bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 Drivers of genetic diversity in secondary metabolic gene clusters in a fungal population 5 6 7 8 Abigail L. Lind1, Jennifer H. Wisecaver2, Catarina Lameiras3, Philipp Wiemann4, Jonathan M. 9 Palmer5, Nancy P. Keller4, Fernando Rodrigues6,7, Gustavo H. Goldman8, Antonis Rokas1,2 10

11 12 1. Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, 13 Tennessee, USA.

14 2. Department of Biology, Vanderbilt University, Nashville, Tennessee, USA.

15 3. Department of Microbiology, Portuguese Oncology Institute of Porto, Porto, Portugal

16 4. Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, 17 Madison, Wisconsin, USA

18 5. Center for Forest Mycology Research, Northern Research Station, US Forest Service, Madison, 19 Wisconsin, USA

20 6. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 21 Braga, Portugal

22 7. ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.

23 8. Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São 24 Paulo, Brazil

25 †Corresponding author and lead contact: [email protected]

26 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

27 Summary 28 29 Filamentous fungi produce a diverse array of secondary metabolites (SMs) critical for defense, 30 virulence, and communication. The metabolic pathways that produce SMs are found in 31 contiguous gene clusters in fungal genomes, an atypical arrangement for metabolic pathways in 32 other eukaryotes. Comparative studies of filamentous fungal species have shown that SM gene 33 clusters are often either highly divergent or uniquely present in one or a handful of species, 34 hampering efforts to determine the genetic basis and evolutionary drivers of SM gene cluster 35 divergence. Here we examined SM variation in 66 cosmopolitan strains of a single species, the 36 opportunistic human pathogen Aspergillus fumigatus. Investigation of genome-wide 37 population-level variation showed that A. fumigatus strains contained five general types of 38 variation in SM gene clusters: non-functional gene polymorphisms, gene gain and loss 39 polymorphisms, whole cluster gain and loss polymorphisms, allelic polymorphisms where 40 different alleles corresponded to distinct, non-homologous clusters, and location 41 polymorphisms in which a cluster was found to differ in its genomic location across strains. 42 These polymorphisms affect the function of representative A. fumigatus SM gene clusters, such 43 as those involved in the production of gliotoxin, fumigaclavine, and helvolic acid, as well as the 44 function of clusters with undefined products. In addition to enabling the identification of 45 polymorphisms whose detection requires extensive genome-wide synteny conservation (e.g., 46 mobile gene clusters and non-homologous cluster alleles), our population genomics approach 47 also implicated multiple underlying genetic drivers, including point mutations, recombination, 48 genomic deletion and insertion events, as well as horizontal gene transfer from distant fungi. 49 Finally, most of the population variants that we uncover have been previously hypothesized to 50 contribute to SM gene cluster diversity across entire fungal classes and phyla. We suggest that 51 the drivers of genetic diversity operating within a fungal population shown here are sufficient 52 to explain SM cluster macroevolutionary patterns. 53 54 Keywords: chemodiversity, specialized metabolism, genome evolution, genome architecture, 55 gene loss, genomic rearrangement bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

56 Introduction 57 Filamentous fungi produce a diverse array of small molecules that function as toxins, 58 antibiotics, and pigments [1]. Though by definition these so-called specialized or secondary 59 metabolites (SMs) are not strictly necessary for growth and development, they are critical to 60 the lifestyle of filamentous fungi [2]. For example, antibiotic SMs gain give their fungal 61 producers a competitive edge in environments crowded with other microbes [3]. SMs can 62 additionally mediate communication between and within species, as well as contribute to 63 virulence on animal and plant hosts in pathogenic fungi [4,5]. 64 65 A genomic hallmark of SMs in filamentous fungi is that the biosynthetic pathways that 66 produce them are typically organized into contiguous gene clusters in the genome [6]. These 67 gene clusters contain the chemical backbone synthesis genes whose enzymatic products 68 produce a core metabolite, such as non-ribosomal peptide synthases (NRPS) and polyketide 69 synthases (PKS), tailoring enzymes that chemically modify the metabolite, transporters involved 70 in product export, and transcription factors that control the expression of the clustered genes 71 [6]. Filamentous fungal genomes, particularly those in the phylum Ascomycota [6], typically 72 contain dozens of SM gene clusters. However, most individual SM gene clusters appear to be 73 either species-specific or narrowly taxonomically distributed in only a handful of species [6,7]. 74 SM gene clusters that are more broadly distributed show discontinuous taxonomic distributions 75 and are often highly divergent between species. Consequently, the identity and total number of 76 SM gene clusters can vary widely even between very closely related species whose genomes 77 exhibit very high sequence and synteny conservation [8,9]. 78 79 In the last decade, several comparative studies have described macroevolutionary 80 patterns of SM gene cluster diversity. For example, studies centered on genomic comparisons 81 of closely related species, such as members of the same genus, have identified several different 82 types of inter-species divergence, from single nucleotide substitutions (e.g., differences in 83 fumonisins produced by Fusarium species are caused by variants in one gene [10]), to gene gain 84 / loss events (e.g., the trichothecene gene clusters in Fusarium species and the aflatoxin family 85 SM gene clusters in Aspergillus species) [11–16], and genomic rearrangements (e.g., the bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

86 trichothecene gene clusters in Fusarium) [11]. Additionally, genetic and genomic comparisons 87 across fungal orders and classes have identified several instances of gene gain or loss [17–19] 88 and horizontal gene transfer [13,20–23] acting on individual genes or on entire gene clusters, 89 providing explanations for the diversity and discontinuity of the taxonomic distribution of 90 certain SM gene clusters across fungal species. 91 92 Although inter-species comparative studies have substantially contributed to our 93 understanding of SM diversity, the high levels of evolutionary divergence of SM clusters make 94 inference of the genetic drivers of SM gene cluster evolution challenging; simply put, it has 95 been difficult to “catch” the mechanisms that generate SM gene cluster variation “in the act”. 96 Several previous studies have examined intra-species or population-level differences in 97 individual SM gene clusters, typically focusing on the presence and frequency of non-functional 98 alleles of clusters involved in production of mycotoxins. Examples of clusters exhibiting such 99 polymorphisms include the gibberellin gene cluster in Fusarium oxysporum [24], the fumonisin 100 gene cluster in Fusarium fujikuroi [25], the aflatoxin and cyclopiazonic gene clusters in 101 Aspergillus flavus [26], and the bikaverin gene cluster in Botrytis cinerea [27]. While these 102 studies have greatly advanced our understanding of SM gene cluster genetic variation and 103 highlighted the importance of population-level analyses, studies examining the entirety of SM 104 gene cluster polymorphisms in fungal populations are so far lacking. We currently do not 105 understand the types and frequency of SM gene cluster polymorphisms in populations, 106 whether these polymorphisms affect all types of SM gene clusters, as well as the genetic drivers 107 of SM gene cluster evolution. 108 109 To address these questions, we investigated the genetic diversity of all 36 known and 110 predicted SM gene clusters in whole genome sequence data from 66 strains, 8 of which were 111 sequenced in this study, of the opportunistic human pathogen Aspergillus fumigatus, a species 112 with cosmopolitan distribution and panmictic population structure [28]. We found that 13 SM 113 gene clusters were generally conserved and harbored low amounts of variation. In contrast, the 114 remaining 23 SM gene clusters were highly variable and contained one or more of five different bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

115 types of genetic variation: single-nucleotide polymorphisms including nonsense and frameshift 116 variants, individual gene gain and loss polymorphisms, entire cluster gain and loss 117 polymorphisms, polymorphisms associated with changes in cluster genomic location, and 118 clusters with non-homologous alleles resembling the idiomorphs of fungal mating loci. Many 119 clusters contained interesting combinations of these different polymorphisms, such as 120 pseudogenization in some strains and entire cluster loss in others. The types of variants we find 121 are likely generated by a combination of DNA replication and repair errors, recombination, 122 genomic insertions and deletions, and horizontal transfer. We additionally find an enrichment 123 for transposable elements (TEs) around horizontally transferred clusters, clusters that change in 124 genomic locations, and idiomorphic clusters. Taken together, our results provide a guide to 125 both the types of polymorphisms and the genetic drivers of SM gene cluster diversification in 126 filamentous fungi. As most of the genetic variants that we observe have been previously 127 associated with SM gene cluster diversity across much larger evolutionary distances and 128 timescales, we argue that population-level processes influencing SM gene cluster diversity are 129 sufficient to explain SM cluster macroevolutionary patterns. 130

131 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

132 Results 133 We analyzed the genomes of 66 globally distributed strains of Aspergillus fumigatus for 134 polymorphisms in SM gene clusters. We performed whole-genome sequencing on 8 strains, and 135 collected the remaining 58 strains from publicly available databases including NCBI Genome 136 and the NCBI Short Read Archive (Figure 1, Table S1) [28–32]. We analyzed all strains for 137 polymorphisms in 33 curated SM gene clusters present in the reference Af293 genome and 138 additionally searched for novel SM gene clusters (see Methods). These examinations revealed 139 five distinct types of polymorphisms which influence SM gene cluster variation (Table 1):

140 a) Single nucleotide and short indel polymorphisms. 33 / 33 SM gene clusters (present in 141 the reference Af293 strain) contained multiple genes with missense SNPs and short 142 indel variants in at least one strain in the population. 23 / 33 SM gene clusters contained 143 one or more genes with frameshift or nonsense variants in the population. 144 b) Gene content polymorphisms involving loss or gain of one or more genes. 6 / 33 SM 145 gene clusters contained a gene content polymorphism in the population. 146 c) Whole SM gene cluster gain and loss polymorphisms. 3 / 33 SM gene clusters present in 147 the genome of the reference Af293 strain were absent in at least one strain in the 148 population and an additional 3 previously unknown SM gene clusters were present in 149 the population. 150 d) Idiomorphic polymorphisms. One locus contained multiple non-homologous SM gene 151 cluster alleles in different strains of the population. 152 e) Genomic location polymorphisms. 2 / 33 SM gene clusters were found in different 153 genomic locations (e.g., different chromosomes) between strains.

154

155 Single-nucleotide and indel polymorphisms 156 It is well established that single nucleotide polymorphisms (SNPs) and short indel 157 polymorphisms are caused by errors in DNA replication and repair, and are a major source of 158 genomic variation [33]. Non-synonymous SNPs and indels with missense, frameshift, and 159 nonsense effects were widespread across the 33 SM reference gene clusters (Table S2). Every bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

160 strain contained numerous missense mutations and at least one nonsense or frameshift 161 mutation in its SM gene clusters. Although missense mutations are likely to influence SM 162 production, the functional effects of nonsense and frameshift mutations are comparatively 163 easier to infer from genomic sequence data because they often lead to truncated proteins 164 lacking a significant portion of their amino acid sequence. For example, a frameshift mutation in 165 the polyketide synthase (PKS) of the trypacidin gene cluster in the A1163 strain results in loss of 166 trypacidin production [34]. Interestingly, we identified a premature stop codon (Gln273*) in a 167 transcription factor required for trypacidin production, tpcD, in a strain sequenced in this study 168 (MO79587EXP). These data suggest that function of this SM gene cluster has been lost through 169 at least two independent genetic events in A. fumigatus. 170 171 Individual nonsense or frameshift variants ranged from very common in the population 172 to rarer variants present in one or a handful of strains. For example, the non-ribosomal peptide 173 synthase (NRPS) pes3 gene (Afu5g12730) in SM gene cluster 21 harbors 16 nonsense or 174 frameshift polymorphisms in 55 strains. Seven of these polymorphisms are common and 175 present in 10 or more strains, while seven are rarer and found in 5 or fewer strains. Strains with 176 lab-mutated null alleles of the pes3 gene are more virulent than strains with functional copies 177 [35], which may explain the widespread occurrence of null pes3 alleles in the Aspergillus 178 population. 179

180 Gene content polymorphisms 181 We additionally identified several SM gene clusters that gained or lost genes in some 182 strains. These gene content polymorphisms were most likely generated through genomic 183 deletion or insertion events and were often present in high frequencies in the population (Table 184 1). In three cases, these polymorphisms impact backbone synthesis genes, rendering the SM 185 gene cluster non-functional. One example involves SM gene cluster 14, whose standard 186 composition includes a pyoverdine synthase gene, an NRPS-like gene, an NRPS backbone gene, 187 and several additional modification genes (Figure 2A). We discovered that 4 / 66 strains lack an 188 11-kb region on the 3’ end of the cluster which normally contains an NRPS gene and two bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

189 additional cluster genes, and the first non-SM genes on the 3’ end flanking the cluster. All A. 190 fumigatus strains contain a copia family transposable element [36,37] at the 3’ end of the 191 cluster, suggesting that transposable elements may have been involved in the generation of this 192 polymorphism. Three of the strains lacking the 11-Kb region form a clade in the SNP-based 193 phylogeny (the fourth, RP-2014 was not included in this phylogeny as short read data were not 194 available). While this polymorphism could have arisen through a deletion event, a homologous 195 cluster lacking the 11-kb region is also present in the reference genomes of Aspergillus lentulus 196 and Aspergillus fischerianus, close relatives of A. fumigatus (Figure 2). The most parsimonious 197 explanation is that the genome of the A. fumigatus ancestor contained an SM gene cluster that 198 lacked the 11-kb region, and that this genomic region was subsequently gained and increased in 199 frequency in the A. fumigatus population. 200 201 Two additional gene content polymorphisms affecting SM backbone genes were 202 restricted to one strain each and appear to have arisen through genomic deletion events. 203 Specifically, strain IF1SWF4 lacks an 8-Kb region near the helvolic acid SM gene cluster, 204 resulting in the loss of the backbone oxidosqualene cyclase gene as well an upstream region 205 containing two non-SM genes (Figure S1). Strain LMB35Aa lacks a 54-kb region on the end of 206 chromosome 2, which includes five genes from the telomere-proximal fumigaclavine C cluster 207 (Figure S1). 208 209 In three other cases, gene content polymorphisms involved gene loss or truncation 210 events of non-backbone structural genes. We found that the second half of the ORF of the gliM 211 O-methyltransferase gene in the gliotoxin gene cluster has been lost in 2 / 66 strains (Figure 212 S1), and that the first half of the permease fmqE in the fumiquinazoline gene cluster has been 213 lost in 4 / 66 strains (Figure S1). Finally, an ABC transporter gene in SM cluster 21 has been 214 almost entirely lost in 21 / 66 strains (Figure S2). This deletion event is found in strains that are 215 related in the SNP phylogeny but does not perfectly mirror population structure (Figure S2). 216 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

217 Whole gene cluster gain and loss polymorphisms 218 Several SM gene clusters were gained or lost entirely in 13 / 66 strains. We observed 219 instances where a cluster present in the genome of the reference Af293 strain was absent or 220 pseudogenized in other strains as well as cases in which SM clusters present in other strains 221 were absent from the reference Af293 strain. 222 223 The most notable example of an SM gene cluster that was present in the Af293 224 reference genome but absent or pseudogenized in others was SM cluster 4. This cluster 225 contains 5 genes on the tip of the Af293 chromosome 1 and contains orthologs to five of the six 226 genes in the fusarielin gene cluster in Fusarium graminearum [38]. This cluster is also present in 227 several other Aspergillus species, including A. clavatus and A. niger [38]. Phylogenetic analysis 228 of the genes in this SM gene cluster is consistent with horizontal gene transfer between fungi in 229 the class Sordariomycetes and fungi in the class Eurotiomycetes, or alternatively with extensive 230 gene loss in both Sordariomycetes and Eurotiomycetes (Figure S3). This gene cluster is entirely 231 absent in 4 / 66 strains, and its genes are undergoing pseudogenization in an additional 43 232 strains via multiple independent mutational events (Figure 3A). These four strains form a single 233 clade on the SNP-based phylogeny, suggesting that this cluster was lost in a single deletion 234 event (Figure 3B). Further, 19 strains shared a single frameshift variant in the polyketide 235 synthase gene (4380_4381insAATGGGCT; frameshift at Glu1461 in Afu1g17740) and an 236 additional 13 strains shared a single frameshift variant (242delG; frameshift at Gly81) in an 237 aldose 1-epimerase gene (Afu1g17723). Eleven other strains each contained one to several 238 frameshift or nonsense polymorphisms involving nine unique mutational sites, suggesting that 239 this pathway is undergoing multiple independent pseudogenization events. Five of these strains 240 contained multiple distinct frameshifts and premature stop codons in more than one gene in 241 the cluster, indicating that the entire pathway is pseudogenized in these strains. The 242 distributions of pseudogenizing variants only in the aldose 1-epimerase gene and of 243 pseudogenizing variants in multiple genes in the cluster roughly correlate with population 244 structure, while the distribution of pseudogenizing variants in the PKS backbone gene does not 245 (Figure 3B). 246 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

247 By searching for novel SM gene clusters in the genomes of the other 65 A. fumigatus 248 strains, we found three SM gene clusters that were absent from the genome of the Af293 249 reference strain. As SM gene clusters are often present in repeat-rich and subtelomeric regions 250 that are challenging to assemble [39,40], these strains might harbor additional novel SM gene 251 clusters. 252 253 One of the novel SM gene clusters that we identified, cluster 34, was present in all but 254 two of the strains (Af293 and F7763). This cluster contains a PKS backbone gene, one PKS-like 255 gene with a single PKS associated domain, nine genes with putative biosynthetic functions 256 involved in secondary metabolism, and six hypothetical proteins (Figure 3C). The two strains 257 that lack this cluster contain a likely non-functional cluster fragment that includes the PKS-like 258 gene, two biosynthetic genes, and three hypothetical proteins. Interestingly, the 3’ region 259 flanking this cluster is syntenic across all 66 strains but the 5’ region is not, suggesting that a 260 recombination or deletion event may have resulted in the loss of this cluster in the Af293 and 261 F7763 strains. 262 263 The other SM gene clusters that were absent from the Af293 genome are present at 264 lower frequencies in the population; cluster 35 is present in 2 / 66 strains and cluster 36 in 4 / 265 66 strains. Cluster 35 is located in a region syntenic with an Af293 chromosome 4 region and is 266 flanked on both sides by transposable elements (Figure S4). The two strains that contain this 267 novel cluster are not sister to each other in the SNP-based phylogeny (Figure S4). Eight of the 268 14 genes in this SM gene cluster are homologous to genes in an SM gene cluster in the genome 269 of the insect pathogenic fungus Metarhizium anisopliae (Figure S4). Phylogenetic analysis of 270 these 8 genes is consistent with a horizontal transfer event (Figure S5). Cluster 36 is an NRPS 271 containing cluster located on genomic scaffolds that lack homology to either the Af293 or 272 A1163 genomes, making it impossible to determine on which chromosome this cluster is 273 located (Figure S3). Two of the strains containing this novel cluster are sister to each other in 274 the SNP-based phylogeny, while the third is distant to these two (Figure S4). The evolutionary bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

275 histories of the genes in the cluster are consistent with vertical inheritance and are present in 276 multiple Aspergillus species. 277

278 Idiomorph polymorphisms 279 One of the most peculiar types of polymorphisms that we identified is a locus containing 280 different unrelated alleles of SM gene clusters, reminiscent of the idiomorph alleles at the 281 fungal mating loci [41]. This locus, which resides on chromosome 3 and corresponds to cluster 282 10 in the Af293 genome (Figure 4), was previously described as being strain-specific in a 283 comparison between Af293 and A1163 [30] and is thought to reside in a recombination hot 284 spot [28]. Our analysis showed that there are at least 6 different alleles of this cluster in the 285 population, containing 4 different types of key enzymes involved in natural product 286 biosynthesis: a polyketide synthase (PKS) non-ribosomal peptide synthetase (NRPS) hybrid, a 287 highly reducing (HR) PKS, a non-reducing (NR) PKS and an NRPS-like enzyme (Figure 4). Two 288 additional alleles were present in only one strain each (Figure S6). 289 290 In the Af293 reference genome, the cluster present at this locus contains one NR-PKS 291 along with an NRPS-like gene (Allele B). In the A1163 reference genome and 17 other strains, 292 there is a PKS-NRPS and an HR-NRPS (Allele E). These alleles show an almost complete lack of 293 sequence similarity except for a conserved hypothetical protein and a fragment of the HR-PKS 294 in the Af293 allele; in contrast, the upstream and downstream flanking regions of the two 295 alleles, which do not contain any backbone genes, are syntenic. Remarkably, another allele, 296 present in 12 strains, contains all of the genes from both the Af293 and A1163 clusters (Allele 297 D). The remaining three alleles contain various combinations of these genes. One allele found in 298 22 strains contains some A1163-specific genes including the HR-PKS and no Af293-specific 299 genes (Allele F), while another allele found in 3 strains contains some Af293-specific genes 300 including the NRPS-like gene but no A1163 genes (Allele A). The final allele, present in 8 strains, 301 contains the entire Af293 allele as well as part of the A1163 allele containing the HR-PKS (Allele 302 C). Every allele is littered with long terminal repeat sequence fragments from gypsy and copia 303 TE families as well as with sequence fragments from DNA transposons from the mariner family bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

304 [36]. In some cases, these TEs correspond with breakpoints in synteny between alleles, 305 suggesting that the diverse alleles of this SM gene cluster may arise via TE-driven 306 recombination. Further, both of the alleles that are restricted to a single strain had an insertion 307 event of several genes near a TE, though the rest of the locus is highly similar to one of the 308 more common alleles (Figure S6). 309 310 Preliminary metabolite analysis comparing one of the strains (08-19-02-30) that 311 contains the allele harboring all four key enzymes (Allele D) to two strains (08-12-12-13 and 08- 312 19-02-10) that only codes for the putative cluster the HR-PKS backbone gene (Allele F) by 313 untargeted XCMS analysis [42] revealed the presence of 2 unique masses in 08-19-02-30 (Table 314 S4; Fig. 4B). Further analysis is underway to investigate whether any of these m/z can be 315 directly linked to the allele present in 08-19-02-30. 316 317 The evolutionary history of this highly diverse locus is unclear (Figure 4C). While it is 318 tempting to speculate that the largest allele containing all observed genes represents the 319 ancestral state, it does not explain the presence of a shared hypothetical protein and PKS gene 320 fragment between the Af293 locus (Allele C) and the A1163 locus (Allele B). The distributions of 321 Alleles A, B, C, and F roughly correlate with the population structure of the strains, while the 322 distribution of Alleles D and E do not (Figure 4C). 323

324 Genomic location polymorphisms 325 The final type of polymorphism that we observed is associated with SM gene clusters 326 that are located in different genomic locations in different strains, suggesting that these SM 327 gene clusters are behaving like mobile elements. This type of polymorphism was observed in 328 SM gene clusters 1 and 33, both of which produce as yet identified products, and are present at 329 low frequencies in the population. 330 331 SM gene cluster 1, which is present in six strains at three different genomic locations 332 (Figure 5A), consists of a PKS and four other modification genes that are always flanked by a 15 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

333 Kb region (upstream) and a 43 Kb region (downstream) containing TEs. In the reference Af293 334 strain and in strain F7763, this SM gene cluster and its flanking regions are located on 335 chromosome 1, while in strains dutch3, F13619, and Z5 they are located between Afu4g07320 336 and Afu4g07340 on chromosome 4. In contrast, in strain JCM_10253, the cluster and flanking 337 regions are located on chromosome 8 immediately adjacent to the 3’ end of the intertwined 338 fumagillin and pseurotin SM gene supercluster [43]. The strains containing the allele on 339 Chromosome 1 are sister to each other in the SNP-based phylogeny, while the other strains are 340 scattered across the tree and do not reflect the underlying population structure (Figure 5C). 341 342 In 5 / 6 strains, the cluster appears to be functional and does not contain nonsense SNPs 343 or indels. However, the cluster found on chromosome 1 in strain F7763 contains two stop 344 codons in the oxidoreductase gene (Gln121* and Gln220*) and two premature stop codons in 345 the polyketide synthase (Gln1156* and Gln1542*), suggesting this strain contains a null allele. 346 347 This “jumping” gene cluster is not present in any other sequenced Aspergillus genus, 348 and phylogenetic analysis of its constituent genes is consistent with horizontal gene transfer 349 between fungi (Figure S7). Specifically, this gene cluster is also present in Phaeosphaeria 350 nodorum, a plant pathogen from the class Dothideomycetes, Pseudogymnoascus pannorum, a 351 fungus isolated from permafrost from the Leotiomycetes, Escovopsis weberi, and a fungal 352 parasite of fungus-growing ants from the Sordariomycetes (Figure 5B). One additional species, 353 the endophyte Hypoxylon sp. CI4A from the class Sordariomycetes, contains four of the five 354 cluster genes but is missing Afu1g00970, an MFS drug transporter. However, this species 355 contains an unrelated gene annotated as an MFS drug transporter immediately adjacent to this 356 cluster, so this species may be using a different transporter (Figure 5B). None of these fungi 357 contain the upstream or downstream TE-rich flanking regions present in A. fumigatus, and each 358 fungus contains additional unique genes with putative biosynthetic functions adjacent to the 359 transferred cluster. The most likely explanation for this change in flanking regions is that this 360 SM gene cluster was transferred into A. fumigatus once and has subsequently moved its 361 location in the genome. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

362 363 The second SM gene cluster that shows variation in its genomic location across strains, 364 cluster 33, contains a terpene synthase. This cluster is present in only 5 strains at 3 distinct 365 locations (Figure S8). Similar to cluster 1, this cluster is also flanked by TEs, and in one strain the 366 clusters is located 58 Kb from another SM gene cluster. Two strains that contain the cluster in 367 the same genomic location are sister to each other in the SNP-based phylogeny, while the other 368 three strains containing the cluster do not reflect the underlying population structure (Figure 369 S8). In contrast to cluster 1, this cluster does not appear to have been horizontally transferred 370 between fungi and its genes are present in other sequenced Aspergillus species. As this mobile 371 cluster was not horizontally transferred, it is possible that the horizontal transfer of cluster 1 372 and its mobile nature throughout the A. fumigatus genome are driven by different mechanisms. 373 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

374 Discussion 375 Our examination of 66 genomes from strains of Aspergillus fumigatus revealed that five 376 general types of polymorphisms describe variation in SM gene clusters in a fungal population. 377 These polymorphisms include variation in single nucleotides, gene and gene cluster gains and 378 losses, non-homologous clusters at the same genomic position, and changes in genomic 379 locations of clusters (Figure 6). In several cases, the genetic mechanisms that gave rise to these 380 polymorphisms and their functional consequences are clear. Polymorphisms in single- 381 nucleotides most likely arose from errors in DNA replication or repair, while gene loss 382 polymorphisms likely arose from genomic deletion or recombination events. Others, such as SM 383 gene clusters that exist as non-homologous alleles or that entered the population through 384 horizontal transfer, could arise through multiple genetic mechanisms. Transposable elements 385 are common in polymorphic SM gene clusters and are found flanking mobile and horizontally 386 transferred clusters, as well as in regions adjacent to non-homologous alleles and where gene 387 gain has occurred, suggesting they contribute to SM gene cluster diversity. Using a population 388 genomics approach to identify SM gene cluster variants allowed us to also capture and describe 389 novel polymorphisms, including mobile gene clusters and idiomorphic clusters, which are 390 difficult to identify in comparative genomic studies between species whose conservation of 391 genome-wide synteny is low. Strikingly, the variants and genetic drivers we observe at the 392 population level are also implicated as driving SM gene cluster variation between fungal 393 species, suggesting that the observed microevolutionary processes are sufficient to explain 394 macroevolutionary patterns of SM gene cluster evolution. 395 396 The first novel type of polymorphism was observed in SM gene clusters 1 and 33, which 397 by occupying different genomic locations in different strains, appear to behave in a manner 398 similar to mobile genetic elements. Interestingly, both clusters are located near or immediately 399 adjacent to other SM gene clusters in some strains. For example, cluster 1 is located 400 immediately adjacent to the intertwined fumagillin and pseurotin supercluster [43] in one 401 strain. This supercluster is regulated by the transcriptional factor fapR and is located in a 402 chromosomal region controlled by the master SM regulators laeA and veA [43,44], raising the 403 hypothesis that these mobile gene clusters might be co-opting the regulatory machinery bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

404 already in place. Previous work has hypothesized that the fumagillin and pseurotin supercluster 405 formed through genomic rearrangement events placing the once-independent gene clusters in 406 close proximity to each other [43]. Our observation that this mobile gene cluster is located in 407 this same region not only supports this hypothesis but also implicates transposable elements as 408 one of the mechanisms by which such genomic rearrangements are formed. These 409 superclusters may also represent an intermediate stage in the formation of new SM gene 410 clusters. Supercluster formation, potentially mediated by mobile gene clusters, and followed 411 by gene loss, could explain macroevolutionary patterns of SM gene clusters have shown that 412 clustered genes in one species can be dispersed over multiple gene clusters in other species 413 [9,11]. 414 415 The second novel type of polymorphism was the presence of multiple non-homologous 416 alleles at the cluster 10 locus, echoing the structure of the idiomorphic mating type locus [41]. 417 This region has previously been reported to be a recombination hotspot in A. fumigatus [28] 418 and all alleles contain numerous transposable elements. Thus, it is possible that polymorphism 419 at this locus originated via SM gene cluster fusion or splitting events driven by transposable 420 elements. Interestingly, two other previously described instances of SM gene cluster variation 421 bear close resemblance to the A. fumigatus idiomorphic SM gene cluster 10 locus. The first is 422 the presence of two non-homologous Aspergillus flavus alleles, where some strains contain a 9- 423 gene sesquiterpene-like SM gene cluster and others contain a non-homologous 6-gene SM gene 424 cluster at the same genomic location [45]. The second is the presence of two non-homologous 425 SM gene clusters at the same, well-conserved, locus in a comparison of six species of 426 dermatophyte fungi [46]. Based on these results, we hypothesize that idiomorphic clusters may 427 be common in fungal populations and contribute to the broad diversity of SM gene clusters 428 across filamentous fungi. 429 430 The remaining types of SM polymorphism in the A. fumigatus population have 431 previously been described at the species level. For example, null alleles have been reported for 432 many individual SM gene clusters across diverse fungal species [10,17,26,27] and our results bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

433 show that it is a widespread phenomenon, affecting two-thirds of the A. fumigatus SM gene 434 clusters. Consistent with previous literature reporting the presence of additional SM gene 435 clusters not present in the reference strain [24,25], we also identify four low-frequency SM 436 gene clusters in the A. fumigatus population, two of which are horizontally transferred from 437 distantly related fungi and two that appear vertically inherited. We find numerous cases of 438 gene loss and relatively fewer cases of gene gain; previous studies have implicated these 439 processes across numerous types of SM gene clusters and reported their effects on metabolite 440 production [47]. Previous work has indicated that gene and gene cluster gain and loss tends to 441 occur near telomeres, suggesting that higher rates of genetic events like recombination lead to 442 loss [48]. While we do find several cases of gene and gene cluster loss in subtelomeric clusters, 443 including the fumigaclavine gene cluster and the helvolic acid gene cluster, we also find gene 444 loss in clusters that are not located near telomeres, such as the gliotoxin gene cluster and the 445 fumiquinazoline gene cluster. These findings suggest that while gene and gene cluster loss can 446 occur in telomeric regions, clusters in other genomic regions also experience high rates of loss. 447 448 Previous work has demonstrated that SM gene clusters, like the metabolites that they 449 produce, are highly divergent between fungal species. Our examination of genome-wide 450 variation shows that these SM gene clusters are correspondingly diverse within individual 451 strains of a single fungal species. Furthermore, the observed genetic changes in SM gene 452 clusters are widespread across different types of gene clusters and are caused by many 453 underlying genetic drivers, including gene and gene cluster gain, loss, non-homologous cluster 454 alleles, and mobile gene clusters. The net effect of these substitutions, gains, losses, and 455 rearrangements raises the hypothesis that fungal SM gene clusters are likely in a state of 456 evolutionary flux, constantly altering their SM gene cluster repertoire, and consequently 457 modifying and diversifying their chemodiversity. 458 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

459 Methods 460 Strains analyzed 461 Eight strains of A. fumigatus were isolated from four patients with recurrent cases of 462 aspergillosis in the Portuguese Oncology Institute in Porto, Portugal. Each strain was 463 determined to be A. fumigatus using macroscopic features of the culture and microscopic 464 morphology observed in the slide preparation from the colonies with lactophenol solution [49]. 465 Based on the morphological characterization, all clinical strains were classified as A. fumigatus 466 complex-Fumigati. After whole genome sequencing, retrieval and examination of the beta 467 tubulin and calmodulin sequences of each strain confirmed that all strains belonged to A. 468 fumigatus (see Phylogenetic Analysis and Figure S9). The genomes of all eight strains were 469 sequenced using 150bp Illumina paired-end sequence reads at the Genomic Services Lab of 470 Hudson Alpha (Huntsville, Alabama, USA). Genomic libraries were constructed with the Illumina 471 TruSeq library kit and sequenced on an Illumina HiSeq 2500 sequencer. Samples of all eight 472 strains were sequenced at greater than 180X coverage or depth (Table S1). Short read 473 sequences for these 8 strains are available in the Short Read Archive under accession 474 SRP109032.

475

476 In addition to the 8 strains sequenced in this study, we retrieved 58 A. fumigatus strains 477 with publicly available whole genome sequencing data, resulting in a population genomics 478 dataset of 66 strains (Table S1). The strains used included both environmental and clinical 479 strains and were isolated from multiple continents. Genome assemblies for 10 of these strains, 480 including the Af293 and A1163 reference strains, were available for download from GenBank 481 [28–32,50]. For 6 of these strains, short read sequences were also available from the NCBI Short 482 Read Archive (SRA), which were used for variant discovery only (see Single nucleotide variant 483 (SNV) and indel discovery) and not for genome assembly. Short read sequences were not 484 available for the remaining 4 strains. Short read sequences were downloaded for an additional 485 48 strains from the Short Read Archive if they were sequenced with paired-end reads and at 486 greater than 30x coverage. 487 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

488 Single nucleotide variant (SNV) and indel discovery 489 All strains with available short read data (62 of 66 strains) were aligned to both the 490 Af293 and A1163 reference genomes using BWA mem version 0.7.12-r1044 [51]. Coverage of 491 genes present in the reference genome was calculated using bedtools v2.25.0 [52]. SNV and 492 indel discovery and genotyping was performed relative to the Af293 reference genome and was 493 conducted across all samples simultaneously using the Genome Analysis Toolkit version 3.5-0- 494 g36282e4 with recommended hard filtering parameters [53–55] and annotated using snpEff 495 version 4.2 [56]. 496

497 De novo genome assembly and gene annotation 498 All 56 strains without publicly available genome assemblies were de novo assembled 499 using the iWGS pipeline [57]. Specifically, all strains were assembled using SPAdes v3.6.2 and 500 MaSuRCA v3.1.3 and resulting assemblies were evaluated using QUAST v3.2 [58–60]. The 501 average N50 of assemblies constructed with this strategy was 463 KB (Table S1). Genes were 502 annotated in these assemblies as well as in five GenBank assemblies with no predicted genes 503 using augustus v3.2.2 trained on A. fumigatus gene models [61]. Repetitive elements were 504 annotated in all assemblies using RepeatMasker version open-4.0.6 [62]. 505

506 Secondary metabolic gene cluster annotation and discovery 507 Secondary metabolic gene clusters in the Af293 reference genome were taken from two 508 recent reviews, both of which considered computational and experimental data to delineate 509 cluster boundaries [63,64] (Table S3). The genomes of the other 65 strains were scanned for 510 novel SM gene clusters using identified using antiSMASH v3.0.5.1 [65]. To prevent potential 511 assembly errors from confounding the analysis, any inference about changes in genomic 512 locations of genes or gene clusters was additionally verified by manually inspecting alignments 513 and ensuring that paired end reads supported an alternative genomic location (see SNV and 514 indel discovery). Cases where paired end reads did not support the change in genomic location 515 or where mapping was ambiguous or low quality were discarded. 516 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

517 Phylogenetic analysis 518 To confirm all strains in this analysis belonged to the species Aspergillus fumigatus, the 519 genomic sequences of the beta tubulin and calmodulin genes were extracted from the 520 assembled genomes of all strains. Gene phylogenies were constructed using Aspergillus 521 fischerianus as an outgroup using RAxML v8.0.25 with the GTRGAMMA substitution model [66]. 522 The tree was midpoint rooted and all branches with bootstrap support less than 80% were 523 collapsed (Figure S9). 524 525 To construct a SNP-based strain phylogeny, biallelic SNPs with no missing data were 526 pruned using SNPRelate v1.8.0 with a linkage disequilibrium threshold of 0.8 [67]. A total of 527 15,274 SNVs were used to create a phylogeny using RAxML v8.0.25 with the ASC_BINGAMMA 528 substitution model [66]. The tree was midpoint rooted and all branches with bootstrap support 529 less than 80% were collapsed. 530 531 To understand the evolutionary histories of specific SM gene clusters showing unusual 532 taxonomic distributions, we reconstructed the phylogenetic trees of their SM genes. 533 Specifically, SM cluster protein sequences were queried against a local copy of the NCBI non- 534 redundant protein database (downloaded May 30, 2017) using phmmer, a member of the 535 HMMER3 software suite [68] using acceleration parameters --F1 1e-5 --F2 1e-7 --F3 1e-10. A 536 custom perl script sorted the phmmer results based on the normalized bitscore (nbs), where 537 nbs was calculated as the bitscore of the single best-scoring domain in the hit sequence divided 538 by the best bitscore possible for the query sequence (i.e., the bitscore of the query aligned to 539 itself). No more than five hits were retained for each unique NCBI Taxonomy ID. Full-length 540 proteins corresponding to the top 100 hits (E-value < 1 × 10-10) to each query sequence were 541 extracted from the local database using esl-sfetch [68]. Sequences were aligned with MAFFT 542 v7.310 using the E-INS-i strategy and the BLOSUM30 amino acid scoring matrix [69] and 543 trimmed with trimAL v1.4.rev15 using its gappyout strategy [70]. The topologies were inferred 544 using maximum likelihood as implemented in RAxML v8.2.9 [66] using empirically determined 545 substitution models and rapid bootstrapping (1000 replications). The phylogenies were bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

546 midpoint rooted and branches with less than 80% bootstrap support were collapsed using the 547 ape and phangorn R packages [71,72]. Phylogenies were visualized using ITOL version 3.0 [73]. 548

549 Differential metabolite analysis 550 For natural product analysis, 5 x 106 spores/mL for the indicated strains were grown in 551 50 mL liquid GMM [74] for five days at 25 °C and 250 rpm in duplicates. Supernatants were 552 extracted with equal volumes of ethyl acetate, dried down and resuspened in 20% acetonitrile 553 (ACN). Each sample was analyzed by ultra-high performance liquid chromatography (UHPLC) 554 coupled with mass spectrometry (MS). The samples were separated on a ZORBAX Eclipse XDB- 555 C18 column (Agilent, 2.1 x 150 mm with a 1.8 µM particle size using a binary gradient of 0.5 % 556 (v/v) formic acid (FA) as solvent A and 0.5 % (v/v) FA in ACN as solvent B that was delivered by a 557 VanquishTM UHPLC system (Thermo Scientific) with a flow rate of 0.2 mL/min. The binary 558 gradient started with 20% B that was increased with a linear gradient to 100% B in 15 min 559 followed by an isocratic step at 100% B for 5 min. Before every run, the system was equilibrated 560 for 5 min at 20% B. The UHPLC system was coupled to a Q Exactive hybrid quadrupole 561 OritrapTM MS (Thermo Scientific). For electrospray ionization, the ion voltage was set at +/-3.5 562 kV in positive and negative mode, respectively. Nitrogen was used as sheath gas at a flow rate 563 of 45 and as sweep gas at a flow rate of 2. Data analysis was performed using XCMS [42] and 564 Maven [75] software. 565 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

566

567 Acknowledgements 568 A.L.L. was supported by the U.S. National Library of Medicine training grant 2T15LM007450. 569 This work was supported in part by the National Science Foundation (DEB-1442113 to A.R.) and 570 by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 571 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) 572 (NORTE-01-0145-FEDER-000013 to F.R.). G.H.G. thanks the Fundação de Amparo à Pesquisa do 573 Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e 574 Tecnológico (CNPq), both from Brazil, for funding and support. This work was conducted in part 575 using the resources of the Advanced Computing Center for Research and Education at 576 Vanderbilt University (http://www.accre.vanderbilt.edu/).

577

578 Author contributions 579 Conceptualization, A.R., G.H.G., A.L.L.; Methodology, A.L.L., J.H.W, C.L, P.W., J.M.P.; 580 Investigation, A.L.L.; Visualization, A.L.L., J.H.W, P.W.; Resources, G.H.G., F.R., N.P.K., C.L.; 581 Writing, A.L.L, A.R.

582 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

583 Table 1. Types and rates of SM gene cluster variants in A. fumigatus strains.

584 Description Phenotype Drivers Frequency at Frequency at Previous cluster level strain level reports Single- Potential for DNA replication 100% (33/33 Every strain Bikaverin in nucleotide protein function errors; relaxation clusters; affected Botrytis [17,27], polymorphisms change (missense); of purifying missense); aflatoxin in and indels abrogation of selection 70% (23/33 Aspergillus protein function clusters; oryzae and (nonsense and nonsense and Aspergillus frameshift) frameshift) flavus [26], fumonisins in Fusarium [10], 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 sterigmatocysti in cluster elements n in Aspergillus expression or [11–15] metabolite transport 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; [24,25] transposable elements fCluster Changes in Transposable 1 gene 8 unique Putative SM idiomorphs metabolites elements; cluster identified gene clusters in produced or recombination; alleles dermatophytes; structure of other putative SM metabolites mechanisms? gene cluster in Aspergillus flavus and Aspergillus oryzae [45,46] Mobile gene Potential for Transposable 2 gene 8 / 66 None clusters change in gene elements; clusters strains regulation horizontal gene transfer; other mechanisms?

585 586 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

587 588 Table S1. Summary of strains, sequence data, and assemblies used. 589 590 Table S2. All nonsynonymous variants in SM gene cluster genes. 591 592 Table S3. Description, locus tags, and gene annotations of all reference Af293 SM gene 593 clusters. 594 595 Table S4. XCMS analysis of extracted supernatants from 08-19-02-30, 08-12-12-13, and 08-19- 596 02-10. 597 598 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27

F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 MO78722EXP F16216 F17729 F17582 F17729 W F14946G 12-7504652F12636 ISSF21 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

0.2

Sequenced in this study Knox et al 2016 [31]

Nierman et al 2005 [28] Paul et al 2017 [32] No publication; Fedorova et al 2008 [29] sequenced by JCVI No publication; Abdolrasouli et al 2015 [30] sequenced by RIKEN

Figure 1. SNP-based phylogeny of A. fumigatus strains. The phylogeny was constructed using 15,274 biallelic SNPs with no missing data. The tree is midpoint rooted and all branches with bootstrap support less than 80% are collapsed. This phylogeny does not include strains Af10, Af210, Z5, or RP-2014 as short read data were not available. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Reference & most strains

CYP450Copper trasporter Oxidoreductase Iron transporter pyoverdine/dityrosine synthase Oxidoredctuase Transcription factor Alcohol acyltransferase NRPS-like transferase NRPS Copia transposon

Afu3g13600 610 620 630 640 650 660 670 680 690 700 710 720 730 F15927, RP-2014, & MO79587EXP

Copia transposon

LMB35Aa

275 KB Copia transposon

Aspergillus lentulus 18 KB

Aspergillus fischerianus

Protein kinase

PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27

F15927 12-7505054 LMB35Aa JCM10253 B F16311 F18085 AF90 MO78722EXP F16216 F17729 F17582 F17729 W F14946G 12-7504652F12636 ISSF21 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

Lacks 11-Kb region of SM gene cluster 14

Figure 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. (A) 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. (B) Strains without 11-Kb region highlighted in red. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

zinc binding enoyl reductase PKS A Transcription factor Oxidoreductase aldose 1-epimerase familyCYP450 protein

Afu1g17710 720 723 725 730 740

AF90 LMB35Aa, F15927 MO78722EXP

12 strains F7763 MO54056EXP Strains with LMB35Aa, MO79587EXP, F15927 F14946G pseudogenizing SNVs 19 strains JCM10253 MO78722EXP, IF1SWF4 09-7500806

PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27

F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 MO78722EXP F16216 F17729 B F17729 W F17582 F14946G 12-7504652F12636 ISSF21 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

Fusarilein-like cluster deleted

Pseudogenizing variants only in PKS

Pseudogenizing variants only in aldose 1-epimerase C Pseudogenizing variants in multiple genes in cluster

Reference Af293 & F7763 Chrom 1 5’ nucleotidase Pectate lyasePKS-like Chrom 1 (no synteny)

Afu1g 160 140 130 120 110 01170 A1163 & all other strains

MonooxygenaseMonooxygenaseMFS transporter EOS Methyltransferase Alpha/betaMFS hydrolase transporterRibonuclease Chrom1 Transcription factor PKS 35 KB

Afu1g 160 140 130 120110 1100 AFUB_079710 720 730 740 750 760 770 790780 800 01170

Figure 3. Pseudogenization and gene loss in SM gene clusters. (A) Positions of frameshift variants and nonsense variants in the fusarielin-like SM gene cluster 4. (B) Strains lacking the fusarielin-like cluster or containing frameshift and nonsense variants in the cluster. (C) 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. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

NRPS-like A Allele A; 3 strains HR-PKS fragment NR-PKS Allele B; Reference Af293 strain

Allele C; 8 strains

Allele D; 12 strains

Allele E; A1163 & 17 strains

PKS-NRPS Allele F; 22 strains B 8 104

HR-PKS PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11 6 Afu 591/12 08-19-02-30 (Allele D) Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11 104 08-12-12-13 (Allele F) Afu 1042/09Afu 942/09 08-19-02-10 (Allele F) 10-01-02-27

F15927 12-7505054 LMB35Aa 4 JCM10253 F16311 mA U 4 F18085 10 AF90 MO78722EXP mA U C F16216 F17729 F17582 F17729 W F14946G 12-7504652F12636 2 ISSF21 IF1SWF4 4 10 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 0 MO89263LAB F14513G 0 5 10 15 20 100 MO76959EXP ]nim[ emit ]nim[ 80 100 MO69250EXP 6 104 F13535 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-19-02-30 (Allele D) 08-31-08-91 100 F14403 100 100 08-12-12-13 (Allele F) 87 100 MO68507EXP 08-12-12-13 100 4 08-19-02-10 (Allele F) 4 10 100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 4 08-19-02-10 100 97 12-7504462 2 10 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61 Allele A Allele D

Allele B Allele E 0 0 5 10 15 20 ]nim[ emit ]nim[ Allele C Allele F

Figure 4. Six alleles of an idiomorphic SM gene cluster. (A) Alleles of SM gene cluster 10 on chromosome 3. Red boxes denote transposable elements. Green arrows denote backbone genes (PKS or NRPS). (B) Extracted ion chromatograms for the two m/z identified in negative mode from XCMS analysis comparing extracts from strains with Alleles D and F. (C) Distribution of the six cluster alleles across a SNP-based phylogeny of strains. Bolded and italicized strains have unique alleles but appear derived from another allele (see also Figure S5). bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Af293 & F7763

FAD-dependentoxidase Chrom 1 Mariner Ant1 transposonLINE transposon ORF derivedMFS transporterreverse transcriptase Dehydrogenase/reductaseOxidoreductase PKS Reverse transcriptase

Afu1g00940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1070

08-31-08-91, Z5 & F13619 Chrom 4

JCM10253 Fumagillin/Pseurotin Chrom 8 Supercluster

FAD-dependentoxidase B Aspergillus fumigatus Af293 MFS transporter Dehydrogenase/reductaseOxidoreductase PKS

970 980 990 1000 1010

EOS Phaeosphaeria nodorum SN15 O-methyltransferaseCYP450 O-methyltransferase class Dothideomycete

Short chain EOS dehdyrogenase CYP450 Pseudogymnoascus pannorum EOS class Leotiomycete

Escovopsis weberi PKS Transporter CYP450 class Sordariomycete

Hypoxylon sp. CI4A class Sordariomycete

MFS drug transporter (unrelated to Afu1g00970)

PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27 Cluster on Chromosome 1

F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 C MO78722EXP Cluster on Chromosome 4 F16216 F17729 F17582 F17729 W F14946G 12-7504652F12636 ISSF21 Cluster on Chromosome 8 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

Figure 5. Multiple genomic locations of a horizontally transferred SM gene cluster. (A) SM gene cluster 1 (Afu1g00970-01010) and flanking region is found in different genomic locations.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. (C) Strains containing SM gene cluster 1 in SNP-based phylogeny. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

High frequency polymorphisms Single-nucleotide Gene gain Gene loss Gene cluster loss polymorphism

Secondary metabolic gene cluster

Allele A Location A

Allele B Location B

Idiomorphic cluster Mobile gene cluster

Allele A

Allele B

Cluster fusion

Low frequency polymorphisms

Figure 6. Types and frequencies of all SM gene cluster variants in the A. fumigatus population. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxivIF1SWF4 a license helvolic to displayacid deletion the preprint in perpetuity. It is made available under A aCC-BY-NC-ND 4.0 International license. Af293 genes

IF1SWF4 coverage

B LMB35Aa fumigaclavine deletion Af293 genes

LMB35Aa coverage

C gliM partial deletion Af293 genes F14532 coverage F15390 coverage

fmqE partial deletion D Af293 genes

MO79587EXP coverage

F15927 coverage

LMB35Aa coverage

Figure S1. 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. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A cluster 21 gene deletion part 1

Af293 genes AF65 Afu124 Afu166 Afu218 Afu257 Afu591 Afu942 Afu1042 F5211G F12041 F12636 F13952 F14513G F14532 B cluster 21 gene deletion part 2 Af293 genes

F13535

F15390

F16216

F17582

12-7504652

10-01-02-27

08-12-12-13

PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27

F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 MO78722EXP F16216 F17729 F17582 F17729 W F14946G C 12-7504652F12636 ISSF21 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

Cluster 21 partial gene deletion

Figure S1. Partial deletion of ABC transporter gene in SM gene cluster 21. (A,B) Coverage of 21 strains with partial deletion of SM gene cluster 21. (C) Position of strains with partial gene deletion in SNP based phylogeny. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review)Figure is S2.the author/funder, Gene phylogenies who has granted of the bioRxivfusarielin-like a license toSM display gene the cluster preprint 4. in Theseperpetuity. phylogenies It is made available are under consistent with horizontal atransferCC-BY-NC-ND between 4.0 International Eurotiomycete license .and Sordariomycete fungi or with extensive gene loss.

S2A

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2B

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2C

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2D

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2E

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S2F

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certifiedJCM10253 by peer review) & F13619 is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A PKS Chr 4 MFS transporter Gypsy transposon CYP450 Transporter ProteinO-methyltransferase kinase AldehydeAmidohydrolase reductaseCYP450MarinerGypsy transposonChr transposon 4

Afu4g01560 AFUB_102040

Metarhizium anisopliae B MO54056EXP CYP450AminotransferaseReverse transcriptase NRPS Aminotransferase

12-7504462

08-19-02-30

Z5

C SM cluster SM cluster start end NRPS MO54056EXP predicted genes on entire cluster scaffold

08-19-02-30 coverage

12-7504462 coverage

MO54056EXP coverage

No short read data available for Z5 PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27 Has novel SM cluster from (A) F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 MO78722EXP F16216 F17729 Has novel SM cluster from (B) F17582 F17729 W F14946G 12-7504652F12636 D ISSF21 IF1SWF4 F5211G F12219 AF41

F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

Figure S3. 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 marked). (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.(D) Strains with novel clusters on SNP phylogeny. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under Figure S4. Gene phylogeniesaCC-BY-NC-ND of SM 4.0gene International cluster license 24. The. phylogenies of several genes in this cluster are consistent with horizontal transfer between Aspergillus fumigatus and Metarhizium fungi.

S4A

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4B

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 99 100 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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4C

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4D

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 OQD95763.1-29845-Penicillium vulpinum-Eurotiomycetes 100 100 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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4E

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4F

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4G

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4H

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4I

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4J

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4L

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S4M

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review)Figure is the S6.author/funder, Gene whophylogenies has granted bioRxiv of the a license mobile to display SM thegene preprint cluster in perpetuity. 1. These It is made phylogenies available under are consistent with horizontalaCC-BY-NC-ND transfer 4.0 International between license Eurotiomycete,. Dothidiomycete, Leotiomycete, and Sordariomycete fungi.

S6A

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S6B

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S6C

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S6D

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

S6E

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 bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under A aCC-BY-NC-ND 4.0 International license.

09-7500806

AFUT-1 LTRAfu6g09300Afu6g09307 - C2H2Afu6g09317 -fingerAfu6g09330 chitinase domain- LysM domainAfu6g09390Afu6g09395AFUB_092660Afu6g09400Afu6g09410Afu6g09420Afu6g09430Afu6g09440 - folylpoolyglutamate synthase

Reference Af293 strain; Allele B

02450

NR- PKS NRPS-iike oxidoreductaseoxidoreductase PKS fragment MFS transporter Gypsy transposon Mariner transposon FAD monoxygenasetranscription factor C6 transcription factor

B

F17582 AFUB_044180Gypsy transposonAFUB_044190 AFUB_044200AFUB_044210AFUB_044230AFUB_044240AFUB_044280AFUB_044290AFUB_044510 partialAFUB_044400AFUB_044420AFUB_045620Gypsy transposon HR-PKS Mariner transposon

Allele F

Gypsy transposon Mariner transposon

Figure S5. 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 main text Figure 4). 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 main text Figure 4). None of these genes are likely SM gene cluster backbone genes. One additional transposable element is contained in this insertion. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Reference Af293 & F7763

EOS Integral membrane protein Terpene cyclaseCYP450 PolysaccharideC6 transcription biosynthesisMariner-1 factor protein transposon Chr5 A1163 & 12-7504462 Afu5g00100 110 120 130 135

Multiple Gypsy transposons transposons Chr 4 32-57 KB 75-98 KB Multiple Gypsy & MarinerChr 4

JCM10253

SM gene EOS Multiple transposonsGypsy & Mariner Mariner-3 transposon Chr 1 cluster 11 KB 42 KB

PXE78597OM

Afu 257/E11

Afu 343/P/11 Afu 218/E11

Afu 591/12 Afu 166/E11 AF65 (NCPF 7097) Afu 124/E11

Afu 1042/09Afu 942/09 10-01-02-27

F15927 12-7505054 LMB35Aa JCM10253 F16311 F18085 AF90 MO78722EXP F16216 F17729 F17582 F17729 W F14946G 12-7504652F12636 ISSF21 IF1SWF4 F5211G F12219 AF41

B F13619 100 AF72 F14532 F17764 F15390 100 MO89263LAB F14513G 100 MO76959EXP 80 F13535 100 MO69250EXP 100 Reference A1163 F13952 90 95 100 98 F16134 F12041 80 84

08-31-08-91 100 F14403 100 100 87 100 MO68507EXP 08-12-12-13 100

100 08-19-02-30 08-36-03-25 100 100 86 MO54056EXP 93 12-7505446 100 F7763 100 94 12-7505220 86 100 Reference Af293 100 82 100 08-19-02-10 100 97 12-7504462 100 100 87 97 08-19-02-46 09-7500806 100 87 87 MO91298SB 08-19-02-61

SM gene cluster on Chromosome 5

SM gene cluster on Chromosome 4

SM gene cluster on Chromosome 8

Figure S7. Genomic contexts of mobile SM gene cluster 5. (A) 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. (B) Strains containing this cluster marked on SNP based phylogeny. bioRxiv preprint doi: https://doi.org/10.1101/149856; this version posted July 11, 2017. The copyright holder for this preprint (which was not

certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under85 08-36-03-25 aCC-BY-NC-ND 4.0 International license. 08-31-08-91 08-12-12-13 F16216 F16134 Z5 Afu 257/E11 A Afu 942/09 Afu 124/E11 Afu 166/E11 10-01-02-27 Afu 343/P/11 Afu 591/12 Afu 1042/09 Afu 218/E11 F18085 MO78722EXP F14403 F16311 ISSFT021 AF90 MO89263LAB F7763 12-7505220 Reference A1163 Af210 08-19-02-46 MO68507EXP F17729 12-7505054 90 F17729 W F12041 IF1SWF4 MO91298SB MO76959EXP 08-19-02-61 08-19-02-10 12-7505446 F15390 09-7500806 F14513G 08-19-02-30 MO69250EXP MO54056EXP F17764 95 F14946G F13952 F13619 F12636 Af10 F13535 F12219 JCM10253 AF41 F17582 F5211G 12-7504462 12-7504652 AF72 F14532 AF65 (NCPF 7097) Af293 MO79587EXP 96 LMB35Aa RP2014 F15927 A_fisherianus

0.004

B 12-7505220 F16216 Afu 257/E11 F17582 08-19-02-46 08-36-03-25 08-19-02-61 Afu 343/P/11 10-01-02-27 88 F13619 08-19-02-10 Afu 218/E11 Afu 124/E11 12-7504652 Afu 942/09 12-7505446 08-12-12-13 Afu 591/12 Afu 1042/09 Afu 166/E11 F15927 Af210 AF65 (NCPF 7097) MO79587EXP F14513G F13952 RP2014 100 F5211G F12041 F12636 F14532 F15390 12-7505054 LMB35Aa Z5 F13535 08-31-08-91 96 ISSFT021 IF1SWF4 F17729 MO54056EXP F16134 F17764 F12219 F14946G 12-7504462 F17729 W F14403 08-19-02-30 AF90 AF72 JCM10253 Af293 F7763 MO68507EXP AF41 MO78722EXP 09-7500806 A1163_ref MO76959EXP F18085 MO91298SB MO89263LAB MO69250EXP Af10 F16311 A_fischerianus

0.007

Figure S1. Marker gene phylogenies of all strains and Aspergillus fischerianus. (A) Phylogeny of beta tubulin gene. (B) Phylogeny of calmodulin gene.