The Mitochondrial Genome of Ophiostoma Himal-Ulmi and Comparison with Other Dutch Elm Disease Causing Fungi

Canadian Journal of Microbiology

The mitochondrial genome of Ophiostoma himal-ulmi and comparison with other Dutch elm disease causing fungi.

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2020-0589.R1

Manuscript Type: Article

Date Submitted by the 04-Feb-2021 Author:

Complete List of Authors: Wai, Alvan; University of Manitoba, Hausner, Georg; University of Manitoba, Buller Building 213

Keyword: Introns, Ophiostomatales, homing endonucleases, Dutch Elm Disease

Is the invited manuscript for Draft consideration in a Special Not applicable (regular submission) Issue? :

1 The mitochondrial genome of Ophiostoma himal-ulmi and comparison with other Dutch

2 elm disease causing fungi.

4 Alvan Wai and Georg Hausner#

5 Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2

6 #Corresponding author: [email protected]

Draft

1 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 2 of 42

9 Abstract

10 The mitochondrial genome of Ophiostoma himal-ulmi, a species endemic to the Western

11 Himalayas and a member of the Dutch elm disease-causing fungi, has been sequenced and

12 characterized. The mitochondrial genome was compared with other available genomes for

13 members of the Ophiostomatales, including other agents of Dutch elm disease (Ophiostoma ulmi,

14 Ophiostoma novo-ulmi subspecies novo-ulmi and Ophiostoma novo-ulmi subspecies americana)

15 and it was observed that gene synteny is highly conserved and variability among members of the

16 Dutch-elm disease-causing fungi is primarily due to the number of intron insertions. Among the

17 Dutch elm disease-causing fungi examined, O. himal-ulmi has the largest mitochondrial genomes

18 ranging from 94 934 bp to 111 712 bp due to the expansion of the number of introns.

19 Draft

20 Keywords: introns, Ophiostomatales, homing endonucleases, Dutch elm disease

2 © The Author(s) or their Institution(s) Page 3 of 42 Canadian Journal of Microbiology

22 Introduction

23 Brasier and Mehrotra (1995) conducted a mycological survey in northern Himachal

24 Pradesh (Western Himalayas) with the focus on Ophiostoma species isolated from breeding

25 galleries of scolytid beetles present within the bark of Ulmus wallichiana. They isolated an

26 Ophiostoma species that resembled Ophiostoma ulmi but had a set of physiological and

27 morphological features that set it apart from O. ulmi and Ophiostoma novo-ulmi. In addition,

28 interfertility tests demonstrated reproductive isolation, therefore this new species was designated

29 as Ophiostoma himal-ulmi. Brasier and Mehrotra hoped that the discovery of this potential

30 “sibling species” may contribute towards a better understanding of the origins of Dutch elm

31 disease. Historically Ophiostoma ulmi (Buisman) Melin & Nannf. (1934) was viewed to be the

32 causative agent of Dutch elm disease (DED).Draft However, more recently it has been recognized that

33 O. ulmi has been replaced by more aggressive forms assigned to Ophiostoma novo-ulmi (Brasier

34 1991). Members of the O. ulmi species complex are vectored by bark beetles and are agents of a

35 devastating wilt disease that had and continues to have significant impact on urban forests.

36 Ophiostoma novo-ulmi based on cultural and molecular characters has been separated into two

37 subspecies: O. novo-ulmi subsp. novo-ulmi and O. novo-ulmi subsp. americana (Brasier and Kirk

38 2001; Harrington et al. 2001). Both subspecies are well established in Europe but so far O. novo-

39 ulmi subsp. novo-ulmi has only been reported from Europe and Eurasia (Brasier 2001; Brasier

40 and Buck 2001).

41 Most fungi are obligate aerobes so functional mitochondria are essential for their survival

42 and fitness. With regards to structure, mitochondrial DNA can be circular, linear, and in a few

43 cases segmented (i.e., multi-chromosomal) (Valach et al. 2011; Lang 2018). For most fungi,

44 mitochondrial genomes exist as multiple copies and are represented as circular molecules, and

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45 are compacted into mitochondrial nucleoids possibly composed of multimeric (concatemers)

46 molecules and DNA-binding complexes (Miyakawa 2017). The mitochondrial DNA

47 concatemers might be due to DNA replication mechanisms that involve rolling circles (Bendich

48 1996; Hausner et al. 2006, Chen and Clark-Walker 2018). Fungal mitochondrial genomes can

49 vary greatly in size from 12.055 kbp to > 500 kbp (James et al. 2013; Liu et al. 2020). The size

50 variation can be explained, in part, by the presence of introns, intergenic spacers, duplications,

51 proliferation of repeats, and insertions of plasmid components (Hausner 2003; Himmelstrand et

52 al. 2014; Freel et al. 2015; Deng et al. 2018; 2020; Lang 2018; Sandor et al. 2018; Medina et al.

53 2020).

54 Although fungal mitochondrial genomes show great variation in size, filamentous

55 Ascomycetes fungi encode a similar geneDraft complement: genes involved in translation [small and

56 large ribosomal subunit RNAs (rns and rnl)] plus a set of tRNAs; genes coding for components

57 of the respiratory chain such as subunits for Complexes III and IV (cob, cox1, cox2, and cox3),

58 subunits of NADH dehydrogenase (nad1 to nad6 and nad4L), and subunits for the ATP synthase

59 (atp6, atp8, and usually atp9) (Lang 2018; Zardoya 2020). In addition, most members of the

60 Ascomycota encode the ribosomal protein RPS3 (rps3; Hausner 2003; Freel et al. 2015), where it

61 can be encoded within an intron or positioned as a free-standing gene (Wai et al. 2019).

62 Fungal mtDNA introns are unique as they are potential mobile elements that encode so

63 called intron-encoded proteins (IEPs) that catalyze their mobility to cognate intron-less alleles

64 and the IEPs, in some instances, enhance (maturase activity) the intron RNAs ability to self-

65 splice from the transcripts of host genes they have invaded (Belfort 2003; Lang et al. 2007;

66 Hausner 2012). Based on the splicing mechanism and secondary structures mitochondrial introns

67 can be assigned to either group I or group II introns (Belfort et al. 2002). Homing endonucleases

4 © The Author(s) or their Institution(s) Page 5 of 42 Canadian Journal of Microbiology

68 (HEs) or maturases, which are DNA-cutting enzymes or proteins that facilitate splicing and

69 reverse transcriptases are IEPs associated usually with group I and group II introns, respectively.

70 Although, there are examples of group II introns that encode HEs that have the potential to

71 catalyze the mobility of their host group II introns (Toor and Zimmerly 2002; Mullineux et al.

72 2010). In fungal mtDNAs, two families of HEs are noted to be encoded by homing endonuclease

73 genes (HEGs), named by the presence of conserved amino acid motifs: the LAGLIDADG and

74 the GIY-YIG families of HEs (Stoddard 2014). Although it has been noted that HEGs can move

75 independently of their ribozyme partners (Mota and Collins 1988), more frequently they appear

76 to have co-evolved with their intron counterparts that encode them (Megarioti and Kouvelis

77 2020). The gain and loss of introns and intron mobility appears to promote mitochondrial DNA

78 size polymorphisms and rearrangementsDraft by promoting intra- and inter mitochondrial genome

79 recombination events (Kanzi et al. 2016; Franco et al. 2017; Wu and Hao 2019; Deng et al.

80 2020).

81 Previously, we characterized the mitogenome for Ophiostoma novo-ulmi subsp. novo-

82 ulmi (Abboud et al. 2018). Here, we report the complete mitogenome of Ophiostoma himal-ulmi

83 and compare it with those of other members of the DED-causing fungal species complex (Brasier

84 and Buck 2001; Hessenauer et al. 2020). The data may provide some insight in understanding the

85 origin of O. novo-ulmi and provide a resource for developing markers that allow for

86 distinguishing the various species (or subspecies) that can cause Dutch elm disease.

88 Material and Methods

89 Source of culture and culturing methods

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90 Ophiostoma himal-ulmi Brasier & M.D. Mehrotra (CBS 374.67; Mycobank: 363234,

91 isolated from an Ulmus wallichiana twig, India, Kashmir, Baba Reshi) was obtained from The

92 Westerdijk Fungal Biodiversity Institute (Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands).

93 An approximately 5 mm3 block of stock culture maintained on malt extract agar (MEA;

94 supplemented with yeast extract) slant (30 g/L malt extract, 1 g/L yeast extract, 2 g/L agar) was

95 transferred to a malt extract agar plate (100 mm diameter Petri plate containing approximately 40

96 mL MEA) and incubated in the dark at 20 °C for up to four weeks. Five 250 mL Erlenmeyer

97 flask containing 100 mL PYG broth (1 g/L peptone, 1 g/L yeast extract, 3 g/L glucose) were

98 each inoculated with ten agar blocks (cut from the edge of the mycelium; approximate

99 dimensions = 2 mm x 2 mm x 1 mm) and incubated in the dark at 20 °C for up to two weeks. Draft 100

101 Mitochondria and mitochondrial DNA extraction

102 Fungal mass was collected by vacuum filtration. Briefly, the liquid culture was filtered

103 through a Whatman® Grade 1 qualitative filter paper placed in a Büchner funnel under vacuum

104 until most of the liquid broth was removed. The fungal mass was transferred to a prechilled (-20

105 °C) mortar and mixed, per 1 g fungal mass, with 1.5 g acid-washed, autoclaved sand and 2 mL

106 isolation buffer [10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 440 mM sucrose]. The

107 mixture was ground until it formed a “slurry” after which it was transferred into a 15 mL

108 Corning® polypropylene conical centrifuge tube and centrifuged in a Sorvall™ Legend™ XTR

109 centrifuge (Thermo ScientificTM) at 3 000 g for 15 min. The supernatant was transferred to a new

110 30 mL Corning® glass centrifuge tube and centrifuged in a Sorvall™ RC 5B Plus centrifuge

111 (Mandel) using a SS-34 fixed-angle rotor at 20 000 g for 30 min. The goal of the differential

112 centrifugation step was to remove cellular debris and nuclei at the lower speed centrifugation and

6 © The Author(s) or their Institution(s) Page 7 of 42 Canadian Journal of Microbiology

113 obtained an enriched fraction of mitochondrial DNA at the higher speed centrifugation step. The

114 supernatant was decanted and the pellet was resuspended with 3 mL preheated (65 °C) 2X

115 CTAB buffer [2% (w/v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M

116 NaCl]. The suspension was transferred to a new 15 mL Corning® polypropylene conical

117 centrifuge tube and incubated in a 65 °C water bath for 60 min. with mixing (by gently inverting

118 the tube) every 20 min. The tube was cooled to room temperature after which equal volume of (3

119 mL) chloroform was added and mixed by gentle inversion until an emulsion was formed. The

120 tube was centrifuged at 3 000 g for 15 min. (until an aqueous layer formed). The aqueous layer

121 was transferred to a new 15 mL Corning® polypropylene conical centrifuge tube, mixed with

122 RNase, and incubated in a 37 °C water bath for 30 min. A second chloroform extraction was

123 performed, and the tube was centrifugedDraft at 3 000 g for 30 min. (until a clear aqueous layer was

124 formed). The aqueous layer was transferred into a new 15 mL Corning® polypropylene conical

125 centrifuge tube, mixed with 2.5 volumes of precooled (-20 °C) 95% ethanol, and placed in a

126 freezer (-20 °C) for 3 h. The precipitate was pelleted by centrifuging at 3 000 g for 15 min. The

127 supernatant was decanted, and the pellet was washed with 1 mL precooled (-20 °C) 70% ethanol

128 and centrifuged at 3 000 g for 5 min. The supernatant was decanted, and the pellet was dried and

129 resuspended in 200 mL 10 mM Tris-HCl (pH 8.0).

130

131 DNA preparation and next generation sequencing

132 The enriched mtDNA preparation of O. himal-ulmi was sent to MicrobesNG (Units 1-2

133 First Floor, The BioHub, Birmingham Research Park, 97 Vincent Drive, Birmingham, B15 2SQ,

134 UK) for Illumina sequencing. Briefly, with regards to DNA preparation for MicrobesNG,

135 quantification and quality assessment of the crude mtDNA extract were performed by NanoDrop

7 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 8 of 42

136 2000c UV-Vis spectrophotometer and agarose gel electrophoresis analyses. The extract was

137 aliquoted into a 1.5 mL polypropylene conical microcentrifuge tube (labeled based on

138 instructions provided by MicrobesNG) and diluted to 30 ng/µL DNA in a final volume of 100

139 µL.

140

141 Reassembly and analyses of next generation sequencing data

142 MicrobesNG returned the sequencing reads, the adapters of which were trimmed using

143 Trimmomatic version 0.30 (Bolger et al. 2014) using sliding window with a quality cutoff of 15.

144 A draft assembly was also provided, assembled using SPAdes version 3.7 (Bankevich et al.

145 2012); and annotated contigs, annotations generated by Prokka version 1.14.3 (Seemann 2014).

146 In order to enhance the assemblyDraft and recover as many mitochondrial genome-derived

147 reads, we reassembled the data. The reads from MicrobesNG were assessed using FastQC

148 v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The assembly from

149 MicrobesNG was then evaluated by reassembling the reads using two approaches, SPAdes and

150 A5-miseq (Tritt et al. 2012; Coil et al. 2015). The SPAdes assembly was generated using SPAdes

151 version 3.14.0, setting the “--careful” option, which was used to obtain an assembly minimizing

152 indels and mismatches. With regards to the A5-miseq pipeline, the “--end” option was set to “5”,

153 which specifies for a final scaffolding step. All other parameters were set at default. BLASTn

154 (Altschul et al. 1990; Johnson et al. 2008; Camacho et al. 2009) was used to search for

155 contigs/scaffolds corresponding to the mtDNA in all assemblies. The contigs/scaffolds were

156 ordered based on O. novo-ulmi subspecies novo-ulmi mtDNA sequence (GenBank accession

157 number: MG020143.1; Abboud et al. 2018) using the Mauve Contig Mover (Darling et al. 2004;

158 Rissman et al. 2009).

8 © The Author(s) or their Institution(s) Page 9 of 42 Canadian Journal of Microbiology

159 Gaps in the O. himal-ulmi mtDNA assembly, which were identified based on

160 comparative sequence analyses with related DED-causing fungi sequences assembled in this

161 study (see further below), that could not be resolved using in silico methods, were filled by

162 Sanger sequencing using custom-designed primers. Purified PCR products were sent to the DNA

163 Sequencing Services of the Research Institute in Oncology & Hematology (RIOH; Room

164 ON6042, 675 McDermot Avenue, Winnipeg, Manitoba, Canada) for Sanger sequencing.

165 Resulting reads from Sanger sequencing were mapped to the assembled contigs/scaffolds and the

166 gaps were closed based on at least a 10 bp unambiguous overlap of paired forward and reverse

167 reads. The resulting mtDNA assembly was deposited in GenBank (GenBank accession number:

168 MW250274).

169 Draft

170 Recovery and assembly of mitochondrial genomes for other DED-causing fungi

171 Additional DED fungal mtDNAs were assembled using a similar strategy as for O. himal-

172 ulmi with reads data deposited in the Sequence Read Archive (SRA; Shumway et al. 2010;

173 Leinonen et al. 2011). The dataset was part of a larger dataset by Hessenauer et al. (2020). Only

174 a sample of strains where complete/near complete mtDNA assemblies could be obtained were

175 used for this study. These included three O. himal-ulmi (HP30, HP31, and HP32; SRA accession

176 numbers: SRR10139865, SRR10139864, and SRR10139863, respectively) three O. novo-ulmi

177 subspecies americana (DDS25, DDS100, and US137, SRA accession numbers: SRR10139932,

178 SRR10139938, and SRR10139915, respectively), one O. novo-ulmi subspecies novo-ulmi (R108,

179 SRA accession number: SRR10139923), one O. ulmi sensu lato (DDS296; SRA accession

180 number: SRR10139930), and three O. ulmi (H868, H873, and PG40; SRA accession numbers:

181 SRR10139873, SRR10139869, and SRR10139855, respectively). In addition to the O. novo-ulmi

9 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 10 of 42

182 subspecies novo-ulmi (R108) mtDNA sequence, an additional sequence for O. novo-ulmi

183 subspecies novo-ulmi (GenBank accession number: MG020143.1; Abboud et al. 2018) was

184 obtained from GenBank (Sayers et al. 2020). The complete annotation of the assembled

185 mitogenomes for the DED-causing pathogens are provided in the supplementary data file

186 (Supplementary data file 1).

187

188 Annotation of the mitochondrial genomes

189 A preliminary annotation of the O. himal-ulmi mtDNA was performed using the

190 MFannot tool (https://megasun.bch.umontreal.ca/cgi-bin/dev_mfa/mfannotInterface.pl; setting

191 “Genetic Code” to “4 Mold, Protozoan, and Coelenterate Mitochondrial;

192 Mycoplasma/Spiroplasma”) and RNAweaselDraft (https://megasun.bch.umontreal.ca/cgi-

193 bin/RNAweasel/RNAweaselInterface.pl; set to predict all RNA molecules that can be predicted

194 by RNAweasel and setting “Genetic Code” to “4 Mold, Protozoan, and Coelenterate

195 Mitochondrial; Mycoplasma/Spiroplasma”). Predictions of tRNAs were verified/supplemented

196 with tRNAscan-SE 2.0 (Chan and Lowe 2019). Annotations of the conserved protein-coding

197 genes (atp6, atp8, atp9, cob, cox1-3, nad1-6, nad4L) and, to a lesser extent, nonstructural genes

198 (i.e. rnl and rns, and the tRNAs), were performed manually and verified by comparative

199 sequence analyses using sequences assembled in this study, and related sequences from members

200 of the Ophiostomatales obtained from GenBank. Briefly, this involved aligning each set of gene

201 sequences using MAFFT version 7 (Katoh and Standley 2013), setting the iterative refinement

202 method to “E-INS-i”, to account for long gaps created by introns. The resulting alignments were

203 manually curated using the alignment viewer and editor, AliView version 1.25 (Larsson 2014).

204 Introns that were not predicted by the MFannot tool/RNAweasel were, where possible, manually

10 © The Author(s) or their Institution(s) Page 11 of 42 Canadian Journal of Microbiology

205 identified based on conserved core sequences and secondary structure elements (Michel and

206 Westhof 1990; reviewed in Michel et al. 1989). Unidentifiable introns were annotated as

207 “Unidentifiable” (Table 1). Final annotation of the mtDNA was performed in Artemis

208 (Rutherford et al. 2000). The mtDNA was visualized and represented using Circos (Krzywinski

209 et al. 2009). Represented features include the conserved protein-coding genes, nonstructural

210 genes, and introns. The GC plot was generated using a window size of 100 bp and a step size of

211 20 bp. The intron landscape for members of causative agents of DED was visualized with Circos.

212

213 Phylogenic analysis of mitochondrial protein coding regions

214 In order to assess the relationship among the DED-causing fungi, a phylogenetic tree was

215 constructed based on Bayesian analysis.Draft A total of 61 concatenated amino acid sequences,

216 derived from thirteen conserved protein-coding genes (atp6, atp8, cob, cox1-3, nad1-6, nad4L),

217 were analyzed, including O. himal-ulmi, the eight DED-causing fungi assembled in this study, O.

218 novo-ulmi subspecies novo-ulmi mtDNA sequence (GenBank accession number: MG020143.1;

219 Abboud et al. 2018), and 48 other sequences used in Abboud et al. (2018), Zubaer et al. (2018),

220 and recently analyzed by our lab (O. minus, GenBank accession number: MW122509; O.

221 piliferum, GenBank accession number: MW122508; Zubaer et al. 2021). The amino acid

222 sequences were aligned using MAFFT with all settings at default and the resulting alignment was

223 manually adjusted using AliView. MrBayes 3.2.7a (Huelsenbeck and Ronquist 2001; Ronquist et

224 al. 2012) was used for building the phylogenetic tree. A fixed-rate amino acid substitution model

225 was estimated using the model jumping (or mixed model) function implemented in MrBayes.

226 Rate variation among sites was modeled with a combination of invariable sites model and

227 gamma model (i.e., rates from a gamma distribution). The priors for the proportion of invariant

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228 sites and shape parameters (alpha) were left at their default values (i.e., a uniform distribution

229 between 0.0 and 1.0 and an exponential distribution with a mean value of 1.0, respectively). The

230 analysis was run with 1 000 000 generations and a sampling frequency of 1 000. The remaining

231 parameters were left at default. Based on model jumping, the cpREV model was estimated with

232 the highest probability. The initial 25% of the samples were discarded as burn-in and the

233 remaining samples were used to construct the 50% majority rule consensus tree. The tree was

234 visualized and re-rooted on the Eurotiales branch (split between Eurotiales and Sordariomycetes)

235 using FigTree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

236

237 Results

238 Organisation and features of the mitochondrialDraft genome for O. himal-ulmi and other DED-

239 causing agents

240 The mitochondrial genome of O. himal-ulmi (CBS 374.67) can be represented as a

241 circular molecule of 111 712 bp (GenBank accession number: MW250274; Fig. 1). All genes are

242 encoded on one strand of the mitochondrial DNA; the protein-coding genes are in the following

243 order: cox1, nad1, nad4, atp8, atp6, cox3, nad6, nad2, nad3, cox2, nad4L, nad5, and cob. The

244 nad2 and nad3 genes are fused (in frame) and the nad4L gene overlaps with nad5 by one

245 nucleotide. The atp9 gene could not be identified. The rRNA genes are located as follows: rns

246 between atp6 and cox3 and rnl between nad6 and nad2. Twenty-six tRNA genes, covering all 20

247 amino acids, as illustrated in Fig. 1 are dispersed across the mitochondrial genome but the bulk

248 (16 out of 26) of the tRNAs are located upstream (V, I, S, and P) and downstream (T, E, M, M,

249 L, G, A, F, L, Q, H, and M) of the rnl gene.

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250 Additional sequences for DED-causing pathogens, including three O. himal-ulmi

251 sequences were recovered from Sequence Read Archive [part of a larger dataset by Hessenauer

252 et al. (2020)], and the mitochondrial genome components were assembled and annotated. For the

253 additional O. himal-ulmi strains, the sequences were essentially identical to the strain sequenced

254 in this study, except their sizes ranged from 94 934 to 98 082 bp. The size polymorphism is

255 largely due to the variable number of introns among the genomes sampled. Similarly, sequences

256 for three recovered O. ulmi mitochondrial genomes ranged in size from 77 596 to 77 608 bp, and

257 for the three O. novo-ulmi subsp. americana sequences, sizes ranged from 56 451 to 60 420 bp.

258 Finally, for the two strains of O. novo-ulmi subsp. novo-ulmi, the sizes are the same at 64 848 bp.

259 Mauve progressive alignment of the mitochondrial genomes for the various members of

260 the DED-causing species showed that theDraft mitochondrial genomes are co-linear and therefore

261 showed no evidence of rearrangements (Fig. 2). The gene synteny maps for the DED-causing

262 fungi within the context of other members of the Order Ophiostomatales showed that gene order

263 across the Ophiostomatales is highly conserved (Fig. 3), with some minor variations noted with

264 regards to the tRNA loci. As shown in Fig. 3, the mitochondrial gene synteny for all examined

265 DED-causing fungi are identical and all are missing the atp9 gene; the variations in

266 mitochondrial genome sizes are due to intron polymorphism and intergenic spacers.

267

268 DED-causing pathogens and their mitogenome intron complement

269 The mitochondrial genome of O. himal-ulmi (CBS 374.67) contains 51 introns, 41 are

270 group I introns, 5 are group II introns and 5 introns could not be categorized. For O. himal-ulmi

271 (CBS 374.67), introns and intron-encoded ORFs comprise 73% of the mitochondrial genome

272 (Fig. 4). Table 1 summarizes the location of all introns, their classification and intron-encoded

13 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 14 of 42

273 ORFs. Group I introns encoded either GIY-YIG or LAGLIDADG type ORFs with two group I

274 introns showing no evidence for an ORF (nad1-145 and cox2-234). Among the group II introns,

275 two encoded reverse transcriptases, two encoded LAGLIDADG type ORFs and one group II

276 intron (cox1-216) lacked an ORF (Table 1). As noted in other members of the Ascomycota, rps3

277 is intron-encoded (mL2450) and as previously reported, rps3 is part of a gene fusion where the

278 rps3 coding segment is fused in frame with a double-motif LAGLIDADG coding region

279 (Sethuraman et al. 2009). The cox1 gene has the most intron insertions (17) followed by the rnl

280 (6) and cox2 (6) genes (Table 1).

281 Among the DED-causing pathogens, O. himal-ulmi is intron-rich with numbers ranging 282 from 42 to 51 intron insertions among theDraft four examined mitochondrial genomes. Conversely, O. 283 novo-ulmi subsp. americana mitochondrial genomes contained the lowest numbers of introns,

284 with either 24 or 25 in the genomes sampled. Twenty-seven introns were noted in the two O.

285 novo-ulmi subsp. novo-ulmi mitochondrial genomes. The mitochondrial genomes for the three O.

286 ulmi sequences contained either 35 or 36 introns. The mitochondrial intron landscape for the

287 DED-causing fungi is represented in Fig. 5, showing that among these fungi, the cox1 gene is

288 intron-rich (18 intron insertion sites) followed by the rnl (8 insertion sites), cox2 (7 insertions

289 sites) and cob (6 insertion sites) genes.

290 For the mitochondrial genome of O. himal-ulmi (CBS 374.67), evidence of potential free-

291 standing homing endonuclease genes (HEGs) were noted in the following locations for double-

292 motif LAGLIDADG HEGs: between trnY(gua) and trnN(guu), between trnD(guc) and trnS(gcu),

293 and between trnE(uuc) and trnM(cau). In addition, a GIY-YIG type HEG was located between

294 trnL(uaa) and trnG(ucc). Partial 5′ and 3′ duplications were noted for atp6 where these

295 duplicated segments were embedded within the atp6-572 intron (IC2 type intron). Partial 3′ gene

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296 duplications were noted for cox1, nad3, nad5 and nad6 and were associated with intron-like

297 insertions (at various state of degeneration). Similar 3′ gene duplication events were previously

298 observed in O. novo-ulmi subsp. novo-ulmi and other fungi and these are probably due to mobile

299 introns moving flanking regions during transposition into new sites (Abboud et al. 2018).

300

301 Phylogeny of the DED-causing agents vs mitogenome size and intron numbers

302 The phylogenetic analysis of 61 concatenated mitochondrial protein sequences including

303 38 species that belong to the Ophiostomatales yielded a topology showing monophyletic

304 groupings for the Microascales, Hypocreales, Glomerellales, Sordariales, and Ophiostomatales 305 (Fig. 6). Within the Ophiostomatales, severalDraft lineages could be identified representing the 306 following genera: Ceratocystiopsis, Graphilbum, Hawksworthiomyces, Raffaelea sensu stricto,

307 Leptographium, Esteya, Ophiostoma sensu stricto, and Sporothrix (de Beer 2013; 2016) (Fig. 6).

308 The DED-causing fungi form a monophyletic grouping within the Ophiostoma sensu stricto

309 clade. Within the DED-causing fungi clade the O. himal-ulmi sequences share a node with high

310 support however, the monophyly of remaining members of this group (O. ulmi and O. novo-

311 ulmi) of fungi cannot be resolved. Mitochondrial sequences show that some species assigned to

312 Sporothrix (S. insectorum) and Raffaelea (R. quercivora, R. quercus-mongolicae, and R.

313 sulphurea) maybe need to be reevaluated as they failed to group within the Sporothrix sensu

314 stricto and Raffaelea sensu stricto clades.

315 Within the Ophiostoma sensu stricto clade mitochondrial genome sizes and intron

316 content are quite variable and not necessarily correspond do the phylogenetic position of the

317 species examined (Fig. 6; Table 2). For example, Ophiostoma piceae has the smallest genome

318 within the grouping at 33 599 bp and 6 introns and O. himal-ulmi has the largest mitochondrial

15 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 16 of 42

319 genome at 111 712 bp and 51 introns. Genome sizes do appear to correspond to the number of

320 introns they contain, with smaller mitochondrial genomes containing lower number of introns

321 and the larger genomes being intron-rich. In general, mitochondrial genome size variation and

322 polymorphisms among the DED-causing fungi appears to be mostly due to intron content.

323 The combined sizes of intergenic regions have been tabulated for the DED fungi

324 (Supplementary Table 1S) and these range from 6300 bp to 11 119 bp as found within the

325 smallest mitochondrial genome (59 003 bp) and the largest mitochondrial genomes (111 712 bp,

326 respectively. In contrast, the combined intronic contribution towards the sizes for these

327 mitochondrial genomes range from 33 618 bp to 81 510 bp (Table 1S).

328

329 Discussion Draft

330 The O. himal-ulmi mitochondrial genome for strain CBS 374.67, at 111 712 bp, is the

331 largest reported so far among the DED-causing fungi and the largest among the characterized

332 sequences for species belonging to the genus Ophiostoma sensu stricto. The expansion of the O.

333 himal-ulmi mitochondrial DNA sequence and size variation observed among the various DED-

334 causing fungi studied is primarily due to intron (including intronic ORFs) content. Similar

335 observations have been made in other fungal groups where mitochondrial DNA variations among

336 members of the same genus were in part due to the presence or absence of introns (Jalalzadeh et

337 al. 2015; Kanzi et al. 2016; Franco et al. 2017; Zubaer et al. 2018).

338 Although mitochondrial gene synteny is highly conserved among the Ophiostomatales, in

339 all members of Ophiostoma sensu lato (including the DED-causing fungi), except of Ophiostoma

340 ips, the atp9 gene is absent. The absence of the atp9 gene has been observed in other fungal taxa

341 and nuclear mitochondrial-derived versions of atp9 could be identified (Franco et al. 2017;

16 © The Author(s) or their Institution(s) Page 17 of 42 Canadian Journal of Microbiology

342 Zubaer et al. 2018) suggesting that in some fungi a copy of the mitochondrial atp9 gene was

343 transferred to the nuclear genome. We examined the nuclear scaffolds available from our study

344 for O. himal-ulmi and those for other DED-causing fungi from the study by Hessenauer et al.

345 (2020) and failed to detect sequences similar to the mitochondrial atp9. Recent work by Fonseca

346 et al. (2020) on members of the Hypocreales also failed to detect a nuclear-encoded version of

347 atp9, but they did detect other mitochondrial derived genes within the nuclear genomes for 18

348 different species such as nad1, nad2, nad3, nad4L, nad4, nad5, cob, cox1, rnl, rns, rps3, atp6,

349 and cox2. The latter demonstrates that mitochondrial genes can be transferred to the nuclear

350 genome and it might suggest that in some instances the loss of atp9 can be compensated by the

351 presence of a nuclear analog.

352 The mitochondrial DNA-based phylogenyDraft confirmed the monophyletic origin of the

353 DED-causing fungi and their position within Ophiostoma sensu stricto. Within this genus,

354 mitochondrial genome sizes and intron content are quite variable and do not correspond to their

355 respective phylogenetic position. Within the basal branching members of Ophiostoma sensu

356 stricto, O. ips and O. piliferum have 42 and 29 introns, respectively, and O. piceae, a common

357 blue-stain fungus, stands out with its small genome and low (6) intron number, suggesting that

358 the O. piceae mitochondrial genome underwent streamlining whereby introns appear to have

359 been lost. Previously, we noted streamlining of mitochondrial genomes for members of

360 Sporothrix sensu stricto (Abboud et al. 2018). Conversely, among the DED-causing fungi

361 sampled, we noticed a trend whereby the O. novo-ulmi subspecies had the lowest intron numbers

362 (24-27), followed by O. ulmi (35-36), and with the O. himal-ulmi mitochondrial genomes

363 containing the highest number of introns (42-51). This demonstrates that introns contribute

17 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 18 of 42

364 towards mitochondrial genome size variation and the mitochondrial intron landscape (Fig. 5)

365 shows their contribution towards genetic diversity with regards to gene architecture.

366 Mobile introns can generate mitochondrial genome diversity as they have the potential to

367 move to new locations (ectopic integration) are invade cognate alleles that lack introns. There is

368 also evidence that mobile introns can move horizontally and invade new genomes (reviewed by

369 Hausner 2012). For example, transient hyphal fusion may allow cytoplasm to be exchanged

370 between fungal strains and this would make it possible for mitochondria to fuse facilitating

371 mtDNA recombination events, including the movement of introns and HEGs. Intron loss is still

372 viewed to be an event that can occur gradually by mutations (including precise deletions) or by

373 recombination events whereby a reverse-transcribed version of an allele replaces a genomic-

374 intron containing version. It is assumed Draftthat introns evolve in a neutral pattern, whereby the lack

375 of selection leads to the accumulation of mutations eventually leading first to the loss of mobility

376 and eventually decreasing splicing efficiency and thus leading to loss of the intron (Goddard and

377 Burt 1999). In order for mobile introns to persist, they have to outpace the loss of introns (due to

378 drift) by invading new alleles or new sites or reinvade sites where introns have been lost

379 (including by means of vertical and horizontal transmission) (Wu and Hao 2014; Guha et al.

380 2018). One way for mobile introns and their HEGs to escape this homing endonuclease life cycle

381 of invasion and degeneration (loss) is by acquiring beneficial functions that impact fitness

382 (Goddard and Burt 1999; Guha et al. 2018).

383 There might be indirect ways for mobile introns to maintain their numbers such as the

384 requirement of the intron complement for modulating or fine tuning the gene expression of the

385 mitochondrial genome for Saccharomyces cerevisiae (Rudan et al. 2018) or as suggested

386 viewing introns as sensors whose splicing efficiency can be influenced by environmental

18 © The Author(s) or their Institution(s) Page 19 of 42 Canadian Journal of Microbiology

387 conditions (Belfort 2017). In one instance, the insertion of a group II intron into the rns gene has

388 been linked to hypovirulence in the Chestnut blight fungus Cryphonectria parasitica, potentially

389 benefitting the pathogen as the host species can avoid extinction (Baidyaroy et al. 2011). Cinget

390 and Bélanger (2020) showed a more specific impact of an intron towards the phenotype of some

391 fungi where a group ID intron located in the cob gene can interfere with fungicide resistance.

392 Although, it is still unknown what controls the size of the mitochondrial genome intron

393 complement, it is probably impacted by nuclear genes such as those that can promote splicing

394 (maturase-acting proteins), outcrossing, or impact DNA repair and recombination (Contamine

395 and Picard 2000; Hausner 2012; Yan et al. 2018).

396 Comparative mitochondrial intron analysis for the DED-causing fungi will require

397 exhaustive sampling of mitochondrial genomesDraft in order to gain a better understanding of the

398 evolutionary dynamics of introns and their persistence in fungal populations. However, based on

399 comparative nuclear genome analysis, there is strong evidence for hybridization and

400 introgression events among the DED-causing fungi (Hessenauer et al. 2020), that combined with

401 drift (gain and loss of introns) could make these types of deeper analyses very challenging.

402 Finally, the characterization of mitochondrial genomes does provide an opportunity to

403 discover new genetic elements that have applications in biotechnology such as homing

404 endonucleases with novel cutting sites and ribozymes such as group I and II intron RNAs

405 (Stoddard 2014; Guha et al. 2017; Belfort et al. 2019). For example, I-OnuI, a homing

406 endonuclease discovered in O. novo-ulmi subsp. americana (Gibb and Hausner 2005) has been

407 adopted by a variety of research groups as a valuable meganuclease for applications in genome

408 editing (McMurrough et al. 2018).

409

19 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 20 of 42

410 Acknowledgements

411 GH would like to acknowledge funding from an NSERC Discovery grant. The authors

412 declare that the research was conducted in the absence of any conflict of interest. AW and GH

413 contributed to the design of this study; AW performed the experiments and analyzed the data;

414 GH and AW wrote the manuscript, and both read and approved the submitted version.

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647 Zardoya, R. 2020. Recent advances in understanding mitochondrial genome diversity. F1000Res.

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650 resinifera is intron rich. Sci. Rep. 8(1): 17591. doi: 10.1038/s41598-018-35926-y.

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654 Figure legends:

655

656 Figure 1. Circular representation of the mitochondrial genome of Ophiostoma himal-ulmi (CBS

657 374.67). Genes, introns, and GC plot are shown on the outer, middle, and inner tracks,

658 respectively. The purple line of the GC plot corresponds to the average GC content of the

659 mitochondrial genome of O. himal-ulmi.

660

661 Figure 2. Progressive MAUVE alignment for the mitochondrial genomes of the sampled Dutch

662 elm disease-causing fungi.

663

664 Figure 3. Gene synteny of a sample of 38Draft fungi of the Ophiostomatales. Amino acids are

665 represented with the single-letter code. “C.” = Ceratocystiopsis, “E.” = Esteya, “F.” =

666 Fragosphaeria, “G.” = Graphilbum, “Gr.” = Grosmannia, “L.” = Leptographium, “O.” =

667 Ophiostoma, “R.” = Raffaelea, “S.” = Sporothrix, “DED-causing fungi” = O. himal-ulmi (CBS

668 374.67, HP30, HP31, HP32), O. novo-ulmi subspecies americana (DDS25, DDS100, and

669 US137), O. novo-ulmi subspecies novo-ulmi (MG020143.1 and R108), O. ulmi sensu lato

670 (DDS296), and O. ulmi (H868, H873, and PG40). “N/A” = Not applicable; atp8 gene missing

671 due to partial assembly; nad4 and atp6 genes were partially complete. “-“ = absence of gene. “.”

672 = presence of gene.

673

674 Figure 4. Composition of the Ophiostoma himal-ulmi (CBS 374.67) mitochondrial genome;

675 showing the proportion of protein-coding segments (exons), rDNA, tRNAs, intergenic spacers,

676 and introns (including intron-encoded ORFs).

677

678 Figure 5. Intron distribution in the mitochondrial genome of the 13 sampled Dutch elm disease-

679 causing fungi. The sample included four Ophiostoma himal-ulmi strains, three O. novo-ulmi

680 subspecies americana, two O. novo-ulmi subspecies novo-ulmi, one O. ulmi sensu lato, and three

681 O. ulmi. See text and Hessenauer et al. (2020) for more information on DED-causing fungi.

682 Intron insertion sites for the NADH dehydrogenase genes were based on the respective coding

683 sequence of Saccharomyces cerevisiae (NCBI Reference Sequence accession number:

684 NC_001224.1) and Neurospora crassa (NCBI Reference Sequence accession number:

685 NC_026614.1) for all other protein-coding genes, and Escherichia coli (GenBank accession

686 number: AB035922.1) for the ribosomal genes (see Table 1). Group I introns are represented in

687 blue, group II introns in red, and unidentifiableDraft introns in yellow.

688

689 Figure 6. Phylogenetic tree of the Sordariomycetes with emphasis on the Dutch elm disease

690 (DED)-causing fungi. The tree was constructed based on Bayesian inference using concatenated

691 amino acid sequences, composed of atp6, atp8, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4,

692 nad4L, nad5, and nad6, from 61 fungal sequences; including 13 DED-causing fungi. Branch

693 lengths are proportional to the number of substitutions per site. Support values are indicated at

694 the nodes of the tree. NCBI/MitoFun accession numbers for each sequence are indicated in

695 parentheses, and where not available, a strain identifier is used. “Cer.” = “Ceratocystiopsis”,

696 “Est.” = “Esteya”, “Fra.” = “Fragosphaeria”, “Gra.” = “Graphilbum”, “Gro.” =

697 “Grosmannia”, “Haw.” = “Hawksworthiomyces”, “Lep. s. l.” = “Leptographium sensu lato”,

698 “Oph. s. s.” = “Ophiostoma sensu stricto”, “Raf. s. s.” = “Raffaelea sensu stricto”.

699

700 Tables:

701 Table 1: Summary of introns and intron-encoded proteins observed within the mitochondrial

702 genome of Ophiostoma himal-ulmi (CBS 374.67).

703 Table 2: Comparison of the mitochondrial genomes and their intron complement for the studied

704 members of Ophiostoma sensu stricto.

705

706

707 Supplementary Files:

708 cjm-2020-0589.R1suppla

709 Supplementary data file 1: GenBank formatted annotations for the DED-causing fungi

710 mitogenome sequences used in this study.Draft

711

712 cjm-2020-0589.R1supplb

713 Supplementary Table 1S: Comparison of total intron and intergenic region composition of the

714 Dutch elm disease-causing fungi examined in this study.

715

716

717

718

Table 1. Summary of introns and intron-encoded proteins observed within the mitochondrial genome of Ophiostoma himal-ulmi (CBS 374.67).

Number Insertion sitea Intron subgroupb IEPc cox1 1 199 II RT 2 212 IB 2x GIY 3 216 II - 4 281 IB LAG(2) 5 386 IB LAG(2) 6 493 IB GIY 7 540 IC2 LAG(2) 8 615 IB LAG(2) 9 709 ID LAG(2) 10 731 IB LAG(2) 11 867 IB LAG(2) 12 900 IB LAG(2) 13 Draft1057 IB GIY 14 1125 IB LAG(2) 15 1262 IB GIY 16 1281 IB GIY 17 1411 Unidentifiable LAG(2), cox1d nad1 1 145 IA - 2 166 IB GIY 3 291 IC2 LAG(2) 4 636 IB GIY nad4 1 658 IC2 LAG(2) atp6 1 81 IC2 LAG(2) 2 572 IC2 GIY, LAG(2), atp6e rns 1 379 II RT 2 952 II LAG(2) cox3 1 640 IA LAG(2) nad6 1 537 Unidentifiable GIY, nad6d rnl 1 812 IC1 GIY 2 965 IC1 GIY 3 1700 IA LAG(2) 4 1968 IC2 GIY

5 2060 II LAG(2) 6 2450 IA RPS3, LAG(2) nad2 1 1719 IA LAG(2) nad3 1 140 Unidentifiable GIY, nad3d 2 308 Unidentifiable GIY cox2 1 93 IC2 LAG(2) 2 234 IB - 3 324 IA LAG(2) 4 558 IC2 GIY 5 606 IC1 GIY 6 657 IC1 GIY nad5 1 248 ID 2x LAG(2) 2 324 IC2 LAG(2) 3 1301 Unidentifiable GIY, nad5d cob 1 155 IB LAG(2) 2 Draft393 ID GIY 3 437 IB LAG(2) 4 490 IA LAG(2), GIY 5 506 IB LAG(2) aIntron insertion sites were designated based on the coding sequence of reference genes; reference for NADH dehydrogenase genes = Neurospora crassa (NCBI accession number: NC_026614.1), other protein-coding genes = Saccharomyces cerevisiae (NCBI accession number: NC_001224.1), rRNA genes = Escherichia coli (NCBI accession number: AB035922.1) bIntron subgrouping applies only to group I introns; group II introns are annotated as “II”; Unidentifiable = No identifiable group I or II intron based on MFannot/RNAweasel results and manual inspection cIntron-encoded protein (IEP) associated with intron insertion site based on blastx results; LAG(2) = double-motif LAGLIDADG homing endonuclease; RT = reverse transcriptase; GIY = GIY-YIG homing endonuclease; - = no conserved motif detected based on blastx results dBased on BLASTx, beginning of intron encodes a “copy” (< 100% sequence similarity) of downstream exon eBased on BLASTx, GIY-YIG sequence appears to be interrupted by sequences encoding for a double-motif LAGLIDADG and partial sequences of downstream and upstream atp6 coding sequences

Table 2. Comparison of the mitochondrial genomes and their intron complement for the studied members of Ophiostoma sensu stricto.

Number of introns in genes Total NCBI/SRA introns by Name of organism Genome size (bp) accession number atp6 atp8 atp9 cob cox1 cox2 cox3 nad1 nad2 nad3 nad4 nad4L nad5 nad6 rnl rns species or strain

Ophiostoma himal-ulmi (CBS 374.67) MW250274 111 712 2 0 N/A 5 17 6 1 4 1 2 1 0 3 1 6 2 51

Ophiostoma himal-ulmi (HP30) SRR10139865 94 934 2 0 N/A 5 14 6 1 4 1 0 1 0 2 1 4 1 42

Ophiostoma himal-ulmi (HP31) SRR10139864 94 934 2 0 N/A 5 14 6 1 4 1 0 1 0 2 1 4 1 42

Ophiostoma himal-ulmi (HP32) SRR10139863 98 082 2 0 N/A 5 14 6 1 4 1 1 1 0 3 1 4 1 44

Ophiostoma ips (CBS 138721) NTMB01000349.1 97 849 3 N/Aa 0 5 13 4 2 4 3 1 1 0 1 0 4 1 42

Ophiostoma minus [WIN(M)495] MW122509.1 91 847 3 0 N/Ab 1 9 7 2 3 3 0 0 0 3 0 5 2 38

Ophiostoma piceae (UAMH 11346) SRR869560 33 599 0 0 N/A 0 1 1 2 1 0 0 0 0 0 0 1 0 6

Ophiostoma piliferum [WIN(M)959] MW122508.1 69 966 2 0 N/Ab 6 9 4 0 3 1 0 0 1 2 0 1 0 29

Ophiostoma novo-ulmi subspecies americana (DDS25) SRR10139932 56 451 1 0 N/A 3 8 4 1 1 2 0 0 0 1 0 3 0 24

Ophiostoma novo-ulmi subspecies americana (DDS100) SRR10139938 60 420 1 Draft0 N/A 3 8 4 1 1 1 0 0 0 1 0 4 0 24

Ophiostoma novo-ulmi subspecies americana (US137) SRR10139915 59 277 1 0 N/A 4 8 4 0 1 1 0 0 0 1 1 4 0 25

Ophiostoma novo-ulmi subspecies novo-ulmi (IMI 343.101) MG020143.1 64 848 2 0 N/A 3 8 5 0 1 1 0 0 0 2 1 4 0 27

Ophiostoma novo-ulmi subspecies novo-ulmi (R108) SRR10139923 64 848 2 0 N/A 3 8 5 0 1 1 0 0 0 2 1 4 0 27

Ophiostoma ulmi (H868) SRR10139873 77 596 2 0 N/A 5 11 6 0 4 1 0 0 0 1 1 4 1 36

Ophiostoma ulmi (H873) SRR10139869 75 934 1 0 N/A 5 11 6 0 4 1 0 0 0 1 1 4 1 35

Ophiostoma ulmi (PG40) SRR10139855 77 608 2 0 N/A 5 11 6 0 4 1 0 0 0 1 1 4 1 36

Ophiostoma ulmi sensu lato (DDS296) SRR10139930 59 003 1 0 N/A 3 8 5 0 1 1 0 0 0 1 0 4 0 24

Total introns by genes 29 0 0 66 172 85 12 45 21 4 5 1 27 10 64 11 552 aMitochondrial scaffold incomplete; atp8 gene missing, and nad4 and atp6 were partial. batp9 is absent [as previously observed in Ophiostoma novo-ulmi (Abboud et al. 2018)].

Page 37 of 42 Canadian Journal of Microbiology

trnR(ucu)

110

109

11 108

107 0

1 106 2 3 105 4 5

104 6 103 7 8 102 cob 9 101 10 100 1 1

99 12

98 13

97 14 50 nad5 15 96 cox1 nad4L 40 16 trnR(acg) 95 30 17 94 18 93 20 19 92 10 20 91 21 90 22 89 23 88 cox2 24 87 25 86 26 trnI(aau) 85 Draft 27 84 Ophiostoma himal-ulmi 28 83 (CBS 374.67) 111 712 bp nad1 29 82 30 nad3 81 31

80 32 trnM(cau) 79 trnH(gug) nad2 nad4 33 trnQ(uug) 78 34 trnL(uag) 77 35

trnF(gaa) 76 36 trnA(ugc) atp8 75 37 trnG(ucc) atp6 74 38 trnL(uaa) 73 39 trnM(cau) trnM(cau) 72 40 71 41 trnE(uuc) 42 70 rns trnT(ugu) 43 69 44 68 rnl 45

67 cox3 46 66 47 65 48

64 49

nad6 63 50 51 62 52 61 53 trnY(gua) 54

60 59 58

57 trnN(guu)

trnK(uuu) trnD(guc)

trnS(gcu)

trnV(uac)

trnW(uca) Canadian Journal of Microbiology Page 38 of 42

Draft

© The Author(s) or their Institution(s) Page 39 of 42 Canadian Journal of Microbiology cox1 –– nad1 nad4 atp8 atp6 – rns NY cox3 WSD–K nad6 PS–IV rnl –MHQLFAGLMMET – nad2 nad3 – atp9 cox2 R– nad4L nad5 cob C–R

C. brevicomis ...... V ......

C. minuta, H. lignivorus ...... M ......

E. vermicola ......

F. purpurea, S. insectorum ...... S ...... I ......

G. fragrans . . N ...... M ......

Gr. penicillata . R ......

L. lundbergii ...... L ......

O. ips . .. . . N/A ......

O. minus . . I . . . . I ...... – ......

O. piceae, DED-causing fungi . . I ...... Draft...... – ......

O. piliferum ...... – ......

R. albimanens . KK ......

R. ambrosiae . . N ...... N ...... A ......

R. arxii ...... A ......

R. lauricola . . F ...... X . . K ......

R. quercivora ...... AL...... I ......

R. quercus-mongolicae ...... I ......

R. sp...... S ...... M ......

R. sulphurea ...... L ...... L . . R ...... K . .

S. brasiliensis, S. globosa, S. schenckii . . X ......

S. pallida . . F ...... © The. Author(s) or ... their. Institution(s)...... Canadian Journal of Microbiology Page 40 of 42

rRNA Intergenic region tRNA 4% 10% 2% Exon 11%

Draft

Intron 73%

600 0 400 200

400 200 600 800

1000 0 2000 1200

1800 1400

1600 1600 0 nad1 1400 3 200

1200 400

6 600 nad51000 800 800 9 1000 600 12 0 400 200 200 15 400

nad4 0 600

600 800

400 1000

cox2 200 1200

0 1400 nad3 400 Draft 1600 0 200 200 0 400 1600 600 atp6 1400 0 1200 200 nad2 1000 400 800 600 600 800 400 1000 rns 200 1200 0 1400

2800 0

2600 200

400 2400 600 2200 800 2000 0

200 1800 400 cox3 1600 600 Group I intron 0

200 1400 400 1200 Group II intron 800 1000 rnl 600 © The Author(s) or their Institution(s) nad6 Unidentiﬁable intron 100 Beauveria bassiana (NC_010652.2) Canadian Journal of Microbiology Cordyceps bassiana (NC_017842.1) Page 42 of 42 100 100 Cordyceps brongniartii (NC_011194.1) 100 Lecanicillium muscarium (NC_004514.1) Metarhizium anisopliae (NC_008068.1) 100 94 Trichoderma reesei (NC_003388.1)

100 Fusarium graminearum (NC_009493.1) Fusarium oxysporum (AY945289.1) 100 100 Gibberella moniliformis (NC_016687.1) 100 Fusarium solani (NC_016680.1) 100 Ceratocystis cacaofunesta (NC_020430.1) 100 Endoconidiophora resinifera (MH551223.1) Glomerella graminicola (CM001021.1)

100 Verticillium alboatrum (MitoFun: MF000004) 100 Verticillium dahliae (NC_008248.1) 100 Sordaria macrospora (CABT01004783.1) 100 Neurospora crassa (KC683708.1) Podospora anserina (NC_001329.3) Madurella mycetomatis (NC_018359.1) 100 100 Chaetomium thermophilum (NC_015893.1) Gra. 100 Chaetomium globosum (CH670323.1) Graphilbum fragrans (LLKO01000061.1) Raf. s. s. 100 Raffaelea sp. (PCDF01000414.1) 100 100 Raffaelea lauricola (PCDG01000206.1) Raffaelea arxii (PCDH01000124.1) Raffaelea ambrosiae (PCDI01000067.1) 100 100 100 Raffaelea albimanens (PCDJ01000011.1) Gro. 95 Grosmannia penicillata (PCDK01000036.1)

100 Raffaelea sulphurea (PCDD01000156.1) Lep. s. l. Raffaelea quercus-mongolicae (NIPS01000008.1) 100 100 100 Raffaelea quercivora (PCDE01000018.1) Draft Leptographium lundbergii (LDEF01000080.1) 100 Est. Ophiostomatales Haw. Esteya vermicola (KY644696.1) 100 Hawksworthiomyces lignivorus (NTMA01000166.1) 100 Fra. Sporothrix insectorum (AZHD01000037.1) 100 Fragosphaeria purpurea (PCDL01000017.1)

100 Sporothrix brasiliensis (AWTV01000012.1) Sporothrix globosa (LVYW01000010.1) Spo. s. s. 100 100 Sporothrix schenckii (AB568600.1) Sporothrix pallida (CM003773.1) Size (bp) Introns Ophiostoma himal-ulmi (CBS 374.67) 111 712 51 100 Ophiostoma himal-ulmi (HP30) 94 934 42 Ophiostoma himal-ulmi (HP31) 94 934 42

100 Ophiostoma himal-ulmi (HP32) 98 082 44 Ophiostoma novo-ulmi subspecies americana (DDS25) 56 451 24 Ophiostoma novo-ulmi subspecies americana (DDS100) 60 420 24 97 100 Ophiostoma novo-ulmi subspecies americana (US137) 59 277 25 Ophiostoma novo-ulmi subspecies novo-ulmi (R108) 64 848 27 Ophiostoma novo-ulmi subspecies novo-ulmi (MG020143.1) 64 848 27

100 Ophiostoma ulmi sensu lato (DDS296) 59 003 24 Ophiostoma ulmi (H868) 77 596 36 Ophiostoma ulmi (H873) 75 934 35 92 100 100 Ophiostoma ulmi (PG40) 77 608 36 Ophiostoma minus (MW122509) Oph. s. s. 100 Ophiostoma piceae (UAMH 11346) 100 Ophiostoma ips (NTMB01000349.1) Ophiostoma piliferum (MW122508) Cer. Ceratocystiopsis minuta (LZPB01000172.1) 100 Ceratocystiopsis brevicomis (PCDN01000199.1) Aspergillus fumigatus (NC_017016.1) 100 Penicillium digitatum (NC_015080.1)

0.1