over-expressed genes in A. fumigatus were significantly enriched for twelve GO

terms potentially related to cell growth, namely RIBOSOME BIOGENESIS, CELLULAR AMINO ACID

METABOLIC PROCESS, TRANSLATION, RNA METABOLIC PROCESS, STRUCTURAL MOLECULE

ACTIVITY, HELICASE ACTIVITY, RNA BINDING, TRANSFERASE ACTIVITY, NUCLEUS, NUCLEOLUS,

RIBOSOME, and CYTOSOL. Five of these twelve terms were also significantly enriched in A.

nidulans (RIBOSOME BIOGENESIS, CELLULAR AMINO ACID METABOLIC PROCESS, RNA METABOLIC

PROCESS, NUCLEUS, and NUCLEOLUS). Over-expressed genes were present in the remaining seven

terms in A. nidulans but did not show statistically significant enrichment.

MtfA’s regulatory role is smaller in scope in A. fumigatus compared to A. nidulans

We next examined the role of the recently identified SM regulator MtfA (Smith and

Calvo 2014; Ramamoorthy et al. 2013) in A. fumigatus and A. nidulans by performing RNA

sequencing and differential gene expression analysis of ΔmtfA vs WT strains of both species (A.

fumigatus tTDS4.1 ΔmtfA (Smith and Calvo 2014) and CEA10, A. nidulans TRVp ΔmtfA and

TRV50.2 (Ramamoorthy et al. 2013) ). In contrast to our findings with veA, we found a striking

difference in the percentage of genes regulated in both species (Table 2-1). Thirty-six genes were

over-expressed (0.4%) and 63 (0.6%) were under-expressed in the A. fumigatus ΔmtfA vs WT

analysis, whereas in the A. nidulans ΔmtfA vs WT analysis 400 genes were over-expressed

(3.7%) and 568 were under-expressed (5.3%).

To determine the functional categories impacted by mtfA deletion in both species, we

performed GO term enrichment analysis on the genes differentially expressed between ΔmtfA

and WT strains. Under-expressed as well as over-expressed genes in A. nidulans were

significantly enriched for SECONDARY METABOLIC PROCESS, TOXIN METABOLIC PROCESS and

OXIDOREDUCTASE ACTIVITY, suggesting that MtfA is involved in positive and negative regulation

of different secondary metabolites (Figure 2-3). Over-expressed genes in A. nidulans were also

significantly enriched for asexual developmental processes, namely DEVELOPMENTAL PROCESS

and ASEXUAL SPORULATION.

Under-expressed genes in A. fumigatus were significantly enriched for two of the three

processes as in A. nidulans, namely SECONDARY METABOLIC PROCESS and OXIDOREDUCTASE

ACTIVITY. However, over-expressed genes in A. fumigatus were not significantly enriched for

any GO terms; some over-expressed genes were present in the SECONDARY METABOLIC PROCESS

term, though this was not statistically significant.

Regulation of similar processes regardless of gene conservation

Figure 2-4. Orthology of SM and development genes differentially expressed in

ΔveA and ΔmtfA in A. fumigatus and A. nidulans. Circos plots of SM genes (A,B) and developmental genes (C,D) showing change in gene expression patterns under ΔveA

(A,C) and ΔmtfA (B,D) conditions. Outer black track shows the relative gene counts in A.

nidulans (right) and A. fumigatus (left). Inner track shows the relative number of under-

expressed genes (red), over-expressed genes (blue) and not differentially expressed genes

(grey). Links indicate orthologous genes between the two species that are both under-

expressed (red links), both over-expressed (blue links) and both not differentially

expressed (light grey links); purple links indicate that the orthologous genes have

conflicting expression patterns.

To examine whether SM gene conservation correlated with conservation of regulation by

VeA and MtfA, we examined whether orthologous genes in A. nidulans and A. fumigatus showed the same responses in ΔveA vs WT and ΔmtfA vs WT analyses (Figure 2-4). SM gene expression in ΔveA A. nidulans and A. fumigatus was similar in terms of numbers of differentially expressed genes despite the large number of genes without orthologs between these two species (Figure 2-4a; Figure 2-1a). Of the 184 under-expressed SM genes in ΔveA A. nidulans, only 64 genes (34.8%) had an ortholog in A. fumigatus. Of these 64 conserved genes,

45 (70.3%) had at least one differentially expressed ortholog in A. fumigatus, and 37 (57.8%) had at least one similarly under-expressed ortholog in A. fumigatus. Fewer SM genes were over- expressed in either ΔveA A. nidulans or A. fumigatus; of the 67 over-expressed genes in A. nidulans, 14 (20.9%) had orthologs in A. fumigatus. Of these 14 conserved genes, 5 had at least one differentially expressed ortholog in A. fumigatus, and 3 had at least one similarly over- expressed ortholog in A. fumigatus.

When mtfA was deleted, fewer SM genes were differentially expressed in A. fumigatus than in A. nidulans. Of the 107 under-expressed genes in ΔmtfA A. nidulans, 36 (33.6%) had an ortholog in A. fumigatus (Figure 4b). Unlike veA, however, only 6 of these conserved genes had

differentially expressed orthologs in A. fumigatus. Finally, of the 32 over-expressed genes in

ΔmtfA A. nidulans, 2 of the 12 genes with orthologs in A. fumigatus had orthologs that were

differentially expressed.

Apart from their involvement in the global regulation of SM, both VeA and MtfA are

also involved in the regulation of asexual and sexual development. In contrast to genes involved

in SM, genes involved in asexual and sexual development in Aspergillus have been shown to be

highly conserved across the genus (Galagan et al. 2005). Of the 490 genes annotated to the GO

term DEVELOPMENTAL PROCESS, 462 have at least one ortholog among the 478 genes annotated

to this term in A. fumigatus. In ΔveA A. nidulans, 72 developmental genes are under-expressed

and 32 are over-expressed. Of the 72 under-expressed genes, 66 (91.7%) have an ortholog in A. fumigatus, 30 of which have a differentially expressed ortholog (Figure 2-4C). There are fewer over-expressed developmental genes in ΔveA A. nidulans, but they show similar trends; 31 of the

32 over-expressed genes have an ortholog, 15 of which have differentially expressed orthologs in

A. fumigatus and 11 of which have over-expressed orthologs. In contrast with veA, many more developmental genes were differentially expressed in A. nidulans ΔmtfA (35) than in A. fumigatus ΔmtfA (1). While 4 of the 6 under-expressed genes and 28 of the 29 over-expressed genes in A. nidulans had orthologs in A. fumigatus, none of these orthologs were differentially expressed (Figure 2-4D).

Discussion

Here, we examined the interplay between secondary metabolites with narrow taxonomic distributions and their broadly conserved SM global regulators in four Aspergillus species. We found remarkably few conserved SM gene clusters in A. fumigatus, A. nidulans, A. oryzae, and

A. niger (Figure 2-1a). Further, the genes comprising these clusters were significantly more

species-specific than genes involved in primary metabolism (Figure 2-2), and those SM genes

that were conserved were assigned to non-homologous pathways (e.g., genes in the

sterigmatocystin gene cluster; Figure 2-1B). Despite the high level of divergence in SM

pathways, regulators of SM production are conserved throughout filamentous fungi (Brakhage

2013). We assessed the conservation of roles of two of these regulators, veA and mtfA, in A.

fumigatus and A. nidulans by comparing genome-wide gene expression of deletion mutants of

veA and mtfA in both species with wild-type strains. We found that the role of veA in controlling secondary metabolism and development was conserved in both species (Figure 2-3), though the regulated genes were often different (Figure 2-4). In contrast, we found that while deleting mtfA

negatively impacted SM gene expression in both A. fumigatus and A. nidulans, developmental

genes were only impacted in A. nidulans (Figure 2-3).

Aspergillus secondary metabolic genes and gene clusters are largely species-specific

Previous studies have described relatively small numbers of conserved SM gene clusters between the closely related species Aspergillus fumigatus, fisherianus, and clavatus (Khaldi et al.

2010). These studies considered SM gene clusters to be conserved if 80% or more of their genes

were shared. Since the four Aspergillus species under study here are much less closely related

(Gibbons and Rokas 2012), we used a lower threshold of 50% for conservation. Even using this

relaxed threshold, we found no clusters that were conserved in all four species, one cluster

conserved in three species, and a small number conserved between pairs of species (Figure 2-

1A). SM gene clusters have been described as not only species-specific but sometimes strain

specific; two isolates of A. fumigatus differ in one putative SM gene cluster (Fedorova et al.

2008), and multiple SM gene clusters vary in their presence and absence in isolates of A. niger

(Andersen et al. 2011).

In addition to the non-conservation of SM gene clusters, we found that the genes

comprising SM genes are much more likely to be lineage-specific than those involved in primary

metabolic functions (Figure 2-2). Surprisingly, many genes involved in SM clusters had

orthologs in other Aspergilli that were not in an SM gene cluster themselves. This observation

may support the hypothesis that SM gene clusters can be formed or altered by incorporating non-

SM genes through genomic rearrangements and possible co-regulation with other genes in the biosynthetic pathway (Wong and Wolfe 2005). However, many of the SM genes were species- specific even when compared against all genes in the other four Aspergilli (Figure 2-1), and as

many as 21.7% of SM genes were not present in any other sequenced Aspergillus species. This

high number of species-specific genes involved in Aspergilli may be explained by extensive gene

duplication and loss, de novo gene emergence, very high sequence divergence driven by

selection, or horizontal gene transfer.

The evolution of the circuit regulating secondary metabolism and development

Figure 2-5. Model of gene regulatory network evolution in Aspergillus. Generalized gene regulatory networks for VeA (A,B) and MtfA (C,D) in A. fumigatus (A,C) and A. nidulans (B,D). As master transcriptional regulators, VeA and MtfA can both promote the expression of a gene (indicated by an arrow →), or inhibit gene expression (indicated by a bar ). Target genes are either present in both species (green fill) or species-specific

(yellow⟞ fill). 35

Our findings with respect to the genes differentially expressed in the absence of veA

support a conserved role for veA in regulating secondary metabolism and development in A.

fumigatus and A. nidulans; however, the downstream genes regulated by VeA are different

between the two species. Whether lineage-specific or conserved, SM genes are differentially

expressed in veA’s absence in both A. fumigatus and A. nidulans. Interestingly, conserved genes

differentially expressed in one species are often not differentially expressed in the other. We

propose that the transcriptional circuit by which VeA regulates secondary metabolism and

development in both fungi has diverged at the level of both the target genes and the regulatory

signal (Figure 2-5). VeA is known to have many interacting partners (A. M. Calvo 2008); among

these, it is responsible for transporting the Velvet family protein VelB from the cytoplasm to the

nucleus, where both proteins interact with LaeA, forming a trimeric complex that regulates

secondary metabolism production and development (Ö. Bayram et al. 2008). However, each

protein has functions in the cell outside of this complex, which can be seen through the different

effects of individual gene deletion on gene expression and morphogenesis (Ö. Bayram et al.

2008; Sourabh Dhingra et al. 2013; Perrin et al. 2007). VeA interacts with red light-sensing

proteins in the nucleus, and it is speculated that VeA may act as a scaffold protein recruiting

additional transcriptional regulators (O. Bayram and Braus 2012). Finally, recent analysis has

shown that the Velvet domain is a DNA-binding domain, and that Velvet family proteins may act as direct transcriptional regulators (Ahmed et al. 2013). The number and complexity of VeA’s interacting partners, and its putative transcription factor function, offers many degrees of freedom for changes in specific gene regulation in both A. fumigatus and A. nidulans, while preserving its important ecological role in coordinating sexual development and secondary metabolism production in response to dark conditions.

Much less is known about the regulatory partners of the putative C2H2 zinc finger transcription factor MtfA; however, we suggest that MtfA acts downstream of VeA in A. nidulans, but not in A. fumigatus, as its expression is decreased in ΔveA A. nidulans but not ΔveA

A. fumigatus. Our results show that MtfA is involved in regulating secondary metabolism in both

A. fumigatus and A. nidulans, though it regulates fewer clusters in A. fumigatus than in A. nidulans. As was the case with veA, deleting mtfA results in differentially expressed lineage- specific and conserved SM genes, though differentially expressed conserved genes were not necessarily differentially expressed in the other species, indicating a divergence in the signal that targets these genes for regulation by MtfA or its interacting partners. Unlike veA, however, deleting mtfA resulted in developmental gene expression changes exclusively in A. nidulans, suggesting that there has been a loss in A. fumigatus or gain in A. nidulans of the regulatory signal that directs MtfA or its downstream targets to regulate developmental processes. Taken together, our results suggest extensive rewiring in the regulatory circuit governing secondary metabolism and development between A. nidulans and A. fumigatus.

CHAPTER III

REGULATION OF SECONDARY METABOLISM BY THE VELVET COMPLEX IS

TEMPERATURE-RESPONSIVE IN ASPERGILLUS

Authors

Abigail L. Lind, Timothy D. Smith, Timothy Saterlee, Ana M. Calvo, and Antonis Rokas

Introduction

Filamentous fungi produce a diverse array of small molecules collectively known as secondary metabolites (SMs). Much research on SMs has focused on their double-edged impact on humans (Keller, Turner, and Bennett 2005); while many are valued as pharmaceuticals, such as the antibiotic penicillin and the cholesterol-lowering drug lovastatin (Paláez 2004; Kennedy et al. 1999), others are potent toxins, such as the acutely carcinogenic aflatoxin (J. W. Bennett and

Klich 2003). In the fungal natural environment, SMs have a variety of functions; they can operate as signaling molecules (Yim, Wang, and Davies 2007; Rodríguez-Urra et al. 2012), as virulence factors to aid pathogenic lifestyles (R H Proctor, Hohn, and McCormick 1995;

Stanzani et al. 2005; Coméra et al. 2007), as microbial inhibitors to carve out a competitive advantage in environments crowded with other microbes (Losada et al. 2009; König et al. 2013), or as a defense against fungivorous predators (Rohlfs et al. 2007; A. M. Calvo and Cary 2015).

SM production is closely linked with environmental signals (A. Brakhage et al. 2009; Keller

2015). For example, the SM aflatoxin is not produced by Aspergillus parasiticus at 37ºC, the organism’s optimal temperature for growth, but is produced at 28ºC (Feng and Leonard 1998).

Furthermore, the effects of specific environmental conditions on SM production can be varied;

for example, sterigmatocystin is produced in much higher quantities at 37ºC than at 28ºC in

Aspergillus nidulans, a pattern of expression that is the reverse of its close chemical relative

aflatoxin in A. parasiticus (Feng and Leonard 1998).

The expression of genes involved in the synthesis and secretion of SMs is governed by a

hierarchical network of master regulators that respond to multiple environmental cues (Axel a

Brakhage 2013). One such environmentally-responsive complex of master regulators is the

Velvet protein complex, whose constituent proteins are broadly conserved regulators of fungal

development and secondary metabolism (O. Bayram and Braus 2012; A. Calvo et al. 2016). In

the absence of light in A. nidulans, two Velvet complex members, VeA and VelB, enter the

nucleus where VeA interacts with the chromatin modifying protein LaeA (Ö. Bayram et al.

2008). The resulting heterotrimeric protein complex modulates expression of SM gene clusters

and developmental processes in many fungi (Wiemann et al. 2010; Chettri et al. 2012; Hoff et al.

2010; Lind et al. 2015), including the opportunistic human pathogen Aspergillus fumigatus (S

Dhingra, Andes, and Calvo 2012; Perrin et al. 2007; Sourabh Dhingra et al. 2013).

While most master regulators of secondary metabolism are known in the context of the

individual environmental cues that activate them, it is likely that these regulators combinatorially

control SM production to fine-tune the metabolic profile of a fungus to changing environments.

The possibility of combinatorial regulation is supported by recent studies showing that multiple

environmental cues can regulate production of the SM terrein in Aspergillus terreus (Gressler et

al. 2015), that both the light-responsive regulator VeA and the nitrogen regulator AreA are

required for wild-type levels of SM-producing gene transcription in Fusarium oxysporum

(López-Berges et al. 2014), and that glucose concentration can impact SM production in A.

nidulans through changes in the subcellular localization of VeA (Atoui et al. 2010).

The fungal genus Aspergillus is an excellent system to examine the influence of environmental variation in SM regulation, as the mechanisms for SM production have been widely studied in this group of organisms. The SM gene clusters (Inglis et al. 2013) and SM production profiles (Frisvad et al. 2009; Chiang et al. 2008) of several species are described in depth, and several master SM regulators are well characterized (Axel a Brakhage 2013).

Furthermore, although variation of SM production in response to environmental cues including temperature (J. Yu et al. 2011; OBrian et al. 2007), pH (Tilburn et al. 1995; Bignell et al. 2005), light (Ö. Bayram et al. 2008), and hypoxia (Barker et al. 2012; Blatzer et al. 2011) has been observed, it has not been systematically characterized or mechanistically understood. For this study, we chose A. fumigatus, the most common cause of a suite of diseases known collectively as aspergillosis (Latge 1999). A. fumigatus produces a diverse array of SMs including the immune-suppressing SM gliotoxin, which is thought to promote its virulence (Scharf et al.

2012). Additionally, A. fumigatus is highly thermotolerant; it can grow at 55ºC and can survive at temperatures up to 75ºC (Ryckeboer et al. 2003; Beffa et al. 1998; Abad et al. 2010). It is unknown whether changes in temperature affect global patterns of gene expression in the secondary metabolic pathways of this opportunistic pathogen.

To test whether variation in environmental cues other than the known light response can influence Velvet complex based SM regulation in A. fumigatus, we examined global gene expression using RNA sequencing in response to different temperatures in wild-type, ΔveA, and

ΔlaeA backgrounds. We found that change in temperature had a marked impact on the expression of SM genes, and that VeA regulates the genes required for producing at least 4 SMs at 37ºC but not at 30ºC, suggesting a combinatorial interaction of temperature- and light-based regulation of secondary metabolism in Aspergillus.

Methods

Strains and culture conditions

Aspergillus fumigatus wild-type CEA10, ∆veA TDS1.15 (pyrG1 ΔveA::pyrGA. fum) (S

Dhingra, Andes, and Calvo 2012) and TSD62.1 ((pyrG1 ΔveA::pyrGA. fum) (Sourabh Dhingra et

al. 2013), were used in this study. Strains were stored as 30% glycerol stocks at -80°C. Conidia of A. fumigatus wild type, ∆veA and ∆laeA strains were inoculated in 25 ml Czapek-Dox medium (107/ml) and grown as stationary cultures for 72 h at either 30°C or 37°C in the dark.

RNA isolation

Figure 3-1. Expression analysis of veA and laeA in A. fumigatus wild type and

respective mutants at 30° and 37° by qRT-PCR. Strains were grown on Czapek Dox

medium. Expression was normalized to the wild-type. Bars represent standard error.

Asterisks indicate no detection.

Mycelial mats were collected and immediately frozen in liquid nitrogen. Samples were

then lyophilized and ground. Total RNA was extracted using Direct-zolTM RNA MiniPrep Kit

from ZYMO following the manufacturer instructions. RNA was resuspended in autoclaved

ddH2O. Samples were stored at -80°C. Expected veA and laeA expression patterns in the wild type and corresponding deletion mutants were verified by qRT-PCR (Fig 3-1).

RNA sequencing

RNA-seq libraries were constructed and sequenced at the Vanderbilt Technologies for

Advanced Genomics Core Facility at Vanderbilt University using the Illumina Tru-seq RNA

sample prep kit as previously described (S Dhingra, Andes, and Calvo 2012; Lind et al. 2015).

Briefly, total RNA quality was assessed via Bioanalyzer (Agilent). Upon passing quality control,

poly-A RNA was purified from total RNA and second strand cDNA was synthesized from

mRNA. cDNA ends were then blunt repaired and 3’ ends adenylated. Barcoded adapters were

ligated to the adenylated ends and the libraries were PCR enriched, quantified, pooled and

sequenced an on Illumina HiSeq 2500 sequencer. Two biological replicates were generated for

each strain sequenced.

Gene expression analysis

Raw RNA-seq reads were trimmed of low-quality reads and adapter sequences using

Trimmomatic with the suggested parameters for single-end read trimming (Bolger, Lohse, and

Usadel 2014). After read trimming, all samples contained between 9.5-14.1 million reads, with the average sample containing 12 million reads. Trimmed reads were aligned to the A. fumigatus

Af293 version s03_m04_r11 genome from the Aspergillus Genome Database (Arnaud et al.

2010; Arnaud et al. 2012). Read alignment was performed with Tophat2 using the reference gene annotation to guide alignment and without attempting to detect novel transcripts (parameter –no- novel-juncs) (D. Kim et al. 2013). Reads aligning to each gene were counted using HTSeq-count with the union mode (Anders, Pyl, and Huber 2014). Differential expression was determined using the DESeq2 R package (Michael I Love, Huber, and Anders 2014). Genes were considered differentially expressed if their Benjamini-Hochberg adjusted p-value was less than 0.1 and their log2 fold change was greater than 1 or less than -1.

Functional enrichment analysis

Functional category enrichment was determined for over- and under-expressed genes in all conditions tested using the Cytoscape plugin BiNGO (Shannon et al. 2003; Maere, Heymans, and Kuiper 2005). To allow for a high-level view of the types of differentially expressed gene sets, the Aspergillus GOSlim v1.2 term subset was used (The Gene Ontology Consortium 2014).

The Benjamini-Hochberg multiple testing correction was applied and functional categories were considered significantly enriched if the adjusted p-value was less than 0.05.

Gene cluster expression

Aspergillus fumigatus secondary metabolic gene clusters were taken from a combination of computationally predicted and experimentally characterized gene clusters (Inglis et al. 2013;

Lind et al. 2015). SM gene clusters were designated “differentially expressed” if half or more of the genes in the cluster were differentially expressed. As many gene clusters contained genes that were statistically significantly differentially expressed (adjusted p-value < 0.1) but did not meet

our cutoff of a log2 fold-change greater than 1 or less than -1, gene clusters with half or more

genes meeting the statistical significance cutoff were considered “weakly differentially

expressed”. Clusters containing a mix of over- and under-expressed genes were considered to

have “mixed” expression.

Data availability

All RNA sequence data files are available from the NCBI's Short Read Archive database

(accession number: SRP080951).

Results

Temperature shift changes the expression of 10% of all genes and of more than half of the genes

in SM gene clusters

To investigate the effect of temperature on gene expression, we compared the

transcriptomes of A. fumigatus wild-type (WT) grown at 37ºC compared to WT grown at 30ºC.

This comparison identified 1,101 differentially expressed genes (|log2 fold-change| >1, adjusted p-value < 0.1), which corresponds to more than 10% of the A. fumigatus transcriptome. Of these genes, 402 were expressed at a higher degree (over-expressed) and 699 genes were expressed at a lower degree (under-expressed) at 37ºC than at 30ºC. Genes over-expressed at 37ºC were enriched (adjusted p-value < 0.05) for the functional categories CARBOHYDRATE METABOLIC

PROCESS and EXTRACELLULAR REGION; genes under-expressed at 37ºC were enriched for the

categories CELL ADHESION, SECONDARY METABOLIC PROCESS, TOXIN METABOLIC PROCESS, and

OXIDOREDUCTASE ACTIVITY (Figure 3-2A).

Figure 3-2. Comparison of enriched functional categories and overlapping differential gene expression. (A) Enriched functional categories for genes differentially expressed under variable temperature conditions. Red boxes indicate GOSlim terms enriched in under-expressed genes, blue boxes indicate categories enriched in over- expressed genes, and purple boxes indicate categories enriched in both over- and under- expressed genes. (B,C) Overlap between genes differentially expressed in ΔveA vs. WT at

30º and 37º. (D,E) Overlap between genes differentially expressed in ΔlaeA vs. WT at 30º and 37º.

As functional category enrichment analysis indicated that genes involved in secondary

metabolism were expressed at lower levels in WT at 37ºC than at 30ºC, we next investigated the

impact of temperature on expression of each of the 37 previously identified secondary metabolic

gene clusters (Inglis et al. 2013; Lind et al. 2015). We found that half or more of the genes in 13 gene clusters were expressed at lower levels at 37ºC than at 30ºC, including the clusters encoding the conidial melanin pigment, fumigaclavine, endocrocin, trypacidin, fumipyrrole, gliotoxin,

fumiquinazoline, fumitremorgin, fumagillin, pseurotin and three gene clusters that do not encode

known products (cluster 15, cluster 30, and cluster 35) (Figure 3-4). As previous analysis has

shown that endocrocin is not produced at temperatures above 35ºC, these results indicate that this

is attributable to changes in gene expression (Berthier et al. 2013).Three other gene clusters that

do not encode known products, namely cluster 21, cluster 25, and cluster 36, were over-

expressed at 37ºC (Figure 3-4). Additionally, half or more genes in 6 gene clusters (cluster 5,

cluster 6, cluster 18, cluster 23, cluster 28, and cluster 31) were differentially expressed, but

contained a mixture of both over- and under-expressed genes; none of these gene clusters encode

known products.

VeA regulates a much large number of genes at 37ºC than at 30ºC

To investigate how temperature influences VeA’s role in controlling gene expression, we compared the transcriptomes of a ΔveA strain with WT grown at either 37ºC or 30ºC. In agreement with previous studies (Sourabh Dhingra et al. 2013; Lind et al. 2015), we found a very

large number (3,404) of differentially expressed genes in ΔveA at 37ºC, with 1,821 over-

expressed genes and 1,583 under-expressed genes. Many fewer genes were differentially

expressed in the ΔveA strain at 30ºC. Specifically, 1,986 genes were differentially expressed in

ΔveA, with 1,026 genes over-expressed and 960 genes under-expressed. A comparison of the

3,404 differentially expressed genes at 37ºC with the 1,986 differentially expressed genes at

30ºC revealed that a subset of 1,439 genes was differentially expressed in ΔveA at both

temperatures, suggesting that their regulation by VeA is temperature independent (Figure 3-2B).

However, while 518 genes were differentially expressed solely at 30ºC, almost four times as

many genes (1,935) were differentially expressed solely at 37ºC; these results indicate that the

regulatory impact of VeA is much greater at 37ºC than at 30ºC.

To determine the functions of differentially expressed genes in ΔveA vs WT at 30ºC and

37ºC we performed functional category enrichment analyses. Over-expressed genes in ΔveA

were enriched for functional categories relating to transcription and translation activity at both

30ºC and 37ºC, while the categories DNA-DEPENDENT TRANSCRIPTION, TRANSFERASE ACTIVITY,

NUCLEOTIDYLTRANSFERASE ACTIVITY, REGULATION OF BIOLOGICAL PROCESS, and DNA BINDING

were only enriched at 37ºC (Figure 3-2A). Further, the number of over-expressed genes in each category was higher for all significantly enriched categories at 37ºC, with the exceptions of

LYASE ACTIVITY, CYTOSKELETAL ORGANIZATION, and MOTOR ACTIVITY which were unchanged.

Genes under-expressed in ΔveA were enriched for functional categories related to

secondary metabolism, including SECONDARY METABOLIC PROCESS and TOXIN METABOLIC

PROCESS. The only significantly enriched category for genes under-expressed in ΔveA at 37ºC that was not enriched for genes under-expressed at 30ºC was CARBOHYDRATE METABOLIC

PROCESS (Figure 3-2A). The number of under-expressed genes annotated to each functional

category was higher at 37ºC, with the exception of RIBOSOME, CYTOSKELETAL ORGANIZATION,

and CELL ADHESION which remained unchanged. These enrichment analyses indicate that though

many more genes are differentially expressed in ΔveA at 37ºC, VeA is regulating similar

categories of genes at both temperatures.

LaeA regulates similar numbers and types of genes at 30ºC and 37ºC

To investigate how temperature influences LaeA’s role in gene regulation, we compared

the transcriptomes of a ΔlaeA strain with WT grown at either 37ºC or 30ºC. While ΔveA strains showed temperature-dependent differences in the number of differentially expressed genes,

ΔlaeA strains showed similar numbers of differentially expressed genes at both 30ºC and 37ºC.

In total, 1,971 genes were differentially expressed in ΔlaeA strains as compared to WT at 37ºC

(632 over-expressed and 1,339 under-expressed), while 1,809 genes were differentially expressed at 30ºC (452 over-expressed and 1,357 under-expressed). There was moderate overlap of the sets of differentially expressed genes at the two temperatures; 1,109 genes were differentially expressed in ΔlaeA at both temperatures, while 770 and 862 genes were only differentially expressed at 30ºC and 37ºC, respectively (Figure 3-2C).

To identify the functions of genes differentially expressed in the ΔlaeA strain compared

to WT at 37ºC and 30ºC, we performed functional category enrichment analyses. Genes over-

expressed at both 37ºC and 30ºC in ΔlaeA were enriched for the categories TRANSPORT,

TRANSPORTER ACTIVITY, MEMBRANE, and PLASMA MEMBRANE. However, genes over-expressed

at 37ºC were enriched for an additional 14 functional categories related to cell division,

filamentous growth, and DNA metabolism that were not enriched in genes over-expressed at

30ºC (Figure 3-2A). Under-expressed genes at both 30ºC and 37ºC were enriched for categories

relating to secondary metabolism, in agreement with LaeA’s well-documented role as a master

regulator of secondary metabolism (Bok, Hoffmeister, et al. 2006; O. Bayram and Braus 2012).

Two functional categories, CARBOHYDRATE METABOLISM and CELL WALL were enriched for under-expressed genes at 30ºC but not 37ºC.

Figure 3-3. Comparison of enriched functional categories and differential gene

expression in ΔveA and ΔlaeA strains at 30º and 37º. (A) Enriched functional categories

for genes differentially expressed in both ΔveA vs. WT and in ΔlaeA vs. WT at either 30º

or 37º. Red boxes indicate GOSlim terms enriched in under-expressed genes, blue boxes

indicate categories enriched in over-expressed genes, and purple boxes indicate categories

enriched in both over- and under-expressed genes. (B) Overlap between genes over-

expressed in ΔveA vs. WT and ΔlaeA vs. WT at 30º. (C) Overlap between genes over-

expressed in ΔveA vs. WT and ΔlaeA vs. WT at 37º. (D) Overlap between genes under-

expressed in ΔveA vs. WT and ΔlaeA vs. WT at 30º. (E) Overlap between genes under-

expressed in ΔveA vs. WT and ΔlaeA vs. WT at 37º.

VeA and LaeA have greater regulatory overlap at 37ºC than at 30ºC

As VeA and LaeA are both members of the Velvet complex and are known to interact, it

is very likely that they exhibit substantial overlap in the genes they regulate (A. M. Calvo 2008).

To examine the effect of temperature on this regulatory overlap, we determined the intersection

of genes differentially expressed in ΔveA versus WT and in ΔlaeA versus WT at 30ºC and 37ºC.

In total, 741 genes were under-expressed in both ΔveA and ΔlaeA at 37ºC (this number corresponds to 41% of all under-expressed genes in ΔveA and to 55% of all under-expressed genes in ΔlaeA) and 579 genes were under-expressed in both ΔveA and ΔlaeA at 30ºC (41% of all under-expressed genes in ΔveA and 55% of all under-expressed genes in ΔlaeA) (Figure 3-

2B,C). The 741 genes under-expressed at 37ºC were significantly enriched for the functional categories SECONDARY METABOLIC PROCESS, OXIDOREDUCTASE ACTIVITY, EXTRACELLULAR

REGION, TOXIN METABOLIC PROCESS, CELL WALL, and CARBOHYDRATE METABOLIC PROCESS

(Figure 3-3A). The 579 genes under-expressed at 30ºC were also enriched for the functional categories SECONDARY METABOLIC PROCESS, OXIDOREDUCTASE ACTIVITY, EXTRACELLULAR

REGION, and TOXIN METABOLIC PROCESS, but not for the CELL WALL and CARBOHYDRATE

METABOLIC PROCESS categories (Figure 3-3A).

In total, 274 genes were over-expressed in both ΔveA and ΔlaeA at 37ºC (17% of all genes over-expressed in ΔveA and 43% of all genes over-expressed in ΔlaeA), while 104 genes were over-expressed in both ΔveA and ΔlaeA at 30ºC (10% of all genes over-expressed in ΔveA and 23% of all genes over-expressed in ΔlaeA) (Figure 3-2C,D). Enrichment of functional categories in genes that were over-expressed in ΔveA and ΔlaeA was strikingly different at 30ºC and 37ºC. Although the categories RNA METABOLIC PROCESS, DNA BINDING, HELICASE

ACTIVITY, NUCLEUS, DNA-DEPENDENT TRANSCRIPTION, REGULATION OF BIOLOGICAL PROCESS,

PROTEIN KINASE ACTIVITY, CHROMOSOME, HYDROLASE ACTIVITY, and DNA METABOLIC PROCESS

were significantly enriched in genes over-expressed in both ΔveA and ΔlaeA at 37ºC (Figure 3-

2A), no functional categories were significantly enriched at 30ºC. Because fewer genes were over-expressed in both ΔveA and ΔlaeA at either temperature than were under-expressed, and many more genes were over-expressed in VeA’s absence than in LaeA’s absence, these results suggest that LaeA may primarily function as a positive regulator of gene expression.

Figure 3-4. Differential expression of SM gene clusters in all conditions. Dark red boxes

indicate half or more genes are underexpressed, dark blue boxes indicate half or more genes are overexpressed, and dark purple boxes indicate that half or more genes are a combination of overexpressed and underexpressed genes. Light-colored boxes indicate that half or more genes in that gene cluster meet the statistical significance cutoff for differential expression but have less than a twofold change in expression.

Figure 3-5. Expression and differential expression of the fumagillin and pseurotin

clusters in wild-type and ΔveA at 37º and 30º. (A, B) Expression in panels A and B is

shown as the regularized log transformation of the number of RNA-seq reads

aligning to that gene as implemented in DESeq2. Genes that are differentially expressed

between conditions are separated by a red line if under-expressed in ΔveA or yellow line

if over-expressed in ΔveA. (C) log2 fold change of all genes in the fumagillin and pseurotin

gene clusters between ΔveA vs. WT at 37º and 30º.

Many SM gene clusters are regulated by both VeA and LaeA at 37ºC, but only by LaeA at 30ºC

We expected that SM clusters regulated by the Velvet complex, comprised of the VelB,

VeA, and LaeA proteins (Ö. Bayram et al. 2008), would require both VeA and LaeA for wild-

type levels of expression. SM gene clusters not controlled by this protein complex, however,

may not show differential gene expression in ΔveA or ΔlaeA strains or may be differentially expressed in only one strain. At 37ºC, 12 SM gene clusters were under-expressed in both ΔveA and ΔlaeA, suggesting that they may be regulated by the Velvet protein complex; these clusters include DHN melanin pigment, fumigaclavine, endocrocin, trypacidin, fumipyrrole, gliotoxin, fumiquinazoline, cluster 28, cluster 30, fumitremorgin, fumagillin, and pseurotin (Figure 3-4).

Interestingly, 6 of these SM gene clusters were either normally expressed in ΔveA at 30ºC or had much less of a change from wild-type expression, suggesting that VeA’s regulatory role may be temperature-dependent. These clusters include DHN melanin pigment, fumiquinazoline, cluster

28, fumitremorgin, fumagillin, and pseurotin (Figure 3-4; Figure 3-5; Figure 3-6). Furthermore, clusters expressed more highly at 37ºC than at 30ºC in WT A. fumigatus were also often under-

53 expressed in ΔveA and ΔlaeA strains; of the 12 clusters under-expressed in both ΔveA and ΔlaeA strains at 37ºC (Figure 3), 11 were expressed at higher levels in WT at 30ºC than at 37ºC. The exception was cluster 28, which was under-expressed in both ΔveA and ΔlaeA strains at 37ºC but was expressed at similar levels in WT at 30ºC and 37ºC.

Figure 3-6. Expression and differential expression of gene clusters showing a higher

change in gene expression in ΔveA 37º than at 30º. (A,B) Fumitremorgin cluster, (C,D)

cluster 28, (E,F), fumiquinazoline cluster, and (G,H) DHN melanin pigment clusters in

wild-type and ΔveA at 37º and 30º. Expression (A,C,E,G) is represented as the regularized

log transformation of the number of RNA-seq reads aligning to that gene as implemented

in DESeq2. Genes that are differentially expressed between conditions are separated by a

red line if under-expressed in ΔveA or yellow line if over-expressed in ΔveA. Differential

expression between ΔveA and wild-type is also shown as log2 fold change (B,D,F,H).

Several clusters were differentially expressed in either ΔlaeA or ΔveA, but not in both.

The hexadehydroastechrome cluster, while under-expressed in ΔlaeA at both 30ºC and 37ºC, was over-expressed in ΔveA at 30ºC and showed mixed expression in ΔveA at 37ºC (Figure 3-4).

Two gene clusters, cluster 14 and cluster 21, were under-expressed in ΔveA at both temperatures but showed mixed expression in ΔlaeA. Finally, the neosartoricin/fumicycline cluster, which was very lowly expressed in WT at both 30ºC and 37ºC, contained some up-regulated genes in ΔveA at both temperatures, but showed no change in expression in ΔlaeA strains. The expression patterns of these gene clusters indicate that, although VeA and LaeA play roles in their regulation, these proteins may in some cases be acting independently of each other.

Discussion

Production of secondary metabolites in A. fumigatus and other filamentous fungi is triggered by diverse environmental cues such as temperature, pH, and nutrient sources, and several master SM regulators that respond to these cues have been identified. However, the extent to which master SM regulators can integrate multiple environmental cues to regulate SM production is not known. Considered together, our findings that temperature regulates global SM

production in A. fumigatus and that the light-responsive master SM regulator VeA is also responsive to changes in temperature, provide support for the hypothesis that regulation of SM production in response to environmental cues is combinatorial.

Growth at 37ºC compared to growth to 30ºC had a marked impact on gene expression in

A. fumigatus WT, significantly changing the expression levels of ~10% of its genes. Importantly,

genes involved in secondary metabolism were disproportionately affected (Fig 3-2A); 13 of the total 37 SM gene clusters were expressed at higher levels at 30ºC than 37ºC, while three clusters were expressed at lower levels at 30ºC (Fig 3-4A). These results are in accordance with studies in A. flavus that find a global pattern of higher SM cluster expression at 30ºC than at 37ºC, the optimal temperature for growth in both fungi (J. Yu et al. 2011). Our findings indicate that temperature plays a significant role in secondary metabolism expression, though as A. fumigatus grows more rapidly at 37ºC than at 30ºC we cannot rule out that other growth-related factors

(e.g., differential cell density) might also play a role in the gene expression changes we observe.

To elucidate the possible interactions between environmental conditions including temperature change to light-responsive SM production, we exposed deletion strains of genes encoding two key members of the Velvet protein complex, veA and laeA, to different temperature conditions. At 37ºC, the optimal temperature for A. fumigatus growth, we find that

VeA and LaeA are both involved in regulating genes in many SM gene clusters. While the lists of which genes are parts of the known SM gene clusters are not identical to the lists used in previous analyses of LaeA’s regulatory role of controlling secondary metabolism, our RNA-seq results generally agree with previously published microarray data (Perrin et al. 2007). One notable difference from previous reports is our finding that a putative terpene-producing cluster on chromosome 5 (Afu5g00100-00135) is under LaeA regulation. Further, our findings that VeA

transcriptionally regulates many gene clusters agrees with chemical data that shows that VeA is

required for the synthesis of fumagillin, fumitremorgin, and fumigaclavine at 37ºC (Sourabh

Dhingra et al. 2013).

The sets of genes that are increased in VeA and LaeA’s absence do not show broad

overlap in their functions (Figure 3-1A), suggesting that VeA and LaeA’s regulatory roles are

distinct from each other. This inference is further supported by the observation of 6 SM gene

clusters that are differentially regulated by VeA but not by LaeA at different temperatures. The

Velvet protein complex formed by LaeA, VeA, and VelB has been implicated as a regulator of

secondary metabolism in many fungi (O. Bayram and Braus 2012; Chettri et al. 2012; Wiemann

et al. 2010; Ö. Bayram et al. 2008; A. M. Calvo 2008), and our findings provide additional

evidence that the LaeA and VeA have functionally distinct roles in regulating SM clusters (O.

Bayram and Braus 2012; Lin et al. 2013).

Our finding that VeA’s regulation of SM gene clusters is temperature-dependent raises the hypothesis that in addition to its critical role in controlling dark-responsive secondary metabolism by localizing in the nucleus under dark conditions and to a lesser degree under light conditions (Ö. Bayram et al. 2008), VeA may also be involved in controlling the response to temperature. Interestingly, previous work in A. nidulans has shown that glucose concentration influences both VeA’s subcellular localization and sterigmatocystin production, altering the effect of light on the biosynthesis of this mycotoxin (Atoui et al. 2010); thus, light and temperature might just be two of the many environmental cues that VeA responds to.

How might VeA, a single protein, mediate such a diversity of regulatory controls on multiple SM gene clusters in response to several different environmental cues? One possibility is that VeA’s regulatory diversity is mediated through the protein’s multiple interaction partners.

VeA forms a heterodimer with another velvet family protein, VelB, and both proteins are

necessary for sexual fruiting body formation in A. nidulans. Many of VeA’s interacting partners impact its subcellular localization. For example, in A. nidulans VeA interacts with the methyltransferases LlmF and the VipC-VapB heterodimer, which respectively increase and repress VeA’s nuclear import (Palmer et al. 2013; O. Sarikaya-Bayram et al. 2014). VeA is also known to interact directly with the red light sensing protein FphA and therefore indirectly with the blue light sensing White Collar homologs LreA and LreB, which may modulate VeA’s light responsive capabilities and subcellular location as well as potentially playing roles in glucose response (Purschwitz et al. 2008; Purschwitz, Müller, and Fischer 2009; Atoui et al. 2010; Ö.

Sarikaya-Bayram et al. 2015). Another possible mechanism explaining VeA’s multi-faceted role is offered by recent experiments in A. nidulans showing that phosphorylation of different combinations of residues of VeA generates distinct phenotypes, including changes in sterigmatocystin production (Rauscher et al. 2015).

Irrespective of what the precise molecular mechanism(s) contribute to VeA’s diverse array of regulatory controls, the emerging picture from recent studies including this one is that

VeA is integrating multiple environmental signals including light (Ö. Bayram et al. 2008), glucose (Atoui et al. 2010), nitrogen source (López-Berges et al. 2014), and temperature (this study), allowing filamentous fungi to modulate cellular processes including secondary metabolism in response to changing environments.

CHAPTER IV

TRANSCRIPTIONAL REGULATORS OF ASEXUAL DEVELOPMENT CONTROL BOTH

SPORE AND HYPHAL SECONDARY METABOLIC PATHWAYS IN ASPERGILLUS

FUMIGATUS

Authors

Abigail L. Lind, Fang Yun Lim, Nancy P. Keller, Antonis Rokas

Introduction

Filamentous fungi produce a diverse array of small molecule secondary metabolites that with diverse ecological roles in fungal pathogenicity, communication, and defense. These secondary metabolites (SMs) are typically produced by pathways organized into contiguous gene clusters (SMGCs), which is atypical of metabolic pathways in most eukaryotes (Keller, Turner, and Bennett 2005). Secondary metabolites are fast evolving, and they are often shared by only a small number of fungal species (J. Bennett and Bentley 1989). Transcriptional regulators of secondary metabolism, in contrast, are typically well conserved. Master secondary metabolic regulators respond to a variety of environmental signals including pH, temperature, light, and nutrient sources to transcriptionally regulate SMGCs (Axel a Brakhage 2013).

Secondary metabolism and development in Aspergillus species and other fungi are tightly linked (A. M. Calvo et al. 2002). In fact, many of the environmental signals that regulate SM production including temperature, pH, and carbon or nitrogen sources, also trigger the onset of asexual and sexual development (A. M. Calvo et al. 2002). Several SMs are specifically

localized or produced in differentiated asexual tissues, such as the DHN melanin pigment

integrated in the cell wall of Aspergillus fumigatus conidia (Keller 2015).

Asexual developmental tissues in A. fumigatus are linked with multiple secondary metabolites with spore-protective properties, including the DHN melanin pigment, endocrocin,

and fumiquinazoline (Keller 2015; Lim et al. 2014). In Aspergillus, the central asexual

development pathway is controlled by three central regulatory genes expressed at specific time

points in developing tissues (J.-H. Yu 2010). The founding member of this regulatory cascade,

BrlA, accumulates in vegetative cells shortly before asexual development (Park and Yu 2016). In

the middle stages of conidiation, BrlA activates expression of AbaA, which controls

conidiophore development. AbaA then activates WetA in late stages of development, which is

required for critical cell wall components of conidia (Park and Yu 2016). Recent evidence

suggests that these regulators may play roles in the expression of both spore-specific, and in one

case vegetative-cell specific, SM gene clusters (Lim et al. 2014; Shin, Kim, and Yu 2015;

Twumasi-Boateng et al. 2009). Further, evidence suggests all three regulators are critical for vegetative growth, including hyphal branching and cell death (Tao and Yu 2011).

To determine whether the central asexual developmental pathway BrlA→AbaA→WetA is involved in regulating secondary metabolism generally in Aspergillus fumigatus, we performed RNA sequencing and metabolomic analyses of on ΔbrlA, ΔabaA, ΔwetA and wild-

type A. fumigatus. Further, we demonstrate a role for BrlA in regulating secondary metabolites

associated with asexual tissues as well as those that are produced primarily by vegetative cells.

This work adds to the evidence that the fast evolving and species-specific process of secondary metabolism is regulated in tandem with conserved processes by ancient, highly conserved regulatory networks.

Methods

Growth conditions and RNA isolation

All strains were inoculated into liquid YPD media and incubated at 37 oC for 18 hours to

ensure developmental competence and synchronization. Mycelia were harvested, washed, and

transferred to either liquid GMM or liquid RPMI media and incubated at 29 oC. Tissue was

harvested after 48 hours post transfer and RNA was extracted using the Qiagen RNeasy Plant

Mini Kit.

RNA sequencing

RNA-seq libraries were constructed and sequenced at the Genomic Services Lab of

Hudson Alpha (Huntsville, Alabama). Libraries were constructed with the Illumina TruSeq

Stranded mRNA Library Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500

sequencer. Two biological replicates were generated for each strain sequenced.

Differential gene expression analysis

Raw RNA-seq reads were trimmed of low-quality reads and adapter sequences using

Trimmomatic with the suggested parameters for paired-end read trimming (Bolger, Lohse, and

Usadel 2014). After read trimming, all samples contained between 19-49 million read pairs, with

the average sample containing 28 million reads. Trimmed reads were aligned to the A. fumigatus

Af293 version s03_m04_r11 genome from the Aspergillus Genome Database (Arnaud et al.

2010; Arnaud et al. 2012). Read alignment was performed with Tophat2 using the reference gene

annotation to guide alignment and without attempting to detect novel transcripts (parameter –no-

novel-juncs) (D. Kim et al. 2013). Reads aligning to each gene were counted using HTSeq-count with the union mode (Anders, Pyl, and Huber 2014). Differential expression was determined using the DESeq2 software (Michael I Love, Huber, and Anders 2014). Genes were considered differentially expressed if their Benjamani-Hochberg adjusted p-value was less than 0.1.

Functional enrichment analysis

Functional category enrichment was determined for differentially expressed genes in all conditions tested using the Cytoscape plugin BiNGO (Shannon et al. 2003; Maere, Heymans, and Kuiper 2005). To allow for a high-level view of the types of differentially expressed gene sets, the Aspergillus GOSlim v1.2 term subset was used (The Gene Ontology Consortium 2014).

The Benjamini-Hochberg multiple testing correction was applied and functional categories were considered significantly enriched if the adjusted p-value was less than 0.05.

Gene cluster expression

A. fumigatus secondary metabolic gene clusters were taken from a combination of computationally predicted and experimentally characterized gene clusters (Inglis et al. 2013;

Bignell et al. 2016) SM gene clusters were designated differentially expressed if half or more of the genes in the cluster were differentially expressed. Gene clusters were designated over- expressed if half or more of the genes in the cluster were over-expressed or were designated under-expressed if half or more of the genes in the cluster were under-expressed. Gene clusters where half or more of the genes in the cluster were differentially expressed but did not have half or more genes being either over-expressed or under-expressed were designated mixed expression.

Results

Genome-wide transcriptional impact of BrlA, AbaA, and WetA

Table 4-1. Differential gene expression in ΔbrlA, ΔabaA, ΔwetA and wild-type during

growth on GMM and RPMI media

Expression WT RPMI ΔbrlA ΔbrlA ΔabaA ΔabaA ΔwetA ΔwetA

vs. GMM GMM RPMI GMM RPMI GMM RPMI

Over-expressed 3305 3358 2391 1148 309 1192 1241

Under-expressed 3429 3380 2474 747 208 966 996

To determine the genome-wide regulatory roles of asexual developmental regulators,

RNA sequencing was performed on ΔbrlA, ΔabaA, ΔwetA A. fumigatus strains grown on both

GMM and RPMI media. Growth on these different media sources caused dramatic differences in

gene expression, particularly with respect to secondary metabolism, in wild-type fungi. Strains

grown on GMM grow in the vegetative state for a prolonged period, while strains grown on

RPMI begin producing spores faster than strains grown on GMM. In total, 3305 genes were

expressed more highly during RPMI growth and 3429 genes were expressed more highly during

GMM growth, comprising 69% of all protein-coding genes in A. fumigatus (Table 4-1). Genes

expressed more highly during growth on GMM than on RPMI were significantly enriched for 45

functional categories related to processes including growth, development, and lipid metabolism.

Genes expressed more highly during growth on RPMI than on GMM were enriched for

significantly enriched for six functional categories, including oxidoreductase activity,

peroxisome, hydrolase activity, carbohydrate metabolic process, lipase activity, and DNA

binding.

Figure 4-1. Expression of all secondary metabolic gene clusters in Aspergillus fumigatus

in all conditions tested.

A large portion of the 32 secondary metabolic gene clusters (SMGCs) were expressed at different levels on different media conditions (Figure 4-1). Several SMGCs associated with

asexual reproduction, including DHN melanin, fumigaclavine, and endocrocin, were expressed

more highly during growth on RPMI. As A. fumigatus sporulates rapidly on RPMI media, these

differences are likely caused by a higher amount of conidia present in strains grown on RPMI.

Other metabolites, such as gliotoxin and fumisoquin, are expressed at much higher levels on

GMM than on RPMI media. During growth on GMM, all genes in the gliotoxin and fumisoquin

gene clusters are expressed on average 32-fold more highly than they are on RPMI media.

A large number of the 9,784 genes in the A. fumigatus genome were differentially expressed in ΔbrlA strains during growth on GMM and during growth on RPMI, comprising

6,738 differentially expressed genes (3,358 over-expressed and 3,380 under-expressed) and

4,865 genes (2,391 over-expressed and 2,474 under-expressed) respectively (Table 4-1). Fewer genes were differentially expressed in the ΔabaA and ΔwetA strains, where roughly 20% of all protein-coding genes were differentially expressed. The ΔabaA RPMI biological replicates were less similar to each other than all other pairs of biological replicates (Figure 4-2), and it is an outlier with only 517 differentially expressed genes due to lower statistical power (Figure 4-2).

Figure 4-2. Heatmap of Euclidean distance between all RNA-seq samples.

Genes under-expressed in ΔbrlA during growth on GMM and on RPMI were enriched for similar functional categories, including SECONDARY METABOLISM, DEVELOPMENTAL PROCESS, and ASEXUAL SPORULATION (Figure 4-3A). Categories only enriched during growth on GMM included RIBOSOME BIOGENESIS, TRANSLATION, CELLULAR RESPIRATION, RIBOSOME, and

STRUCTURAL MOLECULE ACTIVITY. Categories only enriched during growth on RPMI included

PEROXISOME and SIGNAL TRANSDUCER ACTIVITY. Genes under-expressed in ΔabaA during growth on GMM and on RPMI were enriched for SECONDARY METABOLIC PROCESS and

OXIDOREDUCTASE ACTIVITY (Figure 4-3B). No additional functional categories were enriched for

genes under-expressed in ΔabaA during growth on RPMI, but four additional categories were

enriched during growth on GMM (CELLULAR AMINO ACID METABOLIC PROCESS, RESPONSE TO

CHEMICAL STIMULUS, TOXIN METABOLIC PROCESS, and TRANSFERASE ACTIVITY). Three

categories, including SECONDARY METABOLIC PROCESS, TOXIN METABOLIC PROCESS, and

OXIDOREDUCTASE ACTIVITY were enriched for genes under-expressed in ΔwetA during growth on

GMM, but no functional categories were enriched for genes under-expressed during growth on

RPMI (Figure 4-3C).

67 v

Figure 4-3. Gene Ontology enrichment analysis for genes under-expressed in (A) ΔbrlA,

(B) ΔabaA, and (C) ΔwetA strains as compared to wild-type.

Media-specific effects of asexual regulators

Figure 4-4. Overlap of genes under-expressed in A) ΔbrlA, (B) ΔabaA, and (C) ΔwetA as

compared to wild-type during growth on GMM media versus growth on RPMI media.

To determine the extent that BrlA, AbaA, and WetA gene regulation is dependent on

media conditions, we compared under-expressed genes in both GMM and RPMI conditions. In total, 1,203 genes were under-expressed in ΔbrlA during growth on both GMM and RPMI

(Figure 4-4A). However, many genes were only under-expressed relative to wild-type during growth on GMM (2,177 genes) or on RPMI (1,271 genes). Similar results were observed for

ΔabaA and ΔwetA (Figure 4-4B,C). In total, 40 genes were under-expressed in ΔabaA during growth on RPMI and on GMM, while 707 were only differentially expressed during growth on

GMM and 168 were only differentially expressed during growth on RPMI. Finally, 291 genes were under-expressed in ΔwetA during growth on both types of media, while 675 were only under-expressed on GMM and 705 were only under-expressed during growth on RPMI.

Shared and specific targets of BrlA, AbaA, and WetA

Figure 4-5. Shared and specific targets of BrlA, AbaA, and WetA. (A) Genes under-

expressed in ΔbrlA, ΔabaA, and ΔwetA compared to wild-type on GMM media. (B) Genes

under-expressed in ΔbrlA, ΔabaA, and ΔwetA compared to wild-type on RPMI media.

As BrlA, AbaA, and WetA constitute a core transcriptional cascade that controls asexual

development in Aspergillus species, we expect that some genes will be under-expressed in the

absence of each regulator. We found that 229 genes were under-expressed in all strains during

growth on GMM and 116 genes were under-expressed in all strains during growth on RPMI

(Figure 4-5). Relatively few genes were only differentially expressed in ΔabaA, where 125 genes

(16.7%) were under-expressed exclusively in the ΔabaA strain during growth on GMM and 24 genes (11.5%) during growth on RPMI. More genes were uniquely under-expressed in ΔwetA strains, with 399 genes (41.3%) were under-expressed exclusively in ΔwetA during growth on

GMM and 461 genes (46.3%) were under-expressed exclusively in ΔwetA during growth on

RPMI. However, a strikingly large number of genes were only differentially expressed in ΔbrlA;

2,590 genes (76.6%) were differentially expressed only on ΔbrlA GMM and 1,899 genes

(76.7%) were differentially expressed only on ΔbrlA RPMI.

Secondary metabolic gene clusters and their products are regulated by BrlA, AbaA, and WetA

Nine SMGCs were under-expressed in ΔbrlA strains during growth on both GMM and

RPMI media, including ferricrocin, fusarine C, DHN melanin, fumigaclavine, endocrocin,

helvolic acid, gliotoxin, fumiquinazoline, and a cluster encoding an unknown product (Cluster

31) (Figure 4-1). Several SMGCs showed media-dependent changes in expression; the

hexadehydroastechrome cluster, the fumitremorgin cluster, and the supercluster encoding the

fumagillin and pseurotin gene clusters were up-regulated in the ΔbrlA strain during growth on

RPMI but not on GMM. Additionally, the fumisoquin gene cluster and the pyripyropene gene

cluster were under-expressed on GMM media but showed mixed regulation on RPMI media.

SMGCs were also differentially expressed in ΔabaA and ΔwetA strains, though fewer

than in the ΔbrlA strain. The SMGC encoding ferricrocin was under-expressed in ΔabaA on both

GMM and RPMI media, and the gene clusters encoding DHN melanin, fumigaclavine, endocrocin, and helvolic acid were under-expressed in ΔwetA on both media types.

As seen for BrlA, several SMGCs showed media-dependent regulation by AbaA and by WetA.

The gene clusters encoding DHN melanin, endocrocin, fumisoquin, fumiquinazoline, and a cluster encoding an unknown product (Cluster 31) were under-expressed in ΔabaA on GMM but not RPMI, and the clusters encoding fumiquinazoline and pyropyripene A were only under- expressed in ΔwetA on GMM and not RPMI.

Some SMGCs showed patterns of repression by WetA and AbaA on RPMI media but activation on GMM; the gliotoxin gene cluster was under-expressed in ΔabaA and ΔwetA on

GMM but over-expressed in ΔabaA and ΔwetA on RPMI. Additionally, as for ΔbrlA, the

supercluster encoding fumagillin and pseurotin was under-expressed in ΔwetA on GMM but over-expressed on RPMI.

BrlA regulates secondary metabolites both associated with asexual tissues and associated with

hyphal growth

Figure 4-6. Gene expression and fold changes of spore and hyphal associated secondary

metabolic gene clusters in all strains and conditions. (A,B) Gene expression and log2 fold

change of the endocrocin gene cluster, a spore-associated secondary metabolite. (C,D)

Gene expression and log2 fold change of the fumigaclavine gene cluster, a spore-associated

ergot alkaloid. (E, F) Gene expression and log2 fold change of the gliotoxin gene cluster,

a metabolite produced throughout filamentous growth. (G,H) Gene expression and log2

fold change of the fumisoquin gene cluster, a metabolite produced throughout filamentous

growth.

The small molecule products of many of the SM gene clusters under-expressed in ΔbrlA,

ΔabaA, and ΔwetA are known to be spore-associated and are either only produced in asexual

tissues or are produced at higher levels during the onset of asexual development. These include

endocrocin, DHN melanin, fumigaclavine, and others (Berthier et al. 2013; Tsai et al. 1999;

Coyle et al. 2007). The ΔbrlA strain does not undergo any form of asexual development while

the ΔabaA and ΔwetA strains are halted at later points in asexual development. Accordingly, SM

gene clusters that are associated with asexual tissues are under-expressed in ΔbrlA, ΔabaA, and

ΔwetA strains in at least one of the media conditions tested, but the largest change in gene

expression compared to wild-type is seen in the ΔbrlA strain (Figure 4-6A-D).

In addition to these developmentally associated SMs, BrlA shows a pattern of influencing

non-developmental associated secondary metabolites. These include ferricrocin, helvolic acid,

gliotoxin, and fumisoquin. These non-developmental associated secondary metabolites were

typically produced at higher levels in the wild-type when grown on GMM media, and in some

cases were very lowly expressed on RPMI media. These gene clusters were strongly under-

expressed in the ΔbrlA mutant on GMM and somewhat under-expressed in ΔabaA and ΔwetA

(Figure 4-6 E-H).

Discussion

The onset of asexual development and changes in secondary metabolite production occur simultaneously in filamentous fungi. Here we investigate how these processes are transcriptionally linked by analyzing the genome wide transcriptional roles of the central regulators of asexual development. The three regulators, BrlA, AbaA, and WetA, are required for the early, middle, and late stages of asexual development, respectively. These regulators

73 collectively coordinate structural changes as the fungus moves from the vegetative growth stage to the spore-producing and dispersing stage. Here, we find significant overlap in the identities and the types of genes controlled by these regulators across different environmental conditions.

However, we find a distinct role for BrlA in regulating secondary metabolism, and notably in regulating secondary metabolites associated with spore protection as well as those associated with vegetative growth. These results suggest that BrlA plays a central role in coordinating both the physical and chemical changes associated with secondary metabolism.

Remarkably, BrlA regulates the highly conserved process of asexual development as well as the fast evolving and often species-specific process of secondary metabolism. How this regulator bridges that gap is unclear, though transcriptional rewiring may play a role, as other regulators of secondary metabolism and development undergo extensive transcriptional rewiring both of their developmental and transcriptional targets (Lind et al. 2015).

Interestingly, while many other secondary metabolic regulators have been lost in yeasts that lack true hyphae as well as secondary metabolism (Roze et al. 2010), both BrlA and AbaA are conserved. In yeast, the AbaA homologue TEC1 retains part of its developmental role, controlling the expression of genes involved in filamentation (Chou, Lane, and Liu 2006). The

BrlA homologue MSN2, however, controls parts of the stress response and does not play an identified role in development (Gorner et al. 1998). Further elucidation of the transcriptional network controlled by BrlA and comparative studies will be required to understand how this asexual regulatory network has changed and been rewired across fungi.

CHAPTER V

DRIVERS OF GENETIC DIVERSITY IN SECONDARY METABOLIC GENE CLUSTERS

IN A FUNGAL POPULATION

Authors

Abigail L. Lind, Jennifer H. Wisecaver, Catarina Lameiras, Fernando Rodrigues, Gustavo H. Goldman, Antonis Rokas

Introduction

Filamentous fungi produce a diverse array of small molecules that function as toxins,

antibiotics, and pigments (Vining 1990). Though by definition secondary metabolites (SMs) are

not strictly necessary for growth and development, they are critical to the lifestyle of filamentous

fungi (Schimek 2011). For example, antibiotic SMs give their fungal producers a competitive

edge in environments crowded with other microbes (Fox and Howlett 2008). SMs can additionally mediate communication between and within species, as well as contribute to virulence on animal and plant hosts in pathogenic fungi (Scharf, Heinekamp, and Brakhage

2014; Yim, Wang, and Davies 2007).

A genomic hallmark of SMs in filamentous fungi is that the biosynthetic pathways that produce them are typically organized into contiguous gene clusters in the genome (Keller,

Turner, and Bennett 2005). These gene clusters contain the chemical backbone synthesis genes

whose enzymatic products produce a core metabolite, such as non-ribosomal peptide synthases

(NRPS) and polyketide synthases (PKS), tailoring enzymes that chemically modify the metabolite, transporters involved in product export, and transcription factors that control the

75 expression of the clustered genes (Keller, Turner, and Bennett 2005). Filamentous fungal genomes, particularly those in the phylum Ascomycota (Keller, Turner, and Bennett 2005), typically contain dozens of SM gene clusters. However, most individual SM gene clusters appear to be either species-specific or narrowly taxonomically distributed in only a handful of species

(J. Bennett and Bentley 1989; Keller, Turner, and Bennett 2005). SM gene clusters that are more broadly distributed show discontinuous taxonomic distributions and are often highly divergent between species. Consequently, the identity and total number of SM gene clusters can vary widely even between very closely related species whose genomes exhibit very high sequence and synteny conservation (Khaldi et al. 2010; Lind et al. 2015).

In the last decade, several comparative studies have described macroevolutionary patterns of SM gene cluster diversity. For example, studies centered on genomic comparisons of closely related species, such as members of the same genus, have identified several different types of inter-species divergence, including single nucleotide substitutions, gene gain / loss events, and genomic rearrangements. Single nucleotide substitutions in one gene result in structural differences in fumonisins produced by Fusarium species (Robert H. Proctor et al. 2008)), while gene gain and loss events have altered trichothecene gene clusters in Fusarium species and aflatoxin family SM gene clusters in Aspergillus species (Robert H Proctor et al. 2009; Berry et al. 2011; Slot and Rokas 2011; Ehrlich et al. 2004; Carbone et al. 2007; J. Yu et al. 2004).

Additionally, trichothecene gene clusters in Fusarium have been altered by genome rearrangement events (Robert H Proctor et al. 2009). Genetic and genomic comparisons across fungal orders and classes have further identified several instances of gene gain or loss

(Campbell, Rokas, and Slot 2012; Kroken et al. 2003; Bushley and Turgeon 2010) and horizontal gene transfer (Slot and Rokas 2011; Patron et al. 2007; Khaldi et al. 2008; Khaldi and Wolfe

2011; Reynolds et al. 2017) acting on individual genes or on entire gene clusters, providing

explanations for the diversity and discontinuity of the taxonomic distribution of certain SM gene

clusters across fungal species.

Although inter-species comparative studies have substantially contributed to our

understanding of SM diversity, the high levels of evolutionary divergence of SM clusters make

inference of the genetic drivers of SM gene cluster evolution challenging. Simply put, it has been

difficult to “catch” the mechanisms that generate SM gene cluster variation “in the act”. Several previous studies have examined intra-species or population-level differences in individual SM gene clusters, typically focusing on the presence and frequency of non-functional alleles of clusters involved in production of mycotoxins. Examples of clusters exhibiting such

polymorphisms include the gibberellin gene cluster in Fusarium oxysporum (Wiemann, Sieber,

et al. 2013), the fumonisin gene cluster in Fusarium fujikuroi (Chiara et al. 2015), the aflatoxin

and cyclopiazonic gene clusters in Aspergillus flavus (P.-K. Chang, Horn, and Dorner 2005), and

the bikaverin gene cluster in Botrytis cinerea (Schumacher et al. 2013). While these studies have

greatly advanced our understanding of SM gene cluster genetic variation and highlighted the

importance of population-level analyses, studies examining the entirety of SM gene cluster

polymorphisms in fungal populations are so far lacking. We currently do not understand the

types and frequency of SM gene cluster polymorphisms in populations, whether these

polymorphisms affect all types of SM gene clusters, as well as the genetic drivers of SM gene

cluster evolution.

To address these questions, we investigated the genetic diversity of all 36 known and

predicted SM gene clusters in whole genome sequence data from 66 strains of the opportunistic

human pathogen Aspergillus fumigatus, 8 of which were sequenced in this study. We found that

13 SM gene clusters were generally conserved and harbored low amounts of variation. In contrast, the remaining 23 SM gene clusters were highly variable and contained one or more of

five different types of genetic variation: single-nucleotide polymorphisms including nonsense

and frameshift variants, individual gene gain and loss polymorphisms, entire cluster gain and

loss polymorphisms, polymorphisms associated with changes in cluster genomic location, and

clusters with non-homologous alleles resembling the idiomorphs of fungal mating loci. Many

clusters contained interesting combinations of these different polymorphisms, such as

pseudogenization in some strains and entire cluster loss in others. The types of variants we find

are likely generated by a combination of DNA replication and repair errors, recombination,

genomic insertions and deletions, and horizontal transfer. We additionally find an enrichment for

transposable elements (TEs) around horizontally transferred clusters, clusters that change in

genomic locations, and idiomorphic clusters. Taken together, our results provide a guide to both

the types of polymorphisms and the genetic drivers of SM gene cluster diversification in

filamentous fungi. As most of the genetic variants that we observe have been previously

associated with SM gene cluster diversity across much larger evolutionary distances and

timescales, we argue that population-level processes influencing SM gene cluster diversity are sufficient to explain SM cluster macroevolutionary patterns.

Methods

Strains analyzed

Eight strains of A. fumigatus were isolated from four patients with recurrent cases of

aspergillosis in the Portuguese Oncology Institute in Porto, Portugal. Each strain was determined

to be A. fumigatus using macroscopic features of the culture and microscopic morphology

78 observed in the slide preparation from the colonies with lactophenol solution (de Hoog et al.

2001). All clinical strains were classified as A. fumigatus complex-Fumigati based on morphology. The genomes of all eight strains were sequenced using 150bp Illumina paired-end sequence reads at the Genomic Services Lab of Hudson Alpha (Huntsville, Alabama, USA).

Genomic libraries were constructed with the Illumina TruSeq library kit and sequenced on an

Illumina HiSeq 2500 sequencer. Samples of all eight strains were sequenced at greater than

180X coverage or depth.

We retrieved 58 additional A. fumigatus strains with publicly available whole genome sequencing data, resulting in a population genomics dataset of 66 strains (Appendix B). The strains used included both environmental and clinical strains and were isolated from multiple continents. Genome assemblies for 10 of these strains were available for download from

GenBank (Paul et al. 2017; Liu et al. 2011; Nierman et al. 2005; Fedorova et al. 2008; Knox et al. 2016; Abdolrasouli et al. 2015). For 6 of these strains, short read sequences were also available from the NCBI Short Read Archive (SRA), which were used for variant discovery only

(see Single nucleotide variant (SNV) and indel discovery) and not for genome assembly. Short read sequences were not available for the remaining 4 strains. Short read sequences were downloaded for an additional 48 strains from the Short Read Archive if they were sequenced with paired-end reads and at greater than 30x coverage.

Single nucleotide variant (SNV) and indel discovery

All strains with available short read data (62 of 66 strains) were aligned to both the Af293 and A1163 reference genomes using BWA mem version 0.7.12-r1044 (Li and Durbin 2009).

Coverage of genes present in the reference genome was calculated using bedtools v2.25.0

(Quinlan and Hall 2010). SNV and indel discovery and genotyping was performed relative to the

Af293 reference genome and was conducted across all samples simultaneously using the

Genome Analysis Toolkit version 3.5-0-g36282e4 with recommended hard filtering parameters

(McKenna et al. 2010; Van der Auwera et al. 2013; DePristo et al. 2011) and annotated using snpEff version 4.2 (Cingolani et al. 2012).

De novo genome assembly and gene annotation

All 56 strains without publicly available genome assemblies were de novo assembled using the iWGS pipeline (Zhou et al. 2016). Specifically, all strains were assembled using

SPAdes v3.6.2 and MaSuRCA v3.1.3 and resulting assemblies were evaluated using QUAST v3.2 (Bankevich et al. 2012; Zimin et al. 2013; Gurevich et al. 2013). The average N50 of assemblies constructed with this strategy was 463 KB (Appendix B). Genes were annotated in these assemblies as well as in five GenBank assemblies with no predicted genes using augustus v3.2.2 trained on A. fumigatus gene models (Stanke and Morgenstern 2005). Repetitive elements were annotated in all assemblies using RepeatMasker version open-4.0.6 (Smit, Hubley, and

Green, n.d.).

Secondary metabolic gene cluster annotation and discovery

Secondary metabolic gene clusters in the Af293 reference genome were taken from two recent reviews, both of which considered computational and experimental data to delineate cluster boundaries (Inglis et al. 2013; Bignell et al. 2016). The genomes of the other 65 strains were scanned for novel SM gene clusters using identified using antiSMASH v3.0.5.1 (Medema et al. 2011). To prevent potential assembly errors from confounding the analysis, any inference

about changes in genomic locations of genes or gene clusters was additionally verified by

manually inspecting alignments and ensuring that paired end reads supported an alternative

genomic location (see SNV and indel discovery). Cases where paired end reads did not support

the change in genomic location or where mapping was ambiguous or low quality were discarded.

Phylogenetic analysis

To construct a SNP-based strain phylogeny, biallelic SNPs with no missing data were

pruned using SNPRelate v1.8.0 with a linkage disequilibrium threshold of 0.8 (Zheng et al.

2012). A phylogeny was constructed using RAxML v8.0.25 using the ASC_BINGAMMA

substitution model (Stamatakis 2014). The tree was midpoint rooted and all branches with

bootstrap support less than 80% were collapsed.

To understand the evolutionary histories of specific SM gene clusters showing unusual

taxonomic distributions, we reconstructed the phylogenetic trees of their SM genes. Specifically,

SM cluster protein sequences were queried against a local copy of the NCBI non-redundant

protein database (downloaded May 30, 2017) using phmmer, a member of the HMMER3

software suite (Eddy 2009) using acceleration parameters --F1 1e-5 --F2 1e-7 --F3 1e-10. A

custom perl script sorted the phmmer results based on the normalized bitscore (nbs), where nbs

was calculated as the bitscore of the single best-scoring domain in the hit sequence divided by the best bitscore possible for the query sequence (i.e., the bitscore of the query aligned to itself).

No more than five hits were retained for each unique NCBI Taxonomy ID. Full-length proteins corresponding to the top 100 hits (E-value < 1 × 10-10) to each query sequence were extracted from the local database using esl-sfetch (Eddy 2009). Sequences were aligned with MAFFT v7.310 using the E-INS-i strategy and the BLOSUM30 amino acid scoring matrix (Katoh and

Standley 2013) and trimmed with trimAL v1.4.rev15 using its gappyout strategy (Capella-

Gutierrez, Silla-Martinez, and Gabaldon 2009). The topologies were inferred using maximum likelihood as implemented in RAxML v8.2.9 (Stamatakis 2014) using empirically determined substitution models and rapid bootstrapping (1000 replications). The phylogenies were midpoint rooted and branches with less than 80% bootstrap support were collapsed using the ape and phangorn R packages (Paradis, Claude, and Strimmer 2004; Schliep 2011). Phylogenies were visualized using ITOL version 3.0 (Letunic and Bork 2016).

Results

Figure 5-1. SNP-based phylogeny of A. fumigatus strains. The phylogeny was

constructed using biallelic SNPs with no missing data. The tree is midpoint rooted and all

branches with bootstrap support less than 80% are collapsed.

We analyzed the genomes of 66 globally distributed strains of Aspergillus fumigatus for polymorphisms in SM gene clusters. We performed whole-genome sequencing on 8 strains, and collected the remaining 58 strains from publicly available databases including NCBI Genome and the NCBI Short Read Archive (Figure 5-1, Appendix B) (Nierman et al. 2005; Fedorova et al. 2008; Abdolrasouli et al. 2015; Knox et al. 2016; Paul et al. 2017). We analyzed all strains for polymorphisms in 33 curated SM gene clusters present in the reference Af293 genome and additionally searched for novel SM gene clusters (see Methods). These examinations revealed five distinct types of polymorphisms which influence SM gene cluster variation (Table 5-1):

a) Single nucleotide and short indel polymorphisms. 33 / 33 SM gene clusters (present in

the reference Af293 strain) contained multiple genes with missense SNPs and short indel

variants in at least one strain in the population. 23 / 33 SM gene clusters contained one or

more genes with frameshift or nonsense variants in the population.

b) Gene content polymorphisms involving loss or gain of one or more genes. 6 / 33 SM

gene clusters contained a gene content polymorphism in the population.

c) Whole SM gene cluster gain and loss polymorphisms. 3 / 33 SM gene clusters present in

the genome of the reference Af293 strain were absent in at least one strain in the

population and an additional 3 previously unknown SM gene clusters were present in the

population.

d) Idiomorphic polymorphisms. One locus contained multiple non-homologous SM gene

cluster alleles in different strains of the population.

e) Genomic location polymorphisms. 2 / 33 SM gene clusters were found in different

genomic locations (e.g., different chromosomes) between strains.

Table 5-1. Types and rates of SM gene cluster variants in A. fumigatus strains.

Description Phenotype Drivers Frequency at Frequency Previous cluster level in strains reports

Single- Potential for protein DNA replication 100% (33/33 Every strain Bikaverin in nucleotide function change errors; relaxation clusters; affected Botrytis polymorphisms (missense); of purifying missense); (Schumacher et and indels abrogation of selection 70% (23/33 al. 2013; protein function clusters; Campbell, (nonsense and nonsense and Rokas, and Slot frameshift) frameshift) 2012), aflatoxin in Aspergillus oryzae and Aspergillus flavus (P.-K. Chang, Horn, and Dorner 2005), fumonisins in Fusarium (Robert H. Proctor et al. 2008), many others

Gene content Loss of gene cluster Deletion and 6 clusters 27 / 66 Trichothecene polymorphisms function; structural insertion events; strains in Fusarium, changes in the recombination; aflatoxin and metabolite; change transposable sterigmatocystin in cluster elements in Aspergillus expression or (Robert H metabolite transport Proctor et al. 2009; Berry et al. 2011; Slot and Rokas 2011; Ehrlich et al. 2004; Carbone et al. 2007)

Whole gene Loss or gain of Deletion and 6 clusters 13 / 66 Gibberellin and cluster novel metabolites insertion events; strains fumonisin in polymorphisms horizontal gene Fusarium transfer; (Chiara et al. transposable 2015; Wiemann, elements Sieber, et al. 2013)

Cluster Changes in Transposable 1 gene cluster 8 unique Putative SM idiomorphs metabolites elements; identified gene clusters in produced or recombination; alleles dermatophytes; structure of other putative SM metabolites mechanisms? gene cluster in Aspergillus

flavus and Aspergillus oryzae (Zhang, Rokas, and Slot 2012; Gibbons, Salichos, et al. 2012) Mobile gene Potential for change Transposable 2 gene 8 / 66 strains None clusters in gene regulation elements; clusters horizontal gene transfer; other mechanisms?

Single-nucleotide and indel polymorphisms

It is well established that single nucleotide polymorphisms (SNPs) and short indel

polymorphisms are caused by errors in DNA replication and repair, and are a major source of

genomic variation (Roberts and Kunkel 1996). Non-synonymous SNPs and indels with missense, frameshift, and nonsense effects were widespread across the 33 SM reference gene clusters.

Every strain contained numerous missense mutations and at least one nonsense or frameshift

mutation in its SM gene clusters. Although missense mutations are likely to influence SM

production, the functional effects of nonsense and frameshift mutations are comparatively easier

to infer from genomic sequence data because they often lead to truncated proteins lacking a

significant portion of their amino acid sequence. For example, a frameshift mutation in the

polyketide synthase (PKS) of the trypacidin gene cluster in the A1163 strain results in loss of

trypacidin production (Throckmorton et al. 2015). Interestingly, we identified a premature stop

codon (Gln273*) in a transcription factor required for trypacidin production, tpcD, in a strain

sequenced in this study (MO79587EXP). These data suggest that function of this SM gene

cluster has been lost through at least two independent genetic events in A. fumigatus.

Individual nonsense or frameshift variants ranged from very common in the population to rarer variants present in one or a handful of strains. For example, the non-ribosomal peptide synthase (NRPS) pes3 gene (Afu5g12730) in SM gene cluster 21 harbors 16 nonsense or frameshift polymorphisms in 55 strains. Seven of these polymorphisms are common and present in 10 or more strains, while seven are rarer and found in 5 or fewer strains. Strains with lab- mutated null alleles of the pes3 gene are more virulent than strains with functional copies

(O’Hanlon et al. 2011), which may explain the widespread occurrence of null pes3 alleles in the

Aspergillus population.

Gene content polymorphisms

Figure 5-2. Differences in gene content in SM gene cluster 14 in A. fumigatus strains

and closely related species. Four A. fumigatus strains lack an 11-Kb region in this cluster,

including an NRPS backbone gene. Regions upstream and downstream of this cluster are

syntenic. LMB35Aa also contains a large inversion that moves a transcription factor,

oxidoreductase, and hypothetical protein 275 kb away from the cluster. Aspergillus

fischerianus and Aspergillus lentulus, close relatives of A. fumigatus, contain a cluster

lacking the 11-kb region.

We additionally identified several SM gene clusters that gained or lost genes in some

strains. These gene content polymorphisms were most likely generated through genomic deletion

or insertion events and were often present in high frequencies in the population (Table 5-1). In

three cases, these polymorphisms impact backbone synthesis genes, rendering the SM gene cluster non-functional. One example involves SM gene cluster 14, whose standard composition

includes a pyoverdine synthase gene, an NRPS-like gene, an NRPS backbone gene, and several

additional modification genes (Figure 5-2). We discovered that 4 / 66 strains lack an 11-kb

region on the 3’ end of the cluster which normally contains an NRPS gene and two additional

cluster genes, and the first non-SM genes on the 3’ end flanking the cluster. All A. fumigatus

strains contain a copia family transposable element (Kapitonov and Jurka 2008) at the 3’ end of

the cluster, suggesting that transposable elements may have been involved in the generation of

this polymorphism. While this polymorphism could have arisen through a deletion event, a

homologous cluster lacking the 11-kb region is also present in the reference genomes of

Aspergillus lentulus and Aspergillus fischerianus, close relatives of A. fumigatus (Figure 5-2).

The most parsimonious explanation is that the genome of the A. fumigatus ancestor contained an

SM gene cluster that lacked the 11-kb region, and that this genomic region was subsequently

gained and increased in frequency in the A. fumigatus population.

Figure 5-3. Alignments showing deletion of genes in SM gene clusters. (A) Deletion of

helvolic acid genes in IF1SWF4. (B) Deletion of fumigaclavine genes in LMB35Aa. (C)

Partial deletion of gliM in the gliotoxin gene cluster in two strains. (D) Partial deletion of

fmqE in the fumiquinazoline gene cluster in 3 strains. (E) Partial deletion of Afu5g12720

in SM gene cluster 21 in 14 strains. (F) Partial deletion of Afu5g12720 in SM gene cluster

21 in 7 strains.

Two additional gene content polymorphisms affecting SM backbone genes were

restricted to one strain each and appear to have arisen through genomic deletion events.

Specifically, strain IF1SWF4 lacks an 8-Kb region near the helvolic acid SM gene cluster,

resulting in the loss of the backbone oxidosqualene cyclase gene as well an upstream region containing two non-SM genes (Figure 5-3A). Strain LMB35Aa lacks a 54-kb region on the end of chromosome 2, which includes five genes from the telomere-proximal fumigaclavine C cluster (Figure 5-3B).

In three other cases, gene content polymorphisms involved gene loss or truncation events of non-backbone structural genes. We found that the second half of the ORF of the gliM O- methyltransferase gene in the gliotoxin gene cluster has been lost in 2 / 66 strains (Figure 5-3C), that the first half of the permease fmqE in the fumiquinazoline gene cluster has been lost in 4 / 66 strains (Figure 5-3D), and that an ABC transporter gene in SM cluster 21 has been almost entirely lost in 21 / 66 strains (Figure 5-3E,F).

Whole gene cluster gain and loss polymorphisms

Figure 5-4. Pseudogenization and gene loss in SM gene clusters. (A) SM gene cluster

found in most A. fumigatus strains but absent from the Af293 reference and from the F7763

strain. EOS denotes end of scaffold. (B) Positions of frameshift variants and nonsense

variants in the fusarielin-like SM gene cluster 4.

Several SM gene clusters were gained or lost entirely in 13 / 66 strains. We observed instances where a cluster present in the genome of the reference Af293 strain was absent or pseudogenized in other strains as well as cases in which SM clusters present in other strains were absent from the reference Af293 strain. 90

The most notable example of an SM gene cluster that was present in the Af293 reference genome but absent or pseudogenized in others was SM cluster 4. This cluster contains 5 genes on the tip of the Af293 chromosome 1 and contains orthologs to five of the six genes in the fusarielin gene cluster in Fusarium graminearum (Sørensen et al. 2012). This cluster is also present in several other Aspergillus species, including A. clavatus and A. niger (Sørensen et al.

2012). Phylogenetic analysis of the genes in this SM gene cluster is consistent with horizontal gene transfer between fungi in the class Sordariomycetes and fungi in the class Eurotiomycetes, or alternatively with extensive gene loss in both Sordariomycetes and Eurotiomycetes (Appendix

C). This gene cluster is entirely absent in 4 / 66 strains, and its genes are undergoing pseudogenization in an additional 44 strains via multiple independent mutational events (Figure

5-4A). Specifically, 19 strains shared a single frameshift variant in the polyketide synthase gene

(4380_4381insAATGGGCT; frameshift at Glu1461 in Afu1g17740) and an additional 13 strains shared a single frameshift variant (242delG; frameshift at Gly81) in an aldose 1-epimerase gene

(Afu1g17723). Twelve other strains each contained one to several frameshift or nonsense polymorphisms involving nine unique mutational sites, suggesting that this pathway is undergoing multiple independent pseudogenization events. Five of these strains contained multiple distinct frameshifts and premature stop codons in more than one gene in the cluster, indicating that the entire pathway is pseudogenized in these strains.

By searching for novel SM gene clusters in the genomes of the other 65 A. fumigatus strains, we found three SM gene clusters that were absent from the genome of the Af293 reference strain. As SM gene clusters are often present in repeat-rich and subtelomeric regions that are challenging to assemble (Treangen and Salzberg 2011; Palmer and Keller 2010), these strains might harbor additional novel SM gene clusters.

One of the novel SM gene clusters that we identified, cluster 34, was present in all but

two of the strains (Af293 and F7763). This cluster contains a PKS backbone gene, one PKS-like

gene with a single PKS associated domain, nine genes with putative biosynthetic functions

involved in secondary metabolism, and six hypothetical proteins (Figure 5-4B). The two strains

that lack this cluster contain a likely non-functional cluster fragment that includes the PKS-like gene, two biosynthetic genes, and three hypothetical proteins. Interestingly, the 3’ region

flanking this cluster is syntenic across all 66 strains but the 5’ region is not, suggesting that a recombination or deletion event may have resulted in the loss of this cluster in the Af293 and

F7763 strains.

Figure 5-5. Novel SM gene clusters in A. fumigatus strains. (A) Synteny between a novel

PKS containing cluster in two strains with an SM gene cluster in Metarhizium anisopliae.

This novel PKS cluster is located between transposable elements in a region syntenic with the reference Af293 chromosome 4. (B) Novel SM gene cluster in MO54056EXP and

3 additional strains. This cluster is only located on one scaffold in MO540556EXP and is

fragmented across the other strains (ends of scaffolds are denoted). (C) Coverage data from

short read alignments for MO54056EXP, 12-7504462, and 08-19-02-30 relative to the

MO54056EXP scaffold containing the novel SM gene cluster.

The other SM gene clusters that were absent from the Af293 genome are present at lower frequencies in the population; cluster 35 is present in 2 / 66 strains and cluster 36 in 4 / 66 strains. Cluster 35 is located in a region syntenic with an Af293 chromosome 4 region and is flanked on both sides by transposable elements (Figure 5-5A). Eight of the 14 genes in this SM gene cluster are homologous to genes in an SM gene cluster in the genome of the insect pathogenic fungus Metarhizium anisopliae (Figure 5-5A). Phylogenetic analysis of these 8 genes is consistent with a horizontal transfer event (Appendix C). Cluster 36 is an NRPS containing

cluster located on genomic scaffolds that lack homology to either the Af293 or A1163 genomes, making it impossible to determine on which chromosome this cluster is located (Figure 5-5B,C).

The evolutionary histories of the genes in the cluster are consistent with vertical inheritance and are present in multiple Aspergillus species.

Idiomorph polymorphisms

Figure 5-6. Six alleles of an idiomorphic SM gene cluster. Alleles of SM gene cluster

10 on chromosome 3. Red boxes denote transposable elements. Green arrows denote

backbone genes (PKS or NRPS) or genes containing domains associated with PKS or

NRPS genes. Purple arrows denote genes likely involved in SM biosynthesis, transport, or

cluster regulation. Gray arrows denote genes without a putative function or with functions

unrelated to SM.

One of the most peculiar types of polymorphisms that we identified is a locus containing

different unrelated alleles of SM gene clusters, reminiscent of the idiomorph alleles at the fungal

mating loci (Metzenberg and Glass 1990). This locus, which resides on chromosome 3 and

corresponds to cluster 10 in the Af293 genome (Figure 5-6), was previously described as being

strain-specific in a comparison between Af293 and A1163 (Fedorova et al. 2008) and is thought to reside in a recombination hot spot (Abdolrasouli et al. 2015). Our analysis showed that this locus contained at least 6 different alleles present in two or more of the 66 strains as well as 2 additional alleles that were each only present in one strain (Figure 5-7).

Figure 5-7. Two alleles of the idiomorphic SM gene cluster 10 present in one strain

each. (A) This allele contains an insertion of genes from Chromosome 6 immediately

upstream of Allele C (see Figure 5-6). None of these genes are likely SM gene cluster

backbone genes. An additional transposable element is found flanking this insertion. (B)

This allele contains an insertion of genes present in the A1163 reference but not in the

Af293 reference in the middle of Allele A (see Figure 5-6). None of these genes

are likely SM gene cluster backbone genes. One additional transposable element is

contained in this insertion.

In the Af293 reference genome, the cluster present at this locus contains one full-length

PKS gene along with multiple genes that contain NRPS- or PKS-associated domains (Allele C).

In the A1163 reference genome and 17 other strains, there is a full-length NRPS and a full-length

PKS (Allele B). These alleles show an almost complete lack of sequence similarity except for a

conserved hypothetical protein and a fragment of the full-length A1163 PKS in the Af293 allele; in contrast, the upstream and downstream flanking regions of the two alleles, which do not contain any backbone genes, are syntenic. Remarkably, another allele, present in 12 strains, contains all of the genes from both the Af293 and A1163 clusters (Allele D). The remaining three alleles contain various combinations of these genes. One allele found in 22 strains contains some A1163-specific genes and no Af293-specific genes (Allele A), while another allele found in 3 strains contains some Af293-specific genes but no A1163 genes (Allele F). The final allele, present in 8 strains, contains the entire Af293 allele as well as part of the A1163 allele (Allele E).

Every allele is littered with long terminal repeat sequence fragments from gypsy and copia TE families as well as with sequence fragments from DNA transposons from the mariner family

(Kapitonov and Jurka 2008). In some cases, these TEs correspond with breakpoints in synteny between alleles, suggesting that the diverse alleles of this SM gene cluster may arise via TE- driven recombination. Further, both of the alleles that are restricted to a single strain had an insertion event of several genes near a TE, though the rest of the locus is highly similar to one of

the more common alleles (Figure 5-7). The evolutionary history of this highly diverse locus is

unclear. While it is tempting to speculate that the largest allele containing all observed genes

represents the ancestral state, it does not explain the presence of a shared hypothetical protein

and PKS gene fragment between the Af293 locus (Allele C) and the A1163 locus (Allele B).

Genomic location polymorphisms

Figure 5-8. Multiple genomic locations of a horizontally transferred SM gene cluster.

(A) Genomic location of SM gene cluster 1 (Afu1g00970-01010) and flanking region in

all strains. This cluster is on chromosome 1 in two strains, chromosome 4 in three strains,

and adjacent to the intertwined fumagillin and pseurotin supercluster on chromosome 8 in

one strain. The flanking regions contain transposon-derived open reading frames including

two putative reverse transcriptases. (B) Synteny of A. fumigatus SM gene cluster 1 with

clusters in Phaeosphaeria nodorum, Pseudogymnoascus pannorum, Escovopsis weberi,

and Hypoxylon sp. CI4A. EOS denotes end of scaffold. All species contain non-syntenic

genes predicted by antiSMASH to be part of a biosynthetic gene cluster.

The final type of polymorphism that we observed is associated with SM gene clusters that are located in different genomic locations in different strains, suggesting that these SM gene clusters are behaving like mobile elements. This type of polymorphism was observed in SM gene clusters 1 and 33, both of which produce as yet to be identified products, and are present at low frequencies in the population.

SM gene cluster 1, which is present in six strains at three different genomic locations

(Figure 5-8A), consists of a PKS and four other modification genes that are always flanked by a

15 Kb region (upstream) and a 43 Kb region (downstream) containing TEs. In the reference

Af293 strain and in strain F7763, this SM gene cluster and its flanking regions are located on chromosome 1, while in strains dutch3, F13619, and Z5 they are located between Afu4g07320 and Afu4g07340 on chromosome 4. In contrast, in strain JCM_10253, the cluster and flanking regions are located on chromosome 8 immediately adjacent to the 3’ end of the intertwined fumagillin and pseurotin SM gene supercluster (Wiemann, Guo, et al. 2013).

In 5 / 6 strains, the cluster appears to be functional and does not contain nonsense SNPs or indels. However, the cluster found on chromosome 1 in strain F7763 contains two stop codons in the oxidoreductase gene (Gln121* and Gln220*) and two premature stop codons in the polyketide synthase (Gln1156* and Gln1542*), suggesting this strain contains a null allele.

This “jumping” gene cluster is not present in any other sequenced Aspergillus genus, and phylogenetic analysis of its constituent genes is consistent with horizontal gene transfer between fungi (Appendix C). Specifically, this gene cluster is also present in Phaeosphaeria nodorum, a plant pathogen from the class Dothideomycetes, Pseudogymnoascus pannorum, a fungus isolated from permafrost from the Leotiomycetes, Escovopsis weberi, and a fungal parasite of fungus- growing ants from the Sordariomycetes (Figure 5-8B). One additional species, the endophyte

Hypoxylon sp. CI4A from the class Sordariomycetes, contains four of the five cluster genes but is missing Afu1g00970, an MFS drug transporter. However, this species contains an unrelated gene annotated as an MFS drug transporter immediately adjacent to this cluster, so this species may be using a different transporter (Figure 5-8B). None of these fungi contain the upstream or downstream TE-rich flanking regions present in A. fumigatus, and each fungus contains additional unique genes with putative biosynthetic functions adjacent to the transferred cluster.

The most likely explanation for this change in flanking regions is that this SM gene cluster was transferred into A. fumigatus once and has subsequently moved its location in the genome.

100

Figure 5-9. Genomic contexts of mobile SM gene cluster 5. This gene cluster is located

on Chromosome 5 in two strains, on Chromosome 4 in two strains, and on Chromosome 1

in one strain adjacent to a putative SM gene cluster. Multiple transposable elements flank

the cluster in each strain.

The second SM gene cluster that shows variation in its genomic location across strains, cluster 33, contains a terpene synthase. This cluster is present in only 5 strains at 3 distinct locations (Figure 5-9). Similar to cluster 1, this cluster is also flanked by TEs and in one strain the cluster is located 58 Kb from another SM gene cluster. In contrast to cluster 1, this cluster does not appear to have been horizontally transferred between fungi and its genes are present in other sequenced Aspergillus species. As this mobile cluster was not horizontally transferred, it is possible that the horizontal transfer of cluster 1 and its mobile nature throughout the A. fumigatus genome are driven by different mechanisms.

101

Discussion

Figure 5-10. Types and frequencies of all SM gene cluster variants in the A. fumigatus

population.

Our examination of 66 genomes from strains of Aspergillus fumigatus revealed that five general types of polymorphisms describe variation in SM gene clusters in a fungal population.

These polymorphisms include variation in single nucleotides, gene and gene cluster gains and losses, non-homologous clusters at the same genomic position, and changes in genomic locations of clusters (Figure 5-10). In several cases, the genetic mechanisms that gave rise to these polymorphisms and their functional consequences are clear. Polymorphisms in single- nucleotides most likely arose from errors in DNA replication or repair, while gene loss

102 polymorphisms likely arose from genomic deletion or recombination events. Others, such as SM gene clusters that exist as non-homologous alleles or that entered the population through horizontal transfer, could arise through multiple genetic mechanisms. Transposable elements are common in polymorphic SM gene clusters and are found flanking mobile and horizontally transferred clusters, as well as in regions adjacent to non-homologous alleles and where gene gain has occurred, suggesting they contribute to SM gene cluster diversity. Using a population genomics approach to identify SM gene cluster variants allowed us to also capture and describe novel polymorphisms, including mobile gene clusters and idiomorphic clusters, which are difficult to identify in comparative genomic studies between species whose conservation of genome-wide synteny is low. Strikingly, the variants and genetic drivers we observe at the population level are also implicated as driving SM gene cluster variation between fungal species, suggesting that the observed microevolutionary processes are sufficient to explain macroevolutionary patterns of SM gene cluster evolution. Below, we discuss our key results and place them in the broader context of SM gene cluster evolution.

The first novel type of polymorphism was observed in SM gene clusters 1 and 33, which by occupying different genomic locations in different strains, appear to behave in a manner similar to mobile genetic elements. Interestingly, both clusters are located near or immediately adjacent to other SM gene clusters in some strains. For example, cluster 1 is located immediately adjacent to the intertwined fumagillin and pseurotin supercluster (Wiemann, Guo, et al. 2013) in one strain. This supercluster is regulated by the transcriptional factor fapR and is located in a chromosomal region controlled by the master SM regulators laeA and veA (Wiemann, Guo, et al.

2013; Lin et al. 2013), raising the hypothesis that these mobile gene clusters might be co-opting the regulatory machinery already in place. Previous work has hypothesized that the fumagillin

103 and pseurotin supercluster formed through genomic rearrangement events placing the once- independent gene clusters in close proximity to each other (Wiemann, Guo, et al. 2013). Our observation that this mobile gene cluster is located in this same region not only supports this hypothesis but also implicates transposable elements as one of the mechanisms by which such genomic rearrangements are formed. These superclusters may also represent an intermediate stage in the formation of new SM gene clusters. Supercluster formation, potentially mediated by mobile gene clusters, and followed by gene loss, could explain macroevolutionary patterns of

SM gene clusters have shown that clustered genes in one species can be dispersed over multiple gene clusters in other species (Robert H Proctor et al. 2009; Lind et al. 2015).

The second novel type of polymorphism was the presence of multiple non-homologous alleles at the cluster 10 locus, echoing the structure of the idiomorphic mating type locus

(Metzenberg and Glass 1990). This region has previously been reported to be a recombination hotspot in A. fumigatus (Abdolrasouli et al. 2015) and all alleles contain numerous transposable elements. Thus, it is possible that polymorphism at this locus originated via SM gene cluster fusion or splitting events driven by transposable elements. Interestingly, two other previously described instances of SM gene cluster variation bear close resemblance to the A. fumigatus idiomorphic SM gene cluster 10 locus. The first is the presence of two non-homologous

Aspergillus flavus alleles, where some strains contain a 9-gene sesquiterpene-like SM gene cluster and others contain a non-homologous 6-gene SM gene cluster at the same genomic location (Gibbons, Salichos, et al. 2012). The second is the presence of two non-homologous SM gene clusters at the same, well-conserved, locus in a comparison of six species of dermatophyte fungi (Zhang, Rokas, and Slot 2012). Based on these results, we hypothesize that idiomorphic

104

clusters may be common in fungal populations and contribute to the broad diversity of SM gene

clusters across filamentous fungi.

The remaining types of SM polymorphism in the A. fumigatus population have previously been described at the species level. For example, null alleles have been reported for many individual SM gene clusters across diverse fungal species (Schumacher et al. 2013;

Campbell, Rokas, and Slot 2012; P.-K. Chang, Horn, and Dorner 2005; Robert H. Proctor et al.

2008) and our results show that it is a widespread phenomenon, affecting two-thirds of the A. fumigatus SM gene clusters. Consistent with previous literature reporting the presence of additional SM gene clusters not present in the reference strain (Wiemann, Sieber, et al. 2013;

Chiara et al. 2015), we also identify four low-frequency SM gene clusters in the A. fumigatus population, two of which are horizontally transferred from distantly related fungi and two that appear vertically inherited. We find numerous cases of gene loss and relatively fewer cases of gene gain; previous studies have implicated these processes across numerous types of SM gene clusters and reported their effects on metabolite production (Schardl et al. 2013). Previous work has indicated that gene and gene cluster gain and loss tends to occur near telomeres, suggesting that higher rates of genetic events like recombination lead to loss (Young et al. 2015). While we do find several cases of gene and gene cluster loss in subtelomeric clusters, including the fumigaclavine gene cluster and the helvolic acid gene cluster, we also find gene loss in clusters that are not located near telomeres, such as the gliotoxin gene cluster and the fumiquinazoline gene cluster. These findings suggest that while gene and gene cluster loss can occur in telomeric regions, clusters in other genomic regions also experience high rates of loss.

Previous work has demonstrated that SM gene clusters, like the metabolites that they produce, are highly divergent between fungal species. Our examination of genome-wide

105 variation shows that these SM gene clusters are correspondingly diverse within individual strains of a single fungal species. Furthermore, the observed genetic changes in SM gene clusters are widespread across different types of gene clusters and are caused by many underlying genetic drivers, including gene and gene cluster gain, loss, non-homologous cluster alleles, and mobile gene clusters. The net effect of these substitutions, gains, losses, and rearrangements raises the hypothesis that fungal SM gene clusters are likely in a state of evolutionary flux, constantly altering their SM gene cluster repertoire, and consequently modifying and diversifying their chemodiversity.

106

CHAPTER VI

SUMMARY

In this dissertation, I examine how secondary metabolism regulation changes across species, across different stages of the fungal lifestyle, and across environmental conditions. This work reveals that secondary metabolism regulation has been extensively rewired across different fungi, that regulators of development also play roles in controlling secondary metabolism production, and that environmentally responsive regulators can integrate multiple environmental signals to fine tune the metabolites they regulate. I additionally examine the genetic drivers of secondary metabolic gene cluster diversity at the population level and discover numerous types of genetic variants that explain species-level distributions of these clusters. This dissertation contributes to our understanding of how transcriptional networks controlling species-specific processes evolve, as well as genetic factors that drive the diversification of fast evolving, clustered metabolic pathways.

In Chapter II, I examined how the transcriptional networks controlling secondary metabolism and development have evolved in Aspergillus. I demonstrated that no secondary metabolic gene clusters are conserved across the Aspergillus genus and that when any genes in these clusters are conserved, they are fragmented across different clusters and chromosomes in other species. By examining the genome-wide regulatory role of two master regulators of secondary metabolism, VeA and MtfA, in Aspergillus nidulans and Aspergillus fumigatus, I showed that VeA has been extensively transcriptionally rewired both with respect to secondary metabolism and development, and that MtfA’s regulatory role has changed between A. fumigatus and A. nidulans. This works shows that conserved secondary metabolic regulatory networks

107 control highly species-specific secondary metabolic pathways through extensive transcriptional rewiring. Under this bottom-up model of regulatory evolution, fast-evolving and novel secondary metabolic gene clusters are “plugged in” to an existing, environmentally responsive regulatory network.

Future directions. While this study demonstrated that the VeA regulator has been rewired across Aspergillus species, it is not clear how the structure of the regulatory network itself has changed. Studies of regulatory rewiring in other organisms, primarily yeast, have collectively demonstrated that different regulatory networks often experience common evolutionary constraints and share similar trajectories (Sorrells and Johnson 2015). However, these studies focus on the evolution of core biological processes that tend to be well conserved.

Because the targets of secondary metabolic regulatory networks are so fast evolving, the structure and evolution of these regulatory networks may have unique features that have not yet been described. In particular, it is not clear how horizontally transferred gene clusters, which occur at relatively high frequencies in fungal genomes, are integrated into the regulatory machinery of the accepting fungus. These questions could be addressed in the future using methods such as ChIP-seq and regulatory network reconstruction.

In Chapter III, I hypothesized that master regulators of secondary metabolism may be able to sense and respond to multiple different environmental cues. To test this, I examined the transcriptome-wide regulatory role of two members of the light-responsive Velvet regulatory complex, VeA and LaeA, at different temperatures. The regulatory role of LaeA, a putative methyltransferase, did not change at different temperatures, but the regulatory role of VeA changed at different temperatures. Notably, VeA regulated three SM gene clusters on

108

Chromosome 8 at 37 oC but not at 30 oC. These results, along with previous work that shows

carbon source concentration can affect VeA’s subcellular localization (Atoui et al. 2010), show

that this regulator responds to environmental stimuli other than light and can integrate multiple

environmental signals into its regulation of secondary metabolism.

Future directions. This work demonstrates that the VeA master regulator of secondary metabolism is capable of responding to multiple environmental signals. It is unclear if this is a general phenomenon of master secondary metabolic regulators, as LaeA showed no change in function at different temperatures. Further work could be done to test different VeA with different environmental conditions, or on testing different master regulators at different temperatures. Understanding cross-talk between different master regulators as well as combinatorial effects of environmental signals will likely reveal interesting links between the ecological roles of fungal secondary metabolites and their regulation.

In Chapter IV, I investigated how asexual development and secondary metabolism are coordinated at the transcriptional level. In Aspergillus fumigatus and many other fungi, asexual development and secondary metabolism production occur simultaneously. I examined how these processes are coordinated by determining the impact of central asexual regulatory pathways in

Aspergillus fumigatus on secondary metabolism production. I demonstrated that the asexual developmental regulator BrlA plays previously unknown roles in regulating both spore-specific secondary metabolites as well as secondary metabolites with no roles in development.

Future directions. BrlA is a highly conserved regulator across filamentous and non- filamentous fungi, and it was previously unknown that this regulator contributed broadly to secondary metabolism production. It is not clear whether BrlA regulates secondary metabolism

109

in other filamentous fungi or whether this is restricted to Aspergillus fumigatus. Examining

BrlA’s role in secondary metabolism regulation in other fungi and gaining a more complete

understanding of the regulatory network it controls will contribute to our understanding of

secondary metabolism regulation and its evolution.

I determined in Chapter II that secondary metabolic gene clusters are not conserved

across different species of Aspergillus. Attempting to understand the genetic mechanisms that

changed these clusters over time at this species-level comparison was difficult, as there was

almost no overlap in clusters. To understand what genetic factors drive variation in secondary

metabolic gene clusters, in Chapter V I compared the genomes of 66 strains of Aspergillus

fumigatus. This analysis revealed that secondary metabolic gene clusters vary dramatically at the

population level, but by using a population based analysis I was able to determine the likely

genetic events that gave rise to this variation. I found that SNVs and indels were extremely

common in secondary metabolic gene clusters, including nonsense and frameshift variants that

render the gene product nonfunctional. Other common types of genetic variation in SM gene

clusters included gene deletion and insertion events and entire gene cluster deletion and gain

events. I was further able to identify two mobile gene clusters that change location throughout

the genome, and a cluster with non-homologous alleles that resemble the “idiomorphic” mating

type locus in fungi. These types of genetic variants are sufficient to explain species-level differences in secondary metabolic gene clusters. The extent of this genetic variation within a single species suggests that these secondary metabolic gene clusters and metabolites that they produce are in a state of evolutionary flux where change and variation are the rule rather than the exception.

110

Future directions. The major limitation to this analysis is that the effects of these genetic

variants on fitness is not clear, as the majority of secondary metabolites in Aspergillus fumigatus

lack well understood ecological roles. While some contribute to the virulence of the fungus,

others likely play roles in other common secondary metabolite functions like self-protection and

microbial communication. Future work on understanding the contributions of these variants to

fitness and what constraints exist on secondary metabolite evolution will require more functional

work characterizing the metabolites themselves. Other experiments that would elucidate the

fitness costs and benefits of variants in secondary metabolic gene clusters would be experimental evolution tests and fitness tests of strains containing different variants.

111

REFERENCES

Abad, Ana, Jimena Victoria Fernández-Molina, Joseba Bikandi, Andoni Ramírez, Javier Margareto, Javier Sendino, Fernando Luis Hernando, Jose Pontón, Javier Garaizar, and Aitor Rementeria. 2010. “What Makes Aspergillus Fumigatus a Successful Pathogen? Genes and Molecules Involved in Invasive Aspergillosis.” Revista Iberoamericana de Micología 27 (4): 155–82. doi:10.1016/j.riam.2010.10.003. Abdolrasouli, Alireza, Johanna Rhodes, Mathew A Beale, Ferry Hagen, Thomas R Rogers, Anuradha Chowdhary, Jacques F Meis, Darius Armstrong-James, and Matthew C Fisher. 2015. “Genomic Context of Azole Resistance Mutations in Aspergillus Fumigatus Determined Using Whole-Genome Sequencing.” mBio 6 (3). doi:10.1128/mBio.00536-15. Ahmed, Yasar Luqman, Jennifer Gerke, Hee-Soo Park, Özgür Bayram, Piotr Neumann, Min Ni, Achim Dickmanns, et al. 2013. “The Velvet Family of Fungal Regulators Contains a DNA- Binding Domain Structurally Similar to NF-κB.” Edited by Ann M. Stock. PLoS Biology 11 (12). Public Library of Science: e1001750. doi:10.1371/journal.pbio.1001750. Anders, S., P. T. Pyl, and W. Huber. 2014. “HTSeq - A Python Framework to Work with High- Throughput Sequencing Data.” bioRxiv, February. Cold Spring Harbor Labs Journals. doi:10.1101/002824. Andersen, Mikael R, Jakob B Nielsen, Andreas Klitgaard, Lene M Petersen, Mia Zachariasen, Tilde J Hansen, Lene H Blicher, et al. 2013. “Accurate Prediction of Secondary Metabolite Gene Clusters in Filamentous Fungi.” Proceedings of the National Academy of Sciences of the United States of America 110 (1): E99-107. doi:10.1073/pnas.1205532110. Andersen, Mikael R, Margarita P Salazar, Peter J Schaap, Peter J I van de Vondervoort, David Culley, Jette Thykaer, Jens C Frisvad, et al. 2011. “Comparative Genomics of Citric-Acid- Producing Aspergillus Niger ATCC 1015 versus Enzyme-Producing CBS 513.88.” Genome Research 21 (6): 885–97. doi:10.1101/gr.112169.110. Arnaud, Martha B, Gustavo C Cerqueira, Diane O Inglis, Marek S Skrzypek, Jonathan Binkley, Marcus C Chibucos, Jonathan Crabtree, et al. 2012. “The Aspergillus Genome Database (AspGD): Recent Developments in Comprehensive Multispecies Curation, Comparative Genomics and Community Resources.” Nucleic Acids Research 40 (Database issue): D653- 9. doi:10.1093/nar/gkr875. Arnaud, Martha B, Marcus C Chibucos, Maria C Costanzo, Jonathan Crabtree, Diane O Inglis, Adil Lotia, Joshua Orvis, et al. 2010. “The Aspergillus Genome Database, a Curated Comparative Genomics Resource for Gene, Protein and Sequence Information for the Aspergillus Research Community.” Nucleic Acids Research 38 (Database issue): D420-7. doi:10.1093/nar/gkp751. Arst, Herbert N. 1996. “Biochemistry and Molecular Microbiology.” In The Mycota, edited by R Brable and G.A. Marzluf, 235–40. Springer. Asami, Yukihiro, Hideaki Kakeya, Rie Onose, Yie-Hwa Chang, Masakazu Toi, and Hiroyuki

112

Osada. 2004. “RK-805, an Endothelial-Cell-Growth Inhibitor Produced by Neosartorya Sp., and a Docking Model with Methionine Aminopeptidase-2.” Tetrahedron 60 (33): 7085–91. doi:10.1016/j.tet.2003.09.104. Ashburner, M, C A Ball, J A Blake, D Botstein, H Butler, J M Cherry, A P Davis, et al. 2000. “Gene Ontology: Tool for the Unification of Biology. The Gene Ontology Consortium.” Nature Genetics 25 (1). Nature America Inc.: 25–29. doi:10.1038/75556. Atoui, a., C. Kastner, C. M. Larey, R. Thokala, O. Etxebeste, E. a. Espeso, R. Fischer, and a. M. Calvo. 2010. “Cross-Talk between Light and Glucose Regulation Controls Toxin Production and Morphogenesis in Aspergillus Nidulans.” Fungal Genetics and Biology 47 (12). Elsevier Inc.: 962–72. doi:10.1016/j.fgb.2010.08.007. Bankevich, Anton, Sergey Nurk, Dmitry Antipov, Alexey A Gurevich, Mikhail Dvorkin, Alexander S Kulikov, Valery M Lesin, et al. 2012. “SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing.” Journal of Computational Biology : A Journal of Computational Molecular Cell Biology 19 (5). Mary Ann Liebert, Inc.: 455–77. doi:10.1089/cmb.2012.0021. Barker, Bridget M, Kristin Kroll, Martin Vödisch, Aurélien Mazurie, Olaf Kniemeyer, and Robert A Cramer. 2012. “Transcriptomic and Proteomic Analyses of the Aspergillus Fumigatus Hypoxia Response Using an Oxygen-Controlled Fermenter.” BMC Genomics 13 (1): 62. doi:10.1186/1471-2164-13-62. Bayram, Ozgür, and Gerhard H Braus. 2012. “Coordination of Secondary Metabolism and Development in Fungi: The Velvet Family of Regulatory Proteins.” FEMS Microbiology Reviews 36 (1): 1–24. doi:10.1111/j.1574-6976.2011.00285.x. Bayram, Özgür, Sven Krappmann, Min Ni, Jin Woo Bok, Kerstin Helmstaedt, Jae-Hyuk Yu, Gerhard H Braus, et al. 2008. “VelB/VeA/LaeA Complex Coordinates Light Signal with Fungal Development and Secondary Metabolism.” Science 320 (5882): 1504–6. doi:10.1126/science.1155888. Beffa, T, F Staib, J Lott Fischer, P F Lyon, P Gumowski, O E Marfenina, S Dunoyer-Geindre, et al. 1998. “Mycological Control and Surveillance of Biological Waste and Compost.” Medical Mycology 36 Suppl 1 (January): 137–45. Benjamini, Yoav, and Yosef Hochberg. 1995. “Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing.” Journal of the Royal Statistical Society. Series B (Methodological) 57 (1). Wiley for the Royal Statistical Society: 289–300. Bennett, J W, and M Klich. 2003. “Mycotoxins.” Clinical Microbiology Reviews 16 (3): 497– 516. Bennett, JW, and R Bentley. 1989. “What’s in a name?—Microbial Secondary Metabolism.” Advances in Applied Microbiology 34. Bergmann, Sebastian, Alexander N Funk, Kirstin Scherlach, Volker Schroeckh, Ekaterina Shelest, Uwe Horn, Christian Hertweck, and Axel A Brakhage. 2010. “Activation of a

113

Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic Nonribosomal Peptide Synthetase Gene Cluster.” Applied and Environmental Microbiology 76 (24): 8143–49. doi:10.1128/AEM.00683-10. Berry, David B., Qiaoning Guan, James Hose, Suraiya Haroon, Marinella Gebbia, Lawrence E. Heisler, Corey Nislow, et al. 2011. “Multiple Means to the Same End: The Genetic Basis of Acquired Stress Resistance in Yeast.” Edited by Gregory P. Copenhaver. PLoS Genetics 7 (11). Public Library of Science: e1002353. doi:10.1371/journal.pgen.1002353. Berthier, Erwin, Fang Yun Lim, Qing Deng, Chun-Jun Guo, Dimitrios P Kontoyiannis, Clay C C Wang, Julie Rindy, David J Beebe, Anna Huttenlocher, and Nancy P Keller. 2013. “Low- Volume Toolbox for the Discovery of Immunosuppressive Fungal Secondary Metabolites.” Edited by Stuart M. Levitz. PLoS Pathogens 9 (4). Public Library of Science: e1003289. doi:10.1371/journal.ppat.1003289. Bignell, Elaine, Timothy C. Cairns, Kurt Throckmorton, William C. Nierman, and Nancy P. Keller. 2016. “Secondary Metabolite Arsenal of an Opportunistic Pathogenic Fungus.” Philosophical Transactions of the Royal Society B: Biological Sciences 371 (1709). Bignell, Elaine, Susana Negrete-Urtasun, Ana Maria Calcagno, Ken Haynes, Herbert N Arst, and Tom Rogers. 2005. “The Aspergillus pH-Responsive Transcription Factor PacC Regulates Virulence.” Molecular Microbiology 55 (4): 1072–84. doi:10.1111/j.1365- 2958.2004.04472.x. Blatzer, Michael, Bridget M Barker, Sven D Willger, Nicola Beckmann, Sara J Blosser, Elizabeth J Cornish, Aurelien Mazurie, Nora Grahl, Hubertus Haas, and Robert A Cramer. 2011. “SREBP Coordinates Iron and Ergosterol Homeostasis to Mediate Triazole Drug and Hypoxia Responses in the Human Fungal Pathogen Aspergillus Fumigatus.” PLoS Genetics 7 (12): e1002374. doi:10.1371/journal.pgen.1002374. Böhnert, Heidi U, Isabelle Fudal, Waly Dioh, Didier Tharreau, Jean-Loup Notteghem, and Marc- Henri Lebrun. 2004. “A Putative Polyketide Synthase/peptide Synthetase from Magnaporthe Grisea Signals Pathogen Attack to Resistant Rice.” The Plant Cell 16 (9). American Society of Plant Biologists: 2499–2513. doi:10.1105/tpc.104.022715. Bok, Jin Woo, DaWoon Chung, S Arunmozhi Balajee, Kieren A Marr, David Andes, Kristian Fog Nielsen, Jens C Frisvad, Katharine A Kirby, and Nancy P Keller. 2006. “GliZ, a Transcriptional Regulator of Gliotoxin Biosynthesis, Contributes to Aspergillus Fumigatus Virulence.” Infection and Immunity 74 (12). American Society for Microbiology: 6761–68. doi:10.1128/IAI.00780-06. Bok, Jin Woo, Dirk Hoffmeister, Lori A. Maggio-Hall, Renato Murillo, Jeremy D. Glasner, and Nancy P. Keller. 2006. “Genomic Mining for Aspergillus Natural Products.” Chemistry & Biology 13 (1): 31–37. Bolger, Anthony M, Marc Lohse, and Bjoern Usadel. 2014. “Trimmomatic: A Flexible Trimmer for Illumina Sequence Data.” Bioinformatics (Oxford, England) 30 (15): 2114–20. doi:10.1093/bioinformatics/btu170.

114

Brakhage, A., S. Bergmann, J. Schuemann, K. Scherlach, V. Schroeckh, and C. Hertweck. 2009. “Fungal Genome Mining and Activation of Silent Gene Clusters.” In The Mycota XV, edited by K Esser, 297–303. Heidelberg: Sprigner-Verlag. Brakhage, Axel a. 2013. “Regulation of Fungal Secondary Metabolism.” Nature Reviews. Microbiology 11 (1). Nature Publishing Group: 21–32. doi:10.1038/nrmicro2916. Brakhage, Axel A. 1998. “Molecular Regulation of Beta -Lactam Biosynthesis in Filamentous Fungi.” Microbiol. Mol. Biol. Rev. 62 (3): 547–85. Brakhage, Axel A, and Volker Schroeckh. 2011. “Fungal Secondary Metabolites - Strategies to Activate Silent Gene Clusters.” Fungal Genetics and Biology : FG & B 48 (1): 15–22. doi:10.1016/j.fgb.2010.04.004. Brown, Daren W, Robert A E Butchko, Mark Busman, and Robert H Proctor. 2007. “The Fusarium Verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production.” Eukaryotic Cell 6 (7): 1210–18. doi:10.1128/EC.00400-06. Brown, D W, J H Yu, H S Kelkar, M Fernandes, T C Nesbitt, N P Keller, T H Adams, and T J Leonard. 1996. “Twenty-Five Coregulated Transcripts Define a Sterigmatocystin Gene Cluster in Aspergillus Nidulans.” Proceedings of the National Academy of Sciences of the United States of America 93 (4): 1418–22. Bushley, Kathryn E, and B Gillian Turgeon. 2010. “Phylogenomics Reveals Subfamilies of Fungal Nonribosomal Peptide Synthetases and Their Evolutionary Relationships.” BMC Evolutionary Biology 10 (1): 26. doi:10.1186/1471-2148-10-26. Caddick, M X, and H N Arst. 1998. “Deletion of the 389 N-Terminal Residues of the Transcriptional Activator AREA Does Not Result in Nitrogen Metabolite Derepression in Aspergillus Nidulans.” Journal of Bacteriology 180 (21). American Society for Microbiology: 5762–64. Calvo, AM, JM Lohmar, B Ibarra, and T Satterlee. 2016. “Velvet Regulation of Fungal Development.” In Mycota Volume 1, edited by Jürgen Wendland, 3rded., 475–97. Springer International Publishing. Calvo, Ana M. 2008. “The VeA Regulatory System and Its Role in Morphological and Chemical Development in Fungi.” Fungal Genetics and Biology 45 (7): 1053–61. doi:10.1016/j.fgb.2008.03.014. Calvo, Ana M, and Jeffrey W Cary. 2015. “Association of Fungal Secondary Metabolism and Sclerotial Biology.” Frontiers in Microbiology 6 (January): 62. doi:10.3389/fmicb.2015.00062. Calvo, Ana M, Richard A Wilson, Jin Woo Bok, and Nancy P Keller. 2002. “Relationship between Secondary Metabolism and Fungal Development.” Microbiology and Molecular Biology Reviews : MMBR 66 (3): 447–59, table of contents. Campbell, Matthew a, Antonis Rokas, and Jason C Slot. 2012. “Horizontal Transfer and Death

115

of a Fungal Secondary Metabolic Gene Cluster.” Genome Biology and Evolution 4 (3): 289–93. doi:10.1093/gbe/evs011. Capella-Gutierrez, S., J. M. Silla-Martinez, and T. Gabaldon. 2009. “trimAl: A Tool for Automated Alignment Trimming in Large-Scale Phylogenetic Analyses.” Bioinformatics 25 (15): 1972–73. doi:10.1093/bioinformatics/btp348. Carbon, Seth, Amelia Ireland, Christopher J Mungall, ShengQiang Shu, Brad Marshall, and Suzanna Lewis. 2009. “AmiGO: Online Access to Ontology and Annotation Data.” Bioinformatics (Oxford, England) 25 (2): 288–89. doi:10.1093/bioinformatics/btn615. Carbone, Ignazio, Jorge H Ramirez-Prado, Judy L Jakobek, and Bruce W Horn. 2007. “Gene Duplication, Modularity and Adaptation in the Evolution of the Aflatoxin Gene Cluster.” BMC Evolutionary Biology 7 (1): 111. doi:10.1186/1471-2148-7-111. Chanda, Anindya, Ludmila V Roze, Suil Kang, Katherine A Artymovich, Glenn R Hicks, Natasha V Raikhel, Ana M Calvo, and John E Linz. 2009. “A Key Role for Vesicles in Fungal Secondary Metabolism.” Proceedings of the National Academy of Sciences of the United States of America 106 (46): 19533–38. doi:10.1073/pnas.0907416106. Chang, P K, K C Ehrlich, J Yu, D Bhatnagar, and T E Cleveland. 1995. “Increased Expression of Aspergillus Parasiticus aflR, Encoding a Sequence-Specific DNA-Binding Protein, Relieves Nitrate Inhibition of Aflatoxin Biosynthesis.” Applied and Environmental Microbiology 61 (6): 2372–77. Chang, Perng-Kuang, Bruce W. Horn, and Joe W. Dorner. 2005. “Sequence Breakpoints in the Aflatoxin Biosynthesis Gene Cluster and Flanking Regions in Nonaflatoxigenic Aspergillus Flavus Isolates.” Fungal Genetics and Biology 42 (11): 914–23. doi:10.1016/j.fgb.2005.07.004. Chang, Perng Kuang, and Kenneth C. Ehrlich. 2013. “Genome-Wide Analysis of the Zn(II)2Cys6 Zinc Cluster-Encoding Gene Family in Aspergillus Flavus.” Applied Microbiology and Biotechnology. doi:10.1007/s00253-013-4865-2. Chettri, Pranav, Ana M Calvo, Jeffrey W Cary, Sourabh Dhingra, Yanan Guo, Rebecca L McDougal, and Rosie E Bradshaw. 2012. “The veA Gene of the Pine Needle Pathogen Dothistroma Septosporum Regulates Sporulation and Secondary Metabolism.” Fungal Genetics and Biology : FG & B 49 (2): 141–51. doi:10.1016/j.fgb.2011.11.009. Chiang, Yi-Ming, Edyta Szewczyk, Tania Nayak, Ashley D Davidson, James F Sanchez, Hsien- Chun Lo, Wen-Yueh Ho, et al. 2008. “Molecular Genetic Mining of the Aspergillus Secondary Metabolome: Discovery of the Emericellamide Biosynthetic Pathway.” Chemistry & Biology 15 (6): 527–32. doi:10.1016/j.chembiol.2008.05.010. Chiara, Matteo, Francesca Fanelli, Giuseppina Mulè, Antonio F. Logrieco, Graziano Pesole, John F. Leslie, David S. Horner, and Christopher Toomajian. 2015. “Genome Sequencing of Multiple Isolates Highlights Subtelomeric Genomic Diversity within Fusarium Fujikuroi.” Genome Biology and Evolution 7 (11): 3062–69. doi:10.1093/gbe/evv198.

116

Chou, S., S. Lane, and H. Liu. 2006. “Regulation of Mating and Filamentation Genes by Two Distinct Ste12 Complexes in Saccharomyces Cerevisiae.” Molecular and Cellular Biology 26 (13): 4794–4805. doi:10.1128/MCB.02053-05. Christensen, Martha, J.C. Frisvad, and Dorothy Tuthill. 1999. “Taxonomy of the Penicillium Miczynskii Group Based on Morphology and Secondary Metabolites.” Mycological Research 103 (5): 527–41. doi:10.1017/S0953756298007515. Cingolani, Pablo, Adrian Platts, Le Lily Wang, Melissa Coon, Tung Nguyen, Luan Wang, Susan J Land, Xiangyi Lu, and Douglas M Ruden. 2012. “A Program for Annotating and Predicting the Effects of Single Nucleotide Polymorphisms, SnpEff: SNPs in the Genome of Drosophila Melanogaster Strain w1118; Iso-2; Iso-3.” Fly 6 (2). Taylor & Francis: 80– 92. doi:10.4161/fly.19695. Coméra, Christine, Karine André, Joëlle Laffitte, Xavier Collet, Pierre Galtier, and Isabelle Maridonneau-Parini. 2007. “Gliotoxin from Aspergillus Fumigatus Affects Phagocytosis and the Organization of the Actin Cytoskeleton by Distinct Signalling Pathways in Human Neutrophils.” Microbes and Infection / Institut Pasteur 9 (1): 47–54. doi:10.1016/j.micinf.2006.10.009. Coyle, Christine M, Shawn C Kenaley, William R Rittenour, and Daniel G Panaccione. 2007. “Association of Ergot Alkaloids with Conidiation in Aspergillus Fumigatus.” Mycologia 99 (6). Mycological Society of America: 804–11. doi:10.3852/MYCOLOGIA.99.6.804. Crabtree, Jonathan, Samuel V Angiuoli, Jennifer R Wortman, and Owen R White. 2007. “Sybil: Methods and Software for Multiple Genome Comparison and Visualization.” Methods in Molecular Biology (Clifton, N.J.) 408 (January): 93–108. doi:10.1007/978-1-59745-547- 3_6. de Hoog, GS, J Guarro, J Gené, and MJ Figueras. 2001. Atlas of Clinical Fungi. Washington, DC: ASM Press. de Weert, Sandra, Irene Kuiper, Ellen L. Lagendijk, Gerda E. M. Lamers, and Ben J. J. Lugtenberg. 2004. “Role of Chemotaxis Toward Fusaric Acid in Colonization of Hyphae of Fusarium Oxysporum F. Sp. Radicis-Lycopersici by Pseudomonas Fluorescens WCS365.” Molecular Plant-Microbe Interactions 17 (11): 1185–91. doi:10.1094/MPMI.2004.17.11.1185. Delaye, Luis, Graciela García-Guzmán, and Martin Heil. 2013. “Endophytes versus Biotrophic and Necrotrophic Pathogens—are Fungal Lifestyles Evolutionarily Stable Traits?” Fungal Diversity 60 (1). Springer Netherlands: 125–35. doi:10.1007/s13225-013-0240-y. Demain, A L, and A Fang. 2000. “The Natural Functions of Secondary Metabolites.” Advances in Biochemical Engineering/biotechnology 69 (January): 1–39. DePristo, Mark A, Eric Banks, Ryan Poplin, Kiran V Garimella, Jared R Maguire, Christopher Hartl, Anthony A Philippakis, et al. 2011. “A Framework for Variation Discovery and Genotyping Using next-Generation DNA Sequencing Data.” Nature Genetics 43 (5): 491– 98. doi:10.1038/ng.806.

117

Dhingra, S, D Andes, and A M Calvo. 2012. “VeA Regulates Conidiation, Gliotoxin Production and Protease Activity in the Opportunistic Human Pathogen Aspergillus Fumigatus.” Eukaryotic Cell 11 (12): 1531–43. doi:10.1128/EC.00222-12. Dhingra, Sourabh, Abigail L Lind, Hsiao-Ching Lin, Yi Tang, Antonis Rokas, and Ana M Calvo. 2013. “The Fumagillin Gene Cluster, an Example of Hundreds of Genes under veA Control in Aspergillus Fumigatus.” Edited by Brett Neilan. PloS One 8 (10). Public Library of Science: e77147. doi:10.1371/journal.pone.0077147. Dolan, Stephen K., Grainne O’Keeffe, Gary W. Jones, and Sean Doyle. 2015. “Resistance Is Not Futile: Gliotoxin Biosynthesis, Functionality and Utility.” Trends in Microbiology. doi:10.1016/j.tim.2015.02.005. Dowzer, C E, and J M Kelly. 1991. “Analysis of the creA Gene, a Regulator of Carbon Catabolite Repression in Aspergillus Nidulans.” Molecular and Cellular Biology 11 (11). American Society for Microbiology (ASM): 5701–9. Eddy, Sean R. 2009. “A New Generation of Homology Search Tools Based on Probabilistic Inference.” Genome Informatics. International Conference on Genome Informatics 23 (1): 205–11. Ehrlich, K. C., P.-K. Chang, J. Yu, and P. J. Cotty. 2004. “Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation.” Applied and Environmental Microbiology 70 (11): 6518–24. doi:10.1128/AEM.70.11.6518-6524.2004. Enright, A J, S Van Dongen, and C A Ouzounis. 2002. “An Efficient Algorithm for Large-Scale Detection of Protein Families.” Nucleic Acids Research 30 (7): 1575–84. Espeso, E. A., and M. A. Penalva. 1996. “Three Binding Sites for the Aspergillus Nidulans PacC Zinc-Finger Transcription Factor Are Necessary and Sufficient for Regulation by Ambient pH of the Isopenicillin N Synthase Gene Promoter.” Journal of Biological Chemistry 271 (46): 28825–30. doi:10.1074/jbc.271.46.28825. Fedorova, Natalie D, Nora Khaldi, Vinita S Joardar, Rama Maiti, Paolo Amedeo, Michael J Anderson, Jonathan Crabtree, et al. 2008. “Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus Fumigatus.” Edited by Paul M. Richardson. PLoS Genetics 4 (4). Public Library of Science: e1000046. doi:10.1371/journal.pgen.1000046. Feng, Guo Hong, and Thomas J. Leonard. 1998. “Culture Conditions Control Expression of the Genes for Aflatoxin and Sterigmatocystin Biosynthesis in Aspergillus Parasiticus and A. Nidulans.” Appl. Envir. Microbiol. 64 (6): 2275–77. Fox, Ellen M, and Barbara J Howlett. 2008. “Secondary Metabolism: Regulation and Role in Fungal Biology.” Current Opinion in Microbiology 11 (6): 481–87. doi:10.1016/j.mib.2008.10.007. Frisvad, Jens C, Birgitte Andersen, and Ulf Thrane. 2008. “The Use of Secondary Metabolite Profiling in Chemotaxonomy of Filamentous Fungi.” Mycological Research 112 (Pt 2): 231–40. doi:10.1016/j.mycres.2007.08.018.

118

Frisvad, Jens C, Christian Rank, Kristian F Nielsen, and Thomas O Larsen. 2009. “Metabolomics of Aspergillus Fumigatus.” Medical Mycology 47 Suppl 1 (January). Informa UK Ltd UK: S53-71. doi:10.1080/13693780802307720. Galagan, James E, Sarah E Calvo, Christina Cuomo, Li-Jun Ma, Jennifer R Wortman, Serafim Batzoglou, Su-In Lee, et al. 2005. “Sequencing of Aspergillus Nidulans and Comparative Analysis with A. Fumigatus and A. Oryzae.” Nature 438 (7071): 1105–15. doi:10.1038/nature04341. Gardiner, Donald M, and Barbara J Howlett. 2005. “Bioinformatic and Expression Analysis of the Putative Gliotoxin Biosynthetic Gene Cluster of Aspergillus Fumigatus.” FEMS Microbiology Letters 248 (2): 241–48. doi:10.1016/j.femsle.2005.05.046. Gibbons, John G, Anne Beauvais, Remi Beau, Kriston L McGary, Jean-Paul Latgé, and Antonis Rokas. 2012. “Global Transcriptome Changes Underlying Colony Growth in the Opportunistic Human Pathogen Aspergillus Fumigatus.” Eukaryotic Cell 11 (1): 68–78. doi:10.1128/EC.05102-11. Gibbons, John G, and Antonis Rokas. 2012. “The Function and Evolution of the Aspergillus Genome.” Trends in Microbiology 21 (1): 14–22. doi:10.1016/j.tim.2012.09.005. Gibbons, John G, Leonidas Salichos, Jason C Slot, David C Rinker, Kriston L McGary, Jonas G King, Maren a Klich, David L Tabb, W Hayes McDonald, and Antonis Rokas. 2012. “The Evolutionary Imprint of Domestication on Genome Variation and Function of the Filamentous Fungus Aspergillus Oryzae.” Current Biology : CB 22 (15): 1403–9. doi:10.1016/j.cub.2012.05.033. Giuliano Garisto Donzelli, Bruno, Donna M. Gibson, and Stuart B. Krasnoff. 2015. “Intracellular Siderophore but Not Extracellular Siderophore Is Required for Full Virulence in Metarhizium Robertsii.” Fungal Genetics and Biology 82 (September): 56–68. doi:10.1016/j.fgb.2015.06.008. Gorner, W., E Durchschlag, M T Martinez-Pastor, F Estruch, G Ammerer, B Hamilton, H Ruis, and C. Schuller. 1998. “Nuclear Localization of the C2H2 Zinc Finger Protein Msn2p Is Regulated by Stress and Protein Kinase A Activity.” Genes & Development 12 (4): 586–97. doi:10.1101/gad.12.4.586. Greene, George H., Kriston L. McGary, Antonis Rokas, and Jason C. Slot. 2014. “Ecology Drives the Distribution of Specialized Tyrosine Metabolism Modules in Fungi.” Genome Biology and Evolution 6 (1). Sinauer Associates, Sunderland (MA): 121–32. doi:10.1093/gbe/evt208. Gressler, Markus, Florian Meyer, Daniel Heine, Peter Hortschansky, Christian Hertweck, and Matthias Brock. 2015. “Phytotoxin Production in Aspergillus Terreus Is Regulated by Independent Environmental Signals.” eLife 4 (January). eLife Sciences Publications Limited: e07861. doi:10.7554/eLife.07861. Gurevich, A., V. Saveliev, N. Vyahhi, and G. Tesler. 2013. “QUAST: Quality Assessment Tool for Genome Assemblies.” Bioinformatics 29 (8): 1072–75.

119

doi:10.1093/bioinformatics/btt086. Heinekamp, Thorsten, Andreas Thywißen, Juliane Macheleidt, Sophia Keller, Vito Valiante, and Axel A. Brakhage. 2012. “Aspergillus Fumigatus Melanins: Interference with the Host Endocytosis Pathway and Impact on Virulence.” Frontiers in Microbiology. doi:10.3389/fmicb.2012.00440. Hibbett, David S, and P Brandon Matheny. 2009. “The Relative Ages of Ectomycorrhizal Mushrooms and Their Plant Hosts Estimated Using Bayesian Relaxed Molecular Clock Analyses.” BMC Biology 7 (1): 13. doi:10.1186/1741-7007-7-13. Hoff, Birgit, Jens Kamerewerd, Claudia Sigl, Rudolf Mitterbauer, Ivo Zadra, Hubert Kürnsteiner, and Ulrich Kück. 2010. “Two Components of a Velvet-like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium Chrysogenum.” Eukaryotic Cell 9 (8): 1236–50. doi:10.1128/EC.00077-10. Hoffmeister, Dirk, and Nancy P Keller. 2007. “Natural Products of Filamentous Fungi: Enzymes, Genes, and Their Regulation.” Natural Product Reports 24 (2): 393–416. doi:10.1039/b603084j. Inglis, Diane O, Jonathan Binkley, Marek S Skrzypek, Martha B Arnaud, Gustavo C Cerqueira, Prachi Shah, Farrell Wymore, Jennifer R Wortman, and Gavin Sherlock. 2013. “Comprehensive Annotation of Secondary Metabolite Biosynthetic Genes and Gene Clusters of Aspergillus Nidulans, A. Fumigatus, A. Niger and A. Oryzae.” BMC Microbiology 13 (1): 91. doi:10.1186/1471-2180-13-91. Kapitonov, Vladimir V., and Jerzy Jurka. 2008. “A Universal Classification of Eukaryotic Transposable Elements Implemented in Repbase.” Nature Reviews Genetics 9 (5). Nature Publishing Group: 411–12. doi:10.1038/nrg2165-c1. Kato, Naoki, Wilhelmina Brooks, and Ana M Calvo. 2003. “The Expression of Sterigmatocystin and Penicillin Genes in Aspergillus Nidulans Is Controlled by veA, a Gene Required for Sexual Development.” Eukaryotic Cell 2 (6): 1178–86. Katoh, K., and D. M. Standley. 2013. “MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability.” Molecular Biology and Evolution 30 (4): 772–80. doi:10.1093/molbev/mst010. Kazan, Kemal, Donald M. Gardiner, and John M. Manners. 2012. “On the Trail of a Cereal Killer: Recent Advances in Fusarium Graminearum Pathogenomics and Host Resistance.” Molecular Plant Pathology. doi:10.1111/j.1364-3703.2011.00762.x. Keller, Nancy P. 2015. “Translating Biosynthetic Gene Clusters into Fungal Armor and Weaponry.” Nature Chemical Biology 11 (9). Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.: 671–77. doi:10.1038/nchembio.1897. Keller, Nancy P., and Thomas M. Hohn. 1997. “Metabolic Pathway Gene Clusters in Filamentous Fungi.” Fungal Genetics and Biology 21 (1): 17–29. Keller, Nancy P, Geoffrey Turner, and Joan W Bennett. 2005. “Fungal Secondary Metabolism -

120

from Biochemistry to Genomics.” Nature Reviews. Microbiology 3 (12): 937–47. doi:10.1038/nrmicro1286. Kennedy, J, K Auclair, S G Kendrew, C Park, J C Vederas, and C R Hutchinson. 1999. “Modulation of Polyketide Synthase Activity by Accessory Proteins during Lovastatin Biosynthesis.” Science (New York, N.Y.) 284 (5418): 1368–72. Khaldi, Nora, Jérôme Collemare, Marc-Henri Lebrun, and Kenneth H Wolfe. 2008. “Evidence for Horizontal Transfer of a Secondary Metabolite Gene Cluster between Fungi.” Genome Biology 9 (1). BioMed Central: R18. doi:10.1186/gb-2008-9-1-r18. Khaldi, Nora, Fayaz T Seifuddin, Geoff Turner, Daniel Haft, William C Nierman, Kenneth H Wolfe, and Natalie D Fedorova. 2010. “SMURF: Genomic Mapping of Fungal Secondary Metabolite Clusters.” Fungal Genetics and Biology 47 (9): 736–41. doi:10.1016/j.fgb.2010.06.003. Khaldi, Nora, and Kenneth H Wolfe. 2011. “Evolutionary Origins of the Fumonisin Secondary Metabolite Gene Cluster in Fusarium Verticillioides and Aspergillus Niger.” International Journal of Evolutionary Biology 2011. Hindawi Publishing Corporation: 423821. doi:10.4061/2011/423821. Kim, Daehwan, Geo Pertea, Cole Trapnell, Harold Pimentel, Ryan Kelley, and Steven L Salzberg. 2013. “TopHat2: Accurate Alignment of Transcriptomes in the Presence of Insertions, Deletions and Gene Fusions.” Genome Biology 14 (4): R36. doi:10.1186/gb- 2013-14-4-r36. Kim, Hee-Seo, Kyu-Yong Han, Kyung-Jin Kim, Dong-Min Han, Kwang-Yeop Jahng, and Keon- Sang Chae. 2002. “The veA Gene Activates Sexual Development in Aspergillus Nidulans.” Fungal Genetics and Biology 37 (1): 72–80. doi:10.1016/S1087-1845(02)00029-4. Kiyota, Takuro, Ryoko Hamada, Kazutoshi Sakamoto, Kazuhiro Iwashita, Osamu Yamada, and Shigeaki Mikami. 2011. “Aflatoxin Non-Productivity of Aspergillus Oryzae Caused by Loss of Function in the aflJ Gene Product.” Journal of Bioscience and Bioengineering 111 (5): 512–17. doi:10.1016/j.jbiosc.2010.12.022. Knox, Benjamin P., Adriana Blachowicz, Jonathan M. Palmer, Jillian Romsdahl, Anna Huttenlocher, Clay C. C. Wang, Nancy P. Keller, and Kasthuri Venkateswaran. 2016. “Characterization of Aspergillus Fumigatus Isolates from Air and Surfaces of the International Space Station.” mSphere 1 (5). König, Claudia C, Kirstin Scherlach, Volker Schroeckh, Fabian Horn, Sandor Nietzsche, Axel A Brakhage, and Christian Hertweck. 2013. “Bacterium Induces Cryptic Meroterpenoid Pathway in the Pathogenic Fungus Aspergillus Fumigatus.” Chembiochem : A European Journal of Chemical Biology 14 (8): 938–42. doi:10.1002/cbic.201300070. Krings, Michael, Thomas N. Taylor, and Nora Dotzler. 2012. “Fungal Endophytes as a Driving Force in Land Plant Evolution: Evidence from the Fossil Record.” In Biocomplexity of Plant-Fungal Interactions, 5–27. Oxford, UK: Wiley-Blackwell. doi:10.1002/9781118314364.ch1.

121

Kroken, Scott, N Louise Glass, John W Taylor, O C Yoder, and B Gillian Turgeon. 2003. “Phylogenomic Analysis of Type I Polyketide Synthase Genes in Pathogenic and Saprobic Ascomycetes.” Proceedings of the National Academy of Sciences of the United States of America 100 (26): 15670–75. doi:10.1073/pnas.2532165100. Krzywinski, Martin, Jacqueline Schein, Inanç Birol, Joseph Connors, Randy Gascoyne, Doug Horsman, Steven J Jones, and Marco A Marra. 2009. “Circos: An Information Aesthetic for Comparative Genomics.” Genome Research 19 (9): 1639–45. doi:10.1101/gr.092759.109. Latge, Jean-Paul. 1999. “Aspergillus Fumigatus and Aspergillosis.” Clin. Microbiol. Rev. 12 (2): 310–50. Letunic, Ivica, and Peer Bork. 2016. “Interactive Tree of Life (iTOL) v3: An Online Tool for the Display and Annotation of Phylogenetic and Other Trees.” Nucleic Acids Research 44 (W1): W242–45. doi:10.1093/nar/gkw290. Li, H., and R. Durbin. 2009. “Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform.” Bioinformatics 25 (14): 1754–60. doi:10.1093/bioinformatics/btp324. Lim, Fang Yun, Brian Ames, Christopher T Walsh, and Nancy P Keller. 2014. “Co-Ordination between BrlA Regulation and Secretion of the Oxidoreductase FmqD Directs Selective Accumulation of Fumiquinazoline C to Conidial Tissues in Aspergillus Fumigatus.” Cellular Microbiology 16 (8): 1267–83. doi:10.1111/cmi.12284. Lin, Hsiao-Ching, Yit-Heng Chooi, Sourabh Dhingra, Wei Xu, Ana M Calvo, and Yi Tang. 2013. “The Fumagillin Biosynthetic Gene Cluster in Aspergillus Fumigatus Encodes a Cryptic Terpene Cyclase Involved in the Formation of β-Trans-Bergamotene.” Journal of the American Chemical Society 135 (12): 4616–19. doi:10.1021/ja312503y. Lind, Abigail L, Jennifer H Wisecaver, Timothy D Smith, Xuehuan Feng, Ana M Calvo, and Antonis Rokas. 2015. “Examining the Evolution of the Regulatory Circuit Controlling Secondary Metabolism and Development in the Fungal Genus Aspergillus.” PLoS Genetics 11 (3). Public Library of Science: e1005096. doi:10.1371/journal.pgen.1005096. Liu, Dongyang, Ruifu Zhang, Xingming Yang, Hongsheng Wu, Dabing Xu, Zhu Tang, and Qirong Shen. 2011. “Thermostable Cellulase Production of Aspergillus Fumigatus Z5 under Solid-State Fermentation and Its Application in Degradation of Agricultural Wastes.” International Biodeterioration & Biodegradation 65 (5): 717–25. doi:10.1016/j.ibiod.2011.04.005. López-Berges, Manuel S., Katja Schäfer, Concepción Hera, and Antonio Di Pietro. 2014. “Combinatorial Function of Velvet and AreA in Transcriptional Regulation of Nitrate Utilization and Secondary Metabolism.” Fungal Genetics and Biology 62: 78–84. Losada, Liliana, Olufinmilola Ajayi, Jens C Frisvad, Jiujiang Yu, and William C Nierman. 2009. “Effect of Competition on the Production and Activity of Secondary Metabolites in Aspergillus Species.” Medical Mycology 47 Suppl 1 (January): S88-96. doi:10.1080/13693780802409542.

122

Love, M. I., W. Huber, and S. Anders. 2014. “Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2.” bioRxiv, February. Cold Spring Harbor Labs Journals. doi:10.1101/002832. Love, Michael I, Wolfgang Huber, and Simon Anders. 2014. “Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2.” Genome Biology 15 (12): 550. doi:10.1186/s13059-014-0550-8. Macheleidt, Juliane, Derek J. Mattern, Juliane Fischer, Tina Netzker, Jakob Weber, Volker Schroeckh, Vito Valiante, and Axel A. Brakhage. 2016. “Regulation and Role of Fungal Secondary Metabolites.” Annual Review of Genetics 50 (1). Annual Reviews 4139 El Camino Way, PO Box 10139, Palo Alto, California 94303-0139, USA: 371–92. doi:10.1146/annurev-genet-120215-035203. Machida, Masayuki, Kiyoshi Asai, Motoaki Sano, Toshihiro Tanaka, Toshitaka Kumagai, Goro Terai, Ken-Ichi Kusumoto, et al. 2005. “Genome Sequencing and Analysis of Aspergillus Oryzae.” Nature 438 (7071): 1157–61. doi:10.1038/nature04300. Maere, Steven, Karel Heymans, and Martin Kuiper. 2005. “BiNGO: A Cytoscape Plugin to Assess Overrepresentation of Gene Ontology Categories in Biological Networks.” Bioinformatics (Oxford, England) 21 (16): 3448–49. doi:10.1093/bioinformatics/bti551. McCowen, M C, M E Callender, and J F Lawlis. 1951. “Fumagillin (H-3), a New Antibiotic with Amebicidal Properties.” Science (New York, N.Y.) 113 (2930): 202–3. McKenna, Aaron, Matthew Hanna, Eric Banks, Andrey Sivachenko, Kristian Cibulskis, Andrew Kernytsky, Kiran Garimella, et al. 2010. “The Genome Analysis Toolkit: A MapReduce Framework for Analyzing next-Generation DNA Sequencing Data.” Genome Research 20 (9). Cold Spring Harbor Laboratory Press: 1297–1303. doi:10.1101/gr.107524.110. Medema, Marnix H, Kai Blin, Peter Cimermancic, Victor de Jager, Piotr Zakrzewski, Michael a Fischbach, Tilmann Weber, Eriko Takano, and Rainer Breitling. 2011. “antiSMASH: Rapid Identification, Annotation and Analysis of Secondary Metabolite Biosynthesis Gene Clusters in Bacterial and Fungal Genome Sequences.” Nucleic Acids Research 39 (Web Server issue): W339-46. doi:10.1093/nar/gkr466. Metzenberg, Robert L., and N. Louise Glass. 1990. “Mating Type and Mating Strategies inNeurospora.” BioEssays 12 (2). Wiley Subscription Services, Inc., A Wiley Company: 53–59. doi:10.1002/bies.950120202. Nierman, William C, Arnab Pain, Michael J Anderson, Jennifer R Wortman, H Stanley Kim, Javier Arroyo, Matthew Berriman, et al. 2005. “Genomic Sequence of the Pathogenic and Allergenic Filamentous Fungus Aspergillus Fumigatus.” Nature 438 (7071): 1151–56. doi:10.1038/nature04332. Notz, Regina, Monika Maurhofer, Helen Dubach, Dieter Haas, and Geneviève Défago. 2002. “Fusaric Acid-Producing Strains of Fusarium Oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas Fluorescens CHA0 in Vitro and in the Rhizosphere of Wheat.” Applied and Environmental Microbiology 68 (5). American Society

123

for Microbiology: 2229–35. doi:10.1128/AEM.68.5.2229-2235.2002. O’Brien, Jonathan, and Gerard D Wright. 2011. “An Ecological Perspective of Microbial Secondary Metabolism.” Current Opinion in Biotechnology 22 (4). Elsevier Ltd: 552–58. doi:10.1016/j.copbio.2011.03.010. O’Hanlon, K. A., T. Cairns, D. Stack, M. Schrettl, E. M. Bignell, K. Kavanagh, S. M. Miggin, G. O’Keeffe, T. O. Larsen, and S. Doyle. 2011. “Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus Fumigatus.” Infection and Immunity 79 (10): 3978–92. doi:10.1128/IAI.00192-11. OBrian, G.R., D.R. Georgianna, J.R. Wilkinson, J. Yu, H.K. Abbas, D. Bhatnagar, T.E. Cleveland, W. Nierman, and G.A. Payne. 2007. “The Effect of Elevated Temperature on Gene Transcription and Aflatoxin Biosynthesis.” Mycologia 99 (2): 232–39. doi:10.3852/mycologia.99.2.232. Paláez, Fernando. 2004. “Biological Activities of Fungal Metabolites.” In Handbook of Industrial Mycology, edited by Zhiqiang An, 49–92. New York: Marcel Dekker. Palmer, Jonathan M, and Nancy P Keller. 2010. “Secondary Metabolism in Fungi: Does Chromosomal Location Matter?” Current Opinion in Microbiology 13 (4): 431–36. doi:10.1016/j.mib.2010.04.008. Palmer, Jonathan M, Jeffrey M Theisen, Rocio M Duran, W Scott Grayburn, Ana M Calvo, and Nancy P Keller. 2013. “Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus Nidulans.” PLoS Genetics 9 (1). Public Library of Science: e1003193. doi:10.1371/journal.pgen.1003193. Paradis, E., J. Claude, and K. Strimmer. 2004. “APE: Analyses of Phylogenetics and Evolution in R Language.” Bioinformatics 20 (2). Oxford University Press: 289–90. doi:10.1093/bioinformatics/btg412. Park, Hee-Soo, and Jae-Hyuk Yu. 2016. “Developmental Regulators in Aspergillus Fumigatus.” Journal of Microbiology (Seoul, Korea) 54 (3): 223–31. doi:10.1007/s12275-016-5619-5. Patron, Nicola J, Ross F Waller, Anton J Cozijnsen, David C Straney, Donald M Gardiner, William C Nierman, and Barbara J Howlett. 2007. “Origin and Distribution of Epipolythiodioxopiperazine (ETP) Gene Clusters in Filamentous Ascomycetes.” BMC Evolutionary Biology 7 (1): 174. doi:10.1186/1471-2148-7-174. Paul, Sujay, Angel Zhang, Yvette Ludeña, Gretty K. Villena, Fengan Yu, David H. Sherman, and Marcel Gutiérrez-Correa. 2017. “Insights from the Genome of a High Alkaline Cellulase Producing Aspergillus Fumigatus Strain Obtained from Peruvian Amazon Rainforest.” Journal of Biotechnology 251: 53–58. doi:10.1016/j.jbiotec.2017.04.010. Perrin, Robyn M, Natalie D Fedorova, Jin Woo Bok, Robert A Cramer, Jennifer R Wortman, H Stanley Kim, William C Nierman, and Nancy P Keller. 2007. “Transcriptional Regulation of Chemical Diversity in Aspergillus Fumigatus by LaeA.” Edited by Scott G Filler. PLoS Pathogens 3 (4). Public Library of Science: e50. doi:10.1371/journal.ppat.0030050.

124

Pfaller, MA, PG Pappas, and JR Wingard. 2006. “Invasive Fungal Pathogens: Current Epidemiological Trends.” Clinical Infectious … 52242 (Suppl 1): 3–14. Proctor, R H, T M Hohn, and S P McCormick. 1995. “Reduced Virulence of Gibberella Zeae Caused by Disruption of a Trichothecene Toxin Biosynthetic Gene.” Molecular Plant- Microbe Interactions : MPMI 8 (4): 593–601. Proctor, Robert H., Mark Busman, Jeong-Ah Seo, Yin Won Lee, and Ronald D. Plattner. 2008. “A Fumonisin Biosynthetic Gene Cluster in Fusarium Oxysporum Strain O-1890 and the Genetic Basis for B versus C Fumonisin Production.” Fungal Genetics and Biology 45 (6): 1016–26. doi:10.1016/j.fgb.2008.02.004. Proctor, Robert H, Daren W Brown, Ronald D Plattner, and Anne E Desjardins. 2003. “Co- Expression of 15 Contiguous Genes Delineates a Fumonisin Biosynthetic Gene Cluster in Gibberella Moniliformis.” Fungal Genetics and Biology : FG & B 38 (2): 237–49. Proctor, Robert H, Susan P McCormick, Nancy J Alexander, and Anne E Desjardins. 2009. “Evidence That a Secondary Metabolic Biosynthetic Gene Cluster Has Grown by Gene Relocation during Evolution of the Filamentous Fungus Fusarium.” Molecular Microbiology 74 (5): 1128–42. doi:10.1111/j.1365-2958.2009.06927.x. Purschwitz, Janina, Sylvia Müller, and Reinhard Fischer. 2009. “Mapping the Interaction Sites of Aspergillus Nidulans Phytochrome FphA with the Global Regulator VeA and the White Collar Protein LreB.” Molecular Genetics and Genomics : MGG 281 (1): 35–42. doi:10.1007/s00438-008-0390-x. Purschwitz, Janina, Sylvia Müller, Christian Kastner, Michelle Schöser, Hubertus Haas, Eduardo A Espeso, Ali Atoui, Ana M Calvo, and Reinhard Fischer. 2008. “Functional and Physical Interaction of Blue- and Red-Light Sensors in Aspergillus Nidulans.” Current Biology : CB 18 (4): 255–59. doi:10.1016/j.cub.2008.01.061. Quinlan, Aaron R, and Ira M Hall. 2010. “BEDTools: A Flexible Suite of Utilities for Comparing Genomic Features.” Bioinformatics (Oxford, England) 26 (6). Oxford University Press: 841–42. doi:10.1093/bioinformatics/btq033. R Core Team. 2014. “R: A Language and Environment for Statistical Computing.” Vienna, Austria. Ramamoorthy, Vellaisamy, Sourabh Dhingra, Alexander Kincaid, Sourabha Shantappa, Xuehuan Feng, and Ana M. Calvo. 2013. “The Putative C2H2 Transcription Factor MtfA Is a Novel Regulator of Secondary Metabolism and Morphogenesis in Aspergillus Nidulans.” Edited by Gustavo Henrique Goldman. PLoS ONE 8 (9). Public Library of Science: e74122. doi:10.1371/journal.pone.0074122. Ramamoorthy, Vellaisamy, Sourabha Shantappa, Sourabh Dhingra, and Ana M Calvo. 2012. “veA-Dependent RNA-Pol II Transcription Elongation Factor-like Protein, RtfA, Is Associated with Secondary Metabolism and Morphological Development in Aspergillus Nidulans.” Molecular Microbiology 85 (4): 795–814. doi:10.1111/j.1365- 2958.2012.08142.x.

125

Rauscher, Stefan, Sylvia Pacher, Maren Hedtke, Olaf Kniemeyer, and Reinhard Fischer. 2015. “A Phosphorylation Code of the Aspergillus Nidulans Global Regulator VelvetA (VeA) Determines Specific Functions.” Molecular Microbiology, November. doi:10.1111/mmi.13275. Reynolds, Hannah T., Jason C. Slot, Hege H. Divon, Erik Lysøe, Robert H. Proctor, and Daren W. Brown. 2017. “Differential Retention of Gene Functions in a Secondary Metabolite Cluster.” Molecular Biology and Evolution 10. Oxford University Press (OUP): e1004816. doi:10.1093/molbev/msx145. Roberts, J.D., and T.A. Kunkel. 1996. “Fidelity of DNA Replication.” In DNA Replication in Eukaryotic Cells, edited by Melvin L DePamphilis, 217–47. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Rodríguez-Urra, Ana Belén, Carlos Jiménez, María Isabel Nieto, Jaime Rodríguez, Hideo Hayashi, and Unai Ugalde. 2012. “Signaling the Induction of Sporulation Involves the Interaction of Two Secondary Metabolites in Aspergillus Nidulans.” ACS Chemical Biology 7 (3). American Chemical Society: 599–606. doi:10.1021/cb200455u. Rohlfs, Marko, Martin Albert, Nancy P Keller, and Frank Kempken. 2007. “Secondary Chemicals Protect Mould from Fungivory.” Biology Letters 3 (5): 523–25. doi:10.1098/rsbl.2007.0338. Rohlfs, Marko, and Alice C L Churchill. 2011. “Fungal Secondary Metabolites as Modulators of Interactions with Insects and Other Arthropods.” Fungal Genetics and Biology : FG & B 48 (1): 23–34. doi:10.1016/j.fgb.2010.08.008. Roilides, E, H Katsifa, and T J Walsh. 1998. “Pulmonary Host Defences against Aspergillus Fumigatus.” Research in Immunology. doi:10.1016/S0923-2494(98)80769-4. Roze, Ludmila V, Anindya Chanda, Maris Laivenieks, Randolph M Beaudry, Katherine A Artymovich, Anna V Koptina, Deena W Awad, Dina Valeeva, Arthur D Jones, and John E Linz. 2010. “Volatile Profiling Reveals Intracellular Metabolic Changes in Aspergillus Parasiticus: veA Regulates Branched Chain Amino Acid and Ethanol Metabolism.” BMC Biochemistry 11 (1): 33. doi:10.1186/1471-2091-11-33. Roze, Ludmila V, Anindya Chanda, and John E Linz. 2011. “Compartmentalization and Molecular Traffic in Secondary Metabolism: A New Understanding of Established Cellular Processes.” Fungal Genetics and Biology : FG & B 48 (1). Elsevier Inc.: 35–48. doi:10.1016/j.fgb.2010.05.006. Ryckeboer, J, J Mergaert, J Coosemans, K Deprins, and J Swings. 2003. “Microbiological Aspects of Biowaste during Composting in a Monitored Compost Bin.” Journal of Applied Microbiology 94 (1): 127–37. Sarikaya-Bayram, Ozlem, Ozgür Bayram, Kirstin Feussner, Jong-Hwa Kim, Hee-Seo Kim, Alexander Kaever, Ivo Feussner, et al. 2014. “Membrane-Bound Methyltransferase Complex VapA-VipC-VapB Guides Epigenetic Control of Fungal Development.” Developmental Cell 29 (4): 406–20. doi:10.1016/j.devcel.2014.03.020.

126

Sarikaya-Bayram, Özlem, Jonathan M Palmer, Nancy Keller, Gerhard H Braus, and Özgür Bayram. 2015. “One Juliet and Four Romeos: VeA and Its Methyltransferases.” Frontiers in Microbiology 6 (January): 1. doi:10.3389/fmicb.2015.00001. Schardl, Christopher L., Carolyn A. Young, Uljana Hesse, Stefan G. Amyotte, Kalina Andreeva, Patrick J. Calie, Damien J. Fleetwood, et al. 2013. “Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci.” Edited by Joseph Heitman. PLoS Genetics 9 (2). Public Library of Science: e1003323. doi:10.1371/journal.pgen.1003323. Scharf, Daniel H., Thorsten Heinekamp, and Axel A. Brakhage. 2014. “Human and Plant Fungal Pathogens: The Role of Secondary Metabolites.” Edited by Joseph Heitman. PLoS Pathogens 10 (1). Public Library of Science: e1003859. doi:10.1371/journal.ppat.1003859. Scharf, Daniel H., Nicole Remme, Thorsten Heinekamp, Peter Hortschansky, Axel A. Brakhage, and Christian Hertweck. 2010. “Transannular Disulfide Formation in Gliotoxin Biosynthesis and Its Role in Self-Resistance of the Human Pathogen Aspergillus Fumigatus.” Journal of the American Chemical Society 132 (29): 10136–41. doi:10.1021/ja103262m. Scharf, Daniel H, Thorsten Heinekamp, Nicole Remme, Peter Hortschansky, Axel a Brakhage, and Christian Hertweck. 2012. “Biosynthesis and Function of Gliotoxin in Aspergillus Fumigatus.” Applied Microbiology and Biotechnology 93 (2): 467–72. doi:10.1007/s00253- 011-3689-1. Schimek, C. 2011. “Evolution of Special Metabolism in Fungi : Concepts , Mechanisms , and Pathways.” In Evolution of Fungi and Fungal-Like Organisms, The Mycota, edited by S. Pöggler and J. Wöstmeyer, XIV, 293–328. Berlin, Heidelberg: Springer-Verlag. Schliep, Klaus Peter. 2011. “Phangorn: Phylogenetic Analysis in R.” Bioinformatics (Oxford, England) 27 (4). Oxford University Press: 592–93. doi:10.1093/bioinformatics/btq706. Schrettl, Markus, Elaine Bignell, Claudia Kragl, Yasmin Sabiha, Omar Loss, Martin Eisendle, Anja Wallner, Herbert N. Arst, Ken Haynes, and Hubertus Haas. 2007. “Distinct Roles for Intra- and Extracellular Siderophores during Aspergillus Fumigatus Infection.” PLoS Pathogens 3 (9). Cold Spring Harbor Laboratory Press: e128. doi:10.1371/journal.ppat.0030128. Schumacher, Julia, Angélique Gautier, Guillaume Morgant, Lena Studt, Paul-Henri Ducrot, Pascal Le Pêcheur, Saad Azeddine, et al. 2013. “A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis Cinerea Is Positively Controlled by VELVET.” Edited by Sung-Hwan Yun. PLoS ONE 8 (1). Public Library of Science: e53729. doi:10.1371/journal.pone.0053729. Shaaban, Mona, Jonathan M Palmer, Wael A El-Naggar, M A El-Sokkary, El-Sayed E Habib, and Nancy P Keller. 2010. “Involvement of Transposon-like Elements in Penicillin Gene Cluster Regulation.” Fungal Genetics and Biology : FG & B 47 (5): 423–32. doi:10.1016/j.fgb.2010.02.006.

127

Shannon, Paul, Andrew Markiel, Owen Ozier, Nitin S Baliga, Jonathan T Wang, Daniel Ramage, Nada Amin, Benno Schwikowski, and Trey Ideker. 2003. “Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks.” Genome Research 13 (11): 2498–2504. doi:10.1101/gr.1239303. Shin, Kwang-Soo, Young Hwan Kim, and Jae-Hyuk Yu. 2015. “Proteomic Analyses Reveal the Key Roles of BrlA and AbaA in Biogenesis of Gliotoxin in Aspergillus Fumigatus.” Biochemical and Biophysical Research Communications 463 (3): 428–33. doi:10.1016/j.bbrc.2015.05.090. Slot, Jason C, and Antonis Rokas. 2011. “Horizontal Transfer of a Large and Highly Toxic Secondary Metabolic Gene Cluster between Fungi.” Current Biology : CB 21 (2). Elsevier Ltd: 134–39. doi:10.1016/j.cub.2010.12.020. Smit, AFA, R Hubley, and P Green. n.d. “Repeatmasker Open-4.0.” http://www.repeatmasker.org. Smith, Timothy D, and Ana M Calvo. 2014. “The mtfA Transcription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus Fumigatus.” Eukaryotic Cell 13 (6). American Society for Microbiology: 766– 75. doi:10.1128/EC.00075-14. Sørensen, Jens Laurids, Frederik Teilfeldt Hansen, Teis Esben Sondergaard, Dan Staerk, T. Verne Lee, Reinhard Wimmer, Louise Graabaek Klitgaard, Stig Purup, Henriette Giese, and Rasmus John Normand Frandsen. 2012. “Production of Novel Fusarielins by Ectopic Activation of the Polyketide Synthase 9 Cluster in Fusarium Graminearum.” Environmental Microbiology 14 (5). Blackwell Publishing Ltd: 1159–70. doi:10.1111/j.1462- 2920.2011.02696.x. Sorrells, Trevor R, and Alexander D Johnson. 2015. “Making Sense of Transcription Networks.” Cell 161 (4). Elsevier: 714–23. doi:10.1016/j.cell.2015.04.014. Spatafora, Joseph W, and Kathryn E Bushley. 2015. “Phylogenomics and Evolution of Secondary Metabolism in Plant-Associated Fungi.” Current Opinion in Plant Biology 26: 37–44. doi:10.1016/j.pbi.2015.05.030. Stamatakis, A. 2014. “RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies.” Bioinformatics 30 (9): 1312–13. doi:10.1093/bioinformatics/btu033. Stanke, Mario, and Burkhard Morgenstern. 2005. “AUGUSTUS: A Web Server for Gene Prediction in Eukaryotes That Allows User-Defined Constraints.” Nucleic Acids Research 33 (Web Server issue). Oxford University Press: W465-7. doi:10.1093/nar/gki458. Stanzani, Marta, Enrico Orciuolo, Russell Lewis, Dimitrios P Kontoyiannis, Sergio L R Martins, Lisa S St John, and Krishna V Komanduri. 2005. “Aspergillus Fumigatus Suppresses the Human Cellular Immune Response via Gliotoxin-Mediated Apoptosis of Monocytes.” Blood 105 (6): 2258–65. doi:10.1182/blood-2004-09-3421. Stinnett, Suzanne M, Eduardo A Espeso, Laura Cobeño, Lidia Araújo-Bazán, and Ana M Calvo.

128

2007. “Aspergillus Nidulans VeA Subcellular Localization Is Dependent on the Importin Alpha Carrier and on Light.” Molecular Microbiology 63 (1): 242–55. doi:10.1111/j.1365- 2958.2006.05506.x. Sugui, Janyce A, H Stanley Kim, Kol A Zarember, Yun C Chang, John I Gallin, Willian C Nierman, and Kyung J Kwon-Chung. 2008. “Genes Differentially Expressed in Conidia and Hyphae of Aspergillus Fumigatus upon Exposure to Human Neutrophils.” PloS One 3 (7). Public Library of Science: e2655. doi:10.1371/journal.pone.0002655. Szewczyk, Edyta, Tania Nayak, C Elizabeth Oakley, Heather Edgerton, Yi Xiong, Naimeh Taheri-Talesh, Stephen A Osmani, Berl R Oakley, and Berl Oakley. 2006. “Fusion PCR and Gene Targeting in Aspergillus Nidulans.” Nature Protocols 1 (6): 3111–20. doi:10.1038/nprot.2006.405. Tao, Li, and Jae-Hyuk Yu. 2011. “AbaA and WetA Govern Distinct Stages of Aspergillus Fumigatus Development.” Microbiology (Reading, England) 157 (Pt 2): 313–26. doi:10.1099/mic.0.044271-0. The Gene Ontology Consortium. 2014. “Gene Ontology Consortium: Going Forward.” Nucleic Acids Research 43 (D1): D1049-1056. doi:10.1093/nar/gku1179. Then Bergh, K, and A A Brakhage. 1998. “Regulation of the Aspergillus Nidulans Penicillin Biosynthesis Gene acvA (pcbAB) by Amino Acids: Implication for Involvement of Transcription Factor PACC.” Applied and Environmental Microbiology 64 (3): 843–49. Throckmorton, Kurt, Fang Yun Lim, Dimitrios P Kontoyiannis, Weifa Zheng, and Nancy P Keller. 2015. “Redundant Synthesis of a Conidial Polyketide by Two Distinct Secondary Metabolite Clusters in Aspergillus Fumigatus.” Environmental Microbiology, August. doi:10.1111/1462-2920.13007. Tilburn, J, S Sarkar, D A Widdick, E A Espeso, M Orejas, J Mungroo, M A Peñalva, and H N Arst. 1995. “The Aspergillus PacC Zinc Finger Transcription Factor Mediates Regulation of Both Acid- and Alkaline-Expressed Genes by Ambient pH.” The EMBO Journal 14 (4): 779–90. Treangen, Todd J, and Steven L Salzberg. 2011. “Repetitive DNA and next-Generation Sequencing: Computational Challenges and Solutions.” Nature Reviews. Genetics 13 (1). NIH Public Access: 36–46. doi:10.1038/nrg3117. Tsai, H F, Y C Chang, R G Washburn, M H Wheeler, and K J Kwon-Chung. 1998. “The Developmentally Regulated alb1 Gene of Aspergillus Fumigatus: Its Role in Modulation of Conidial Morphology and Virulence.” Journal of Bacteriology 180 (12): 3031–38. Tsai, H F, M H Wheeler, Y C Chang, and K J Kwon-Chung. 1999. “A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in Aspergillus Fumigatus.” Journal of Bacteriology 181 (20): 6469–77. Tsuji, Gento, Youki Kenmochi, Yoshitaka Takano, James Sweigard, Leonard Farrall, Iwao Furusawa, Osamu Horino, and Yasuyuki Kubo. 2002. “Novel Fungal Transcriptional

129

Activators, Cmr1p of Colletotrichum Lagenarium and Pig1p of Magnaporthe Grisea, Contain Cys2His2 Zinc Finger and Zn(II)2Cys6 Binuclear Cluster DNA-Binding Motifs and Regulate Transcription of Melanin Biosynthesis Genes in a de.” Molecular Microbiology 38 (5). Blackwell Publishing Ltd: 940–54. doi:10.1046/j.1365- 2958.2000.02181.x. Turgeon, B. Gillian, and Scott E. Baker. 2007. “Genetic and Genomic Dissection of the Cochliobolus Heterostrophus Tox1 Locus Controlling Biosynthesis of the Polyketide Virulence Factor T-Toxin.” Advances in Genetics. doi:10.1016/S0065-2660(06)57006-3. Twumasi-Boateng, Kwame, Yan Yu, Dan Chen, Fabrice N Gravelat, William C Nierman, and Donald C Sheppard. 2009. “Transcriptional Profiling Identifies a Role for BrlA in the Response to Nitrogen Depletion and for StuA in the Regulation of Secondary Metabolite Clusters in Aspergillus Fumigatus.” Eukaryotic Cell 8 (1): 104–15. doi:10.1128/EC.00265- 08. Umemura, Myco, Hideaki Koike, Nozomi Nagano, Tomoko Ishii, Jin Kawano, Noriko Yamane, Ikuko Kozone, et al. 2013. “MIDDAS-M: Motif-Independent de Novo Detection of Secondary Metabolite Gene Clusters through the Integration of Genome Sequencing and Transcriptome Data.” PloS One 8 (12): e84028. doi:10.1371/journal.pone.0084028. Van der Auwera, Geraldine A., Mauricio O. Carneiro, Christopher Hartl, Ryan Poplin, Guillermo del Angel, Ami Levy-Moonshine, Tadeusz Jordan, et al. 2013. “From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline.” In Current Protocols in Bioinformatics, 43:11.10.1-11.10.33. Hoboken, NJ, USA: John Wiley & Sons, Inc. doi:10.1002/0471250953.bi1110s43. Vining, Leo C. 1990. “Functions of Secondary Metabolites.” Annual Review of Microbiology 44 (January). Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303- 0139, USA: 395–427. doi:10.1146/annurev.mi.44.100190.002143. Weber, G., K. Schörgendorfer, E. Schneider-Scherzer, and E. Leitner. 1994. “The Peptide Synthetase Catalyzing Cyclosporine Production in Tolypocladium Niveum Is Encoded by a Giant 45.8-Kilobase Open Reading Frame.” Current Genetics 26 (2): 120–25. doi:10.1007/BF00313798. Wickham, Hadley. 2009. ggplot2: Elegant Graphics for Data Analysis. Springer New York. Wiemann, Philipp, Daren W Brown, Karin Kleigrewe, Jin Woo Bok, Nancy P Keller, Hans- Ulrich Humpf, and Bettina Tudzynski. 2010. “FfVel1 and FfLae1, Components of a Velvet- like Complex in Fusarium Fujikuroi, Affect Differentiation, Secondary Metabolism and Virulence.” Molecular Microbiology, June. doi:10.1111/j.1365-2958.2010.07263.x. Wiemann, Philipp, Chun-Jun Guo, Jonathan M Palmer, Relebohile Sekonyela, Clay C C Wang, and Nancy P Keller. 2013. “Prototype of an Intertwined Secondary-Metabolite Supercluster.” Proceedings of the National Academy of Sciences of the United States of America 110 (42): 17065–70. doi:10.1073/pnas.1313258110. Wiemann, Philipp, Christian M. K. Sieber, Katharina W. von Bargen, Lena Studt, Eva-Maria

130

Niehaus, Jose J. Espino, Kathleen Huß, et al. 2013. “Deciphering the Cryptic Genome: Genome-Wide Analyses of the Rice Pathogen Fusarium Fujikuroi Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites.” Edited by Aaron P. Mitchell. PLoS Pathogens 9 (6). Public Library of Science: e1003475. doi:10.1371/journal.ppat.1003475. Wisecaver, J. H., J.C. Slot, and A Rokas. 2014. “The Evolution of Fungal Metabolic Pathways.” PLoS Genetics. Wisecaver, Jennifer H, and Antonis Rokas. 2015. “Fungal Metabolic Gene Clusters-Caravans Traveling across Genomes and Environments.” Frontiers in Microbiology. Frontiers. doi:10.3389/fmicb.2015.00161. Wong, Simon, and Kenneth H Wolfe. 2005. “Birth of a Metabolic Gene Cluster in Yeast by Adaptive Gene Relocation.” Nature Genetics 37 (7): 777–82. doi:10.1038/ng1584. Yim, Grace, Helena Huimi Wang, and Julian Davies. 2007. “Antibiotics as Signalling Molecules.” Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 362 (1483): 1195–1200. doi:10.1098/rstb.2007.2044. Young, Carolyn A., Christopher L. Schardl, Daniel G. Panaccione, Simona Florea, Johanna E. Takach, Nikki D. Charlton, Neil Moore, Jennifer S. Webb, and Jolanta Jaromczyk. 2015. “Genetics, Genomics and Evolution of Ergot Alkaloid Diversity.” Toxins. doi:10.3390/toxins7041273. Yu, Jae-Hyuk. 2010. “Regulation of Development in Aspergillus Nidulans and Aspergillus Fumigatus.” Mycobiology 38 (4): 229–37. doi:10.4489/MYCO.2010.38.4.229. Yu, Jiujiang, Perng-Kuang Chang, Kenneth C Ehrlich, Jeffrey W Cary, Deepak Bhatnagar, Thomas E Cleveland, Gary A Payne, John E Linz, Charles P Woloshuk, and Joan W Bennett. 2004. “Clustered Pathway Genes in Aflatoxin Biosynthesis.” Applied and Environmental Microbiology 70 (3). American Society for Microbiology: 1253–62. doi:10.1128/AEM.70.3.1253-1262.2004. Yu, Jiujiang, Natalie D Fedorova, Beverly G Montalbano, Deepak Bhatnagar, Thomas E Cleveland, Joan W Bennett, and William C Nierman. 2011. “Tight Control of Mycotoxin Biosynthesis Gene Expression in Aspergillus Flavus by Temperature as Revealed by RNA- Seq.” FEMS Microbiology Letters 322 (2): 145–49. doi:10.1111/j.1574-6968.2011.02345.x. Zhang, Han, Antonis Rokas, and Jason C JC Slot. 2012. “Two Different Secondary Metabolism Gene Clusters Occupied the Same Ancestral Locus in Fungal Dermatophytes of the Arthrodermataceae.” PLoS One 7 (7): e41903. doi:10.1371/journal.pone.0041903. Zheng, X., D. Levine, J. Shen, S. M. Gogarten, C. Laurie, and B. S. Weir. 2012. “A High- Performance Computing Toolset for Relatedness and Principal Component Analysis of SNP Data.” Bioinformatics 28 (24). Oxford University Press: 3326–28. doi:10.1093/bioinformatics/bts606. Zhou, X., D. Peris, J. Kominek, C. P. Kurtzman, C. T. Hittinger, and A. Rokas. 2016. “In Silico

131

Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de Novo Genome Sequencing Studies.” G3: Genes|Genomes|Genetics, September. doi:10.1534/g3.116.034249. Zimin, A. V., G. Marcais, D. Puiu, M. Roberts, S. L. Salzberg, and J. A. Yorke. 2013. “The MaSuRCA Genome Assembler.” Bioinformatics 29 (21): 2669–77. doi:10.1093/bioinformatics/btt476.

132

APPENDIX A

ROLE OF THE STUDENT

I was the primary author on all of the text in this dissertation and carried out the majority

of the research presented in Chapters II-V. In Chapter II, I performed all RNA-seq data analysis and all downstream analyses, and determined the level of conservation of metabolic gene sets and SM gene clusters in all genomes analyzed. In Chapter III and IV, I performed all RNA-seq data analysis as well as all downstream analysis involving Gene Ontology enrichment analysis and SM gene cluster expression analyses. In Chapter V, I performed de novo assembly, gene prediction, SM gene cluster prediction, short read alignment, polymorphism discovery, and SM gene conservation analyses.

133

APPENDIX B

STRAINS USED IN CHAPTER V

Publicly Publicly

Publicly available available

available gene short

Official name Pubmed ID Source assembly predictions reads

IF1SWF4 27830189 Environmental Y N Y

ISSF21 27830189 Environmental Y N Y

AF41 N/A; sequenced by JCVI Clinical N N Y

AF72 N/A; sequenced by JCVI Clinical N N Y

AF90 N/A; sequenced by JCVI Clinical N N Y

F12041 N/A; sequenced by JCVI Clinical N N Y

F12219 N/A; sequenced by JCVI Clinical N N Y

F12636 N/A; sequenced by JCVI Clinical N N Y

F13535 N/A; sequenced by JCVI Clinical N N Y

F13619 N/A; sequenced by JCVI Clinical N N Y

F13952 N/A; sequenced by JCVI Clinical N N Y

F14403 N/A; sequenced by JCVI Clinical N N Y

F14513G N/A; sequenced by JCVI Clinical N N Y

F14532 N/A; sequenced by JCVI Clinical N N Y

F14946G N/A; sequenced by JCVI Clinical N N Y

F15390 N/A; sequenced by JCVI Clinical N N Y

F15927 N/A; sequenced by JCVI Clinical N N Y

134

F16134 N/A; sequenced by JCVI Clinical N N Y

F16216 N/A; sequenced by JCVI Clinical N N Y

F16311 N/A; sequenced by JCVI Clinical N N Y

F17582 N/A; sequenced by JCVI Clinical N N Y

F17729 N/A; sequenced by JCVI Clinical N N Y

F17729W N/A; sequenced by JCVI Clinical N N Y

F17764 N/A; sequenced by JCVI Clinical N N Y

F18085 N/A; sequenced by JCVI Clinical N N Y

F7763 N/A; sequenced by JCVI Clinical N N Y

F5211G N/A; sequenced by JCVI Clinical N N Y

AF65 (NCPF

7097) 26037120 Clinical N N Y

Afu 1042/09 26037120 Clinical N N Y

Afu 124/E11 26037120 Environmental N N Y

Afu 166/E11 26037120 Environmental N N Y

Afu 218/E11 26037120 Environmental N N Y

Afu 257/E11 26037120 Environmental N N Y

Afu 343/P/11 26037120 Clinical N N Y

Afu 591/12 26037120 Clinical N N Y

Afu 942/09 26037120 Clinical N N Y

08-12-12-13 26037120 Clinical N N Y

08-36-03-25 26037120 Clinical N N Y

08-31-08-91 26037120 Clinical N N Y

08-19-02-61 26037120 Environmental N N Y

135

08-19-02-30 26037120 Environmental N N Y

10-01-02-27 26037120 Clinical N N Y

08-19-02-46 26037120 Environmental N N Y

08-19-02-10 26037120 Environmental N N Y

12-7505446 26037120 Clinical N N Y

12-7505220 26037120 Clinical N N Y

09-7500806 26037120 Clinical N N Y

12-7504652 26037120 Clinical N N Y

12-7504462 26037120 Clinical N N Y

12-7505054 26037120 Clinical N N Y

18404212; N/A, sequenced

A1163 by JCVI (resequencing) Clinical Y Y Y

16372009; 26037120

Af293 (resequencing) Clinical Y Y Y

Y (on RIKEN

website, not

N/A; sequenced by RIKEN available

Center for Life Science through

JCM10253 Technologies Unclear Y Genbank) Y

N/A; sequenced by

Universidad Nacional

LMB35Aa Agraria La Molina, Peru Environmental Y N Y

Af10 N/A; sequenced by JCVI Clinical Y N N

Af210 N/A; sequenced by JCVI Clinical Y N N

136

Z5 26076650 Environmental Y Y N

N/A; Sequenced at

Niveus Oklahoma State University Unclear Y N N

Y; this

MO54056EXP N/A; sequenced in this study Clinical N N study

Y; this

MO68507EXP N/A; sequenced in this study Clinical N N study

Y; this

MO69250EXP N/A; sequenced in this study Clinical N N study

Y; this

MO76959EXP N/A; sequenced in this study Clinical N N study

Y; this

MO78722EXP N/A; sequenced in this study Clinical N N study

Y; this

MO79587EXP N/A; sequenced in this study Clinical N N study

Y; this

MO89263LAB N/A; sequenced in this study Clinical N N study

Y; this

MO91298SB N/A; sequenced in this study Clinical N N study

137

APPENDIX C

GENE PHYLOGENIES FROM CHAPTER V

138

Gene phylogenies of the fusarielin-like SM gene cluster 4. These phylogenies are consistent with horizontal transfer between Eurotiomycete and Sordariomycete fungi or with extensive gene loss.

Tree scale: 0.1 CRG84209.1-28573-Talaromyces islandicus-Eurotiomycetes XP 003650246.1-578455-Thielavia terrestris NRRL-Sordariomycetes XP 001937750.1-426418-Pyrenophora tritici repen-Dothideomycetes OJJ29727.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes XP 001557214.1-332648-Botrytis cinerea B05 10-Leotiomycetes Taxonomy KFX41080.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes XP 007832533.1-1229662-Pestalotiopsis fici W106-Sordariomycetes OJD18273.1-1447872-Emergomyces pasteuriana E-Eurotiomycetes Leotiomycetes XP 007828347.1-1229662-Pestalotiopsis fici W106-Sordariomycetes ORY60123.1-1141098-Pseudomassariella vexata-Sordariomycetes KJK79796.1-1291518-Metarhizium anisopliae BR-Sordariomycetes Sordariomycetes CRG86402.1-28573-Talaromyces islandicus-Eurotiomycetes XP 014562255.1-930091-Bipolaris victoriae FI3-Dothideomycetes XP 018188015.1-1328760-Xylona heveae TC161-other Pezizomycotina Eurotiomycetes OSS49487.1-105696-Epicoccum nigrum-Dothideomycetes ODM21989.1-573508-Aspergillus cristatus-Eurotiomycetes XP 003002636.1-526221-Verticillium alfalfae VaM-Sordariomycetes Dothideomycetes 100 XP 009657844.1-498257-Verticillium dahliae VdLs-Sordariomycetes GAM42327.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 96 XP 013327000.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes other_Pezizomycotina XP 018175195.1-33203-Purpureocillium lilacinum-Sordariomycetes 100 ORY60946.1-1141098-Pseudomassariella vexata-Sordariomycetes CCC08895.1-771870-Sordaria macrospora k hel-Sordariomycetes other_Ascomycota 100 XP 009848053.1-510951-Neurospora tetrasperma FG-Sordariomycetes 87 XP 961575.1-367110-Neurospora crassa OR74A-Sordariomycetes CZT02525.1-914238-Rhynchosporium agropyri-Leotiomycetes other_Fungi CZT43001.1-38038-Rhynchosporium secalis-Leotiomycetes 100 CZS89928.1-914237-Rhynchosporium commune-Leotiomycetes KEY72877.1-1280523-Stachybotrys chartarum IB-Sordariomycetes other_Opisthokonta KFA61053.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA74243.1-1283842-Stachybotrys chartarum IB-Sordariomycetes EEH07194.1-447093-Histoplasma capsulatum G1-Eurotiomycetes other_Eukaryota EGE79493.2-653446-Blastomyces dermatitidis-Eurotiomycetes 100 OAT06041.1-559298-Blastomyces gilchristii S-Eurotiomycetes 100 EQL30383.1-447095-Blastomyces dermatitidis-Eurotiomycetes Bacteria OAT00629.1-559297-Blastomyces dermatitidis-Eurotiomycetes XP 003012332.1-663331-Trichophyton benhamiae CB-Eurotiomycetes XP 003232990.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes Archaea 100 EZF34621.1-1215336-Trichophyton interdigital-Eurotiomycetes 98 EGD97308.1-647933-Trichophyton tonsurans CB-Eurotiomycetes 100 EGE01776.1-559882-Trichophyton equinum CBS-Eurotiomycetes Viruses XP 006964072.1-431241-Trichoderma reesei QM6a-Sordariomycetes XP 013957200.1-413071-Trichoderma virens Gv29 8-Sordariomycetes 99 93 XP 013956156.1-413071-Trichoderma virens Gv29 8-Sordariomycetes XP 013940338.1-452589-Trichoderma atroviride IM-Sordariomycetes 100 XP 018665082.1-398673-Trichoderma gamsii-Sordariomycetes XP 008091305.1-645133-Colletotrichum graminicol-Sordariomycetes XP 018165017.1-759273-Colletotrichum higginsian-Sordariomycetes KZL78320.1-1573173-Colletotrichum incanum-Sordariomycetes 100 100 KZL74784.1-708197-Colletotrichum tofieldiae-Sordariomycetes KXH27618.1-1209931-Colletotrichum salicis-Sordariomycetes 99 XP 007600787.1-1445577-Colletotrichum fioriniae-Sordariomycetes 84 KXH36096.1-703756-Colletotrichum simmondsii-Sordariomycetes KJK74809.1-1291518-Metarhizium anisopliae BR-Sordariomycetes XP 011411271.1-655844-Metarhizium robertsii ARS-Sordariomycetes XP 014576485.1-1276143-Metarhizium majus ARSEF 2-Sordariomycetes 100 KID86641.1-1276136-Metarhizium guizhouense A-Sordariomycetes XP 014543403.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes KFG79083.1-5530-Metarhizium anisopliae-Sordariomycetes 98 KID69143.1-1276135-Metarhizium anisopliae AR-Sordariomycetes KEQ62146.1-1043003-Aureobasidium melanogenum-Dothideomycetes XP 001905190.1-515849-Podospora anserina S mat-Sordariomycetes KKP04693.1-5544-Trichoderma harzianum-Sordariomycetes 99 XP 018061899.1-149040-Phialocephala scopiformis-Leotiomycetes KID84879.1-1276136-Metarhizium guizhouense A-Sordariomycetes GAW18025.1-1813822-fungal sp No 14919-other Fungi 82 100 GAP93356.2-77044-Rosellinia necatrix-Sordariomycetes KKK21031.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK17012.1-308745-Aspergillus rambellii-Eurotiomycetes ORY67809.1-1141098-Pseudomassariella vexata-Sordariomycetes XP 011319437.1-229533-Fusarium graminearum PH 1-Sordariomycetes OJJ34617.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes XP 007603124.1-1445577-Colletotrichum fioriniae-Sordariomycetes 80 100 KXH26573.1-703756-Colletotrichum simmondsii-Sordariomycetes XP 001270371.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes OQD93850.1-60172-Penicillium solitum-Eurotiomycetes CRL30866.1-5075-Penicillium camemberti-Eurotiomycetes 100 100 KOS43412.1-229535-Penicillium nordicum-Eurotiomycetes GAA83285.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 EHA18931.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 96 GAO83195.1-91492-Aspergillus udagawae-Eurotiomycetes 90 QUERY-Afu1g17710-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54955.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes XP 002850357.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 003177421.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes OAL69031.1-34388-Trichophyton violaceum-Eurotiomycetes 92 XP 003231747.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes 100 89 OAL67580.1-5551-Trichophyton rubrum-Eurotiomycetes 83 EZF67310.1-1215329-Trichophyton rubrum CBS 2-Eurotiomycetes 100 87 EZF27012.1-1215337-Trichophyton rubrum MR850-Eurotiomycetes 94 EZF46053.1-1215328-Trichophyton rubrum CBS 1-Eurotiomycetes XP 003174142.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes XP 003230896.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes 96 EZF28066.1-1215337-Trichophyton rubrum MR850-Eurotiomycetes 100 92 OAL71455.1-34388-Trichophyton violaceum-Eurotiomycetes EGE04590.1-559882-Trichophyton equinum CBS-Eurotiomycetes EGD93098.1-647933-Trichophyton tonsurans CB-Eurotiomycetes 87 EZF36203.1-1215336-Trichophyton interdigital-Eurotiomycetes 90 KDB19964.1-1215338-Trichophyton interdigital-Eurotiomycetes

139 Tree scale: 0.1 XP 018156681.1-759273-Colletotrichum higginsian-Sordariomycetes 100 ENH79013.1-1213857-Colletotrichum orbiculare-Sordariomycetes CZT02535.1-914238-Rhynchosporium agropyri-Leotiomycetes CZT43006.1-38038-Rhynchosporium secalis-Leotiomycetes 100 CZS89923.1-914237-Rhynchosporium commune-Leotiomycetes Taxonomy XP 013277062.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes XP 007736464.1-1182542-Capronia epimyces CBS 606-Eurotiomycetes 100 XP 016240750.1-91928-Exophiala spinifera-Eurotiomycetes Leotiomycetes 84 XP 016258459.1-215243-Exophiala oligosperma-Eurotiomycetes XP 007827070.1-1229662-Pestalotiopsis fici W106-Sordariomycetes XP 007817533.1-655844-Metarhizium robertsii ARS-Sordariomycetes Sordariomycetes 89 KFG81511.1-5530-Metarhizium anisopliae-Sordariomycetes KJK74649.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 XP 014540250.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes Eurotiomycetes KID84095.1-1276136-Metarhizium guizhouense A-Sordariomycetes XP 014573814.1-1276143-Metarhizium majus ARSEF 2-Sordariomycetes XP 013431313.1-1043004-Aureobasidium namibiae CB-Dothideomycetes Dothideomycetes GAW21476.1-1813822-fungal sp No 14919-other Fungi 80 XP 018155051.1-759273-Colletotrichum higginsian-Sordariomycetes EQL01261.1-911162-Ophiocordyceps sinensis C-Sordariomycetes other_Pezizomycotina 100 CZT13654.1-914237-Rhynchosporium commune-Leotiomycetes KDN72350.1-1173701-Colletotrichum sublineola-Sordariomycetes 100 XP 008100387.1-645133-Colletotrichum graminicol-Sordariomycetes other_Ascomycota XP 002544623.1-336963-Uncinocarpus reesii 1704-Eurotiomycetes XP 013340600.1-1043005-Aureobasidium subglaciale-Dothideomycetes GAM82793.1-1603295-fungal sp No 11243-other Fungi other_Fungi G3XMB7.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 100 100 XP 001393494.1-425011-Aspergillus niger CBS 513-Eurotiomycetes XP 001594902.1-665079-Sclerotinia sclerotiorum-Leotiomycetes other_Opisthokonta XP 001560980.1-332648-Botrytis cinerea B05 10-Leotiomycetes 97 ESZ98313.1-1432307-Sclerotinia borealis F 41-Leotiomycetes XP 008086832.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes other_Eukaryota KIN01422.1-913774-Oidiodendron maius Zn-Leotiomycetes XP 018075682.1-149040-Phialocephala scopiformis-Leotiomycetes OCK95926.1-794803-Cenococcum geophilum 1 58-Dothideomycetes Bacteria 82 100 OCL10303.1-574774-Glonium stellatum-Dothideomycetes XP 007293085.1-1072389-Marssonina brunnea f sp-Leotiomycetes XP 018077409.1-149040-Phialocephala scopiformis-Leotiomycetes Archaea 86 CZT52794.1-38038-Rhynchosporium secalis-Leotiomycetes CZT04210.1-914237-Rhynchosporium commune-Leotiomycetes 100 CZT13906.1-914238-Rhynchosporium agropyri-Leotiomycetes Viruses ORY67810.1-1141098-Pseudomassariella vexata-Sordariomycetes OJJ34618.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes KGQ02700.1-1245745-Beauveria bassiana D1 5-Sordariomycetes XP 011319442.1-229533-Fusarium graminearum PH 1-Sordariomycetes 100 XP 009262582.1-1028729-Fusarium pseudograminearu-Sordariomycetes KZL72807.1-708197-Colletotrichum tofieldiae-Sordariomycetes 99 100 XP 007603123.1-1445577-Colletotrichum fioriniae-Sordariomycetes 90 KXH26574.1-703756-Colletotrichum simmondsii-Sordariomycetes XP 001270372.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes KOS43413.1-229535-Penicillium nordicum-Eurotiomycetes OQD93913.1-60172-Penicillium solitum-Eurotiomycetes 100 CRL30865.1-5075-Penicillium camemberti-Eurotiomycetes GAT21367.1-1069201-Aspergillus luchuensis-Eurotiomycetes 100 GAA83286.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 QUERY-Afu1g17720-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KEY83928.1-1476192-Aspergillus fumigatus var-Eurotiomycetes XP 001275177.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes EYE92016.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes 100 OJJ83693.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes 95 ODM24346.1-573508-Aspergillus cristatus-Eurotiomycetes KOC18776.1-1392242-Aspergillus flavus AF70-Eurotiomycetes XP 001827115.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 100 XP 002384353.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes 94 KJK67560.1-1403190-Aspergillus parasiticus S-Eurotiomycetes XP 015412269.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes XP 020054870.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes OJJ68416.1-767769-Aspergillus brasiliensis-Eurotiomycetes 83 GAA92532.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes OJZ90081.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes 100 OJI84725.1-767770-Aspergillus tubingensis C-Eurotiomycetes GAQ35625.1-5061-Aspergillus niger-Eurotiomycetes EHA20992.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 99 XP 001390020.1-425011-Aspergillus niger CBS 513-Eurotiomycetes GAW19657.1-1813822-fungal sp No 14919-other Fungi EKG13971.1-1126212-Macrophomina phaseolina M-Dothideomycetes XP 007587274.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes 99 XP 020131637.1-236234-Diplodia corticola-Dothideomycetes 93 KKY24657.1-420778-Diplodia seriata-Dothideomycetes 99 OMP82498.1-420778-Diplodia seriata-Dothideomycetes XP 008097989.1-645133-Colletotrichum graminicol-Sordariomycetes 94 OLN87541.1-708187-Colletotrichum chlorophyt-Sordariomycetes XP 018161333.1-759273-Colletotrichum higginsian-Sordariomycetes 100 CCF43873.1-80884-Colletotrichum higginsian-Sordariomycetes KZL77770.1-708197-Colletotrichum tofieldiae-Sordariomycetes 98 KZL83287.1-1573173-Colletotrichum incanum-Sordariomycetes EQB56236.1-1237896-Colletotrichum gloeospori-Sordariomycetes 87 ENH76248.1-1213857-Colletotrichum orbiculare-Sordariomycetes 85 OHF02959.1-1209926-Colletotrichum orchidophi-Sordariomycetes XP 007598459.1-1445577-Colletotrichum fioriniae-Sordariomycetes 97 KXH51170.1-1460502-Colletotrichum nymphaeae-Sordariomycetes 83 KXH33030.1-703756-Colletotrichum simmondsii-Sordariomycetes KXH47137.1-1209931-Colletotrichum salicis-Sordariomycetes XP 018758495.1-334819-Fusarium verticillioides-Sordariomycetes CZR48165.1-1227346-Fusarium proliferatum ET1-Sordariomycetes 100 S0EES1.1-1279085-Fusarium fujikuroi IMI 58-Sordariomycetes OBS18895.1-36050-Fusarium poae-Sordariomycetes 99 XP 009259311.1-1028729-Fusarium pseudograminearu-Sordariomycetes 99 XP 011327621.1-229533-Fusarium graminearum PH 1-Sordariomycetes

140 Tree scale: 0.1 XP 008081690.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes XP 007827071.1-1229662-Pestalotiopsis fici W106-Sordariomycetes 90 KID84094.1-1276136-Metarhizium guizhouense A-Sordariomycetes 100 KJK74648.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 EXU96140.1-568076-Metarhizium robertsii-Sordariomycetes Taxonomy KFG81512.1-5530-Metarhizium anisopliae-Sordariomycetes 100 XP 009262581.1-1028729-Fusarium pseudograminearu-Sordariomycetes 100 EYB31406.1-5518-Fusarium graminearum-Sordariomycetes Leotiomycetes 100 XP 011319441.1-229533-Fusarium graminearum PH 1-Sordariomycetes OJJ34619.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes ORY67811.1-1141098-Pseudomassariella vexata-Sordariomycetes Sordariomycetes 92 100 XP 007919597.1-1286976-Phaeoacremonium minimum U-Sordariomycetes KZL72809.1-708197-Colletotrichum tofieldiae-Sordariomycetes 100 KXH26575.1-703756-Colletotrichum simmondsii-Sordariomycetes Eurotiomycetes 95 91 XP 007603122.1-1445577-Colletotrichum fioriniae-Sordariomycetes XP 001270373.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes KOS43410.1-229535-Penicillium nordicum-Eurotiomycetes Dothideomycetes CRL30864.1-5075-Penicillium camemberti-Eurotiomycetes 100 OQD93854.1-60172-Penicillium solitum-Eurotiomycetes 100 KEY83927.1-1476192-Aspergillus fumigatus var-Eurotiomycetes other_Pezizomycotina 100 QUERY-Afu1g17723-330879-Aspergillus fumigatus Af2-Eurotiomycetes 97 KMK54953.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 94 EHA18932.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes other_Ascomycota 100 GAA83287.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 GAT21368.1-1069201-Aspergillus luchuensis-Eurotiomycetes GAM89934.1-1603295-fungal sp No 11243-other Fungi other_Fungi OCK79255.1-1314670-Lepidopterella palustris-Dothideomycetes KDB17570.1-1159556-Ustilaginoidea virens-Sordariomycetes OLN87181.1-708187-Colletotrichum chlorophyt-Sordariomycetes other_Opisthokonta EME45950.1-675120-Dothistroma septosporum N-Dothideomycetes 100 XP 007925836.1-383855-Pseudocercospora fijiensi-Dothideomycetes XP 016217501.1-253628-Verruconis gallopava-Dothideomycetes other_Eukaryota 100 XP 016217502.1-253628-Verruconis gallopava-Dothideomycetes KPM41344.1-78410-Neonectria ditissima-Sordariomycetes 93 XP 003041148.1-660122-Nectria haematococca mpVI-Sordariomycetes Bacteria XP 003004171.1-526221-Verticillium alfalfae VaM-Sordariomycetes 100 XP 009654134.1-498257-Verticillium dahliae VdLs-Sordariomycetes OQO06661.1-1507870-Rachicladosporium antarct-Dothideomycetes Archaea OQO18097.1-1974281-Rachicladosporium sp CCF-Dothideomycetes 100 OQO10055.1-1507870-Rachicladosporium antarct-Dothideomycetes KND94354.1-1163406-Tolypocladium ophioglosso-Sordariomycetes Viruses 98 KYK61204.1-98403-Drechmeria coniospora-Sordariomycetes 100 ODA80971.1-98403-Drechmeria coniospora-Sordariomycetes OAA79236.1-1081108-Cordyceps confragosa RCEF-Sordariomycetes XP 006670394.1-983644-Cordyceps militaris CM01-Sordariomycetes 100 OAA52451.1-1081107-Cordyceps brongniartii RC-Sordariomycetes XP 013425836.1-1043004-Aureobasidium namibiae CB-Dothideomycetes KEQ67761.1-1043003-Aureobasidium melanogenum-Dothideomycetes 99 XP 013347900.1-1043005-Aureobasidium subglaciale-Dothideomycetes KEQ89864.1-1043002-Aureobasidium pullulans E-Dothideomycetes XP 020127555.1-236234-Diplodia corticola-Dothideomycetes OMP87711.1-420778-Diplodia seriata-Dothideomycetes 91 100 KKY19282.1-420778-Diplodia seriata-Dothideomycetes XP 007582513.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes 94 EKG15688.1-1126212-Macrophomina phaseolina M-Dothideomycetes GAW20816.1-1813822-fungal sp No 14919-other Fungi XP 007789192.1-1287681-Eutypa lata UCREL1-Sordariomycetes 98 OTB04225.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 100 100 OTA91720.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes 100 OTA57144.1-1001937-Hypoxylon sp EC38-Sordariomycetes XP 013950960.1-413071-Trichoderma virens Gv29 8-Sordariomycetes OPB39282.1-1491466-Trichoderma guizhouense-Sordariomycetes XP 018658687.1-398673-Trichoderma gamsii-Sordariomycetes 100 100 XP 013944581.1-452589-Trichoderma atroviride IM-Sordariomycetes OTA03574.1-858221-Trichoderma parareesei-Sordariomycetes 100 ETS05692.1-1344414-Trichoderma reesei RUT C-Sordariomycetes 93 XP 006963461.1-431241-Trichoderma reesei QM6a-Sordariomycetes OAL52377.1-765867-Pyrenochaeta sp DS3sAY3a-Dothideomycetes XP 018386305.1-5599-Alternaria alternata-Dothideomycetes 100 XP 008020889.1-671987-Setosphaeria turcica Et28-Dothideomycetes KNG44838.1-183478-Stemphylium lycopersici-Dothideomycetes 95 XP 007710543.1-930089-Bipolaris zeicola 26 R 13-Dothideomycetes XP 014557659.1-930091-Bipolaris victoriae FI3-Dothideomycetes 100 XP 007695836.1-665912-Bipolaris sorokiniana ND9-Dothideomycetes XP 007688430.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes KHJ30190.1-52586-Erysiphe necator-Leotiomycetes 99 CCU74944.1-546991-Blumeria graminis f sp-Leotiomycetes 100 EPQ61501.1-1268274-Blumeria graminis f sp-Leotiomycetes ESZ92078.1-1432307-Sclerotinia borealis F 41-Leotiomycetes 97 100 CCD49728.1-999810-Botrytis cinerea T4-Leotiomycetes 100 EMR86983.1-1290391-Botrytis cinerea BcDW1-Leotiomycetes CZT04403.1-914238-Rhynchosporium agropyri-Leotiomycetes CZT06225.1-914237-Rhynchosporium commune-Leotiomycetes 100 CZT44003.1-38038-Rhynchosporium secalis-Leotiomycetes KFY73687.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes XP 018131908.1-342668-Pseudogymnoascus verrucos-Leotiomycetes OBT57271.1-1622150-Pseudogymnoascus sp 24MN-Leotiomycetes KFZ08350.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes OBT40144.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes OBT85056.1-1622148-Pseudogymnoascus sp 03VT-Leotiomycetes OBT79024.1-1622149-Pseudogymnoascus sp 05NY-Leotiomycetes 100 KFY20101.1-1420901-Pseudogymnoascus sp VKM-Leotiomycetes KFY26564.1-1420906-Pseudogymnoascus sp VKM-Leotiomycetes KFX89420.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes KFY07860.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes KFY29642.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes OBT62886.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes 87 KFZ11761.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes 93 KFZ01402.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes

141 Tree scale: 0.1 XP 011319440.1-229533-Fusarium graminearum PH 1-Sordariomycetes OJJ34620.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 100 KZL72754.1-708197-Colletotrichum tofieldiae-Sordariomycetes XP 007603120.1-1445577-Colletotrichum fioriniae-Sordariomycetes 99 100 KXH26577.1-703756-Colletotrichum simmondsii-Sordariomycetes Taxonomy ORY67812.1-1141098-Pseudomassariella vexata-Sordariomycetes 97 XP 001270374.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes CRL30864.1-5075-Penicillium camemberti-Eurotiomycetes Leotiomycetes 91 OQD93951.1-60172-Penicillium solitum-Eurotiomycetes 100 KOS43411.1-229535-Penicillium nordicum-Eurotiomycetes 100 EHA18933.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes Sordariomycetes 100 GAT21369.1-1069201-Aspergillus luchuensis-Eurotiomycetes 100 GAA83288.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 93 KEY83926.1-1476192-Aspergillus fumigatus var-Eurotiomycetes Eurotiomycetes 100 QUERY-Afu1g17725-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54952.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes GAD97588.1-1356009-Byssochlamys spectabilis-Eurotiomycetes Dothideomycetes KIV78800.1-1016849-Exophiala sideris-Eurotiomycetes XP 013324239.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes 90 XP 013327701.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes other_Pezizomycotina XP 007787724.1-1263415-Endocarpon pusillum Z0702-Eurotiomycetes XP 018188082.1-1328760-Xylona heveae TC161-other Pezizomycotina SLM39940.1-136370-Umbilicaria pustulata-other Pezizomycotina other_Ascomycota XP 006962774.1-431241-Trichoderma reesei QM6a-Sordariomycetes EKG11783.1-1126212-Macrophomina phaseolina M-Dothideomycetes 100 100 OMP88640.1-420778-Diplodia seriata-Dothideomycetes other_Fungi XP 013270916.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes XP 016227003.1-212818-Exophiala mesophila-Eurotiomycetes XP 016632485.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes other_Opisthokonta 100 XP 018695363.1-1367422-Fonsecaea erecta-Eurotiomycetes XP 016250130.1-569365-Cladophialophora immunda-Eurotiomycetes 100 OQV00536.1-569365-Cladophialophora immunda-Eurotiomycetes other_Eukaryota 100 XP 007739659.1-1182543-Cladophialophora psammoph-Eurotiomycetes 100 XP 016625804.1-1442370-Cladophialophora bantiana-Eurotiomycetes 100 OAL38606.1-856822-Fonsecaea nubica-Eurotiomycetes Bacteria XP 013284061.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes 100 OAG44669.1-254056-Fonsecaea monophora-Eurotiomycetes OJJ30508.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes Archaea 90 ODM14997.1-573508-Aspergillus cristatus-Eurotiomycetes 100 OJJ85962.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes 99 EYE95192.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes Viruses KKK12315.1-308745-Aspergillus rambellii-Eurotiomycetes 100 CEL05234.1-454130-Aspergillus calidoustus-Eurotiomycetes 98 XP 659488.1-227321-Aspergillus nidulans FGSC-Eurotiomycetes 87 OJJ64297.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes 100 OJI98390.1-1036611-Aspergillus versicolor CB-Eurotiomycetes XP 001215107.1-341663-Aspergillus terreus NIH26-Eurotiomycetes XP 015404014.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 90 80 OGM50557.1-109264-Aspergillus bombycis-Eurotiomycetes 100 KJK64619.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 85 KOC13210.1-1392242-Aspergillus flavus AF70-Eurotiomycetes 94 BAE57429.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 99 86 XP 020058705.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes OOF99045.1-602072-Aspergillus carbonarius I-Eurotiomycetes 100 OJJ77694.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 EHA19494.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 100 100 XP 001394301.2-425011-Aspergillus niger CBS 513-Eurotiomycetes 80 GAA83936.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 OJZ90757.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes XP 001272758.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes GAQ07125.1-293939-Aspergillus lentulus-Eurotiomycetes 100 XP 001260060.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes GAO90844.1-91492-Aspergillus udagawae-Eurotiomycetes 98 KEY79284.1-1476192-Aspergillus fumigatus var-Eurotiomycetes XP 749561.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes 98 100 EDP54085.1-451804-Aspergillus fumigatus A11-Eurotiomycetes OGE49342.1-1835702-Penicillium arizonense-Eurotiomycetes 100 OQD80997.1-416450-Penicillium antarcticum-Eurotiomycetes OQE08078.1-29845-Penicillium vulpinum-Eurotiomycetes OQE47134.1-36646-Penicillium coprophilum-Eurotiomycetes 100 CDM35225.1-1365484-Penicillium roqueforti FM-Eurotiomycetes OQE29031.1-254877-Penicillium flavigenum-Eurotiomycetes OQE88303.1-60175-Penicillium nalgiovense-Eurotiomycetes 100 99 XP 002557726.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 KZN85110.1-5076-Penicillium chrysogenum-Eurotiomycetes KGO69212.1-40296-Penicillium italicum-Eurotiomycetes 100 XP 016600192.1-27334-Penicillium expansum-Eurotiomycetes KUM56968.1-48697-Penicillium freii-Eurotiomycetes 100 93 OQD61678.1-60169-Penicillium polonicum-Eurotiomycetes 100 OQD87913.1-60172-Penicillium solitum-Eurotiomycetes KOS46330.1-229535-Penicillium nordicum-Eurotiomycetes OQD59998.1-60169-Penicillium polonicum-Eurotiomycetes OQE15639.1-303698-Penicillium steckii-Eurotiomycetes OQD74193.1-69771-Penicillium decumbens-Eurotiomycetes EPS33859.1-933388-Penicillium oxalicum 114-Eurotiomycetes 100 OKO98199.1-1316194-Penicillium subrubescens-Eurotiomycetes 86 97 CEO60574.1-104259-Penicillium brasilianum-Eurotiomycetes 100 OOQ85244.1-104259-Penicillium brasilianum-Eurotiomycetes XP 003021658.1-663202-Trichophyton verrucosum H-Eurotiomycetes 100 XP 003175950.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes 100 XP 001275058.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes XP 015406159.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 97 GAQ09758.1-293939-Aspergillus lentulus-Eurotiomycetes 100 GAO90499.1-91492-Aspergillus udagawae-Eurotiomycetes 90 XP 001262985.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes 80 EDP52282.1-451804-Aspergillus fumigatus A11-Eurotiomycetes 93 XP 754149.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54265.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 88 KEY83423.1-1476192-Aspergillus fumigatus var-Eurotiomycetes

142 Tree scale: 0.1 XP 008081692.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes XP 007284066.1-1213859-Colletotrichum gloeospori-Sordariomycetes 100 XP 009226576.1-644352-Gaeumannomyces tritici R3-Sordariomycetes OLN95722.1-708187-Colletotrichum chlorophyt-Sordariomycetes ENH79961.1-1213857-Colletotrichum orbiculare-Sordariomycetes Taxonomy 100 XP 018156678.1-759273-Colletotrichum higginsian-Sordariomycetes 100 CCF40672.1-80884-Colletotrichum higginsian-Sordariomycetes XP 007827068.1-1229662-Pestalotiopsis fici W106-Sordariomycetes Leotiomycetes KJK74646.1-1291518-Metarhizium anisopliae BR-Sordariomycetes XP 014540253.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes 99 KID84092.1-1276136-Metarhizium guizhouense A-Sordariomycetes Sordariomycetes 97 XP 007817536.1-655844-Metarhizium robertsii ARS-Sordariomycetes 100 99 EXU96138.1-568076-Metarhizium robertsii-Sordariomycetes KID64554.1-1276135-Metarhizium anisopliae AR-Sordariomycetes Eurotiomycetes 100 KFG81514.1-5530-Metarhizium anisopliae-Sordariomycetes XP 014573819.1-1276143-Metarhizium majus ARSEF 2-Sordariomycetes 99 XP 014573817.1-1276143-Metarhizium majus ARSEF 2-Sordariomycetes Dothideomycetes XP 009262580.1-1028729-Fusarium pseudograminearu-Sordariomycetes 100 EYB31404.1-5518-Fusarium graminearum-Sordariomycetes 100 XP 011319439.1-229533-Fusarium graminearum PH 1-Sordariomycetes other_Pezizomycotina OJJ34621.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 89 XP 007919596.1-1286976-Phaeoacremonium minimum U-Sordariomycetes 100 ORY67813.1-1141098-Pseudomassariella vexata-Sordariomycetes other_Ascomycota KZL72810.1-708197-Colletotrichum tofieldiae-Sordariomycetes 97 100 XP 007603121.1-1445577-Colletotrichum fioriniae-Sordariomycetes 87 KXH26576.1-703756-Colletotrichum simmondsii-Sordariomycetes other_Fungi XP 001270375.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes KOS43404.1-229535-Penicillium nordicum-Eurotiomycetes 100 100 CRL31207.1-5075-Penicillium camemberti-Eurotiomycetes other_Opisthokonta 80 OQD93895.1-60172-Penicillium solitum-Eurotiomycetes 99 EHA18934.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 100 GAA83289.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes other_Eukaryota 95 QUERY-Afu1g17730-330879-Aspergillus fumigatus Af2-Eurotiomycetes KMK54951.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 100 KEY83628.1-1476192-Aspergillus fumigatus var-Eurotiomycetes Bacteria XP 007585874.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes KUM55930.1-48697-Penicillium freii-Eurotiomycetes 100 OQD63677.1-60169-Penicillium polonicum-Eurotiomycetes Archaea OQN98775.1-1507870-Rachicladosporium antarct-Dothideomycetes OQO31264.1-1974281-Rachicladosporium sp CCF-Dothideomycetes 100 OQO32413.1-1974281-Rachicladosporium sp CCF-Dothideomycetes Viruses XP 001221378.1-306901-Chaetomium globosum CBS 1-Sordariomycetes 100 GAP83760.1-77044-Rosellinia necatrix-Sordariomycetes 100 GAW16228.1-1813822-fungal sp No 14919-other Fungi CZT43007.1-38038-Rhynchosporium secalis-Leotiomycetes CZT02537.1-914238-Rhynchosporium agropyri-Leotiomycetes 100 CZS89922.1-914237-Rhynchosporium commune-Leotiomycetes OOQ90648.1-104259-Penicillium brasilianum-Eurotiomycetes 100 OCK87094.1-794803-Cenococcum geophilum 1 58-Dothideomycetes 97 XP 001242735.1-246410-Coccidioides immitis RS-Eurotiomycetes 100 XP 003069899.1-222929-Coccidioides posadasii C7-Eurotiomycetes XP 013324712.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes ORY67421.1-1141098-Pseudomassariella vexata-Sordariomycetes 100 KFY27625.1-1420901-Pseudogymnoascus sp VKM-Leotiomycetes KKY37405.1-1214573-Diaporthe ampelina-Sordariomycetes 97 100 KUI61106.1-694573-Valsa mali var pyri-Sordariomycetes 97 KUI64941.1-105487-Valsa mali-Sordariomycetes XP 016640623.1-563466-Scedosporium apiospermum-Sordariomycetes 90 CRG89195.1-28573-Talaromyces islandicus-Eurotiomycetes KFY65449.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY16430.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes KFY42551.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes 100 99 KFY01186.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes KFY80203.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes XP 018126945.1-342668-Pseudogymnoascus verrucos-Leotiomycetes 100 KFZ13814.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes OIW26266.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes KXX82446.1-100816-Madurella mycetomatis-Sordariomycetes XP 018189444.1-1328760-Xylona heveae TC161-other Pezizomycotina AIP87505.1-1547289-fungal sp NRRL 50135-other Fungi 98 XP 006671675.1-983644-Cordyceps militaris CM01-Sordariomycetes 100 XP 001269054.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes OQE24788.1-254877-Penicillium flavigenum-Eurotiomycetes 100 XP 016593838.1-27334-Penicillium expansum-Eurotiomycetes 100 100 KGO47795.1-27334-Penicillium expansum-Eurotiomycetes XP 018384352.1-5599-Alternaria alternata-Dothideomycetes OJJ60065.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes GAW20888.1-1813822-fungal sp No 14919-other Fungi OAA50381.1-1081105-Metarhizium rileyi RCEF 4-Sordariomycetes KND87974.1-1163406-Tolypocladium ophioglosso-Sordariomycetes 84 OAQ95892.1-1105325-Cordyceps confragosa-Sordariomycetes 100 100 KGO39263.1-27334-Penicillium expansum-Eurotiomycetes 100 XP 016597438.1-27334-Penicillium expansum-Eurotiomycetes AGO86659.1-42747-Fusarium heterosporum-Sordariomycetes 100 BAR40285.1-1608308-Fusarium sp FN080326-Sordariomycetes CCT65288.1-1279085-Fusarium fujikuroi IMI 58-Sordariomycetes 100 KLO99474.1-5127-Fusarium fujikuroi-Sordariomycetes 100 EXL63283.1-1089448-Fusarium oxysporum f sp-Sordariomycetes EWY99914.1-909455-Fusarium oxysporum FOSC 3-Sordariomycetes EWZ45927.1-660027-Fusarium oxysporum Fo47-Sordariomycetes 99 XP 018255233.1-426428-Fusarium oxysporum f sp-Sordariomycetes 100 EGU88867.1-660025-Fusarium oxysporum Fo5176-Sordariomycetes ENH63775.1-1229664-Fusarium oxysporum f sp-Sordariomycetes XP 018760576.1-334819-Fusarium verticillioides-Sordariomycetes EXM16555.1-1089449-Fusarium oxysporum f sp-Sordariomycetes 9893 EMT72751.1-1229665-Fusarium oxysporum f sp-Sordariomycetes CVL06271.1-192010-Fusarium mangiferae-Sordariomycetes 81 CVK99125.1-948311-Fusarium proliferatum-Sordariomycetes 99 KLO92103.1-5127-Fusarium fujikuroi-Sordariomycetes

143 Tree scale: 0.1 GAA82870.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 OOF98050.1-602072-Aspergillus carbonarius I-Eurotiomycetes KXX75968.1-100816-Madurella mycetomatis-Sordariomycetes 100 KFY16432.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes 100 OGM43967.1-109264-Aspergillus bombycis-Eurotiomycetes Taxonomy 100 KFZ13812.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes AFP89391.1-1116209-Cladosporium phlei-Dothideomycetes XP 001274946.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes Leotiomycetes 100 OHE97136.1-1209926-Colletotrichum orchidophi-Sordariomycetes 100 100 XP 008100501.1-645133-Colletotrichum graminicol-Sordariomycetes 100 ENH82918.1-1213857-Colletotrichum orbiculare-Sordariomycetes Sordariomycetes 100 KXH63779.1-1209931-Colletotrichum salicis-Sordariomycetes XP 001394625.2-425011-Aspergillus niger CBS 513-Eurotiomycetes 94 XP 015405462.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes Eurotiomycetes XP 020054826.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes 100 OPB36213.1-1491466-Trichoderma guizhouense-Sordariomycetes 100 KFY96473.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes Dothideomycetes 96 OTB16775.1-1001832-Daldinia sp EC12-Sordariomycetes XP 001258701.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes EKG15173.1-1126212-Macrophomina phaseolina M-Dothideomycetes other_Pezizomycotina 100 OBS22513.1-36050-Fusarium poae-Sordariomycetes 100 ALQ32891.1-36050-Fusarium poae-Sordariomycetes XP 003177417.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes other_Ascomycota AFN68293.1-5599-Alternaria alternata-Dothideomycetes 100 100 XP 018384350.1-5599-Alternaria alternata-Dothideomycetes BAR40283.1-1608308-Fusarium sp FN080326-Sordariomycetes other_Fungi 100 100 AGO86662.1-42747-Fusarium heterosporum-Sordariomycetes ALQ32974.1-70751-Fusarium sp NRRL 25184-Sordariomycetes 100 KLO99472.1-5127-Fusarium fujikuroi-Sordariomycetes other_Opisthokonta ALQ33011.1-42677-Fusarium subglutinans-Sordariomycetes ALQ32847.1-246455-Fusarium foetens-Sordariomycetes 100100 XP 018760578.1-334819-Fusarium verticillioides-Sordariomycetes other_Eukaryota EWZ45929.1-660027-Fusarium oxysporum Fo47-Sordariomycetes 100 EWY99916.1-909455-Fusarium oxysporum FOSC 3-Sordariomycetes 100 EXA51210.1-1080344-Fusarium oxysporum f sp-Sordariomycetes Bacteria 97 EXK85226.1-1089458-Fusarium oxysporum f sp-Sordariomycetes 88 EGU88865.1-660025-Fusarium oxysporum Fo5176-Sordariomycetes KKY30404.1-1214573-Diaporthe ampelina-Sordariomycetes Archaea 100 KUI65083.1-105487-Valsa mali-Sordariomycetes 100 KUI61972.1-694573-Valsa mali var pyri-Sordariomycetes KEQ86259.1-1043002-Aureobasidium pullulans E-Dothideomycetes Viruses 100 100 XP 013343243.1-1043005-Aureobasidium subglaciale-Dothideomycetes ORY67416.1-1141098-Pseudomassariella vexata-Sordariomycetes 98 97 OTA59680.1-1001937-Hypoxylon sp EC38-Sordariomycetes 100 OTA90454.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes KFY84670.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 100 100 KFZ03054.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes XP 018134601.1-342668-Pseudogymnoascus verrucos-Leotiomycetes 100 100 KFY76989.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes OBT61811.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes 93 KFY49789.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY92340.1-1420911-Pseudogymnoascus sp VKM-Leotiomycetes KMM67662.1-454284-Coccidioides posadasii RM-Eurotiomycetes XP 020057498.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes 100 EPS29069.1-933388-Penicillium oxalicum 114-Eurotiomycetes 100 ARF05979.1-69781-Penicillium oxalicum-Eurotiomycetes 100 OTA98397.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 100 SLM36899.1-136370-Umbilicaria pustulata-other Pezizomycotina 100 ORY61265.1-1141098-Pseudomassariella vexata-Sordariomycetes 84 100 KXX75016.1-100816-Madurella mycetomatis-Sordariomycetes XP 007911967.1-1286976-Phaeoacremonium minimum U-Sordariomycetes 100 AFI23577.1-80884-Colletotrichum higginsian-Sordariomycetes KFY06571.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes 93 100 KFY34117.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes KFX98753.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes 100 100 KFY37827.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes 82 KFY92181.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 100 OBT43992.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes AKL78828.1-101852-Glarea lozoyensis-Leotiomycetes XP 018156684.1-759273-Colletotrichum higginsian-Sordariomycetes XP 007827066.1-1229662-Pestalotiopsis fici W106-Sordariomycetes KID84093.1-1276136-Metarhizium guizhouense A-Sordariomycetes 100 100 87 XP 014573816.1-1276143-Metarhizium majus ARSEF 2-Sordariomycetes XP 014540252.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes 100 EXU96139.1-568076-Metarhizium robertsii-Sordariomycetes 10081 XP 007817535.1-655844-Metarhizium robertsii ARS-Sordariomycetes 90 KFG81513.1-5530-Metarhizium anisopliae-Sordariomycetes 90 KJK74647.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 KJK88403.1-1291519-Metarhizium anisopliae BR-Sordariomycetes XP 009262583.1-1028729-Fusarium pseudograminearu-Sordariomycetes 100 XP 011319443.1-229533-Fusarium graminearum PH 1-Sordariomycetes 97 EYB31408.1-5518-Fusarium graminearum-Sordariomycetes OJJ34622.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 100 XP 007919595.1-1286976-Phaeoacremonium minimum U-Sordariomycetes 100 ORY67814.1-1141098-Pseudomassariella vexata-Sordariomycetes 100 KZL72765.1-708197-Colletotrichum tofieldiae-Sordariomycetes 100 KXH26578.1-703756-Colletotrichum simmondsii-Sordariomycetes 99 100 XP 007603119.1-1445577-Colletotrichum fioriniae-Sordariomycetes XP 001270376.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes KOS43405.1-229535-Penicillium nordicum-Eurotiomycetes 100 OQD93852.1-60172-Penicillium solitum-Eurotiomycetes 100 84 CRL31206.1-5075-Penicillium camemberti-Eurotiomycetes GAA83290.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 EHA18935.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 100 GAA83291.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 KEY83630.1-1476192-Aspergillus fumigatus var-Eurotiomycetes 100 KEY83624.1-1476192-Aspergillus fumigatus var-Eurotiomycetes 83 QUERY-Afu1g17740-330879-Aspergillus fumigatus Af2-Eurotiomycetes 99 KMK54950.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes

144 Gene phylogenies of SM gene cluster 24. The phylogenies of several genes in this cluster are consistent with horizontal transfer between Aspergillus fumigatus and Metarhizium fungi.

Tree scale: 0.1 XP 007296427.1-1072389-Marssonina brunnea f sp-Leotiomycetes XP 008077050.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes 100 XP 018072047.1-149040-Phialocephala scopiformis-Leotiomycetes CZT43162.1-38038-Rhynchosporium secalis-Leotiomycetes 100 CZT11580.1-914238-Rhynchosporium agropyri-Leotiomycetes 96 CZS89593.1-914237-Rhynchosporium commune-Leotiomycetes Taxonomy OJJ36038.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes CEL10457.1-454130-Aspergillus calidoustus-Eurotiomycetes XP 002542821.1-336963-Uncinocarpus reesii 1704-Eurotiomycetes Leotiomycetes 99 XP 003065204.1-222929-Coccidioides posadasii C7-Eurotiomycetes 100 XP 001240891.2-246410-Coccidioides immitis RS-Eurotiomycetes ODM15614.1-573508-Aspergillus cristatus-Eurotiomycetes 100 Sordariomycetes 100 EYE92891.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes 95 OJJ78910.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes CBF83180.1-227321-Aspergillus nidulans FGSC-Eurotiomycetes Eurotiomycetes 100 OJJ06615.1-1036611-Aspergillus versicolor CB-Eurotiomycetes 100 OJJ55537.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes OJJ77012.1-767769-Aspergillus brasiliensis-Eurotiomycetes Dothideomycetes EHA27738.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 99 100 XP 001400896.1-425011-Aspergillus niger CBS 513-Eurotiomycetes GAT25150.1-1069201-Aspergillus luchuensis-Eurotiomycetes 100 100 GAA87037.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes other_Pezizomycotina 99 OJZ84412.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes 94 GAQ42891.1-5061-Aspergillus niger-Eurotiomycetes 99 OJI89661.1-767770-Aspergillus tubingensis C-Eurotiomycetes other_Ascomycota XP 001270431.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes GAO81734.1-91492-Aspergillus udagawae-Eurotiomycetes 99 XP 001267442.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes other_Fungi GAQ10144.1-293939-Aspergillus lentulus-Eurotiomycetes 89 QUERY-g6968 Afu4g01560-746128-Aspergillus fumigatus F13619-Eurotiomycetes 99 KMK58224.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 97 XP 746301.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes other_Opisthokonta 100 EDP47212.1-451804-Aspergillus fumigatus A11-Eurotiomycetes XP 001216540.1-341663-Aspergillus terreus NIH26-Eurotiomycetes OGM48739.1-109264-Aspergillus bombycis-Eurotiomycetes other_Eukaryota 88 XP 015410062.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 100 KJK65795.1-1403190-Aspergillus parasiticus S-Eurotiomycetes KOC18044.1-1392242-Aspergillus flavus AF70-Eurotiomycetes Bacteria 86 XP 002375402.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes EIT76050.1-1160506-Aspergillus oryzae 3 042-Eurotiomycetes 99 100 BAE60324.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes Archaea OOO10907.1-5062-Aspergillus oryzae-Eurotiomycetes 98 XP 001727163.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes ORY01044.1-1231657-Clohesyomyces aquaticus-Dothideomycetes OTA20627.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes Viruses XP 016226458.1-212818-Exophiala mesophila-Eurotiomycetes 100 XP 013263454.1-1182545-Exophiala aquamarina CBS-Eurotiomycetes KXJ90614.1-196109-Microdochium bolleyi-Sordariomycetes 100 XP 007794483.1-1287681-Eutypa lata UCREL1-Sordariomycetes XP 018002106.1-1664694-Phialophora attae-Eurotiomycetes 100 XP 008721510.1-1220924-Cyphellophora europaea CB-Eurotiomycetes EKG11171.1-1126212-Macrophomina phaseolina M-Dothideomycetes 97 OMP85362.1-420778-Diplodia seriata-Dothideomycetes 100 XP 020128081.1-236234-Diplodia corticola-Dothideomycetes OQO11280.1-1507870-Rachicladosporium antarct-Dothideomycetes 100 OQO16597.1-1974281-Rachicladosporium sp CCF-Dothideomycetes 88 XP 016760817.1-692275-Sphaerulina musiva SO2202-Dothideomycetes 86 XP 007922748.1-383855-Pseudocercospora fijiensi-Dothideomycetes 100 KXT15555.1-113226-Pseudocercospora musae-Dothideomycetes 90 KXS96288.1-321146-Mycosphaerella eumusae-Dothideomycetes OCF41129.1-1296119-Kwoniella heveanensis CBS-Basidiomycota 100 OCF30869.1-1296120-Kwoniella heveanensis BCC-Basidiomycota 100 XP 019013165.1-1296096-Kwoniella pini CBS 10737-Basidiomycota OCF75561.1-1296123-Kwoniella mangroviensis C-Basidiomycota 100 100 OCF56990.1-1331196-Kwoniella mangroviensis C-Basidiomycota 100 XP 019003966.1-1296122-Kwoniella mangroviensis C-Basidiomycota XP 018033866.1-1460663-Paraphaeosphaeria sporulo-Dothideomycetes OAL45073.1-765867-Pyrenochaeta sp DS3sAY3a-Dothideomycetes OSS49377.1-105696-Epicoccum nigrum-Dothideomycetes 97 XP 001793374.1-321614-Parastagonospora nodorum-Dothideomycetes 81 OAK94798.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 100 XP 018378749.1-5599-Alternaria alternata-Dothideomycetes 81 XP 001932626.1-426418-Pyrenophora tritici repen-Dothideomycetes 99 XP 003302027.1-861557-Pyrenophora teres f tere-Dothideomycetes 97 XP 008027512.1-671987-Setosphaeria turcica Et28-Dothideomycetes XP 007697529.1-665912-Bipolaris sorokiniana ND9-Dothideomycetes 99 XP 007683675.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes XP 014558596.1-930091-Bipolaris victoriae FI3-Dothideomycetes 10099 XP 007712126.1-930089-Bipolaris zeicola 26 R 13-Dothideomycetes XP 014074018.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 100 EMD95091.1-701091-Bipolaris maydis C5-Dothideomycetes XP 003716610.1-242507-Magnaporthe oryzae 70 15-Sordariomycetes OHF04679.1-1209926-Colletotrichum orchidophi-Sordariomycetes 100 EQB56549.1-1237896-Colletotrichum gloeospori-Sordariomycetes 100 100 XP 007286131.1-1213859-Colletotrichum gloeospori-Sordariomycetes KIL85073.1-40199-Fusarium avenaceum-Sordariomycetes CVK92144.1-192010-Fusarium mangiferae-Sordariomycetes 83 100 CCT68210.1-1279085-Fusarium fujikuroi IMI 58-Sordariomycetes CZR43559.1-1227346-Fusarium proliferatum ET1-Sordariomycetes 100 KLP03524.1-5127-Fusarium fujikuroi-Sordariomycetes 93 XP 018747180.1-334819-Fusarium verticillioides-Sordariomycetes KEY68918.1-1280523-Stachybotrys chartarum IB-Sordariomycetes 100 KFA46922.1-1280524-Stachybotrys chartarum IB-Sordariomycetes OAQ82133.1-33203-Purpureocillium lilacinum-Sordariomycetes 92 100 XP 018180896.1-33203-Purpureocillium lilacinum-Sordariomycetes 89 KID90516.1-1276136-Metarhizium guizhouense A-Sordariomycetes KFG78048.1-5530-Metarhizium anisopliae-Sordariomycetes 100 KJK83203.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 83 XP 007821653.1-655844-Metarhizium robertsii ARS-Sordariomycetes XP 014549539.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes

145 Tree scale: 0.1 XP 002845085.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 001273570.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes OJJ58603.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes CEL01231.1-454130-Aspergillus calidoustus-Eurotiomycetes EIT78678.1-1160506-Aspergillus oryzae 3 042-Eurotiomycetes Taxonomy 100 XP 003190831.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 100 EDP47211.1-451804-Aspergillus fumigatus A11-Eurotiomycetes 100 QUERY-g6969-746128-Aspergillus fumigatus F13619-Eurotiomycetes Leotiomycetes 100 KMK58225.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes XP 001398267.2-425011-Aspergillus niger CBS 513-Eurotiomycetes OJJ68624.1-767769-Aspergillus brasiliensis-Eurotiomycetes Sordariomycetes 90 OJZ81931.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes 100 OJI84484.1-767770-Aspergillus tubingensis C-Eurotiomycetes 91 GAQ45479.1-5061-Aspergillus niger-Eurotiomycetes Eurotiomycetes XP 018191062.1-1328760-Xylona heveae TC161-other Pezizomycotina 100 OAL52324.1-765867-Pyrenochaeta sp DS3sAY3a-Dothideomycetes 94 XP 018378722.1-5599-Alternaria alternata-Dothideomycetes Dothideomycetes KZM21115.1-5454-Ascochyta rabiei-Dothideomycetes 97 100 XP 001796022.1-321614-Parastagonospora nodorum-Dothideomycetes EEH11568.1-447093-Histoplasma capsulatum G1-Eurotiomycetes other_Pezizomycotina KMM69569.1-454284-Coccidioides posadasii RM-Eurotiomycetes 85 99 EFW13202.1-443226-Coccidioides posadasii st-Eurotiomycetes 95 XP 003068010.1-222929-Coccidioides posadasii C7-Eurotiomycetes other_Ascomycota 100 KMU80244.1-454286-Coccidioides immitis RMSC-Eurotiomycetes 97 XP 001240477.2-246410-Coccidioides immitis RS-Eurotiomycetes 88 96 KMP06014.1-404692-Coccidioides immitis RMSC-Eurotiomycetes other_Fungi XP 002844998.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 003173465.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes 100 XP 003017135.1-663331-Trichophyton benhamiae CB-Eurotiomycetes other_Opisthokonta 100 100 DAA79278.1-663331-Trichophyton benhamiae CB-Eurotiomycetes EZF73891.1-1215331-Trichophyton soudanense C-Eurotiomycetes 100100 XP 003234940.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes other_Eukaryota KDB20938.1-1215338-Trichophyton interdigital-Eurotiomycetes 100 EGE04025.1-559882-Trichophyton equinum CBS-Eurotiomycetes 100 EGD92514.1-647933-Trichophyton tonsurans CB-Eurotiomycetes Bacteria XP 016759724.1-692275-Sphaerulina musiva SO2202-Dothideomycetes KKY20022.1-158046-Phaeomoniella chlamydospo-Eurotiomycetes GAP91259.2-77044-Rosellinia necatrix-Sordariomycetes Archaea KNG49901.1-183478-Stemphylium lycopersici-Dothideomycetes XP 001265991.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes EEH03636.1-447093-Histoplasma capsulatum G1-Eurotiomycetes Viruses 80 KZM25466.1-5454-Ascochyta rabiei-Dothideomycetes XP 007778467.1-1168221-Coniosporium apollinis CB-Dothideomycetes OTA34654.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes 100 XP 007924137.1-383855-Pseudocercospora fijiensi-Dothideomycetes 100 OAG38885.1-254056-Fonsecaea monophora-Eurotiomycetes 99 XP 008724428.1-1279043-Cladophialophora carrioni-Eurotiomycetes 100 100 OCT54285.1-86049-Cladophialophora carrioni-Eurotiomycetes CZR51218.1-576137-Phialocephala subalpina-Leotiomycetes 98 XP 001594886.1-665079-Sclerotinia sclerotiorum-Leotiomycetes 100 CZT07810.1-914238-Rhynchosporium agropyri-Leotiomycetes 100 CZS93857.1-914237-Rhynchosporium commune-Leotiomycetes 94 XP 008077390.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes XP 018068395.1-149040-Phialocephala scopiformis-Leotiomycetes 100 KFX88714.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY48034.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes 92 100 KFX88222.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes KFY16810.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY34224.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 80 OBT87810.1-1622148-Pseudogymnoascus sp 03VT-Leotiomycetes 86 KFZ11396.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes 99 KFY86108.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes OQE02951.1-29845-Penicillium vulpinum-Eurotiomycetes OJJ65712.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 CEJ62815.1-104259-Penicillium brasilianum-Eurotiomycetes OJJ51154.1-1073090-Penicilliopsis zonata CBS-Eurotiomycetes 92 XP 001272864.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes 100 OKP10478.1-1316194-Penicillium subrubescens-Eurotiomycetes 87 XP 014533754.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes 100 KGO75585.1-40296-Penicillium italicum-Eurotiomycetes 92 KOS46913.1-229535-Penicillium nordicum-Eurotiomycetes XP 013330291.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes 81 XP 002849897.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes KKK24458.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes XP 018188589.1-1328760-Xylona heveae TC161-other Pezizomycotina 98 OOF98851.1-602072-Aspergillus carbonarius I-Eurotiomycetes XP 001269184.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes XP 020054588.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes 96 KKK25535.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK22659.1-308745-Aspergillus rambellii-Eurotiomycetes 100 100 XP 015407646.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 93 GAA93052.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes OJJ70046.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 GAQ45724.1-5061-Aspergillus niger-Eurotiomycetes 100 OJI80326.1-767770-Aspergillus tubingensis C-Eurotiomycetes XP 003177803.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes KKZ65771.1-1247875-Emmonsia crescens UAMH 30-Eurotiomycetes 100 OJD27490.1-1658174-Blastomyces percursus-Eurotiomycetes 98 XP 002844482.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 001536734.1-339724-Histoplasma capsulatum NA-Eurotiomycetes 100 EEH06116.1-447093-Histoplasma capsulatum G1-Eurotiomycetes 99 DAA74701.1-663331-Trichophyton benhamiae CB-Eurotiomycetes 100 XP 003012199.1-663331-Trichophyton benhamiae CB-Eurotiomycetes EGE06895.1-559882-Trichophyton equinum CBS-Eurotiomycetes 99100 KDB20329.1-1215338-Trichophyton interdigital-Eurotiomycetes 97 EZF29251.1-1215336-Trichophyton interdigital-Eurotiomycetes EFW13947.1-443226-Coccidioides posadasii st-Eurotiomycetes KMU75148.1-454286-Coccidioides immitis RMSC-Eurotiomycetes 99 KMP06160.1-404692-Coccidioides immitis RMSC-Eurotiomycetes 99 XP 012213936.1-246410-Coccidioides immitis RS-Eurotiomycetes

146 Tree scale: 0.1 EHA21874.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes ODM24152.1-573508-Aspergillus cristatus-Eurotiomycetes XP 749839.2-330879-Aspergillus fumigatus Af2-Eurotiomycetes EHA21875.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes GAQ02781.1-293939-Aspergillus lentulus-Eurotiomycetes Taxonomy OJJ52319.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes KKK18129.1-308745-Aspergillus rambellii-Eurotiomycetes 94 KGO71756.1-40296-Penicillium italicum-Eurotiomycetes Leotiomycetes OOF90907.1-602072-Aspergillus carbonarius I-Eurotiomycetes 97 XP 001271128.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes OJJ65769.1-767769-Aspergillus brasiliensis-Eurotiomycetes Sordariomycetes 95 CDM37172.1-1365484-Penicillium roqueforti FM-Eurotiomycetes OOO03860.1-5062-Aspergillus oryzae-Eurotiomycetes BAE66189.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes Eurotiomycetes 100 KOC17684.1-1392242-Aspergillus flavus AF70-Eurotiomycetes BAE66187.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 86 XP 001827320.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes Dothideomycetes QUERY-g6970-746128-Aspergillus fumigatus F13619-Eurotiomycetes XP 001263991.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes 89 XP 001258122.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes other_Pezizomycotina XP 749859.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes 82 XP 749857.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes 99 other_Ascomycota KMK54477.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes other_Fungi other_Opisthokonta

other_Eukaryota Bacteria Archaea Viruses

147 Tree scale: 0.1 KFH70213.1-1069443-Mortierella verticillata-other Fungi EXX51121.1-1432141-Rhizophagus irregularis D-other Fungi 99 OCB88219.1-108892-Sanghuangporus baumii-Basidiomycota OAL68712.1-34388-Trichophyton violaceum-Eurotiomycetes 86 XP 001215419.1-341663-Aspergillus terreus NIH26-Eurotiomycetes Taxonomy 99 XP 001216586.1-341663-Aspergillus terreus NIH26-Eurotiomycetes 100 XP 001217170.1-341663-Aspergillus terreus NIH26-Eurotiomycetes 97 XP 001218522.1-341663-Aspergillus terreus NIH26-Eurotiomycetes Leotiomycetes OQE62929.1-60175-Penicillium nalgiovense-Eurotiomycetes KGO69277.1-40296-Penicillium italicum-Eurotiomycetes GAQ08606.1-293939-Aspergillus lentulus-Eurotiomycetes Sordariomycetes XP 002488215.1-441959-Talaromyces stipitatus AT-Eurotiomycetes ODM18440.1-573508-Aspergillus cristatus-Eurotiomycetes OKP03204.1-1316194-Penicillium subrubescens-Eurotiomycetes Eurotiomycetes 100 XP 002480770.1-441959-Talaromyces stipitatus AT-Eurotiomycetes XP 002567108.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 KGO69067.1-40296-Penicillium italicum-Eurotiomycetes Dothideomycetes XP 016596093.1-27334-Penicillium expansum-Eurotiomycetes 100 KGO64397.1-40296-Penicillium italicum-Eurotiomycetes 99 CRL30689.1-5075-Penicillium camemberti-Eurotiomycetes other_Pezizomycotina XP 002488702.1-441959-Talaromyces stipitatus AT-Eurotiomycetes OQD84463.1-60172-Penicillium solitum-Eurotiomycetes 98 XP 015402001.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes other_Ascomycota 100 OKP08625.1-1316194-Penicillium subrubescens-Eurotiomycetes 99 XP 002483205.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 98 OKP01548.1-1316194-Penicillium subrubescens-Eurotiomycetes other_Fungi 96 ODM20081.1-573508-Aspergillus cristatus-Eurotiomycetes 90 OQD94302.1-29845-Penicillium vulpinum-Eurotiomycetes 100 KXG45514.1-5078-Penicillium griseofulvum-Eurotiomycetes other_Opisthokonta OQD74462.1-416450-Penicillium antarcticum-Eurotiomycetes 100 OQE64429.1-60175-Penicillium nalgiovense-Eurotiomycetes 100 OQE11891.1-254877-Penicillium flavigenum-Eurotiomycetes other_Eukaryota 89 XP 002567425.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 CRL30793.1-5075-Penicillium camemberti-Eurotiomycetes 100 KOS37194.1-229535-Penicillium nordicum-Eurotiomycetes Bacteria OOO09542.1-5062-Aspergillus oryzae-Eurotiomycetes OOO09036.1-5062-Aspergillus oryzae-Eurotiomycetes OOO03916.1-5062-Aspergillus oryzae-Eurotiomycetes Archaea 100 OOO11125.1-5062-Aspergillus oryzae-Eurotiomycetes OOO07428.1-5062-Aspergillus oryzae-Eurotiomycetes 100 CRL30062.1-5075-Penicillium camemberti-Eurotiomycetes Viruses CEJ54941.1-104259-Penicillium brasilianum-Eurotiomycetes 100 OQD73537.1-416450-Penicillium antarcticum-Eurotiomycetes 81 100 100 OQD83198.1-60172-Penicillium solitum-Eurotiomycetes 100 OQD93697.1-29845-Penicillium vulpinum-Eurotiomycetes 80 94 CDM36561.1-1365484-Penicillium roqueforti FM-Eurotiomycetes OQD77421.1-416450-Penicillium antarcticum-Eurotiomycetes OQE59292.1-60175-Penicillium nalgiovense-Eurotiomycetes 83 100 CRL30511.1-5075-Penicillium camemberti-Eurotiomycetes 100 ODM14526.1-573508-Aspergillus cristatus-Eurotiomycetes 100 ODM14908.1-573508-Aspergillus cristatus-Eurotiomycetes KZN93944.1-5076-Penicillium chrysogenum-Eurotiomycetes 100 CEJ54944.1-104259-Penicillium brasilianum-Eurotiomycetes 100 OQD77362.1-416450-Penicillium antarcticum-Eurotiomycetes CEJ62802.1-104259-Penicillium brasilianum-Eurotiomycetes 100 CEJ62588.1-104259-Penicillium brasilianum-Eurotiomycetes 100 88 OQE09706.1-254877-Penicillium flavigenum-Eurotiomycetes OQD59907.1-60169-Penicillium polonicum-Eurotiomycetes 100 XP 014536092.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes 96 EKV05644.1-1170229-Penicillium digitatum PHI-Eurotiomycetes 100 OQD59928.1-60169-Penicillium polonicum-Eurotiomycetes 100 XP 016595386.1-27334-Penicillium expansum-Eurotiomycetes 99 OQD93269.1-29845-Penicillium vulpinum-Eurotiomycetes 96 OQE10648.1-254877-Penicillium flavigenum-Eurotiomycetes EKV12561.1-1170229-Penicillium digitatum PHI-Eurotiomycetes 100 OQD95763.1-29845-Penicillium vulpinum-Eurotiomycetes 100 98 KGO48340.1-27334-Penicillium expansum-Eurotiomycetes KOS43721.1-229535-Penicillium nordicum-Eurotiomycetes 81 KUM55737.1-48697-Penicillium freii-Eurotiomycetes CEJ62820.1-104259-Penicillium brasilianum-Eurotiomycetes 100 100 OKP12447.1-1316194-Penicillium subrubescens-Eurotiomycetes OQD94157.1-29845-Penicillium vulpinum-Eurotiomycetes 100 KOS39661.1-229535-Penicillium nordicum-Eurotiomycetes KMK63759.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 97 XP 007842184.1-1229662-Pestalotiopsis fici W106-Sordariomycetes QUERY-g6971-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 GAA93358.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes KUM57015.1-48697-Penicillium freii-Eurotiomycetes 100 OQD78775.1-416450-Penicillium antarcticum-Eurotiomycetes OKP09781.1-1316194-Penicillium subrubescens-Eurotiomycetes 97 KGO49162.1-27334-Penicillium expansum-Eurotiomycetes 100 KGO38396.1-27334-Penicillium expansum-Eurotiomycetes XP 002566879.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 90 100 KZN87744.1-5076-Penicillium chrysogenum-Eurotiomycetes XP 014530679.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes CRL30307.1-5075-Penicillium camemberti-Eurotiomycetes OQE63822.1-60175-Penicillium nalgiovense-Eurotiomycetes 100 OQE59006.1-60175-Penicillium nalgiovense-Eurotiomycetes OGE46542.1-1835702-Penicillium arizonense-Eurotiomycetes OGE46568.1-1835702-Penicillium arizonense-Eurotiomycetes 100 100 OGE46641.1-1835702-Penicillium arizonense-Eurotiomycetes KZN87780.1-5076-Penicillium chrysogenum-Eurotiomycetes KZN93968.1-5076-Penicillium chrysogenum-Eurotiomycetes OQE07396.1-254877-Penicillium flavigenum-Eurotiomycetes 100 OQE05809.1-254877-Penicillium flavigenum-Eurotiomycetes 99 XP 002566875.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 KZN87748.1-5076-Penicillium chrysogenum-Eurotiomycetes CDM32672.1-1365484-Penicillium roqueforti FM-Eurotiomycetes CDM32673.1-1365484-Penicillium roqueforti FM-Eurotiomycetes 89 XP 002566745.1-500485-Penicillium rubens Wiscon-Eurotiomycetes

148 Tree scale: 0.1 KKP06687.1-5544-Trichoderma harzianum-Sordariomycetes EKG13590.1-1126212-Macrophomina phaseolina M-Dothideomycetes OCK74025.1-1314670-Lepidopterella palustris-Dothideomycetes QUERY-g6972-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 KJK94812.1-1291519-Metarhizium anisopliae BR-Sordariomycetes Taxonomy 100 KJK74886.1-1291518-Metarhizium anisopliae BR-Sordariomycetes CCD56079.1-999810-Botrytis cinerea T4-Leotiomycetes 100 EMR80975.1-1290391-Botrytis cinerea BcDW1-Leotiomycetes Leotiomycetes 100 XP 001550804.1-332648-Botrytis cinerea B05 10-Leotiomycetes 100 KJZ75226.1-1043627-Hirsutella minnesotensis-Sordariomycetes KXX79210.1-100816-Madurella mycetomatis-Sordariomycetes Sordariomycetes 99 OCW41141.1-158607-Diaporthe helianthi-Sordariomycetes XP 016643173.1-563466-Scedosporium apiospermum-Sordariomycetes 100 KIM92948.1-913774-Oidiodendron maius Zn-Leotiomycetes Eurotiomycetes GAW20844.1-1813822-fungal sp No 14919-other Fungi 97 KFA69530.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KEY72234.1-1280523-Stachybotrys chartarum IB-Sordariomycetes Dothideomycetes 100 KFA52277.1-1280524-Stachybotrys chartarum IB-Sordariomycetes 92 KKY16571.1-420778-Diplodia seriata-Dothideomycetes 100 XP 020125956.1-236234-Diplodia corticola-Dothideomycetes other_Pezizomycotina XP 008027816.1-671987-Setosphaeria turcica Et28-Dothideomycetes 100 XP 003300446.1-861557-Pyrenophora teres f tere-Dothideomycetes 100100 XP 001937751.1-426418-Pyrenophora tritici repen-Dothideomycetes other_Ascomycota XP 007689110.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes 100 XP 014073566.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 100 EMD94542.1-701091-Bipolaris maydis C5-Dothideomycetes other_Fungi KYK57378.1-98403-Drechmeria coniospora-Sordariomycetes XP 014539081.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes KUM59361.1-48697-Penicillium freii-Eurotiomycetes other_Opisthokonta XP 018075919.1-149040-Phialocephala scopiformis-Leotiomycetes 100 OCK82927.1-1314670-Lepidopterella palustris-Dothideomycetes OCK74982.1-1314670-Lepidopterella palustris-Dothideomycetes other_Eukaryota 100 OCK73526.1-1314670-Lepidopterella palustris-Dothideomycetes XP 018157389.1-759273-Colletotrichum higginsian-Sordariomycetes 92 KZZ96510.1-1081109-Aschersonia aleyrodis RCE-Sordariomycetes Bacteria KDN69202.1-1173701-Colletotrichum sublineola-Sordariomycetes 100 XP 008098964.1-645133-Colletotrichum graminicol-Sordariomycetes XP 007286629.1-1213859-Colletotrichum gloeospori-Sordariomycetes Archaea 100 EQB52534.1-1237896-Colletotrichum gloeospori-Sordariomycetes KDN60187.1-1173701-Colletotrichum sublineola-Sordariomycetes 100 KXH47916.1-1209931-Colletotrichum salicis-Sordariomycetes Viruses OCW42213.1-158607-Diaporthe helianthi-Sordariomycetes 100 KXH50180.1-703756-Colletotrichum simmondsii-Sordariomycetes OIW33137.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes KIM94742.1-913774-Oidiodendron maius Zn-Leotiomycetes 100 KXJ85092.1-196109-Microdochium bolleyi-Sordariomycetes 100 KXJ85939.1-196109-Microdochium bolleyi-Sordariomycetes XP 001260852.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes 98 OJJ40738.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 100 XP 001271874.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes 100 XP 001267016.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes 100 KMK54731.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 100 XP 751828.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes EME39031.1-675120-Dothistroma septosporum N-Dothideomycetes 100 AAC49196.1-162425-Aspergillus nidulans-Eurotiomycetes 100 XP 681087.1-227321-Aspergillus nidulans FGSC-Eurotiomycetes 100 KKK21817.1-308745-Aspergillus rambellii-Eurotiomycetes 100 XP 020053364.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes 100 100 KND91002.1-1163406-Tolypocladium ophioglosso-Sordariomycetes OAL39677.1-856822-Fonsecaea nubica-Eurotiomycetes OAG34162.1-254056-Fonsecaea monophora-Eurotiomycetes 100 XP 013280246.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes 100 OAG44384.1-254056-Fonsecaea monophora-Eurotiomycetes 80 KXT06680.1-321146-Mycosphaerella eumusae-Dothideomycetes XP 018159597.1-759273-Colletotrichum higginsian-Sordariomycetes 100 OLN90223.1-708187-Colletotrichum chlorophyt-Sordariomycetes KZL71764.1-1573173-Colletotrichum incanum-Sordariomycetes 100 KZL63447.1-708197-Colletotrichum tofieldiae-Sordariomycetes XP 008098516.1-645133-Colletotrichum graminicol-Sordariomycetes XP 003717434.1-242507-Magnaporthe oryzae 70 15-Sordariomycetes OOQ86043.1-104259-Penicillium brasilianum-Eurotiomycetes OCK77653.1-1314670-Lepidopterella palustris-Dothideomycetes GAD91552.1-1356009-Byssochlamys spectabilis-Eurotiomycetes 100 OQD73863.1-69771-Penicillium decumbens-Eurotiomycetes 97 CEJ57600.1-104259-Penicillium brasilianum-Eurotiomycetes 94 99 EPS31372.1-933388-Penicillium oxalicum 114-Eurotiomycetes KMU74509.1-454286-Coccidioides immitis RMSC-Eurotiomycetes KMM66554.1-454284-Coccidioides posadasii RM-Eurotiomycetes EFW13723.1-443226-Coccidioides posadasii st-Eurotiomycetes 100 XP 003069072.1-222929-Coccidioides posadasii C7-Eurotiomycetes XP 001240202.2-246410-Coccidioides immitis RS-Eurotiomycetes 95 KMP02584.1-404692-Coccidioides immitis RMSC-Eurotiomycetes KFZ09804.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes OBT43052.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes XP 012740108.1-658429-Pseudogymnoascus destruct-Leotiomycetes KFY12939.1-1420901-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY74280.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes 99 XP 018132394.1-342668-Pseudogymnoascus verrucos-Leotiomycetes KFY92053.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 83 98 KFZ12582.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes OQE06029.1-29845-Penicillium vulpinum-Eurotiomycetes 99 OQE43268.1-36646-Penicillium coprophilum-Eurotiomycetes 100 OGM48142.1-109264-Aspergillus bombycis-Eurotiomycetes KJK61356.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 100 KOC10931.1-1392242-Aspergillus flavus AF70-Eurotiomycetes 81 OOO07101.1-5062-Aspergillus oryzae-Eurotiomycetes EIT78191.1-1160506-Aspergillus oryzae 3 042-Eurotiomycetes 9892 BAE65144.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes BAJ04387.1-5062-Aspergillus oryzae-Eurotiomycetes 83 XP 001826277.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes

149 Tree scale: 0.1 OBT67700.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes APX44019.1-85828-Pestalotiopsis microspora-Sordariomycetes 100 OCL03381.1-574774-Glonium stellatum-Dothideomycetes 100 OAA75818.1-1081108-Cordyceps confragosa RCEF-Sordariomycetes 100 OAR02343.1-1105325-Cordyceps confragosa-Sordariomycetes Taxonomy EKG10413.1-1126212-Macrophomina phaseolina M-Dothideomycetes 100 XP 001212807.1-341663-Aspergillus terreus NIH26-Eurotiomycetes 100 OSS44048.1-105696-Epicoccum nigrum-Dothideomycetes Leotiomycetes 97 100 XP 008100672.1-645133-Colletotrichum graminicol-Sordariomycetes 100 KDN72002.1-1173701-Colletotrichum sublineola-Sordariomycetes ODM17885.1-573508-Aspergillus cristatus-Eurotiomycetes 97 Sordariomycetes 100 XP 002339967.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 100 KIA75609.1-40382-Aspergillus ustus-Eurotiomycetes 100 KUI52981.1-694573-Valsa mali var pyri-Sordariomycetes Eurotiomycetes 100 KKK24813.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK20296.1-308745-Aspergillus rambellii-Eurotiomycetes KUL85087.1-198730-Talaromyces verruculosus-Eurotiomycetes Dothideomycetes 100 GAM35873.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes SLM38803.1-136370-Umbilicaria pustulata-other Pezizomycotina 90 OJJ68593.1-767769-Aspergillus brasiliensis-Eurotiomycetes other_Pezizomycotina 100 100 XP 001905653.1-515849-Podospora anserina S mat-Sordariomycetes 89 KDN67932.1-1173701-Colletotrichum sublineola-Sordariomycetes 100 CEN59741.1-454130-Aspergillus calidoustus-Eurotiomycetes other_Ascomycota 100 KFX41537.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes XP 001245248.2-246410-Coccidioides immitis RS-Eurotiomycetes 100 99 KMU78416.1-454286-Coccidioides immitis RMSC-Eurotiomycetes other_Fungi 100 KMM70187.1-454284-Coccidioides posadasii RM-Eurotiomycetes XP 003071593.1-222929-Coccidioides posadasii C7-Eurotiomycetes 100 EFW13280.1-443226-Coccidioides posadasii st-Eurotiomycetes other_Opisthokonta ADM79462.1-27339-Cladonia grayi-other Pezizomycotina 100 KIM97742.1-913774-Oidiodendron maius Zn-Leotiomycetes KZL84004.1-1573173-Colletotrichum incanum-Sordariomycetes other_Eukaryota OCK79461.1-1314670-Lepidopterella palustris-Dothideomycetes EKG13586.1-1126212-Macrophomina phaseolina M-Dothideomycetes ESZ91464.1-1432307-Sclerotinia borealis F 41-Leotiomycetes Bacteria EMR80977.1-1290391-Botrytis cinerea BcDW1-Leotiomycetes 100 CCD56082.1-999810-Botrytis cinerea T4-Leotiomycetes 100 XP 001550802.1-332648-Botrytis cinerea B05 10-Leotiomycetes Archaea GAW20845.1-1813822-fungal sp No 14919-other Fungi KFA69540.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA52141.1-1280524-Stachybotrys chartarum IB-Sordariomycetes Viruses 100 KFA74445.1-1283842-Stachybotrys chartarum IB-Sordariomycetes 100 KEY72235.1-1280523-Stachybotrys chartarum IB-Sordariomycetes XP 020125916.1-236234-Diplodia corticola-Dothideomycetes 100 100 OMP81764.1-420778-Diplodia seriata-Dothideomycetes 100 KKY16572.1-420778-Diplodia seriata-Dothideomycetes 100 XP 008027817.1-671987-Setosphaeria turcica Et28-Dothideomycetes XP 001937752.1-426418-Pyrenophora tritici repen-Dothideomycetes 100100 XP 003300445.1-861557-Pyrenophora teres f tere-Dothideomycetes XP 007689067.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes 100 XP 014073567.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 100 AAR90276.1-5016-Bipolaris maydis-Dothideomycetes EKG18751.1-1126212-Macrophomina phaseolina M-Dothideomycetes XP 008028244.1-671987-Setosphaeria turcica Et28-Dothideomycetes KNG47856.1-183478-Stemphylium lycopersici-Dothideomycetes 99 XP 003301870.1-861557-Pyrenophora teres f tere-Dothideomycetes 87 AOO87096.1-1187899-Alternaria alternantherae-Dothideomycetes 100 100 XP 018391073.1-5599-Alternaria alternata-Dothideomycetes 98 XP 007690544.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes XP 014552188.1-930091-Bipolaris victoriae FI3-Dothideomycetes 100 100 XP 007709531.1-930089-Bipolaris zeicola 26 R 13-Dothideomycetes XP 014074072.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 100 AAR90277.1-5016-Bipolaris maydis-Dothideomycetes KKP06686.1-5544-Trichoderma harzianum-Sordariomycetes 92 QUERY-g6973-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 KJK94813.1-1291519-Metarhizium anisopliae BR-Sordariomycetes 100 KJK74887.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 OAL00021.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 100 GAQ41879.1-5061-Aspergillus niger-Eurotiomycetes 100 OCK76732.1-1314670-Lepidopterella palustris-Dothideomycetes 100 KXX79217.1-100816-Madurella mycetomatis-Sordariomycetes 89 GAW14687.1-1813822-fungal sp No 14919-other Fungi 100 KJZ75225.1-1043627-Hirsutella minnesotensis-Sordariomycetes XP 016643174.1-563466-Scedosporium apiospermum-Sordariomycetes 83 KFX42009.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes 100 KIM92947.1-913774-Oidiodendron maius Zn-Leotiomycetes KFA60273.1-1283841-Stachybotrys chlorohalona-Sordariomycetes KFA50975.1-1280524-Stachybotrys chartarum IB-Sordariomycetes 100 KFA76431.1-1283842-Stachybotrys chartarum IB-Sordariomycetes 99 100 KEY70385.1-1280523-Stachybotrys chartarum IB-Sordariomycetes CRG91775.1-28573-Talaromyces islandicus-Eurotiomycetes 100 XP 020118539.1-1441469-Talaromyces atroroseus-Eurotiomycetes 100 XP 002340065.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 100 XP 002149742.1-441960-Talaromyces marneffei ATC-Eurotiomycetes 98 GAM33954.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 100 CRG86486.1-28573-Talaromyces islandicus-Eurotiomycetes XP 013957201.1-413071-Trichoderma virens Gv29 8-Sordariomycetes 100 XP 013938921.1-452589-Trichoderma atroviride IM-Sordariomycetes 100 100 XP 018665083.1-398673-Trichoderma gamsii-Sordariomycetes OTA01506.1-858221-Trichoderma parareesei-Sordariomycetes 100 ETS03185.1-1344414-Trichoderma reesei RUT C-Sordariomycetes 100 100 XP 006964226.1-431241-Trichoderma reesei QM6a-Sordariomycetes CCF32372.1-80884-Colletotrichum higginsian-Sordariomycetes 100 XP 018158294.1-759273-Colletotrichum higginsian-Sordariomycetes 100 KPM35785.1-78410-Neonectria ditissima-Sordariomycetes 100 BAV19379.1-80385-Stachybotrys bisbyi-Sordariomycetes 100 KFA69335.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA81444.1-1283842-Stachybotrys chartarum IB-Sordariomycetes 99 KFA51631.1-1280524-Stachybotrys chartarum IB-Sordariomycetes 87 KEY71342.1-1280523-Stachybotrys chartarum IB-Sordariomycetes

150 Tree scale: 0.1 XP 018184752.1-1328760-Xylona heveae TC161-other Pezizomycotina KFY21354.1-1420906-Pseudogymnoascus sp VKM-Leotiomycetes XP 002841013.1-656061-Tuber melanosporum Mel28-other Pezizomycotina QUERY-g6974-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 KJK74880.1-1291518-Metarhizium anisopliae BR-Sordariomycetes Taxonomy GAT55036.1-658473-Mycena chlorophos-Basidiomycota 96 KTB38372.1-221103-Moniliophthora roreri-Basidiomycota 100 XP 007845553.1-1381753-Moniliophthora roreri MCA-Basidiomycota Leotiomycetes AAF64435.2-40559-Botrytis cinerea-Leotiomycetes ESZ95339.1-1432307-Sclerotinia borealis F 41-Leotiomycetes 99 XP 001586264.1-665079-Sclerotinia sclerotiorum-Leotiomycetes Sordariomycetes KIN05050.1-913774-Oidiodendron maius Zn-Leotiomycetes 88 OGE55021.1-1835702-Penicillium arizonense-Eurotiomycetes 100 KFY94063.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes Eurotiomycetes EHL03343.1-1104152-Glarea lozoyensis 74030-Leotiomycetes 84 100 XP 008083356.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes CZR50636.1-576137-Phialocephala subalpina-Leotiomycetes Dothideomycetes 99 XP 018076654.1-149040-Phialocephala scopiformis-Leotiomycetes 93 XP 007292794.1-1072389-Marssonina brunnea f sp-Leotiomycetes 100 CZT47972.1-38038-Rhynchosporium secalis-Leotiomycetes other_Pezizomycotina 100 CZS94684.1-914237-Rhynchosporium commune-Leotiomycetes XP 007915264.1-1286976-Phaeoacremonium minimum U-Sordariomycetes OIW34274.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes other_Ascomycota 87 XP 009852902.1-510951-Neurospora tetrasperma FG-Sordariomycetes 100 XP 011395298.1-367110-Neurospora crassa OR74A-Sordariomycetes OQE41467.1-36646-Penicillium coprophilum-Eurotiomycetes other_Fungi 100 OQD89879.1-416450-Penicillium antarcticum-Eurotiomycetes 100 OGE48087.1-1835702-Penicillium arizonense-Eurotiomycetes 100 GAM38946.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes other_Opisthokonta 84 95 XP 002479167.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 100 XP 002144369.1-441960-Talaromyces marneffei ATC-Eurotiomycetes 100 KFX53693.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes other_Eukaryota ORY66156.1-1141098-Pseudomassariella vexata-Sordariomycetes AEX31648.1-1491472-Trichoderma lixii-Sordariomycetes XP 008031645.1-671987-Setosphaeria turcica Et28-Dothideomycetes Bacteria OTA93836.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes 97 100 OTA69676.1-1001937-Hypoxylon sp EC38-Sordariomycetes GAP91503.1-77044-Rosellinia necatrix-Sordariomycetes Archaea ABR28367.1-167374-Xylaria sp BCC 1067-Sordariomycetes 100 GAW14624.1-1813822-fungal sp No 14919-other Fungi XP 007780309.1-1168221-Coniosporium apollinis CB-Dothideomycetes Viruses EWC44372.1-1043628-Drechslerella stenobrocha-other Pezizomycotina XP 013344082.1-1043005-Aureobasidium subglaciale-Dothideomycetes XP 013432457.1-1043004-Aureobasidium namibiae CB-Dothideomycetes 100 KEQ88423.1-1043002-Aureobasidium pullulans E-Dothideomycetes XP 013325594.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes 91 XP 007581427.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes 97 EKG14558.1-1126212-Macrophomina phaseolina M-Dothideomycetes OQD94582.1-60172-Penicillium solitum-Eurotiomycetes 100 OKP14090.1-1316194-Penicillium subrubescens-Eurotiomycetes 99 CEJ59761.1-104259-Penicillium brasilianum-Eurotiomycetes 100 OOQ82870.1-104259-Penicillium brasilianum-Eurotiomycetes 97 OCK75213.1-1314670-Lepidopterella palustris-Dothideomycetes OCK98761.1-794803-Cenococcum geophilum 1 58-Dothideomycetes 100 OCL13154.1-574774-Glonium stellatum-Dothideomycetes 87 OAL53265.1-765867-Pyrenochaeta sp DS3sAY3a-Dothideomycetes OAL06695.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 100 XP 001794321.1-321614-Parastagonospora nodorum-Dothideomycetes 88 KNG49244.1-183478-Stemphylium lycopersici-Dothideomycetes EME39528.1-675120-Dothistroma septosporum N-Dothideomycetes XP 007928999.1-383855-Pseudocercospora fijiensi-Dothideomycetes XP 016758133.1-692275-Sphaerulina musiva SO2202-Dothideomycetes KJX98136.1-1047168-Zymoseptoria brevis-Dothideomycetes 100 100 XP 003850560.1-336722-Zymoseptoria tritici IPO3-Dothideomycetes 88 AJG42821.1-1047171-Zymoseptoria tritici-Dothideomycetes OTA35326.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes 100 KXL50548.1-245562-Acidomyces richmondensis-Dothideomycetes 95 XP 007681432.1-717646-Baudoinia panamericana UA-Dothideomycetes ODQ69136.1-675824-Lipomyces starkeyi NRRL Y-Saccharomycotina XP 002797035.1-502779-Paracoccidioides lutzii P-Eurotiomycetes 100 EGE77149.2-653446-Blastomyces dermatitidis-Eurotiomycetes 100 KLJ08977.1-1246674-Emmonsia parva UAMH 139-Eurotiomycetes XP 001243393.1-246410-Coccidioides immitis RS-Eurotiomycetes 100 KMU88747.1-396776-Coccidioides immitis H538-Eurotiomycetes 100 KMM68322.1-454284-Coccidioides posadasii RM-Eurotiomycetes XP 003066894.1-222929-Coccidioides posadasii C7-Eurotiomycetes XP 002843435.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 003172129.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes 100 EGD96741.1-647933-Trichophyton tonsurans CB-Eurotiomycetes 99 91 XP 003012776.1-663331-Trichophyton benhamiae CB-Eurotiomycetes 100 XP 003236897.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes 100 XP 003023086.1-663202-Trichophyton verrucosum H-Eurotiomycetes XP 007787617.1-1263415-Endocarpon pusillum Z0702-Eurotiomycetes XP 013255212.1-1182545-Exophiala aquamarina CBS-Eurotiomycetes XP 013267468.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes 83 XP 007720660.1-1182541-Capronia coronata CBS 617-Eurotiomycetes XP 007736804.1-1182542-Capronia epimyces CBS 606-Eurotiomycetes XP 009152324.1-858893-Exophiala dermatitidis NI-Eurotiomycetes 100 XP 016237739.1-91928-Exophiala spinifera-Eurotiomycetes 100 XP 016262934.1-215243-Exophiala oligosperma-Eurotiomycetes OCT46042.1-86049-Cladophialophora carrioni-Eurotiomycetes 100 XP 008724188.1-1279043-Cladophialophora carrioni-Eurotiomycetes XP 018689397.1-1367422-Fonsecaea erecta-Eurotiomycetes 88 XP 016631957.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes XP 016246567.1-569365-Cladophialophora immunda-Eurotiomycetes 100 OQV06046.1-569365-Cladophialophora immunda-Eurotiomycetes 91 XP 013286744.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes 100 OAL34481.1-856822-Fonsecaea nubica-Eurotiomycetes XP 007751748.1-1182543-Cladophialophora psammoph-Eurotiomycetes 98 XP 016621099.1-1442370-Cladophialophora bantiana-Eurotiomycetes

151 Tree scale: 0.1 XP 018138618.1-1380566-Pochonia chlamydosporia 1-Sordariomycetes GAQ08559.1-293939-Aspergillus lentulus-Eurotiomycetes XP 002482893.1-441959-Talaromyces stipitatus AT-Eurotiomycetes XP 018138617.1-1380566-Pochonia chlamydosporia 1-Sordariomycetes ORY69640.1-1141098-Pseudomassariella vexata-Sordariomycetes Taxonomy XP 001217075.1-341663-Aspergillus terreus NIH26-Eurotiomycetes XP 018067965.1-149040-Phialocephala scopiformis-Leotiomycetes XP 018066031.1-149040-Phialocephala scopiformis-Leotiomycetes Leotiomycetes CRG86679.1-28573-Talaromyces islandicus-Eurotiomycetes OJJ40786.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes OJJ77434.1-767769-Aspergillus brasiliensis-Eurotiomycetes Sordariomycetes KKK25665.1-308745-Aspergillus rambellii-Eurotiomycetes XP 007731038.1-1182542-Capronia epimyces CBS 606-Eurotiomycetes XP 008020155.1-671987-Setosphaeria turcica Et28-Dothideomycetes Eurotiomycetes CRG90999.1-28573-Talaromyces islandicus-Eurotiomycetes XP 018188015.1-1328760-Xylona heveae TC161-other Pezizomycotina XP 008026822.1-671987-Setosphaeria turcica Et28-Dothideomycetes Dothideomycetes XP 002485357.1-441959-Talaromyces stipitatus AT-Eurotiomycetes XP 001208364.1-341663-Aspergillus terreus NIH26-Eurotiomycetes XP 020115508.1-1441469-Talaromyces atroroseus-Eurotiomycetes other_Pezizomycotina 96 ESZ98975.1-1432307-Sclerotinia borealis F 41-Leotiomycetes OCL10940.1-574774-Glonium stellatum-Dothideomycetes 96 ORX95400.1-1231657-Clohesyomyces aquaticus-Dothideomycetes other_Ascomycota KFZ05412.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY75262.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes OCK83279.1-1314670-Lepidopterella palustris-Dothideomycetes other_Fungi 99 XP 007685917.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes XP 006669619.1-983644-Cordyceps militaris CM01-Sordariomycetes 100 OAQ99551.1-1105325-Cordyceps confragosa-Sordariomycetes other_Opisthokonta XP 001258910.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes 100 XP 018702657.1-1081104-Isaria fumosorosea ARSEF-Sordariomycetes QUERY-g6975-746128-Aspergillus fumigatus F13619-Eurotiomycetes other_Eukaryota 98 KJK74888.1-1291518-Metarhizium anisopliae BR-Sordariomycetes XP 002478259.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 98 OJJ08318.1-1036611-Aspergillus versicolor CB-Eurotiomycetes Bacteria 99 OJJ53427.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes KFA69539.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA74443.1-1283842-Stachybotrys chartarum IB-Sordariomycetes Archaea 98 KEY72230.1-1280523-Stachybotrys chartarum IB-Sordariomycetes KFY13626.1-1420901-Pseudogymnoascus sp VKM-Leotiomycetes KFY50143.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes Viruses 100 KFY81276.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 98 KFZ03029.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes XP 003836151.1-985895-Leptosphaeria maculans JN-Dothideomycetes 98 OTA33688.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes 97 ELQ40620.1-1143189-Magnaporthe oryzae Y34-Sordariomycetes 100 XP 003709964.1-242507-Magnaporthe oryzae 70 15-Sordariomycetes XP 020127315.1-236234-Diplodia corticola-Dothideomycetes XP 007696884.1-665912-Bipolaris sorokiniana ND9-Dothideomycetes 99 XP 014081844.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 99 XP 018387357.1-5599-Alternaria alternata-Dothideomycetes XP 014558804.1-930091-Bipolaris victoriae FI3-Dothideomycetes 95 XP 007717426.1-930089-Bipolaris zeicola 26 R 13-Dothideomycetes KFX98711.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY00079.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes GAO90615.1-91492-Aspergillus udagawae-Eurotiomycetes 100 XP 003173259.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes 82 89 XP 002846809.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes OTB10323.1-1001832-Daldinia sp EC12-Sordariomycetes OQE43316.1-36646-Penicillium coprophilum-Eurotiomycetes 96 OQD66534.1-60169-Penicillium polonicum-Eurotiomycetes 100 OQE01217.1-29845-Penicillium vulpinum-Eurotiomycetes XP 016594713.1-27334-Penicillium expansum-Eurotiomycetes CRG89870.1-28573-Talaromyces islandicus-Eurotiomycetes 100 XP 002144860.1-441960-Talaromyces marneffei ATC-Eurotiomycetes 100 KFX52360.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes OBT69657.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes 97 KFY41662.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 99 KFY14206.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes OBT82103.1-1622148-Pseudogymnoascus sp 03VT-Leotiomycetes 98 OBT74270.1-1622149-Pseudogymnoascus sp 05NY-Leotiomycetes 93 KFY39125.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes 98 KFY04764.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes 97 KFY01833.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes OBT49351.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes XP 012741383.1-658429-Pseudogymnoascus destruct-Leotiomycetes 81100 OAF54784.1-655981-Pseudogymnoascus destruct-Leotiomycetes XP 018130572.1-342668-Pseudogymnoascus verrucos-Leotiomycetes 99 KFZ09854.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes 89 OBT58818.1-1622150-Pseudogymnoascus sp 24MN-Leotiomycetes KHJ33454.1-52586-Erysiphe necator-Leotiomycetes EPQ66190.1-1268274-Blumeria graminis f sp-Leotiomycetes XP 001225799.1-306901-Chaetomium globosum CBS 1-Sordariomycetes CEJ60096.1-104259-Penicillium brasilianum-Eurotiomycetes XP 002846710.1-554155-Arthroderma otae CBS 1134-Eurotiomycetes XP 006969239.1-431241-Trichoderma reesei QM6a-Sordariomycetes KDN62805.1-1173701-Colletotrichum sublineola-Sordariomycetes 91 100 KJK64082.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 99 XP 002378740.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes 94 KJJ30093.1-1392242-Aspergillus flavus AF70-Eurotiomycetes 83 XP 003190316.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes OQE91512.1-60175-Penicillium nalgiovense-Eurotiomycetes XP 016595100.1-27334-Penicillium expansum-Eurotiomycetes OQE39328.1-36646-Penicillium coprophilum-Eurotiomycetes 100 OQE11539.1-29845-Penicillium vulpinum-Eurotiomycetes XP 002561285.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 86 100 OQE14832.1-254877-Penicillium flavigenum-Eurotiomycetes OQE02657.1-60172-Penicillium solitum-Eurotiomycetes CRL27040.1-5075-Penicillium camemberti-Eurotiomycetes 99 OQD64769.1-60169-Penicillium polonicum-Eurotiomycetes

152 Tree scale: 0.1 XP 003067208.1-222929-Coccidioides posadasii C7-Eurotiomycetes 100 XP 001239644.2-246410-Coccidioides immitis RS-Eurotiomycetes 98 QUERY-g6976-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 CDM31026.1-1365484-Penicillium roqueforti FM-Eurotiomycetes 90 OQE07377.1-29845-Penicillium vulpinum-Eurotiomycetes Taxonomy OGM50384.1-109264-Aspergillus bombycis-Eurotiomycetes GAD99701.1-1356009-Byssochlamys spectabilis-Eurotiomycetes CCX29578.1-1076935-Pyronema omphalodes CBS 1-other Pezizomycotina Leotiomycetes XP 018190638.1-1328760-Xylona heveae TC161-other Pezizomycotina XP 016265392.1-215243-Exophiala oligosperma-Eurotiomycetes 100 XP 007719706.1-1182541-Capronia coronata CBS 617-Eurotiomycetes Sordariomycetes XP 002542375.1-336963-Uncinocarpus reesii 1704-Eurotiomycetes 98 XP 001245277.1-246410-Coccidioides immitis RS-Eurotiomycetes 100 XP 003071570.1-222929-Coccidioides posadasii C7-Eurotiomycetes Eurotiomycetes XP 020119833.1-1441469-Talaromyces atroroseus-Eurotiomycetes XP 002484266.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 99 CRG86649.1-28573-Talaromyces islandicus-Eurotiomycetes Dothideomycetes GAM37920.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 90 XP 002150005.1-441960-Talaromyces marneffei ATC-Eurotiomycetes OJD24174.1-1658174-Blastomyces percursus-Eurotiomycetes other_Pezizomycotina XP 002627299.1-559298-Blastomyces gilchristii S-Eurotiomycetes KLJ05464.1-1246674-Emmonsia parva UAMH 139-Eurotiomycetes 100 OAX83378.1-1658172-Emmonsia sp CAC 2015a-Eurotiomycetes other_Ascomycota KKZ67067.1-1247875-Emmonsia crescens UAMH 30-Eurotiomycetes 98 EGE01764.1-559882-Trichophyton equinum CBS-Eurotiomycetes EZF34609.1-1215336-Trichophyton interdigital-Eurotiomycetes other_Fungi XP 003232978.1-559305-Trichophyton rubrum CBS 1-Eurotiomycetes EZF25477.1-1215337-Trichophyton rubrum MR850-Eurotiomycetes 100 DAA74814.1-663331-Trichophyton benhamiae CB-Eurotiomycetes other_Opisthokonta EGD97296.1-647933-Trichophyton tonsurans CB-Eurotiomycetes XP 003175358.1-535722-Nannizzia gypsea CBS 1188-Eurotiomycetes KIM95126.1-913774-Oidiodendron maius Zn-Leotiomycetes other_Eukaryota ESZ95100.1-1432307-Sclerotinia borealis F 41-Leotiomycetes 100 XP 001591780.1-665079-Sclerotinia sclerotiorum-Leotiomycetes 100 88 XP 001555725.1-332648-Botrytis cinerea B05 10-Leotiomycetes Bacteria CZT50538.1-38038-Rhynchosporium secalis-Leotiomycetes 92 XP 018075789.1-149040-Phialocephala scopiformis-Leotiomycetes 88 CZR67391.1-576137-Phialocephala subalpina-Leotiomycetes Archaea OCK85450.1-1314670-Lepidopterella palustris-Dothideomycetes XP 013431178.1-1043004-Aureobasidium namibiae CB-Dothideomycetes 100 XP 013341200.1-1043005-Aureobasidium subglaciale-Dothideomycetes Viruses XP 018040703.1-1460663-Paraphaeosphaeria sporulo-Dothideomycetes XP 003300409.1-861557-Pyrenophora teres f tere-Dothideomycetes 89 89 95 OAL06049.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 84 OSS43606.1-105696-Epicoccum nigrum-Dothideomycetes KKY19094.1-420778-Diplodia seriata-Dothideomycetes XP 020133117.1-236234-Diplodia corticola-Dothideomycetes 88 EKG17053.1-1126212-Macrophomina phaseolina M-Dothideomycetes XP 007580094.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes KKZ63467.1-1247875-Emmonsia crescens UAMH 30-Eurotiomycetes OAX77823.1-1658172-Emmonsia sp CAC 2015a-Eurotiomycetes OJD11794.1-1447872-Emergomyces pasteuriana E-Eurotiomycetes EGC46119.1-544711-Histoplasma capsulatum H8-Eurotiomycetes XP 001540840.1-339724-Histoplasma capsulatum NA-Eurotiomycetes 80 EEH11142.1-447093-Histoplasma capsulatum G1-Eurotiomycetes 96 KLJ09996.1-1246674-Emmonsia parva UAMH 139-Eurotiomycetes 98 XP 002624675.1-559298-Blastomyces gilchristii S-Eurotiomycetes ODH53684.1-121759-Paracoccidioides brasilie-Eurotiomycetes ODH39903.1-121759-Paracoccidioides brasilie-Eurotiomycetes EEH16171.1-482561-Paracoccidioides brasilie-Eurotiomycetes 97 XP 010763114.1-502780-Paracoccidioides brasilie-Eurotiomycetes XP 015699878.1-502779-Paracoccidioides lutzii P-Eurotiomycetes OOF94419.1-602072-Aspergillus carbonarius I-Eurotiomycetes XP 001216622.1-341663-Aspergillus terreus NIH26-Eurotiomycetes XP 020051878.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes CEN60170.1-454130-Aspergillus calidoustus-Eurotiomycetes OJJ08067.1-1036611-Aspergillus versicolor CB-Eurotiomycetes XP 747931.1-330879-Aspergillus fumigatus Af2-Eurotiomycetes GAQ08997.1-293939-Aspergillus lentulus-Eurotiomycetes XP 001266122.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes XP 001276120.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes KEY75694.1-1476192-Aspergillus fumigatus var-Eurotiomycetes GAO84760.1-91492-Aspergillus udagawae-Eurotiomycetes OQD72719.1-69771-Penicillium decumbens-Eurotiomycetes OJJ33071.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes KKK12354.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK15958.1-308745-Aspergillus rambellii-Eurotiomycetes ODM19926.1-573508-Aspergillus cristatus-Eurotiomycetes 100 EYE95855.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes 97 OQD80537.1-416450-Penicillium antarcticum-Eurotiomycetes 100 OGE57334.1-1835702-Penicillium arizonense-Eurotiomycetes OKP11883.1-1316194-Penicillium subrubescens-Eurotiomycetes EPS34191.1-933388-Penicillium oxalicum 114-Eurotiomycetes 97 OOQ83889.1-104259-Penicillium brasilianum-Eurotiomycetes 89 CEJ62117.1-104259-Penicillium brasilianum-Eurotiomycetes OJJ69307.1-767769-Aspergillus brasiliensis-Eurotiomycetes XP 001393968.1-425011-Aspergillus niger CBS 513-Eurotiomycetes 81 99 EHA28595.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes OJI88301.1-767770-Aspergillus tubingensis C-Eurotiomycetes 98 OJZ82136.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes 94 GAQ43896.1-5061-Aspergillus niger-Eurotiomycetes GAA88176.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes OOO11703.1-5062-Aspergillus oryzae-Eurotiomycetes EIT83343.1-1160506-Aspergillus oryzae 3 042-Eurotiomycetes XP 001822762.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes XP 002382888.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes 98 BAE61629.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes KOC15977.1-1392242-Aspergillus flavus AF70-Eurotiomycetes OGM43570.1-109264-Aspergillus bombycis-Eurotiomycetes 92 XP 015401387.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes

153 Tree scale: 0.1 ESZ92256.1-1432307-Sclerotinia borealis F 41-Leotiomycetes GAW14079.1-1813822-fungal sp No 14919-other Fungi XP 002483014.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 100 KFY21349.1-1420906-Pseudogymnoascus sp VKM-Leotiomycetes OTB02433.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes Taxonomy OTB15543.1-1001832-Daldinia sp EC12-Sordariomycetes 100 OTA55120.1-1001937-Hypoxylon sp EC38-Sordariomycetes 100 OTA88379.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes Leotiomycetes CDM34316.1-1365484-Penicillium roqueforti FM-Eurotiomycetes OQE28987.1-254877-Penicillium flavigenum-Eurotiomycetes OQE03639.1-60172-Penicillium solitum-Eurotiomycetes Sordariomycetes 100 OQE02707.1-29845-Penicillium vulpinum-Eurotiomycetes KXG48478.1-5078-Penicillium griseofulvum-Eurotiomycetes OQE46736.1-36646-Penicillium coprophilum-Eurotiomycetes Eurotiomycetes QUERY-g6977-746128-Aspergillus fumigatus F13619-Eurotiomycetes 100 KID81333.1-1276136-Metarhizium guizhouense A-Sordariomycetes 100 KJK74881.1-1291518-Metarhizium anisopliae BR-Sordariomycetes Dothideomycetes 87 KUL87388.1-198730-Talaromyces verruculosus-Eurotiomycetes 100 CCD50762.1-999810-Botrytis cinerea T4-Leotiomycetes 100 XP 001554299.1-332648-Botrytis cinerea B05 10-Leotiomycetes other_Pezizomycotina SLM40376.1-136370-Umbilicaria pustulata-other Pezizomycotina OQD84609.1-416450-Penicillium antarcticum-Eurotiomycetes 100 OJJ79513.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes other_Ascomycota 100 OJJ55948.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes 95 95 OJI98882.1-1036611-Aspergillus versicolor CB-Eurotiomycetes OTB15976.1-1001832-Daldinia sp EC12-Sordariomycetes other_Fungi XP 007798640.1-1287681-Eutypa lata UCREL1-Sordariomycetes 100 OTA56458.1-1001937-Hypoxylon sp EC38-Sordariomycetes 82 100 OTA82740.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes other_Opisthokonta OAA75951.1-1081108-Cordyceps confragosa RCEF-Sordariomycetes 100 OAA36148.1-1081107-Cordyceps brongniartii RC-Sordariomycetes 99 KGQ02914.1-1245745-Beauveria bassiana D1 5-Sordariomycetes other_Eukaryota 99 XP 008603200.1-655819-Beauveria bassiana ARSEF-Sordariomycetes KKK23147.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK12815.1-308745-Aspergillus rambellii-Eurotiomycetes Bacteria 99 KFX52033.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes 100 KUL81322.1-198730-Talaromyces verruculosus-Eurotiomycetes 89 KUL83650.1-198730-Talaromyces verruculosus-Eurotiomycetes Archaea OTB09705.1-1001832-Daldinia sp EC12-Sordariomycetes XP 001239921.2-246410-Coccidioides immitis RS-Eurotiomycetes EFW19722.1-443226-Coccidioides posadasii st-Eurotiomycetes Viruses 97 100 XP 003067430.1-222929-Coccidioides posadasii C7-Eurotiomycetes XP 013331016.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes 100 CRG86000.1-28573-Talaromyces islandicus-Eurotiomycetes 99 XP 002480148.1-441959-Talaromyces stipitatus AT-Eurotiomycetes 95 KUL85954.1-198730-Talaromyces verruculosus-Eurotiomycetes 88 100 GAM39457.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes OJJ43620.1-1073090-Penicilliopsis zonata CBS-Eurotiomycetes EYE99324.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes OJJ79962.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes 100 ODM14728.1-573508-Aspergillus cristatus-Eurotiomycetes OJJ33439.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes XP 001213574.1-341663-Aspergillus terreus NIH26-Eurotiomycetes 87 GAA85162.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 100 XP 001397938.2-425011-Aspergillus niger CBS 513-Eurotiomycetes 81 OOG00119.1-602072-Aspergillus carbonarius I-Eurotiomycetes 98 96 XP 020058956.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes KJK64871.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 100 OOO10410.1-5062-Aspergillus oryzae-Eurotiomycetes 100 XP 001819693.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes 88 KOS43211.1-229535-Penicillium nordicum-Eurotiomycetes XP 002557457.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 OQE12082.1-29845-Penicillium vulpinum-Eurotiomycetes XP 001272515.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes XP 001211106.1-341663-Aspergillus terreus NIH26-Eurotiomycetes OJJ33579.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 80 OQE22944.1-303698-Penicillium steckii-Eurotiomycetes CEL11526.1-454130-Aspergillus calidoustus-Eurotiomycetes 82 100 OJJ08308.1-1036611-Aspergillus versicolor CB-Eurotiomycetes OGE55730.1-1835702-Penicillium arizonense-Eurotiomycetes 100 OQD90505.1-416450-Penicillium antarcticum-Eurotiomycetes OOF92076.1-602072-Aspergillus carbonarius I-Eurotiomycetes OJJ68031.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 CAK38339.1-5061-Aspergillus niger-Eurotiomycetes 95100 XP 001390458.2-425011-Aspergillus niger CBS 513-Eurotiomycetes GAA90921.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 OJI89131.1-767770-Aspergillus tubingensis C-Eurotiomycetes 96 GAQ47076.1-5061-Aspergillus niger-Eurotiomycetes OJJ78945.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes 100 EYE91325.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes OOF97174.1-602072-Aspergillus carbonarius I-Eurotiomycetes OJJ75164.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 XP 001388700.1-425011-Aspergillus niger CBS 513-Eurotiomycetes 99100 EHA26267.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes GAQ33368.1-5061-Aspergillus niger-Eurotiomycetes 84 98 GAA88775.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 88 OJZ92735.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes CEL03626.1-454130-Aspergillus calidoustus-Eurotiomycetes OQE04218.1-60172-Penicillium solitum-Eurotiomycetes KOS43880.1-229535-Penicillium nordicum-Eurotiomycetes CRL18065.1-5075-Penicillium camemberti-Eurotiomycetes 100 OQD72324.1-60169-Penicillium polonicum-Eurotiomycetes XP 016597175.1-27334-Penicillium expansum-Eurotiomycetes CDM34354.1-1365484-Penicillium roqueforti FM-Eurotiomycetes 98 KXG48446.1-5078-Penicillium griseofulvum-Eurotiomycetes OQE47057.1-36646-Penicillium coprophilum-Eurotiomycetes OQE02745.1-29845-Penicillium vulpinum-Eurotiomycetes XP 002558089.1-500485-Penicillium rubens Wiscon-Eurotiomycetes OQE29095.1-254877-Penicillium flavigenum-Eurotiomycetes 81 OQE95301.1-60175-Penicillium nalgiovense-Eurotiomycetes

154 Tree scale: 0.1 ORY62115.1-1141098-Pseudomassariella vexata-Sordariomycetes XP 008712536.1-1220924-Cyphellophora europaea CB-Eurotiomycetes OCK81425.1-1314670-Lepidopterella palustris-Dothideomycetes XP 008714139.1-1220924-Cyphellophora europaea CB-Eurotiomycetes KKY37949.1-1214573-Diaporthe ampelina-Sordariomycetes Taxonomy 100 OCW42406.1-158607-Diaporthe helianthi-Sordariomycetes QUERY-g6979-746128-Aspergillus fumigatus F13619-Eurotiomycetes 99 KJK74883.1-1291518-Metarhizium anisopliae BR-Sordariomycetes Leotiomycetes 100 KKY35409.1-1214573-Diaporthe ampelina-Sordariomycetes 100 XP 016643171.1-563466-Scedosporium apiospermum-Sordariomycetes XP 016759692.1-692275-Sphaerulina musiva SO2202-Dothideomycetes Sordariomycetes KFY82569.1-1420911-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY51108.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes KXT10224.1-113226-Pseudocercospora musae-Dothideomycetes Eurotiomycetes 99 KXT05579.1-321146-Mycosphaerella eumusae-Dothideomycetes 99 XP 007920973.1-383855-Pseudocercospora fijiensi-Dothideomycetes EME41941.1-675120-Dothistroma septosporum N-Dothideomycetes Dothideomycetes 97 KJY01661.1-1047168-Zymoseptoria brevis-Dothideomycetes 100 XP 003855000.1-336722-Zymoseptoria tritici IPO3-Dothideomycetes 100 SMQ47752.1-1276538-Zymoseptoria tritici ST99-Dothideomycetes other_Pezizomycotina XP 016762379.1-692275-Sphaerulina musiva SO2202-Dothideomycetes KKY28081.1-158046-Phaeomoniella chlamydospo-Eurotiomycetes XP 003350297.1-771870-Sordaria macrospora k hel-Sordariomycetes other_Ascomycota XP 007778793.1-1168221-Coniosporium apollinis CB-Dothideomycetes SLM40999.1-136370-Umbilicaria pustulata-other Pezizomycotina OTA34311.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes other_Fungi 100 KXL49368.1-245562-Acidomyces richmondensis-Dothideomycetes 87 OQO01855.1-1507870-Rachicladosporium antarct-Dothideomycetes XP 007304278.1-721885-Stereum hirsutum FP 91666-Basidiomycota other_Opisthokonta 100 XP 007300043.1-721885-Stereum hirsutum FP 91666-Basidiomycota 95 XP 007308677.1-721885-Stereum hirsutum FP 91666-Basidiomycota 99 XP 007306728.1-721885-Stereum hirsutum FP 91666-Basidiomycota other_Eukaryota OCL12913.1-574774-Glonium stellatum-Dothideomycetes OBW65148.1-5580-Aureobasidium pullulans-Dothideomycetes 97 100 KEQ84071.1-1043002-Aureobasidium pullulans E-Dothideomycetes Bacteria 100 KEQ63473.1-1043003-Aureobasidium melanogenum-Dothideomycetes XP 013424822.1-1043004-Aureobasidium namibiae CB-Dothideomycetes 88 XP 013346325.1-1043005-Aureobasidium subglaciale-Dothideomycetes Archaea ORY25440.1-71784-Naematelia encephala-Basidiomycota KIN08831.1-913774-Oidiodendron maius Zn-Leotiomycetes Viruses XP 008082430.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes 86 CZR62319.1-576137-Phialocephala subalpina-Leotiomycetes 99 XP 018071126.1-149040-Phialocephala scopiformis-Leotiomycetes ESZ89618.1-1432307-Sclerotinia borealis F 41-Leotiomycetes 100 XP 001585790.1-665079-Sclerotinia sclerotiorum-Leotiomycetes XP 015405798.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes XP 016637737.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes 100 OQV09223.1-569365-Cladophialophora immunda-Eurotiomycetes 98 XP 016243778.1-569365-Cladophialophora immunda-Eurotiomycetes 100 OCT46886.1-86049-Cladophialophora carrioni-Eurotiomycetes 100 XP 007759750.1-1182544-Cladophialophora yegresii-Eurotiomycetes OAL34029.1-856822-Fonsecaea nubica-Eurotiomycetes 92 XP 013287719.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes 80 OAG40626.1-254056-Fonsecaea monophora-Eurotiomycetes 99 XP 013946005.1-452589-Trichoderma atroviride IM-Sordariomycetes GAD95884.1-1356009-Byssochlamys spectabilis-Eurotiomycetes KJK65103.1-1403190-Aspergillus parasiticus S-Eurotiomycetes XP 018659219.1-398673-Trichoderma gamsii-Sordariomycetes 100 KJK78284.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 EXV05242.1-568076-Metarhizium robertsii-Sordariomycetes KIW73874.1-5601-Phialophora americana-Eurotiomycetes XP 016634601.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes 100 XP 018691675.1-1367422-Fonsecaea erecta-Eurotiomycetes 99 XP 016244945.1-569365-Cladophialophora immunda-Eurotiomycetes 100 OQU97667.1-569365-Cladophialophora immunda-Eurotiomycetes KFY61715.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes KFY31436.1-1420906-Pseudogymnoascus sp VKM-Leotiomycetes OBT66250.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes KFY91729.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 94 KFY40582.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes KFY11158.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes KFY24850.1-1420906-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY01042.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes 99 KFX87261.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes 94 KFY40191.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes OBT40714.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes 100 OBT73014.1-1622149-Pseudogymnoascus sp 05NY-Leotiomycetes 88 OBT81393.1-1622148-Pseudogymnoascus sp 03VT-Leotiomycetes 93 XP 018128746.1-342668-Pseudogymnoascus verrucos-Leotiomycetes KFY77742.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes 98 OBT57153.1-1622150-Pseudogymnoascus sp 24MN-Leotiomycetes KFZ19681.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes XP 013944411.1-452589-Trichoderma atroviride IM-Sordariomycetes CZR67169.1-576137-Phialocephala subalpina-Leotiomycetes XP 013959314.1-413071-Trichoderma virens Gv29 8-Sordariomycetes XP 018663809.1-398673-Trichoderma gamsii-Sordariomycetes 100 XP 013944800.1-452589-Trichoderma atroviride IM-Sordariomycetes 99 KKP05389.1-5544-Trichoderma harzianum-Sordariomycetes 100 OPB41121.1-1491466-Trichoderma guizhouense-Sordariomycetes OIW24597.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes XP 018185353.1-1328760-Xylona heveae TC161-other Pezizomycotina 100 GAM38058.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 99 KFZ03063.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes KFY17235.1-1420901-Pseudogymnoascus sp VKM-Leotiomycetes 83 KFY11532.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes 100 100 KFY46762.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes OBT46307.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes XP 018130329.1-342668-Pseudogymnoascus verrucos-Leotiomycetes 91 OBT58295.1-1622150-Pseudogymnoascus sp 24MN-Leotiomycetes 98 KFZ07003.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes

155 Tree scale: 0.1 OTB08227.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes KEY73627.1-1280523-Stachybotrys chartarum IB-Sordariomycetes XP 018029975.1-1460663-Paraphaeosphaeria sporulo-Dothideomycetes GAP88297.1-77044-Rosellinia necatrix-Sordariomycetes OJJ67825.1-767769-Aspergillus brasiliensis-Eurotiomycetes Taxonomy G3Y417.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 99 XP 001402406.2-425011-Aspergillus niger CBS 513-Eurotiomycetes 99 CAK49103.1-5061-Aspergillus niger-Eurotiomycetes Leotiomycetes 100 OQE12755.1-254877-Penicillium flavigenum-Eurotiomycetes KXG51721.1-5078-Penicillium griseofulvum-Eurotiomycetes OQD98988.1-29845-Penicillium vulpinum-Eurotiomycetes Sordariomycetes 86 CDM29146.1-1365484-Penicillium roqueforti FM-Eurotiomycetes 86 KGO67466.1-40296-Penicillium italicum-Eurotiomycetes XP 002560462.1-500485-Penicillium rubens Wiscon-Eurotiomycetes Eurotiomycetes 100 KZN83408.1-5076-Penicillium chrysogenum-Eurotiomycetes ESZ91463.1-1432307-Sclerotinia borealis F 41-Leotiomycetes QUERY-g6980-746128-Aspergillus fumigatus F13619-Eurotiomycetes Dothideomycetes 100 KJK74884.1-1291518-Metarhizium anisopliae BR-Sordariomycetes KKP06688.1-5544-Trichoderma harzianum-Sordariomycetes 80 97 KJZ75228.1-1043627-Hirsutella minnesotensis-Sordariomycetes other_Pezizomycotina OCK73150.1-1314670-Lepidopterella palustris-Dothideomycetes CEN59761.1-454130-Aspergillus calidoustus-Eurotiomycetes 100 OJJ57536.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes other_Ascomycota 100 95 100 OJJ05643.1-1036611-Aspergillus versicolor CB-Eurotiomycetes 92 XP 661205.1-227321-Aspergillus nidulans FGSC-Eurotiomycetes 100 CBF75802.1-227321-Aspergillus nidulans FGSC-Eurotiomycetes other_Fungi KXX79207.1-100816-Madurella mycetomatis-Sordariomycetes KFA69547.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA52212.1-1280524-Stachybotrys chartarum IB-Sordariomycetes other_Opisthokonta 100 KEY72232.1-1280523-Stachybotrys chartarum IB-Sordariomycetes 85 KKY16576.1-420778-Diplodia seriata-Dothideomycetes 100 XP 020125930.1-236234-Diplodia corticola-Dothideomycetes other_Eukaryota XP 003300441.1-861557-Pyrenophora teres f tere-Dothideomycetes 100 93 XP 001937756.1-426418-Pyrenophora tritici repen-Dothideomycetes 83 XP 008027821.1-671987-Setosphaeria turcica Et28-Dothideomycetes Bacteria 99 XP 007689107.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes 100 XP 014073545.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes XP 011127785.1-756982-Arthrobotrys oligospora A-other Pezizomycotina Archaea OJZ92826.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes XP 013282149.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes KIJ36684.1-990650-Sphaerobolus stellatus SS-Basidiomycota Viruses GAU94922.1-947166-Ramazzottius varieornatus-Metazoa EKG18972.1-1126212-Macrophomina phaseolina M-Dothideomycetes 100 GAW15911.1-1813822-fungal sp No 14919-other Fungi XP 007795392.1-1287681-Eutypa lata UCREL1-Sordariomycetes 92 XP 008712499.1-1220924-Cyphellophora europaea CB-Eurotiomycetes OGM49889.1-109264-Aspergillus bombycis-Eurotiomycetes 95 OJJ66452.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 OGM45328.1-109264-Aspergillus bombycis-Eurotiomycetes 98 GAD97254.1-1356009-Byssochlamys spectabilis-Eurotiomycetes 100 OJJ37490.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes XP 018061083.1-149040-Phialocephala scopiformis-Leotiomycetes 100 XP 018131309.1-342668-Pseudogymnoascus verrucos-Leotiomycetes OGM45263.1-109264-Aspergillus bombycis-Eurotiomycetes 99 XP 007790916.1-1287681-Eutypa lata UCREL1-Sordariomycetes GAD91854.1-1356009-Byssochlamys spectabilis-Eurotiomycetes 96 XP 018700082.1-1081104-Isaria fumosorosea ARSEF-Sordariomycetes 86 XP 007914153.1-1286976-Phaeoacremonium minimum U-Sordariomycetes 90 XP 003043989.1-660122-Nectria haematococca mpVI-Sordariomycetes XP 015406133.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 100 OGM49559.1-109264-Aspergillus bombycis-Eurotiomycetes OPB36055.1-1491466-Trichoderma guizhouense-Sordariomycetes 100 XP 013938758.1-452589-Trichoderma atroviride IM-Sordariomycetes 95 XP 018664929.1-398673-Trichoderma gamsii-Sordariomycetes KND94247.1-1163406-Tolypocladium ophioglosso-Sordariomycetes 97 KOM23882.1-268505-Ophiocordyceps unilateral-Sordariomycetes CRG89981.1-28573-Talaromyces islandicus-Eurotiomycetes KID82384.1-1276136-Metarhizium guizhouense A-Sordariomycetes 97 XP 014543987.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes 100 XP 007826330.1-655844-Metarhizium robertsii ARS-Sordariomycetes KJK73576.1-1291518-Metarhizium anisopliae BR-Sordariomycetes 100 90 KFG78219.1-5530-Metarhizium anisopliae-Sordariomycetes KIL85477.1-40199-Fusarium avenaceum-Sordariomycetes EMT72782.1-1229665-Fusarium oxysporum f sp-Sordariomycetes 97 EWZ45894.1-660027-Fusarium oxysporum Fo47-Sordariomycetes 100 EXK43331.1-1089452-Fusarium oxysporum f sp-Sordariomycetes 90 100 XP 018255266.1-426428-Fusarium oxysporum f sp-Sordariomycetes 87 CVK96608.1-192010-Fusarium mangiferae-Sordariomycetes CZR45965.1-1227346-Fusarium proliferatum ET1-Sordariomycetes 10097 CVL11161.1-948311-Fusarium proliferatum-Sordariomycetes CCT71951.1-1279085-Fusarium fujikuroi IMI 58-Sordariomycetes 99 KLO92536.1-5127-Fusarium fujikuroi-Sordariomycetes XP 007381777.1-741275-Punctularia strigosozonat-Basidiomycota OAA80406.1-1081108-Cordyceps confragosa RCEF-Sordariomycetes OJJ46451.1-1073090-Penicilliopsis zonata CBS-Eurotiomycetes 83 OQE00380.1-29845-Penicillium vulpinum-Eurotiomycetes 98 OJJ38752.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes XP 001273089.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes 100 OQD99182.1-60172-Penicillium solitum-Eurotiomycetes XP 014535842.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes OQE13089.1-254877-Penicillium flavigenum-Eurotiomycetes 98 OQD65548.1-60169-Penicillium polonicum-Eurotiomycetes CRL29160.1-5075-Penicillium camemberti-Eurotiomycetes KUM63385.1-48697-Penicillium freii-Eurotiomycetes 91 OQE09941.1-29845-Penicillium vulpinum-Eurotiomycetes OQE43328.1-36646-Penicillium coprophilum-Eurotiomycetes KXG50992.1-5078-Penicillium griseofulvum-Eurotiomycetes OQD86089.1-416450-Penicillium antarcticum-Eurotiomycetes AIG62133.1-27334-Penicillium expansum-Eurotiomycetes 99 XP 016595358.1-27334-Penicillium expansum-Eurotiomycetes

156 Tree scale: 0.1 QUERY-g6981-746128-Aspergillus fumigatus F13619-Eurotiomycetes XP 002143162.1-441960-Talaromyces marneffei ATC-Eurotiomycetes 100 KJK74885.1-1291518-Metarhizium anisopliae BR-Sordariomycetes XP 016643170.1-563466-Scedosporium apiospermum-Sordariomycetes XP 018069399.1-149040-Phialocephala scopiformis-Leotiomycetes Taxonomy XP 007585581.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes XP 016211692.1-253628-Verruconis gallopava-Dothideomycetes EKG16098.1-1126212-Macrophomina phaseolina M-Dothideomycetes Leotiomycetes OCL05893.1-574774-Glonium stellatum-Dothideomycetes XP 007595806.1-1445577-Colletotrichum fioriniae-Sordariomycetes OCK73526.1-1314670-Lepidopterella palustris-Dothideomycetes Sordariomycetes KIM95590.1-913774-Oidiodendron maius Zn-Leotiomycetes OCL09156.1-574774-Glonium stellatum-Dothideomycetes KIN00611.1-913774-Oidiodendron maius Zn-Leotiomycetes Eurotiomycetes XP 020058940.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes BAE65144.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 100 XP 001826277.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes Dothideomycetes 100 BAJ04387.1-5062-Aspergillus oryzae-Eurotiomycetes XP 001272463.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes XP 001818593.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes other_Pezizomycotina 100 OOO09520.1-5062-Aspergillus oryzae-Eurotiomycetes 100 XP 002380000.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes GAQ46365.1-5061-Aspergillus niger-Eurotiomycetes other_Ascomycota OJI89939.1-767770-Aspergillus tubingensis C-Eurotiomycetes 100 OJJ66284.1-767769-Aspergillus brasiliensis-Eurotiomycetes 84 EHA23664.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes other_Fungi 94 XP 001391101.1-425011-Aspergillus niger CBS 513-Eurotiomycetes KZL68279.1-708197-Colletotrichum tofieldiae-Sordariomycetes other_Opisthokonta XP 007281288.1-1213859-Colletotrichum gloeospori-Sordariomycetes 98 100 EQB54758.1-1237896-Colletotrichum gloeospori-Sordariomycetes GAP85672.1-77044-Rosellinia necatrix-Sordariomycetes 80 KXX80394.1-100816-Madurella mycetomatis-Sordariomycetes other_Eukaryota 100 ACN71233.1-290576-Colletotrichum lindemuthi-Sordariomycetes 100 XP 007280227.1-1213859-Colletotrichum gloeospori-Sordariomycetes KKY16573.1-420778-Diplodia seriata-Dothideomycetes Bacteria 100 XP 020125921.1-236234-Diplodia corticola-Dothideomycetes XP 001937753.1-426418-Pyrenophora tritici repen-Dothideomycetes 100 98 XP 003300444.1-861557-Pyrenophora teres f tere-Dothideomycetes Archaea 92 XP 008027818.1-671987-Setosphaeria turcica Et28-Dothideomycetes 84 XP 007689109.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes 99 XP 014073535.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes Viruses OBT67983.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes KFY39744.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes KFZ18712.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY98181.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 100 100 OBT50188.1-1622150-Pseudogymnoascus sp 24MN-Leotiomycetes KFZ02893.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes KFY72655.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes 94 XP 018132006.1-342668-Pseudogymnoascus verrucos-Leotiomycetes 94 OBT80254.1-1622149-Pseudogymnoascus sp 05NY-Leotiomycetes 98 OBT86705.1-1622148-Pseudogymnoascus sp 03VT-Leotiomycetes KFY33895.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY11054.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes KFZ09804.1-1420914-Pseudogymnoascus sp VKM-Leotiomycetes OBT43052.1-1622147-Pseudogymnoascus sp WSF-Leotiomycetes OBT76076.1-1622149-Pseudogymnoascus sp 05NY-Leotiomycetes XP 012740108.1-658429-Pseudogymnoascus destruct-Leotiomycetes OBT62545.1-1524831-Pseudogymnoascus sp 2334-Leotiomycetes KFY74280.1-1420912-Pseudogymnoascus sp VKM-Leotiomycetes 100 99 XP 018132394.1-342668-Pseudogymnoascus verrucos-Leotiomycetes KFX97166.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY45029.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes KFY92053.1-1420913-Pseudogymnoascus sp VKM-Leotiomycetes 96 KFZ12582.1-1420915-Pseudogymnoascus sp VKM-Leotiomycetes KFY36094.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY12419.1-1420902-Pseudogymnoascus sp VKM-Leotiomycetes KIM94742.1-913774-Oidiodendron maius Zn-Leotiomycetes XP 007751056.1-1182543-Cladophialophora psammoph-Eurotiomycetes 94 100 XP 016268188.1-215243-Exophiala oligosperma-Eurotiomycetes XP 016257504.1-215243-Exophiala oligosperma-Eurotiomycetes 100 XP 018687752.1-1367422-Fonsecaea erecta-Eurotiomycetes 100 OQV06561.1-569365-Cladophialophora immunda-Eurotiomycetes 100 OAA59705.1-1081102-Sporothrix insectorum RCE-Sordariomycetes OIW33137.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes ENH66031.1-1229664-Fusarium oxysporum f sp-Sordariomycetes 10092 KFA52988.1-1280524-Stachybotrys chartarum IB-Sordariomycetes XP 003665517.1-573729-Thermothelomyces thermoph-Sordariomycetes CDP23371.1-515849-Podospora anserina S mat-Sordariomycetes 100 KXX74268.1-100816-Madurella mycetomatis-Sordariomycetes OJJ40738.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 100 EYE99910.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes 100 ODM23147.1-573508-Aspergillus cristatus-Eurotiomycetes KUM59367.1-48697-Penicillium freii-Eurotiomycetes KJK60354.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 100 BAE60004.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes 100 BAJ04388.1-5062-Aspergillus oryzae-Eurotiomycetes 100 100 XP 001822006.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes OJJ31553.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes KKK15075.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes 100 KKK19335.1-308745-Aspergillus rambellii-Eurotiomycetes XP 001275759.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes GAO87271.1-91492-Aspergillus udagawae-Eurotiomycetes 87 100 100 GAQ06383.1-293939-Aspergillus lentulus-Eurotiomycetes 96 XP 001260852.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes EPS33698.1-933388-Penicillium oxalicum 114-Eurotiomycetes OOQ90151.1-104259-Penicillium brasilianum-Eurotiomycetes 100 100 CEO60361.1-104259-Penicillium brasilianum-Eurotiomycetes 98 CRL17927.1-5075-Penicillium camemberti-Eurotiomycetes 100 OQE30234.1-254877-Penicillium flavigenum-Eurotiomycetes 100 OQE86252.1-60175-Penicillium nalgiovense-Eurotiomycetes

157 Gene phylogenies of the mobile SM gene cluster 1. These phylogenies are consistent with horizontal transfer between Eurotiomycete, Dothidiomycete, Leotiomycete, and Sordariomycete fungi.

Tree scale: 0.1 OIW35691.1-1408157-Coniochaeta ligniaria NRR-Sordariomycetes KUI65771.1-105487-Valsa mali-Sordariomycetes 100 KUI57826.1-694573-Valsa mali var pyri-Sordariomycetes KFY42099.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes KOS21697.1-150374-Escovopsis weberi-Sordariomycetes Taxonomy 100 XP 001803250.1-321614-Parastagonospora nodorum-Dothideomycetes 100 100 QUERY-Afu1g00970-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54466.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes Leotiomycetes XP 003713019.1-242507-Magnaporthe oryzae 70 15-Sordariomycetes KFH48235.1-857340-Acremonium chrysogenum AT-Sordariomycetes GAW13547.1-1813822-fungal sp No 14919-other Fungi Sordariomycetes OLN86531.1-708187-Colletotrichum chlorophyt-Sordariomycetes 98 ENH77543.1-1213857-Colletotrichum orbiculare-Sordariomycetes EQB49150.1-1237896-Colletotrichum gloeospori-Sordariomycetes Eurotiomycetes 100 XP 007277318.1-1213859-Colletotrichum gloeospori-Sordariomycetes 99 OHE99286.1-1209926-Colletotrichum orchidophi-Sordariomycetes 99 KXH26193.1-703756-Colletotrichum simmondsii-Sordariomycetes Dothideomycetes 97 KXH53051.1-1460502-Colletotrichum nymphaeae-Sordariomycetes 91 XP 007600727.1-1445577-Colletotrichum fioriniae-Sordariomycetes 82 KDN68534.1-1173701-Colletotrichum sublineola-Sordariomycetes other_Pezizomycotina 100 XP 008093677.1-645133-Colletotrichum graminicol-Sordariomycetes 99 KZL64706.1-708197-Colletotrichum tofieldiae-Sordariomycetes 100 OHW99315.1-1573173-Colletotrichum incanum-Sordariomycetes other_Ascomycota 100 KZL79639.1-1573173-Colletotrichum incanum-Sordariomycetes EKG19770.1-1126212-Macrophomina phaseolina M-Dothideomycetes XP 020126260.1-236234-Diplodia corticola-Dothideomycetes other_Fungi KJK66792.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 100 KOC17587.1-1392242-Aspergillus flavus AF70-Eurotiomycetes 100 XP 001818821.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes other_Opisthokonta KEQ61239.1-1043003-Aureobasidium melanogenum-Dothideomycetes XP 013428975.1-1043004-Aureobasidium namibiae CB-Dothideomycetes 100 OBW67029.1-5580-Aureobasidium pullulans-Dothideomycetes other_Eukaryota OBW66192.1-5580-Aureobasidium pullulans-Dothideomycetes 100 KEQ79880.1-1043002-Aureobasidium pullulans E-Dothideomycetes OTA36165.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes Bacteria 100 OTA34396.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes 100 XP 007924270.1-383855-Pseudocercospora fijiensi-Dothideomycetes KXT15283.1-113226-Pseudocercospora musae-Dothideomycetes Archaea 100 KXS97744.1-321146-Mycosphaerella eumusae-Dothideomycetes OJJ76878.1-767769-Aspergillus brasiliensis-Eurotiomycetes XP 008599531.1-655819-Beauveria bassiana ARSEF-Sordariomycetes Viruses 100 OGM49724.1-109264-Aspergillus bombycis-Eurotiomycetes 93 XP 015402055.1-1509407-Aspergillus nomius NRRL 1-Eurotiomycetes 100 KFY66583.1-1420909-Pseudogymnoascus sp VKM-Leotiomycetes KFY57765.1-1420910-Pseudogymnoascus sp VKM-Leotiomycetes 100 KFY39884.1-1420908-Pseudogymnoascus sp VKM-Leotiomycetes 100 100 KFX99467.1-1437433-Pseudogymnoascus sp VKM-Leotiomycetes 88 KFX97535.1-1391699-Pseudogymnoascus sp VKM-Leotiomycetes XP 013274790.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes KIW69702.1-5601-Phialophora americana-Eurotiomycetes 100 XP 008723616.1-1279043-Cladophialophora carrioni-Eurotiomycetes 100 80 XP 007755975.1-1182544-Cladophialophora yegresii-Eurotiomycetes XP 018692325.1-1367422-Fonsecaea erecta-Eurotiomycetes 92 XP 016636716.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes XP 016248755.1-569365-Cladophialophora immunda-Eurotiomycetes 96 XP 013278546.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes 100 OAG44093.1-254056-Fonsecaea monophora-Eurotiomycetes XP 016615702.1-1442370-Cladophialophora bantiana-Eurotiomycetes 100 XP 007748458.1-1182543-Cladophialophora psammoph-Eurotiomycetes GAD95758.1-1356009-Byssochlamys spectabilis-Eurotiomycetes OJJ35998.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes ODM17037.1-573508-Aspergillus cristatus-Eurotiomycetes 100 EYE91894.1-1388766-Aspergillus ruber CBS 135-Eurotiomycetes 95 OJJ79785.1-1160497-Aspergillus glaucus CBS 5-Eurotiomycetes CEL10067.1-454130-Aspergillus calidoustus-Eurotiomycetes 80 KKK13230.1-308745-Aspergillus rambellii-Eurotiomycetes 100 KKK14093.1-138278-Aspergillus ochraceoroseu-Eurotiomycetes XP 001273686.1-344612-Aspergillus clavatus NRRL-Eurotiomycetes 100 100 GAO81288.1-91492-Aspergillus udagawae-Eurotiomycetes 100 GAQ05142.1-293939-Aspergillus lentulus-Eurotiomycetes 82 KMK58647.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes 85 XP 001258866.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes OGM42378.1-109264-Aspergillus bombycis-Eurotiomycetes KJK63873.1-1403190-Aspergillus parasiticus S-Eurotiomycetes 100 XP 002381708.1-332952-Aspergillus flavus NRRL33-Eurotiomycetes 96 XP 001825033.2-510516-Aspergillus oryzae RIB40-Eurotiomycetes 90 97 OOO04968.1-5062-Aspergillus oryzae-Eurotiomycetes EIT76281.1-1160506-Aspergillus oryzae 3 042-Eurotiomycetes 99 BAE63900.1-510516-Aspergillus oryzae RIB40-Eurotiomycetes OOF94566.1-602072-Aspergillus carbonarius I-Eurotiomycetes OJJ75218.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 EHA26228.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes 100 XP 001388644.2-425011-Aspergillus niger CBS 513-Eurotiomycetes 100 OJI87224.1-767770-Aspergillus tubingensis C-Eurotiomycetes OJZ92782.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes 100 GAQ33255.1-5061-Aspergillus niger-Eurotiomycetes OQE46937.1-36646-Penicillium coprophilum-Eurotiomycetes OQD97567.1-29845-Penicillium vulpinum-Eurotiomycetes KXG50743.1-5078-Penicillium griseofulvum-Eurotiomycetes CDM34884.1-1365484-Penicillium roqueforti FM-Eurotiomycetes KZN89920.1-5076-Penicillium chrysogenum-Eurotiomycetes 100 OQE15066.1-254877-Penicillium flavigenum-Eurotiomycetes 99 OQE80984.1-60175-Penicillium nalgiovense-Eurotiomycetes XP 016592916.1-27334-Penicillium expansum-Eurotiomycetes CRL25023.1-5075-Penicillium camemberti-Eurotiomycetes OQD91362.1-60172-Penicillium solitum-Eurotiomycetes 90 KGO76725.1-40296-Penicillium italicum-Eurotiomycetes OQD66474.1-60169-Penicillium polonicum-Eurotiomycetes 100 KUM62120.1-48697-Penicillium freii-Eurotiomycetes

158 Tree scale: 0.1 XP 018190871.1-1328760-Xylona heveae TC161-other Pezizomycotina KNG52497.1-183478-Stemphylium lycopersici-Dothideomycetes ESZ91465.1-1432307-Sclerotinia borealis F 41-Leotiomycetes KFA69534.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA74441.1-1283842-Stachybotrys chartarum IB-Sordariomycetes Taxonomy 95 XP 008027822.1-671987-Setosphaeria turcica Et28-Dothideomycetes XP 014073570.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 100 XP 007689106.1-930090-Bipolaris oryzae ATCC 445-Dothideomycetes Leotiomycetes 100 XP 001937757.1-426418-Pyrenophora tritici repen-Dothideomycetes 100 XP 003300440.1-861557-Pyrenophora teres f tere-Dothideomycetes KKY16577.1-420778-Diplodia seriata-Dothideomycetes Sordariomycetes 100 XP 020125929.1-236234-Diplodia corticola-Dothideomycetes GAM36611.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes XP 003654926.1-578455-Thielavia terrestris NRRL-Sordariomycetes Eurotiomycetes GAM40107.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 100 XP 002479463.1-441959-Talaromyces stipitatus AT-Eurotiomycetes EHA28243.1-380704-Aspergillus niger ATCC 10-Eurotiomycetes Dothideomycetes 100 XP 001393507.1-425011-Aspergillus niger CBS 513-Eurotiomycetes 96 XP 013942627.1-452589-Trichoderma atroviride IM-Sordariomycetes 100 XP 018661927.1-398673-Trichoderma gamsii-Sordariomycetes other_Pezizomycotina OAF54392.1-655981-Pseudogymnoascus destruct-Leotiomycetes 97 KUI69403.1-105487-Valsa mali-Sordariomycetes 100 KUI53500.1-694573-Valsa mali var pyri-Sordariomycetes other_Ascomycota OJJ06460.1-1036611-Aspergillus versicolor CB-Eurotiomycetes 100 SLM38091.1-136370-Umbilicaria pustulata-other Pezizomycotina 96 XP 007801361.1-1263415-Endocarpon pusillum Z0702-Eurotiomycetes other_Fungi XP 008086617.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes XP 018142822.1-1380566-Pochonia chlamydosporia 1-Sordariomycetes 100 OCL02959.1-574774-Glonium stellatum-Dothideomycetes other_Opisthokonta OAK94599.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 100 CRG83420.1-28573-Talaromyces islandicus-Eurotiomycetes 100 KUL90120.1-198730-Talaromyces verruculosus-Eurotiomycetes other_Eukaryota 99 OJJ67836.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 OJI89149.1-767770-Aspergillus tubingensis C-Eurotiomycetes 100 ODM21323.1-573508-Aspergillus cristatus-Eurotiomycetes Bacteria 100 XP 018189398.1-1328760-Xylona heveae TC161-other Pezizomycotina KFY42098.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 87 KOS21702.1-150374-Escovopsis weberi-Sordariomycetes Archaea XP 001803249.1-321614-Parastagonospora nodorum-Dothideomycetes 100 97 QUERY-Afu1g00980-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54467.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes Viruses OTA99527.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 100 OTA97076.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes 100 OTA68786.1-1001937-Hypoxylon sp EC38-Sordariomycetes OQE10144.1-254877-Penicillium flavigenum-Eurotiomycetes KXG46525.1-5078-Penicillium griseofulvum-Eurotiomycetes XP 020058182.1-690307-Aspergillus aculeatus ATC-Eurotiomycetes 100 CDM37416.1-1365484-Penicillium roqueforti FM-Eurotiomycetes OQD99008.1-29845-Penicillium vulpinum-Eurotiomycetes KOS46297.1-229535-Penicillium nordicum-Eurotiomycetes 100 OQE96749.1-60175-Penicillium nalgiovense-Eurotiomycetes 81 XP 002564777.1-500485-Penicillium rubens Wiscon-Eurotiomycetes 100 OJZ86826.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes GAA84845.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 GAT24549.1-1069201-Aspergillus luchuensis-Eurotiomycetes GAQ46578.1-5061-Aspergillus niger-Eurotiomycetes 99 OJI83692.1-767770-Aspergillus tubingensis C-Eurotiomycetes 86 CEN59740.1-454130-Aspergillus calidoustus-Eurotiomycetes 100 KIA75595.1-40382-Aspergillus ustus-Eurotiomycetes KUL83490.1-198730-Talaromyces verruculosus-Eurotiomycetes 100 100 GAM39560.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 81 KFX41538.1-1077442-Talaromyces marneffei PM1-Eurotiomycetes 100 XP 002145721.1-441960-Talaromyces marneffei ATC-Eurotiomycetes KKY17669.1-420778-Diplodia seriata-Dothideomycetes 100 OMP84237.1-420778-Diplodia seriata-Dothideomycetes XP 007786093.1-1263415-Endocarpon pusillum Z0702-Eurotiomycetes 80 XP 008720114.1-1220924-Cyphellophora europaea CB-Eurotiomycetes OJJ62960.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes OTA57984.1-1001937-Hypoxylon sp EC38-Sordariomycetes OCW41151.1-158607-Diaporthe helianthi-Sordariomycetes XP 007795874.1-1287681-Eutypa lata UCREL1-Sordariomycetes OOQ89673.1-104259-Penicillium brasilianum-Eurotiomycetes 97 100 OQD81687.1-416450-Penicillium antarcticum-Eurotiomycetes KFA79676.1-1283842-Stachybotrys chartarum IB-Sordariomycetes KEY69109.1-1280523-Stachybotrys chartarum IB-Sordariomycetes 100 KFA45396.1-1280524-Stachybotrys chartarum IB-Sordariomycetes OTB10571.1-1001832-Daldinia sp EC12-Sordariomycetes 100 KLO99253.1-5127-Fusarium fujikuroi-Sordariomycetes 100 XP 018754944.1-334819-Fusarium verticillioides-Sordariomycetes 99 ANH22767.1-696354-Hypocrella siamensis-Sordariomycetes 98 100 KIA75781.1-40382-Aspergillus ustus-Eurotiomycetes 90 KOM23411.1-268505-Ophiocordyceps unilateral-Sordariomycetes KEY71722.1-1280523-Stachybotrys chartarum IB-Sordariomycetes KFA79949.1-1283842-Stachybotrys chartarum IB-Sordariomycetes 100 KFA55988.1-1280524-Stachybotrys chartarum IB-Sordariomycetes KFA68349.1-1283841-Stachybotrys chlorohalona-Sordariomycetes 100 KFA70203.1-1283841-Stachybotrys chlorohalona-Sordariomycetes KFA71244.1-1283842-Stachybotrys chartarum IB-Sordariomycetes 100 KEY71773.1-1280523-Stachybotrys chartarum IB-Sordariomycetes 94 KFA54577.1-1280524-Stachybotrys chartarum IB-Sordariomycetes ORY71423.1-1141098-Pseudomassariella vexata-Sordariomycetes KLU89215.1-644358-Magnaporthiopsis poae ATC-Sordariomycetes 100 XP 003712304.1-242507-Magnaporthe oryzae 70 15-Sordariomycetes 99 100 ELQ69713.1-1143193-Magnaporthe oryzae P131-Sordariomycetes XP 003351972.1-771870-Sordaria macrospora k hel-Sordariomycetes 100 CAQ58437.1-5147-Sordaria macrospora-Sordariomycetes 100 XP 965608.1-367110-Neurospora crassa OR74A-Sordariomycetes 100 EGZ76379.1-510952-Neurospora tetrasperma FG-Sordariomycetes 81 XP 009855463.1-510951-Neurospora tetrasperma FG-Sordariomycetes

159 Tree scale: 0.1 KZL76078.1-708197-Colletotrichum tofieldiae-Sordariomycetes KZL85173.1-1573173-Colletotrichum incanum-Sordariomycetes 100 XP 008089668.1-645133-Colletotrichum graminicol-Sordariomycetes CEJ62862.1-104259-Penicillium brasilianum-Eurotiomycetes 100 KUL90439.1-198730-Talaromyces verruculosus-Eurotiomycetes Taxonomy GAA84860.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 GAQ46564.1-5061-Aspergillus niger-Eurotiomycetes 82 81 OJI83706.1-767770-Aspergillus tubingensis C-Eurotiomycetes Leotiomycetes XP 007738022.1-1182542-Capronia epimyces CBS 606-Eurotiomycetes KIV86245.1-1016849-Exophiala sideris-Eurotiomycetes 100 KIV78870.1-1016849-Exophiala sideris-Eurotiomycetes Sordariomycetes XP 016635763.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes 100 XP 018689742.1-1367422-Fonsecaea erecta-Eurotiomycetes KEQ57958.1-1043003-Aureobasidium melanogenum-Dothideomycetes Eurotiomycetes XP 007582626.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes XP 007919710.1-383855-Pseudocercospora fijiensi-Dothideomycetes 100 KXT10441.1-113226-Pseudocercospora musae-Dothideomycetes Dothideomycetes GAM33346.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 100 KUL83173.1-198730-Talaromyces verruculosus-Eurotiomycetes OCL12203.1-574774-Glonium stellatum-Dothideomycetes other_Pezizomycotina 98 OCK78800.1-1314670-Lepidopterella palustris-Dothideomycetes OQO09931.1-1507870-Rachicladosporium antarct-Dothideomycetes 100 OQO06724.1-1507870-Rachicladosporium antarct-Dothideomycetes other_Ascomycota 95 OQO25097.1-1974281-Rachicladosporium sp CCF-Dothideomycetes KIN03586.1-913774-Oidiodendron maius Zn-Leotiomycetes 94 CZR67742.1-576137-Phialocephala subalpina-Leotiomycetes other_Fungi 99 XP 018070937.1-149040-Phialocephala scopiformis-Leotiomycetes XP 013270009.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes 100 KIV85980.1-1016849-Exophiala sideris-Eurotiomycetes other_Opisthokonta 92 XP 013316661.1-348802-Exophiala xenobiotica-Eurotiomycetes 97 XP 016268262.1-215243-Exophiala oligosperma-Eurotiomycetes 100 XP 016240447.1-91928-Exophiala spinifera-Eurotiomycetes other_Eukaryota OCL02960.1-574774-Glonium stellatum-Dothideomycetes OCK79863.1-1314670-Lepidopterella palustris-Dothideomycetes GAD94648.1-1356009-Byssochlamys spectabilis-Eurotiomycetes Bacteria XP 007361702.1-732165-Dichomitus squalens LYAD-Basidiomycota XP 018068436.1-149040-Phialocephala scopiformis-Leotiomycetes OJJ41604.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes Archaea XP 018067827.1-149040-Phialocephala scopiformis-Leotiomycetes OCK97824.1-794803-Cenococcum geophilum 1 58-Dothideomycetes 99 OCL04641.1-574774-Glonium stellatum-Dothideomycetes Viruses ODM21324.1-573508-Aspergillus cristatus-Eurotiomycetes XP 018189399.1-1328760-Xylona heveae TC161-other Pezizomycotina 100 85 ODM17455.1-573508-Aspergillus cristatus-Eurotiomycetes 99 OJI89150.1-767770-Aspergillus tubingensis C-Eurotiomycetes 100 OJJ67835.1-767769-Aspergillus brasiliensis-Eurotiomycetes GAQ11743.1-293939-Aspergillus lentulus-Eurotiomycetes XP 008086616.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes 87 XP 007798352.1-1287681-Eutypa lata UCREL1-Sordariomycetes CRG83420.1-28573-Talaromyces islandicus-Eurotiomycetes 100 GAM35249.1-1472165-Talaromyces cellulolyticu-Eurotiomycetes 100 KUL90121.1-198730-Talaromyces verruculosus-Eurotiomycetes KFY42097.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes KOS21698.1-150374-Escovopsis weberi-Sordariomycetes XP 003654012.1-578455-Thielavia terrestris NRRL-Sordariomycetes OTA99528.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 96 85 OTA97075.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes 86 OTA68787.1-1001937-Hypoxylon sp EC38-Sordariomycetes XP 001803248.1-321614-Parastagonospora nodorum-Dothideomycetes XP 014072629.1-665024-Bipolaris maydis ATCC 483-Dothideomycetes 82 QUERY-Afu1g00990-330879-Aspergillus fumigatus Af2-Eurotiomycetes XP 018139917.1-1380566-Pochonia chlamydosporia 1-Sordariomycetes XP 018175680.1-33203-Purpureocillium lilacinum-Sordariomycetes GAW20535.1-1813822-fungal sp No 14919-other Fungi XP 007286401.1-1213859-Colletotrichum gloeospori-Sordariomycetes XP 007799272.1-1287681-Eutypa lata UCREL1-Sordariomycetes OAA76696.1-1081108-Cordyceps confragosa RCEF-Sordariomycetes 100 XP 018703534.1-1081104-Isaria fumosorosea ARSEF-Sordariomycetes XP 007599778.1-1445577-Colletotrichum fioriniae-Sordariomycetes OHF00529.1-1209926-Colletotrichum orchidophi-Sordariomycetes KXH66733.1-1209931-Colletotrichum salicis-Sordariomycetes 100 KXH41440.1-1460502-Colletotrichum nymphaeae-Sordariomycetes 89 KXH45168.1-703756-Colletotrichum simmondsii-Sordariomycetes XP 013942446.1-452589-Trichoderma atroviride IM-Sordariomycetes XP 013958002.1-413071-Trichoderma virens Gv29 8-Sordariomycetes OPB36426.1-1491466-Trichoderma guizhouense-Sordariomycetes 100 91 KKP06960.1-5544-Trichoderma harzianum-Sordariomycetes OTA07189.1-858221-Trichoderma parareesei-Sordariomycetes 100 ETR98295.1-1344414-Trichoderma reesei RUT C-Sordariomycetes 100 XP 006968870.1-431241-Trichoderma reesei QM6a-Sordariomycetes KPM35926.1-78410-Neonectria ditissima-Sordariomycetes 99 XP 003041834.1-660122-Nectria haematococca mpVI-Sordariomycetes KIL93926.1-40199-Fusarium avenaceum-Sordariomycetes 96 XP 018760089.1-334819-Fusarium verticillioides-Sordariomycetes CVK86026.1-192010-Fusarium mangiferae-Sordariomycetes KLP07008.1-5127-Fusarium fujikuroi-Sordariomycetes 97 100 KLO82373.1-5127-Fusarium fujikuroi-Sordariomycetes 95 CVK85442.1-948311-Fusarium proliferatum-Sordariomycetes 83 CZR37956.1-1227346-Fusarium proliferatum ET1-Sordariomycetes 99 EXM21264.1-1089449-Fusarium oxysporum f sp-Sordariomycetes EXL74715.1-1089457-Fusarium oxysporum f sp-Sordariomycetes EXL52372.1-1089448-Fusarium oxysporum f sp-Sordariomycetes EWZ80636.1-1080343-Fusarium oxysporum f sp-Sordariomycetes EWY82337.1-909455-Fusarium oxysporum FOSC 3-Sordariomycetes 100 EXA36705.1-1080344-Fusarium oxysporum f sp-Sordariomycetes EWZ31047.1-660027-Fusarium oxysporum Fo47-Sordariomycetes XP 018252497.1-426428-Fusarium oxysporum f sp-Sordariomycetes EXK84015.1-1089458-Fusarium oxysporum f sp-Sordariomycetes 95 EGU83440.1-660025-Fusarium oxysporum Fo5176-Sordariomycetes

160 Tree scale: 0.1 CRG90955.1-28573-Talaromyces islandicus-Eurotiomycetes OCL11392.1-574774-Glonium stellatum-Dothideomycetes OCL02963.1-574774-Glonium stellatum-Dothideomycetes OQD95988.1-29845-Penicillium vulpinum-Eurotiomycetes OAA51374.1-1081107-Cordyceps brongniartii RC-Sordariomycetes Taxonomy XP 008086613.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes 100 EHK97192.1-1104152-Glarea lozoyensis 74030-Leotiomycetes OAK94601.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes Leotiomycetes 91 99 KUL90201.1-198730-Talaromyces verruculosus-Eurotiomycetes 100 CRG83421.1-28573-Talaromyces islandicus-Eurotiomycetes ENH79514.1-1213857-Colletotrichum orbiculare-Sordariomycetes Sordariomycetes EKG13588.1-1126212-Macrophomina phaseolina M-Dothideomycetes OJJ67834.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 OJI89151.1-767770-Aspergillus tubingensis C-Eurotiomycetes Eurotiomycetes 87 98 ODM21322.1-573508-Aspergillus cristatus-Eurotiomycetes 93 XP 018189397.1-1328760-Xylona heveae TC161-other Pezizomycotina KOS21699.1-150374-Escovopsis weberi-Sordariomycetes Dothideomycetes OTA99529.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 100 KFY42096.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes XP 001803247.1-321614-Parastagonospora nodorum-Dothideomycetes other_Pezizomycotina 96 QUERY-Afu1g01000-330879-Aspergillus fumigatus Af2-Eurotiomycetes 99 KMK54470.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes XP 013262530.1-1182545-Exophiala aquamarina CBS-Eurotiomycetes other_Ascomycota XP 008714593.1-1220924-Cyphellophora europaea CB-Eurotiomycetes XP 017995192.1-1664694-Phialophora attae-Eurotiomycetes 100 XP 016219388.1-253628-Verruconis gallopava-Dothideomycetes other_Fungi XP 016626305.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes XP 007755064.1-1182544-Cladophialophora yegresii-Eurotiomycetes 100 OCT50474.1-86049-Cladophialophora carrioni-Eurotiomycetes other_Opisthokonta 82 93 XP 008728080.1-1279043-Cladophialophora carrioni-Eurotiomycetes EQB55257.1-1237896-Colletotrichum gloeospori-Sordariomycetes KKY39798.1-1214573-Diaporthe ampelina-Sordariomycetes other_Eukaryota 100 KUI61446.1-694573-Valsa mali var pyri-Sordariomycetes 89 100 KUI63333.1-105487-Valsa mali-Sordariomycetes OTB10749.1-1001832-Daldinia sp EC12-Sordariomycetes Bacteria 95 OQO03845.1-1507870-Rachicladosporium antarct-Dothideomycetes 100 OQO13276.1-1507870-Rachicladosporium antarct-Dothideomycetes 99 OQO10333.1-1974281-Rachicladosporium sp CCF-Dothideomycetes Archaea 100 OTA35077.1-1157616-Hortaea werneckii EXF 200-Dothideomycetes 99 XP 007673282.1-717646-Baudoinia panamericana UA-Dothideomycetes EME49121.1-675120-Dothistroma septosporum N-Dothideomycetes Viruses 97 KJY00698.1-1047168-Zymoseptoria brevis-Dothideomycetes 100 XP 003853448.1-336722-Zymoseptoria tritici IPO3-Dothideomycetes 100 99SMQ49856.1-1276538-Zymoseptoria tritici ST99-Dothideomycetes XP 007922236.1-383855-Pseudocercospora fijiensi-Dothideomycetes KXT13488.1-113226-Pseudocercospora musae-Dothideomycetes 100 KXS97951.1-321146-Mycosphaerella eumusae-Dothideomycetes XP 009256835.1-1028729-Fusarium pseudograminearu-Sordariomycetes 100 XP 001216325.1-341663-Aspergillus terreus NIH26-Eurotiomycetes OJJ37159.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes GAD94095.1-1356009-Byssochlamys spectabilis-Eurotiomycetes XP 013324705.1-1408163-Rasamsonia emersonii CBS-Eurotiomycetes KZT59844.1-1353952-Calocera cornea HHB12733-Basidiomycota XP 007781013.1-1168221-Coniosporium apollinis CB-Dothideomycetes GAA91749.1-1033177-Aspergillus kawachii IFO-Eurotiomycetes 100 GAQ33294.1-5061-Aspergillus niger-Eurotiomycetes GAO82730.1-91492-Aspergillus udagawae-Eurotiomycetes 89 GAQ09606.1-293939-Aspergillus lentulus-Eurotiomycetes OJI96179.1-1036611-Aspergillus versicolor CB-Eurotiomycetes 100 OJJ62072.1-1036612-Aspergillus sydowii CBS 5-Eurotiomycetes XP 002145015.1-441960-Talaromyces marneffei ATC-Eurotiomycetes 100 XP 002340368.1-441959-Talaromyces stipitatus AT-Eurotiomycetes XP 018063839.1-149040-Phialocephala scopiformis-Leotiomycetes 100 CZR63120.1-576137-Phialocephala subalpina-Leotiomycetes OJZ86812.1-1137211-Aspergillus luchuensis CB-Eurotiomycetes GAT24535.1-1069201-Aspergillus luchuensis-Eurotiomycetes 100 100 GAQ46565.1-5061-Aspergillus niger-Eurotiomycetes OJI83705.1-767770-Aspergillus tubingensis C-Eurotiomycetes OQE32221.1-303698-Penicillium steckii-Eurotiomycetes OKP10497.1-1316194-Penicillium subrubescens-Eurotiomycetes 97 OOQ83193.1-104259-Penicillium brasilianum-Eurotiomycetes 100 CEJ58479.1-104259-Penicillium brasilianum-Eurotiomycetes 99 OQD90155.1-60172-Penicillium solitum-Eurotiomycetes CRL25978.1-5075-Penicillium camemberti-Eurotiomycetes OQE32653.1-254877-Penicillium flavigenum-Eurotiomycetes 88 OQE44909.1-36646-Penicillium coprophilum-Eurotiomycetes CDM30815.1-1365484-Penicillium roqueforti FM-Eurotiomycetes KUM63588.1-48697-Penicillium freii-Eurotiomycetes 96 OQD64120.1-60169-Penicillium polonicum-Eurotiomycetes EXU97138.1-568076-Metarhizium robertsii-Sordariomycetes 100 KID85732.1-1276136-Metarhizium guizhouense A-Sordariomycetes XP 018690608.1-1367422-Fonsecaea erecta-Eurotiomycetes XP 016635219.1-1442371-Fonsecaea multimorphosa C-Eurotiomycetes XP 016254311.1-569365-Cladophialophora immunda-Eurotiomycetes XP 013267126.1-1442369-Rhinocladiella mackenziei-Eurotiomycetes 80 XP 007729240.1-1182542-Capronia epimyces CBS 606-Eurotiomycetes XP 013256604.1-1182545-Exophiala aquamarina CBS-Eurotiomycetes XP 016225318.1-212818-Exophiala mesophila-Eurotiomycetes XP 007740292.1-1182543-Cladophialophora psammoph-Eurotiomycetes 8097 XP 016614130.1-1442370-Cladophialophora bantiana-Eurotiomycetes XP 008729795.1-1279043-Cladophialophora carrioni-Eurotiomycetes XP 007760483.1-1182544-Cladophialophora yegresii-Eurotiomycetes 94 KIW63865.1-5601-Phialophora americana-Eurotiomycetes XP 013285935.1-1442368-Fonsecaea pedrosoi CBS 27-Eurotiomycetes OAG41568.1-254056-Fonsecaea monophora-Eurotiomycetes 94 OAL34722.1-856822-Fonsecaea nubica-Eurotiomycetes XP 013321408.1-348802-Exophiala xenobiotica-Eurotiomycetes 83 XP 016258065.1-215243-Exophiala oligosperma-Eurotiomycetes 99 XP 016233892.1-91928-Exophiala spinifera-Eurotiomycetes

161 Tree scale: 0.1 EGC42541.1-544711-Histoplasma capsulatum H8-Eurotiomycetes 100 EEH02664.1-447093-Histoplasma capsulatum G1-Eurotiomycetes 100 OOF98127.1-602072-Aspergillus carbonarius I-Eurotiomycetes 100 OCK93825.1-794803-Cenococcum geophilum 1 58-Dothideomycetes 85 CDM31411.1-1365484-Penicillium roqueforti FM-Eurotiomycetes Taxonomy 100 OQE22710.1-254877-Penicillium flavigenum-Eurotiomycetes XP 008082817.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes 100 XP 007581912.1-1287680-Neofusicoccum parvum UCRN-Dothideomycetes Leotiomycetes XP 001942161.1-426418-Pyrenophora tritici repen-Dothideomycetes XP 001904308.1-515849-Podospora anserina S mat-Sordariomycetes XP 001216793.1-341663-Aspergillus terreus NIH26-Eurotiomycetes Sordariomycetes 100 OJJ30102.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes 100 KIA75578.1-40382-Aspergillus ustus-Eurotiomycetes 89 XP 001799912.1-321614-Parastagonospora nodorum-Dothideomycetes Eurotiomycetes KDN65693.1-1173701-Colletotrichum sublineola-Sordariomycetes 100 KXH46496.1-1460502-Colletotrichum nymphaeae-Sordariomycetes 100 XP 007598704.1-1445577-Colletotrichum fioriniae-Sordariomycetes Dothideomycetes OCK87084.1-794803-Cenococcum geophilum 1 58-Dothideomycetes 99 XP 014539096.1-1170230-Penicillium digitatum Pd1-Eurotiomycetes 100 XP 001258284.1-331117-Aspergillus fischeri NRRL-Eurotiomycetes other_Pezizomycotina 100 OKP11762.1-1316194-Penicillium subrubescens-Eurotiomycetes SLM33859.1-136370-Umbilicaria pustulata-other Pezizomycotina OCL02964.1-574774-Glonium stellatum-Dothideomycetes other_Ascomycota XP 007919395.1-1286976-Phaeoacremonium minimum U-Sordariomycetes OAL42975.1-765867-Pyrenochaeta sp DS3sAY3a-Dothideomycetes CDP29883.1-515849-Podospora anserina S mat-Sordariomycetes other_Fungi 100 XP 001904579.1-515849-Podospora anserina S mat-Sordariomycetes EHK97193.1-1104152-Glarea lozoyensis 74030-Leotiomycetes 100 XP 008086612.1-1116229-Glarea lozoyensis ATCC 20-Leotiomycetes other_Opisthokonta OTB10641.1-1001832-Daldinia sp EC12-Sordariomycetes 81 OAK94597.1-765868-Stagonospora sp SRC1lsM3-Dothideomycetes 100 CRG83422.1-28573-Talaromyces islandicus-Eurotiomycetes other_Eukaryota 100 KUL90105.1-198730-Talaromyces verruculosus-Eurotiomycetes OTB20523.1-1001832-Daldinia sp EC12-Sordariomycetes 99 XP 018150745.1-759273-Colletotrichum higginsian-Sordariomycetes Bacteria 99 XP 003041774.1-660122-Nectria haematococca mpVI-Sordariomycetes 100 100 ENH66034.1-1229664-Fusarium oxysporum f sp-Sordariomycetes OJJ41603.1-1073089-Aspergillus wentii DTO 13-Eurotiomycetes Archaea 100 OQD95983.1-29845-Penicillium vulpinum-Eurotiomycetes 100 XP 016597047.1-27334-Penicillium expansum-Eurotiomycetes 100 100 KGO67343.1-27334-Penicillium expansum-Eurotiomycetes Viruses 85 KGO38831.1-27334-Penicillium expansum-Eurotiomycetes KXX74576.1-100816-Madurella mycetomatis-Sordariomycetes KZL82819.1-1573173-Colletotrichum incanum-Sordariomycetes 100 OHW95298.1-1573173-Colletotrichum incanum-Sordariomycetes 100 OLN85402.1-708187-Colletotrichum chlorophyt-Sordariomycetes 100 ENH79513.1-1213857-Colletotrichum orbiculare-Sordariomycetes ODM21321.1-573508-Aspergillus cristatus-Eurotiomycetes 99 XP 018189396.1-1328760-Xylona heveae TC161-other Pezizomycotina 100 OJJ67833.1-767769-Aspergillus brasiliensis-Eurotiomycetes 100 OJI89152.1-767770-Aspergillus tubingensis C-Eurotiomycetes XP 016640358.1-563466-Scedosporium apiospermum-Sordariomycetes KJZ71022.1-1043627-Hirsutella minnesotensis-Sordariomycetes 100 89 XP 014540751.1-1276141-Metarhizium brunneum ARSE-Sordariomycetes 99 XP 013956168.1-413071-Trichoderma virens Gv29 8-Sordariomycetes 100 XP 008097951.1-645133-Colletotrichum graminicol-Sordariomycetes 100 XP 007278670.1-1213859-Colletotrichum gloeospori-Sordariomycetes XP 007789027.1-1287681-Eutypa lata UCREL1-Sordariomycetes 100 100 XP 007828360.1-1229662-Pestalotiopsis fici W106-Sordariomycetes 99 OTB12112.1-1001832-Daldinia sp EC12-Sordariomycetes 100 OTB02208.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 98 OTA83804.1-1001938-Hypoxylon sp CO27 5-Sordariomycetes 96 100 OTA56259.1-1001937-Hypoxylon sp EC38-Sordariomycetes XP 003665637.1-573729-Thermothelomyces thermoph-Sordariomycetes OTA99530.1-1001833-Hypoxylon sp CI 4A-Sordariomycetes 100 KFY42107.1-1420907-Pseudogymnoascus sp VKM-Leotiomycetes 100 KOS21700.1-150374-Escovopsis weberi-Sordariomycetes 90 XP 001803246.1-321614-Parastagonospora nodorum-Dothideomycetes 100 QUERY-Afu1g01010-330879-Aspergillus fumigatus Af2-Eurotiomycetes 100 KMK54471.1-1437362-Aspergillus fumigatus Z5-Eurotiomycetes KPM42830.1-78410-Neonectria ditissima-Sordariomycetes KIM99782.1-913774-Oidiodendron maius Zn-Leotiomycetes OQE27687.1-303698-Penicillium steckii-Eurotiomycetes ALQ32841.1-1147111-Fusarium euwallaceae-Sordariomycetes 100 100 XP 003040326.1-660122-Nectria haematococca mpVI-Sordariomycetes CEG03370.1-1318461-Fusarium acuminatum CS590-Sordariomycetes ALQ32810.1-249398-Fusarium commune-Sordariomycetes 100 100 98 XP 018254836.1-426428-Fusarium oxysporum f sp-Sordariomycetes 100 EGU72765.1-660025-Fusarium oxysporum Fo5176-Sordariomycetes 100 XP 018256513.1-426428-Fusarium oxysporum f sp-Sordariomycetes 100 95 EXK26726.1-1089452-Fusarium oxysporum f sp-Sordariomycetes KIL88133.1-40199-Fusarium avenaceum-Sordariomycetes ALQ32988.1-1101274-Fusarium sp NRRL 52700-Sordariomycetes 100 ALQ32947.1-42676-Fusarium sacchari-Sordariomycetes 100 ALQ33027.1-42677-Fusarium subglutinans-Sordariomycetes 100 ALQ32867.1-282569-Fusarium gaditjirri-Sordariomycetes 100 ALQ32976.1-70751-Fusarium sp NRRL 25184-Sordariomycetes ALQ32924.1-48865-Fusarium redolens-Sordariomycetes XP 018761955.1-334819-Fusarium verticillioides-Sordariomycetes 97 100 AAR92219.1-117187-Fusarium verticillioides-Sordariomycetes 100 ALQ32798.1-79018-Fusarium bulbicola-Sordariomycetes 100 ALQ32745.1-48485-Fusarium anthophilum-Sordariomycetes 100 98 ALQ33019.1-42666-Fusarium succisae-Sordariomycetes ALQ32886.1-75915-Fusarium miscanthi-Sordariomycetes 100 CVK94934.1-192010-Fusarium mangiferae-Sordariomycetes 100 KLO90557.1-5127-Fusarium fujikuroi-Sordariomycetes 100 KLP05512.1-5127-Fusarium fujikuroi-Sordariomycetes 95 KLP14354.1-5127-Fusarium fujikuroi-Sordariomycetes 100 CCT74300.1-1279085-Fusarium fujikuroi IMI 58-Sordariomycetes

162