Trollip et al. IMA Fungus (2021) 12:24 https://doi.org/10.1186/s43008-021-00076-w IMA Fungus

RESEARCH Open Access Ophiostomatoid fungi associated with pine bark beetles and infested pines in south‑eastern Australia, including Graphilbum ipis‑grandicollis sp. nov. Conrad Trollip1,2* , Angus J. Carnegie3, Quang Dinh2, Jatinder Kaur2, David Smith4, Ross Mann2, Brendan Rodoni1,2 and Jacqueline Edwards1,2

Abstract The ophiostomatoid fungi are an assemblage of ascomycetes which are arguably best-known for their associations with bark and ambrosia beetles (Curculonidae) and blue stain (sap stain) of many economically important tree species. These fungi are considered a signifcant threat to coniferous forests, which has resulted in numerous studies charac- terising the diversity of bark beetles and their ophiostomatoid associates globally. The diversity of ophiostomatoid fungi present in Australian pine plantations, however, remains largely undetermined. The aims of this study were therefore to reconsider the diversity of ophiostomatoid fungi associated with Pinus in Australia, and to establish the baseline of expected taxa found within these plantation ecosystems. To achieve this, we reviewed Australian plant pathogen reference collections, and analysed samples collected during forest health surveillance programs from the major pine growing regions in south-eastern Australia. In total, 135 ophiostomatoid isolates (15 from reference collections and 120 collected during the current study) were assessed using morphological identifcation and ITS screening which putatively distinguished 15 taxonomic groups. Whole genome sequencing (WGS) of representative isolates from each taxon was performed to obtain high-quality sequence data for multi-locus phylogenetic analysis. Our results revealed a greater than expected diversity, expanding the status of ophiostomatoid fungi associated with Pinus in Australia to include 14 species from six genera in the Ophiostomatales and a single species residing in the Microascales. While most of these were already known to science, our study includes seven frst records for Australia and the description of one new species, Graphilbum ipis-grandicollis sp. nov.. This study also provides an early example of whole genome sequencing (WGS) approaches replacing traditional PCR-based methods for taxonomic surveys. This not only allowed for robust multi-locus sequence extraction during taxonomic assessment, but also permitted the rapid establishment of a curated genomic database for ophiostomatoid fungi which will continue to aid in the development of improved diagnostic resources and capabilities for Australian biosecurity. Keywords: Ceratocystiopsis, Graphilbum, Leptographium, Ophiostoma, Rafaelea, Sporothrix, Graphium, One new taxon

Introduction Fungi within Ophiostomatales and Microascales are best *Correspondence: [email protected] known for their associations with arthropod vectors and 2 Department of Jobs, Precincts and Regions, Agriculture Victoria include examples of some of the most devastating fungal- Research, AgriBio Centre, Bundoora, VIC 3083, Australia Full list of author information is available at the end of the article insect symbioses known to plant pathologists over the past century (Fisher et al. 2012; Wingfeld et al. 2017b;

© The Author(s) 2021, corrected publication 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​ iveco​ mmons.​ org/​ licen​ ses/​ by/4.​ 0/​ . Trollip et al. IMA Fungus (2021) 12:24 Page 2 of 27

Brasier and Webber 2019). Notable examples include the present in exotic pine plantations in Australia, however, Dutch elm disease pathogens, Ophiostoma ulmi and O. remains largely undetermined. novo-ulmi (Santini and Faccoli 2015; Brasier and Web- Since its frst detection in the 1960s (Vaartaja 1967), ber 2019), the laurel wilt pathogen Rafaelea lauricola Ophiostoma ips has been regarded as the most common (Harrington et al. 2008) as well as the numerous patho- fungal species associated with blue stain and pine bark gens belonging to Ceratocystis which cause tree mortality beetles (specifcally Ips grandicollis) in Australia (Stone in natural and agricultural ecosystems (Roux et al. 2007; and Simpson 1987, 1990; Hood and Ramsden 1997; Zhou Wingfeld et al. 2017b; Tsopelas et al. 2017). In a recent et al. 2007; Carnegie et al. 2019). Additionally, surveys of review on novel associations for members of Ophiosto- the fungal associates of Ips grandicollis on Pinus taeda matales and Microascales, Wingfeld et al. (2017b) high- and P. elliottii in New South Wales (NSW) in the late light the numerous biological and anthropogenic factors 1980s serve as the frst reports of a Ceratocystiopsis and that infuence the dispersal of these fungi and their vec- Graphilbum species detected in Australian pine planta- tors globally; a major feature of the ever-increasing threat tions (Stone and Simpson 1987, 1990), while Grosmannia these fungi pose to global biosecurity. huntii was frst reported in NSW in 1998 (Jacobs et al. Despite being formally recognised as two distinct 1998). To date, these serve as the few detailed surveys of orders in the Sordariomycetes, species belonging to Ophi- fungi associated with Australian pine bark beetles. Other, ostomatales and Microascales share a long and compli- somewhat incidental records include the detection of cated taxonomic history and are collectively referred to Ophiostoma foccosum, O. quercus and an unknown spe- as the ophiostomatoid fungi (Wingfeld et al. 1993; Seifert cies reported as a Pesotum af. fragrans, all isolated from et al. 2013). Tis is due to similarities shared across their woodchips of P. radiata from the Tantanoola paper mill biology, particularly in key morphological characters, in South Australia (Harrington et al. 2001; Twaites et al. that is believed to have been driven by convergent evolu- 2005). Evidently, the historical record of ophiostomatoid tion in adaptation to insect-mediated dispersal (De Beer fungi in Australian pine plantations has relied heavily on et al. 2013; Wingfeld et al. 2017b). Ophiostomatoid fungi morphology, and/or the association of blue stain in the are commonly associated with bark (Coleoptera: Scolyti- presence of the pine bark beetle, I. grandicollis (Carnegie nae) and ambrosia (Curculonidae: Scolytinae, Platypodi- and Nahrung 2019; Carnegie et al. 2019). Interestingly, nae) beetles (Kirisits 2004; Hofstetter et al. 2015), where a the introduction of I. grandicollis in 1943 coincides with greater dependency and specifcity is apparent for Ophi- the introduction of two other exotic pine bark beetles, ostomatales compared to Microascales (Wingfeld et al. namely Hylastes ater and Hylurgus ligniperda in 1936 2017b). Ophiostomatoid genera that are most commonly and 1942, respectively (Nahrung et al. 2016). Both are associated with beetles include: Ambrosiella, Endocon- known to also vector ophiostomatoid fungi (Kim et al. idiophora and Graphium in Microascales; and Afroraf- 2011; de Errasti et al. 2018). Te above-mentioned pine faelea, Aureovirgo, Ceratocystiopsis, Fragosphaeria, bark beetles, along with their associated ophiostomatoid Graphilbum, Leptographium, Ophiostoma, Rafaelea, and fungi, are considered as established exotics to Australia. Sporothrix of Ophiostomatales (Hyde et al. 2020). Recent eforts to improve on the capacity of forest bios- While not all ophiostomatoid fungi are responsible for ecurity surveillance, through programs such as the forest tree mortality, many are well recognized as the causal health surveillance program, and the more targeted high- agents of blue stain (sap stain) in the wood of economi- risk site surveillance program (Carnegie et al. 2018), has cally important tree hosts (Kirisits 2004; Seifert et al. led to several detections of cryptic fungal species associ- 2013). Tis is particularly true for pine (Pinus) planta- ated with pine bark beetles and blue stain in NSW (Car- tions globally (Seifert et al. 2013; de Errasti et al. 2018; negie and Nahrung 2019). Tis includes the recent pest Jankowiak et al. 2021). Systematic surveys of bark beetles detections of Graphilbum fragrans, O. angusticollis, O. and ophiostomatoid fungi associated with pine have been pallidulum and Sporothrix cf. abietina, illustrating the completed in North and Central America (Zhou et al. value of targeted surveillance programs for the detection 2004a; Kim et al. 2011; Klepzig and Hofstetter 2011; Tae- of novel pests (Carnegie et al. 2019). Tese fndings also rum et al. 2013; Marincowitz et al. 2020), Europe (Lin- emphasize the need for an updated record of the diversity nakoski et al. 2012; Romón et al. 2014; Jankowiak et al. of established ophiostomatoid fungi associated with Aus- 2012, 2020), Asia (Zhou et al. 2013; Masuya et al. 2013; tralian pine and pine bark beetles. Kirisits et al. 2013), with a signifcant number of surveys Te overall aim of this study was to reconsider the conducted recently in China (Chang et al. 2017, 2019; diversity of ophiostomatoid fungi associated with pine Wang et al. 2018, 2019, 2020), South America (Zhou et al. and pine bark beetles in south-eastern Australia. In order 2004b; de Errasti et al. 2018) and New Zealand (Twaites to achieve this, we looked to: (1) review all available ophi- et al. 2005, 2013). Te diversity of ophiostomatoid fungi ostomatoid reference material previously reported from Trollip et al. IMA Fungus (2021) 12:24 Page 3 of 27

pine and lodged in Australian plant pathogen reference Finally, all wood submissions were screened upon arrival collections; (2) survey the ophiostomatoid fungi found in in the laboratory for any remaining beetles that may have pine plantations during the 2019–2020 forest health sur- been concealed within the galleries. Pine bark beetles veillance period; and (3) use whole genome sequencing present in each sample were sorted into morphospecies, (WGS) of representative taxa to establish a curated data- pooled and then treated as a single submission (repre- base for improved molecular diagnostics of ophiostoma- sentative specimens were morphologically identifed by toid fungi for Australian biosecurity. Crop Health Services diagnostics unit, Agriculture Victo- ria). All samples were stored at 4 °C until they were pro- Materials and methods cessed for fungal isolations. Literature and Australian plant pathogen reference collection review Fungal isolations Ophiostomatoid fungi previously collected from Pinus Fungal isolations from beetle galleries were performed by spp. in Australia were included as references in this study. directly transferring aerial mycelia and/or spore masses Living cultures were recovered from the Victorian Plant found on sporing structures characteristic of ophios- Pathology Herbarium (VPRI) and the New South Wales tomatoid fungi, such as ascomata or synnemata, onto (NSW) Plant Pathology and Mycology Herbarium (DAR) malt extract agar (MEA; Oxoid MEA as per manufac- following database searches using the currently accepted turer instructions; Oxoid, Basingstoke, UK) amended nomenclature (Seifert et al. 2013) and all putative syno- with 0.1 g Tetracycline (Fluka Analytical, Sigma-Aldrich, nyms (MycoBank Database, www.​mycob​ank.​org; Species MO, USA) per 1000 ml of media. When sporing struc- Fungorum, www.​speci​esfun​gorum.​org) of ophiostoma- tures were absent, samples were incubated in moistened toid fungi that were recorded in the respective Austral- plastic containers at room temperature for approximately ian collections and associated with Pinus. Additionally, 21 days to encourage sporulation. When blue stained a literature and GenBank database search (http://​www.​ sapwood was present in a sample, wood chips of approxi- ncbi.​nlm.​nih.​gov) was performed for Australian speci- mately 5 × 5 mm were cut, surface sterilized with 1.5% mens previously reported from Pinus in order to identify sodium hypochlorite for 1 min, and plated onto MEA. additional specimens that had publicly available DNA Beetle isolations followed an amended protocol from sequence data. Alamouti et al. (2006). Beetles from each sample were vortexed in 1 ml of 0.01% Tween80 solution (Nuplex Sample collection during forest health surveillance Industries, South Australia, Australia) for 3 min. Tere- Annual forest health surveillance programs are con- after, spore suspensions were spread onto MEA plates ducted in pine plantations across Australia, includ- and incubated at 22 °C in the dark for 7 d during which ing NSW (Carnegie et al. 2008), Victoria (Smith et al. all germinating single spores and hyphal tips were trans- 2008), Tasmania (Wotherspoon 2008), and South Aus- ferred onto individual MEA plates, producing axenic cul- tralia (Phillips 2008). Tese surveillance programmes tures which were maintained under the same growing capture a broad overview of plantation health, achieved conditions. through aerial and ground surveys across the major growing regions for each state. Taking advantage of this Preliminary identifcation and ITS screening routine surveillance, sampling was concentrated on pine Isolates were preliminarily grouped based on culture trees showing typical symptoms of bark beetle infesta- morphology and growth on MEA. In addition to this, a tion, which included any dead or dying trees, but also Chelex-based internal transcribed spacer (ITS) region tree stumps in recently harvested sections. Samples were sequencing protocol was used to confrm the puta- either collected and sent in by respective state agen- tive identifcation of all ophiostomatoid fungi. In order cies conducting the surveillance, or by the frst author to achieve this, a small amount of mycelia was scraped accompanying forest health surveillance. Samples col- from each isolate using a sterile needle tip and placed lected from May 2019 to March 2020 originated from into individual 200 µl reaction tubes containing 100 µl 40 locations, including collections from NSW (n = 34), of molecular biology grade Chelex 100 resin (Bio-Rad Victoria (n = 2), Tasmania (n = 2), and South Australia Laboratories, Hercules, CA, USA) following a modifed (n = 2) (Additional fle 1: Table S1). Samples of sapwood protocol for Chelex DNA preparation (Walsh et al. 1991). and/or pieces of bark containing beetle galleries were Te ITS region was PCR amplifed using the ITS1F and collected and individually placed into sampling bags to ITS4 primers (White et al. 1990; Gardes and Bruns 1993). retain moisture. Where possible, pine bark beetles were PCR reactions included 3 µl Chelex DNA template, 15 µl collected into 50 ml collection vials on site using forceps of MyTaq Red mix (Bioline, London, UK), 0.4 µM of each and submitted along with their respective wood samples. primer (forward and reverse) and were made up to a Trollip et al. IMA Fungus (2021) 12:24 Page 4 of 27

fnal volume of 30 µl with nuclease free water. PCR cycle check point to ensure cultures were axenic and only a conditions followed those of Duong et al. 2012. PCR single sequence was generated from the consensus of products were sent for purifcation and sequencing at all mapped reads using a minimum of 10 × coverage. Macrogen (Seoul, Rep. of Korea). All resulting sequences Extracted loci were then BLASTn searched to confrm were trimmed, aligned and analysed using Geneious taxonomic afnities and obtain similar sequences from Prime® 2019.1.3 (www.​genei​ous.​com). Sequences were GenBank to be included in phylogenetic analyses along BLASTn searched against the nr/nt database of the NCBI with the sequences of type ophiostomatoid fungi. to confrm placement within either the Ophiostoma- For multi-locus phylogenetic analysis, the ITS and tales or Microascales. Only ophiostomatoid fungi were LSU datasets were used for initial placement of Austral- retained for further analysis. Finally, isolates from a given ian isolates within well-defned species complexes of sample that shared an ITS sequence and belonged to the Ophiostomatales and Microascales. Subsequent phylo- same morphological group were considered the same genetic analyses of the BT, TEF and CAL regions were fungus, with a single axenic culture being chosen as the performed within each species complex where loci were representative isolate in each case. chosen based on availability of reference data from pre- vious studies (e.g. BT and CAL for Sporothrix) which DNA extraction, whole genome sequencing allowed for more accurate delineation of the Austral- and phylogenetic analysis ian taxa. Sequence alignments were performed with Seven to 10 d old cultures were inoculated into 40 mL MAFFT v7.388 using the E-INS-i algorithm and a gap Potato Dextrose Broth (PDB; 9.6 g Oxoid PDB, 400 mL open penalty of 1.53 (Katoh et al. 2019). Te scoring deionized water; Oxoid, Basingstoke, UK) and grown on matrix for alignments spanning across multiple genera a shaking incubator at 150 rpm at room temperature for was 200PAM/k = 2, while for within genus analyses the approximately 72 h. Mycelia were then harvested using scoring matrix was set at 1PAM/k = 2 (Linnakoski et al. autoclaved Miracloth (Merck, Darmstadt, Germany) and 2012; Katoh et al. 2019). All aligned sequence datasets freeze-dried before DNA extraction using the Promega were submitted to TreeBase (No. 27096). Maximum Wizard Genomic DNA Purifcation Kit (Promega, Madi- Likelihood (ML) analysis was performed with RAxML son, WI, USA). Te quality and quantity of extracted v8.2.11 (Stamatakis 2014), using the GTR model with DNA was assessed using a Nanodrop 1000 (Termo optimization for substitution rates and the estimation of Fisher Scientifc, MA USA) and Quantus fuorometer rate heterogeneity (GAMMA) specifed, while the pro- (Promega, Madison, WI, USA), respectively. Librar- portion of invariable sites (+ I) was selected based on ies with an average insert size of 300 bp were generated results of model estimation using Smart Model Selec- using the NextFlex Rapid XP DNA-Seq Kit (Perkin Elmer, tion (SMS; Lefort et al. (2017); available at http://​www.​ Austin, TX, USA). Whole genome sequencing (WGS) atgc-​montp​ellier.​fr/​sms/). Confdence support was esti- was performed on the Novaseq 6000 system (Illumina, mated with bootstrapping of 1000 replicates. Bayesian San Diego, CA, USA). Raw sequencing reads were quality Inference (BI) analyses were done using MrBayes 3.2.6 checked and trimmed using FastP (Chen et al. 2018). Fol- (Huelsenbeck and Ronquist 2001). Te substitution mod- lowing quality trimming, initial de novo genome assem- els and estimated rate parameters, estimated with SMS, blies were produced using SPAdes v3.14.1 (Nurk et al. were then included manually in MrBayes. Four Markov 2013). Assemblies were performed on error-corrected chain Monte Carlo (MCMC) chains were run at the same reads with a kmer range of 33, 55, 77, 97 and 111. time from a random starting tree for 5 000 000 iterations. Assembled genomes provided a platform for sequence Trees were sampled every 100 generations with a burn- extraction of commonly used barcoding loci, including in length of 25%. Posterior probabilities were calculated the ITS, the large subunit of ribosomal DNA (LSU), beta- from a majority rule consensus tree. tubulin (BT), translation elongation factor 1-α (TEF), and the calmodulin (CAL) regions. For each locus, reference Taxonomy sequences for type collections of ophiostomatoid fungi Morphological studies were performed on selected iso- available in GenBank were used to create reference sets. lates belonging to putative novel lineages identifed fol- Sequencing reads for each isolate were subsequently lowing phylogenetic analysis. Cultures were grown at mapped against each reference set using BBMap (Bush- 22 °C on 2% MEA (33 g Oxoid MEA, 10 g Oxoid agar, 1 L nell (2014); sourceforge.net/projects/bbmap/). Locus- deionized water), as well as water agar (WA; 15 g Oxoid specifc binned reads were generated for each isolate, and agar; 1 L deionized water) amended with autoclaved pine these reads were then mapped back to the respective de needles in order to encourage sporulation. Subsequently, novo assembled genome in order to extract the assem- reproductive structures were mounted on glass slides bled locus. Tis mapping step served as an additional with 85% lactic acid and examined using Leica DM6B Trollip et al. IMA Fungus (2021) 12:24 Page 5 of 27

and M205C microscopes (Leica, Heerbrugg, Switzer- P. elliottii (5%), and included a single sample from an land). Measurements of taxonomically characteristic amenity planting of P. ponderosa. Tree species of pine structures (approximately ffty measurements for each bark beetles, namely Ips grandicollis, Hylastes ater and character wherever possible) were made using a mounted Hylurgus ligniperda, and the ambrosia beetle Xyleborus Leica camera operated using the Leica application suite nr. ferrugineus., were recovered from 22 of the samples software v 3.06. Measurements are presented as, (mini- collected (Additional fle 1: Table S1). Ips grandicollis mum-) (mean-standard deviation) – (mean + standard was the most abundant beetle species sampled during deviation) (- maximum). this study, comprising approximately 97% of the beetles included in our dataset. Samples containing H. ater and Genomes of representative species of ophiostomatoid Hy. ligniperda came only from sites in South Australia fungi from Australian pine plantations and Tasmania respectively, while a single sample from Draft genomes of representative isolates for each ophi- northern NSW included the Xyleborus species. ostomatoid taxon collected in this study were subjected Preliminary identifcation and ITS screening charac- to genome quality assessments using QUAST v5.0.2 terised the 120 ophiostomatoid isolates into 15 taxo- (Mikheenko et al. 2018). In order to perform suitable nomic groups, 14 of which resided in , and a single taxon comparisons, the QUAST analyses also included pub- belonged to Microascales (Table 1). Ophiostomatoid iso- licly available genomes of ophiostomatoid fungi that lates were recovered evenly from the sampled pine tissue corresponded to the genera obtained during this study. (56%) and bark beetles (44%), with about two thirds of all Tis was done to update genome completeness assess- isolations associated with a P. radiata host (Additional ments against the latest lineage-specifc datasets avail- fle 2: Table S2). Ophiostoma ips (Taxon 9) and Sporothrix able for BUSCO (Benchmarking Universal Single-Copy pseudoabietina (Taxon 14) were isolated most frequently, Orthologs tool, BUSCO; https://​busco.​ezlab.​org/), as well making up approximately 53% and 19% of the dataset, as to assess gene predictions using a single prediction respectively (Additional fle 2: Table S2). Tis trend was tool (GenMark-ES run in fungal mode). BUSCO models consistent for the abundantly sampled bark beetle vec- were predicted using the Sordariomycetes_odb10 line- tor, Ips grandicollis, where fve additional taxa (taxa 1, 3, age coupled with the Augustus species parameter option 4, 8 and 15) were represented by the 44 fungal isolates set as Neurospora crassa. Draft genome data for the rep- collected from this source. Te remaining taxa were only resentative isolates sequenced in this study has been recovered occasionally, with the host association and iso- deposited at DDBJ/EMBL/GenBank under BioProject lation frequencies recorded in Additional fle 2: Table S2. PRJNA667796. Te accession numbers for each genome Finally, 46 isolates representing all major taxonomic are presented in Table 3. groups were selected for further phylogenetic analysis and taxonomic placement (Table 1). Results Sample collection and fungal isolation Phylogenetic analysis A total of 135 ophiostomatoid isolates were collected Phylogenetic analysis of the ITS (Fig. 1) and LSU (Fig. 2) during this study, 15 of which were obtained from Aus- regions allowed for taxa to be sorted into their respective tralian plant pathogen reference collections (Table 1). Te species complexes, while the additional gene regions of reference isolates available from Australian collections BT, TEF and CAL (Figs. 3, 4, 5, 6, 7, 8, Additional fles included fve Ophiostoma ips, fve Sporothrix sp. (three 4 and 5: Fig. S1, S2) enabled species level resolution and of which were putatively identifed as S. cf. abietina), two more accurate delineation. In Ophiostomatales, the 14 isolates residing within Leptographium s.lat. (one isolate, taxonomic groups were found to encompass six gen- DAR 84705, identifed as Gro. huntii), two identifed as O. era: Ceratocystiopsis (Taxon 1), Graphilbum (Taxa 2–4), angusticollis, and a single G. fragrans isolate. Leptographium s. lat. (Taxa 5–6), Ophiostoma s. lat. Te remaining 120 isolates were obtained from samples (Taxa 7–10), Rafaelea (Taxon 11), and Sporothrix (Taxa received during the 2019–20 forest health surveillance 12–14). Te single taxon residing in Microascales was period, which included isolations from beetles, beetle identifed as belonging to Graphium (Taxon 15). galleries and blue-stained wood chips (Additional fle 1: Table S1). Samples were largely collected from Pinus Ophiostomatales radiata (62.5%), the most common Pinus species grown Taxon 1 comprised of four representative isolates group- across temperate regions of south-eastern Australia, and ing as a well-supported clade within Ceratocystiop- P. caribaea x elliottii hybrids (22.5%), the most commonly sis (Figs. 2, 3). Phylogenetic analysis of the LSU dataset planted species in the subtropical parts of northern revealed Taxon 1 grouped as an independent lineage, NSW. Te remainder were collected from P. taeda (7.5%), close to Ceratocystiopsis (Cop.) ranaculosa and Cop. Trollip et al. IMA Fungus (2021) 12:24 Page 6 of 27 MW075118 MW075128 MW075113 MW075117 MW075127 MW075112 MW075126 MW075122 MW075116 MW075111 MW075131 MW075121 MW075115 MW075125 MW075110 MW075114 MW075123 MW075132 MW075133 MW075124 MW075129 MW075130 MW075119 MW075120 CAL MW066403 MW066413 MW066398 MW066402 MW066412 MW066397 MW066411 MW066407 MW066401 MW066396 MW066416 MW066406 MW066400 MW066410 MW066395 MW066399 MW066408 MW066417 MW066418 MW066409 MW066414 MW066415 MW066404 MW066405 TEF MW066357 MW066367 MW066352 MW066356 MW066366 MW066351 MW066365 MW066361 MW066355 MW066350 MW066370 MW066360 MW066354 MW066364 MW066349 MW066353 MW066362 MW066371 MW066372 MW066363 MW066368 MW066369 MW066358 MW066359 BT d MW046115 MW046125 MW046110 MW046114 MW046124 MW046109 MW046123 MW046119 MW046113 MW046108 MW046128 MW046118 MW046112 MW046122 MW046107 MW046111 MW046120 MW046129 MW046130 MW046121 MW046126 MW046127 MW046116 MW046117 LSU ­ accessions MW046069 MW046079 MW046064 MW046068 MW046078 MW046063 MW046077 MW046073 MW046067 MW046062 MW046082 MW046072 MW046066 MW046076 MW046061 MW046065 MW046074 MW046083 MW046084 MW046075 MW046080 MW046081 MW046070 MW046071 GenBank ITS 2019 2020 2019 2019 2020 2019 2019 2019 2019 2019 2019 2019 2019 2020 2019 2018 2018 2017 2018 2000 2019 2019 Year 2019 2019 Sargeant, D Sargeant, Wotherspoon, K., Wotherspoon, Ramsden, N Carnegie, A. J., A. J., Carnegie, C Trollip, Carnegie, A. J Carnegie, Wotherspoon, K., Wotherspoon, Ramsden, N Carnegie, A. J., A. J., Carnegie, C Trollip, Smith, D Carnegie, A. J., A. J., Carnegie, C Trollip, Sargeant, D Sargeant, Carnegie, A. J., A. J., Carnegie, C Trollip, Carnegie, A. J Carnegie, Carnegie, A. J Carnegie, Sargeant, D Sargeant, Wotherspoon, K., Wotherspoon, Ramsden, N Carnegie, A. J Carnegie, Carnegie, A. J Carnegie, A. J Carnegie, A. J Carnegie, A. J Carnegie, Smith, I D Sargeant, D Sargeant, Collector Carnegie, A. J Carnegie, A. J Carnegie, Tumut, NSW Tumut, Lower Lower Beulah, TAS Urbenville, NSW Urbenville, Inverell, NSW Inverell, Branxholm, TAS Bonalbo, NSW Bonalbo, Nangwarry, SA Urbenville, NSW Urbenville, Rockley, NSW Rockley, Whiporie, NSW Whiporie, Whiporie, NSW Whiporie, Rosewood, NSW Rosewood, Vittoria, NSW Vittoria, Lower Lower Beulah, TAS Moss Vale, MossNSW Vale, Vittoria, NSW Vittoria, Bombala, NSW NSW Neville, Tumbarumba, NSW South Yarra, VIC South Yarra, NSW Rosewood, NSW Vittoria, Location Moss Vale, MossNSW Vale, MossNSW Vale, c Pr Pr Pt Pr Pr Pcxe Pr Pt Pr Pp Pcxe Pr Pr Pr Pr Pr Pr Pr Pr Host Pr Pr Pr Pr Pr G. fragrans G. huntii ips O. ips O. Lodged as Lodged sp. Leptographium angusticollis O. angusticollis O.

M ™ H H H H H H H a,b VPRI43764 VPRI43761 DAR84707 VPRI43760 VPRI43839 VPRI43836 DAR84705 VPRI43759 VPRI43838 DAR84692 VPRI43835 VPRI43523 VPRI43843 VPRI43758 VPRI43834 VPRI43845 VPRI43763 VPRI43756 VPRI22395 DAR84817 VPRI43837 VPRI43765 VPRI43766 VPRI43762 Isolate Isolate ­ number (VPRI43528) (VPRI43530) (VPRI43529) (VPRI43861) - Ophiostoma angusticollis Graphilbum Graphilbum fragrans Grosmannia huntii Grosmannia G. ipis-grandi - collis O. ips O. Gro. radiaticola Gro. O. fasciatum O. G. cf. rectangulo G. cf. sporium sp.Ceratocystiopsis Species Representative isolates of ophiostomatoid fungi associated with pine and bark associated beetles obtained during study fungi the current of ophiostomatoid isolates Representative 7 2 5 3 9 6 8 4 Ophiostomatales 1 1 Table Taxon Trollip et al. IMA Fungus (2021) 12:24 Page 7 of 27 MW075145 MW075144 MW075143 MW075142 MW075155 MW075141 MW075154 MW075140 MW075153 MW075139 MW075152 MW075138 MW075151 MW075137 MW075146 MW075147 MW075148 MW075149 MW075150 MW075134 MW075135 MW075136 CAL MW066430 MW066429 MW066428 MW066427 MW066440 MW066426 MW066439 MW066425 MW066438 MW066424 MW066437 MW066423 MW066436 MW066422 MW066431 MW066432 MW066433 MW066434 MW066435 MW066419 MW066420 MW066421 TEF MW066384 MW066383 MW066382 MW066381 MW066394 MW066380 MW066393 MW066379 MW066392 MW066378 MW066391 MW066377 MW066390 MW066376 MW066385 MW066386 MW066387 MW066388 MW066389 MW066373 MW066374 MW066375 BT d MW046142 MW046141 MW046140 MW046139 MW046152 MW046138 MW046151 MW046137 MW046150 MW046136 MW046149 MW046135 MW046148 MW046134 MW046143 MW046144 MW046145 MW046146 MW046147 MW046131 MW046132 MW046133 LSU ­ accessions MW046096 MW046095 MW046094 MW046093 MW046106 MW046092 MW046105 MW046091 MW046104 MW046090 MW046103 MW046089 MW046102 MW046088 MW046097 MW046098 MW046099 MW046100 MW046101 MW046085 MW046086 MW046087 GenBank ITS 2019 2019 2019 2020 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2018 2013 2013 Year Sargeant, D Sargeant, Sargeant, D Sargeant, Sargeant, D Sargeant, Wotherspoon, K., Wotherspoon, Ramsden, N Carnegie, A. J., A. J., Carnegie, C Trollip, Carnegie, A. J., A. J., Carnegie, C Trollip, Sargeant, D Sargeant, Sargeant, D Sargeant, Sargeant, D Sargeant, Sargeant, D Sargeant, Sargeant, D Sargeant, Sargeant, D Sargeant, Smith, D., Trollip, C Trollip, Smith, D., Carnegie, A. J Carnegie, Carnegie, A. J Carnegie, A. J Carnegie, A. J Carnegie, A. J Carnegie, A. J Carnegie, Smith, D Smith, D Smith, D Collector Bathurst, NSW Batlow, NSW Batlow, Urbenville, NSW Urbenville, Lower Lower Beulah, TAS Whiporie, NSW Whiporie, Urbenville, NSW Urbenville, Whiporie, NSW Whiporie, Batlow, NSW Batlow, Urbenville, NSW Urbenville, Tumut, NSW Tumut, Urbenville, NSW Urbenville, Urbenville, NSW Urbenville, Benalla, VIC Moss Vale, MossNSW Vale, Batlow, NSW Batlow, NSW Whiporie, NSW Whiporie, NSW Whiporie, NSW Whiporie, Chiltern, VIC VIC Shelley, VIC Shelley, Location isolates used for morphological study morphological used for isolates c = Pr Pr Pcxe Pr Pe Pr Pcxe Pr Pcxe Pr Pcxe Pcxe Pr Pr Pr Pcxe Pcxe Pcxe Pcxe Host Pr Pr Pr ex-holotype isolate; M ex-holotype isolate; = S. cf. abietina S. cf. sp . Sporothrix nigrocarpum O. nigrocarpum O. sp . Sporothrix Lodged as Lodged Ophiostoma sp. Ophiostoma sp. ips O.

H H H H H H H H a,b DAR84706 VPRI43755 VPRI43754 VPRI43720 VPRI43846 VPRI43844 VPRI43851 VPRI43752 VPRI43743 VPRI43751 VPRI43738 VPRI43749 VPRI43734 DAR84897 DAR84898 DAR84899 DAR84900 VPRI43721 VPRI42284 VPRI42255 VPRI43731 VPRI43316 Isolate Isolate ­ number (VPRI43531) (VPRI43867) (VPRI43868) (VPRI43869) (VPRI43870) - S. pseudoabietina S. cf. nigrograna S. cf. - euska Sporothrix diensis Rafaelea deltoide Rafaelea ospora O. pallidulum O. Graphium sp. Graphium Species (continued) Isolate obtained from plant pathogen reference collection; T collection; reference pathogen plant obtained from Isolate = H ITS, The internal transcribed spacer; LSU, The large ribosomal subunit (28S); BT, β-tubulin; TEF, Translation elongation factor elongation 1-α; CAL, Calmodulin Translation TEF, β-tubulin; ribosomal subunit (28S); BT, large The spacer; transcribed LSU, internal The ITS, The Victorian Plant Pathology Herbarium (VPRI); The NSW Plant Pathology and Mycology Herbarium (DAR) and Mycology Herbarium Pathology (VPRI); NSW Plant The Pathology Plant Victorian The elliottii; Pt, Pinus taeda; Pe, P. elliottii P. taeda; Pe, x elliottii; Pt, Pinus caribaea P. Pcxe, ponderosa; P. ; Pp, radiata Pinus Host: Pr, 14 13 12 11 10 Microascales 15 1 Table Taxon taxa novel in bold type species name printed represent The a b c d Trollip et al. IMA Fungus (2021) 12:24 Page 8 of 27

O. undulatum CMW19396 NR_137576 T O. quercus CMW2467 AY466626 T ITS O. tsotsi CMW3117 NR_137714 T O. tasmaniense CMW29088 NR_137753 T O. australiae CMW6606 EF408603 T O. catonianum C1084 AF198243 T 73 O. himal-ulmi C1183 AF198233 85 O. novo-ulmi C510 AF198236 70 97 O. ulmi C1182 AF198232 T O. karelicum CMW23099 EU443762 T O. patagonicum_KT362244 T O. tetropii CBS428.94 AY934524 O. kryptum DAOM229701 AY304436 T O. wuyingense T MH144061 O. pseudotsugae 92-634/302/6 AY542502 O. minus AU58.4 AF234834 O. piliferum CBS129.32 AF221070 O. rachisporum CMW23272 HM031490 T O. nitidum CMW38907 NR_147584 T O. canum CBS133.51 HM031489 T O. qinghaiense CMW38902 NR_147587 Ophiostoma s.str. O. xinganense MK748186 O. piceae C1087 AF198226 T O. setosum CMW27833 KU184451 T O. floccosum C1086 AF198231 T O. kunlunense CMW41927 MH121648 T 100 O. longiconidiatum CMW17574 EF408558 T 99 96 O. pluriannulatum MUCL18372 AY934517 T O. multiannulatum AY934512 T O. shangrilae CMW38901 NR_147588 T O. pseudocatenulatum CMW43103 NR_147581 T O. tapionis CMW23265 NR_137625 T 100 O. jiamusiensis CMW40512 MH144064 T O. japonicum CMW44592 MH144086 DAR84692 Taxon 9 77 O. ips CMW7075 AY546704 T 98 O. adjuncti CMW135 AY546696 T CMW30681 MT637227 T 98 O. gilletteae 100 O. pulvinisporum CMW9022 AY546714 T O. ips complex O. fuscum CMW23196 HM031504 T 100 O. pseudobicolor CFCC52683 MK748188 T O. bicolor CBS492.7 DQ268604 T O. montium CMW13221 AY546711 O. manchongi CMW41954 MH121662 T S. prolifera CBS251.88 KX590829 T 72 S. lunata CMW10563 AY280485 T 89 S. fusiformis CMW9968 AY280481 T VPRI43754 Taxon 12 S. euskadiensis CMW27318 DQ674369 T S. gossypina ATCC18999 KX590819 T S. cantabriensis CMW39766 KF951554 T S. gossypina S. rossii CBS116.78 KX590815 T DAR84706 Taxon 14 complex S. pseudoabietina CFCC52626 MH555896 T 98 S. abietina CMW22310 AF484453 T 'S. curviconia 2' CMW17163 KX590836 S. variecibatus CMW23051 DQ821568 T 80 S. uta CMW40316 KU595577 P S. aurorae CMW19362 DQ396796 T S. eucastanea CBS424.77 KX590814 T 79 S. protearum CMW1107 DQ316201 99 100 S. africana CMW823 DQ316199 S. zambiensis CMW28604 EU660453 T S. splendens CMW897 DQ316205 S. narcissi CBS138.50 NR_147512 T 91 S. stenoceras CMW3202 AF484462 T 100 S. pallida CBS131.56 EF127880 T S. stylites CMW14543 EF127883 T S. humicola CMW7618 AF484472 T S. mexicana CBS120341 KX590841 T 93 S. chilensis CBS139891 KP711811 T 98 S. palmiculminata CMW20677 DQ316191 T Sporothrix 84 S. gemella CMW23057 DQ821560 T 99 S. protea-sedis CMW28601 EU660449 T 97 S. cabralii CMW38098 KT362256 T S. itsvo CMW40370 KX590840 T 99 S. aemulophila CMW40381 KT192603 T 92 S. rapaneae CMW40369 KU595583 T S. candida CMW26484 HM051409 T S. 'inflata 2' CMW12526 AY495425 S. dimorphospora CMW12529 AY495428 T S. polyporicola CBS669.88 KX590827 T S. inflata CMW12527 AY495426 T S. guttuliformis CBS437.76 KX590839 T 88 S. dentifunda CMW13016 AY495434 T 87 CMW17210 AB128012 T 94 S. luriei 98 S. brasiliensis CMW29127 KX590832 T S. schenckii CBS359.36 KX590842 T S. globosa CBS120340 KX590838 T S. phasma CMW20676 DQ316219 T S. macroconidia CFCC52628 MH555898 T CMW14487 KX590825 T 87 S. nigrograna 100 VPRI43755 Taxon 13 S. zhejiangensis CFCC52165 KY094071 T A S. nebularis CMW27319 KX590824 T Group G S. curviconia CBS959.73 KX590835 T 92 S. epigloea CBS573.63 KX590817 T 72 80 S. thermarum CMW38930 KR051115 T S. bragantina CBS474.91 FN546965 T S. eucalyptigena CBS139899 KR476721 T 82 O. valdivianum CBS454.83 KX590830 T S. fumea CMW26813 HM051412 T S. dombeyi CBS455.83 KX590826 T 100 VPRI43846 Taxon 10 74 O. pallidulum CMW23278 HM031510 T 99 O. saponiodorum CMW34945 HM031507 T O. lotiforme CFCC52691 MK748185 T O. acarorum CMW41850 MG205657 T 100 O. massoniana CFCC51648 KY094067 T O. jilinense CMW40491 MH144094 T S. brunneoviolacea CMW37443 FN546959 T B Ophiostoma s.lat. 100 O. sejunctum Ophi1A AY934519 T Group A VPRI43764 Taxon 7 100 O. denticulatum ATCC38087 KX590816 T O. angusticollis CBS186.86 AY924383 100 O. coronatum CBS497.77 AY924385 100 O. tenellum CBS189.86 AY934523 USA O. nigricarpum CMW650 AY280489 T 100 O. fasciatum UM56 EU913720 VPRI43845 Taxon 8 85 G. sexdentatum CBS145814 MN548915 T 70 G. crescericum CBS130864 MN548925 T 100 G. furuicola CBS145813 MN548907 T VPRI43761 Taxon 3 Graphilbum sp. C2316 GU129997 84 G. interstitialis CBS145816 MN548909 T 100 G. kesiyae CMW41729 MG205669 T 98 G. fragrans CBS279.54 AF198248 T 88 95 98 DAR84707 Taxon 2 G. microcarpum YCC459 AB506676 G. aff. fragrans C1496 DQ062977 G. sparsum CBS405.77 MN548924 T 100 98 G. anningense CFCC52631 MH555903 T Graphilbum G. puerense CMW41942 MG205671 T G. acuminatum CBS145828 MN548902 T 94 VPRI43763 Taxon 4 G. cf. rectangulosporium C2300 GU393357 G. cf. rectangulosporium C2477 GU129987 G. tsugae UAMH11701 KJ661745 T 100 G. carpaticum CBS145835 KY568116 T G. rectangulosporium TFM FPH7756 AB242825 T G. curvidentis CBS145832 KY568111 T G. gorcense CBS146203 MN548919 T G. nigrum CBS163.61 MH858010 100 Fragosphaeria purpurea AB278192 Fragosphaeria reniformis AB278193

0.2 Fig. 1 ML phylogeny of the ITS region for isolates residing in Ophiostoma, Sporothrix. and Graphilbum. Sequences generated in this study are printed in bold. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. T ex-type ≥ ≥ = cultures. A Group name as described by de Beer et al. (2016). B Group name as described by Chang et al. (2017) Trollip et al. IMA Fungus (2021) 12:24 Page 9 of 27

L. raffai CMW34451 MT637211 T 94 Gro. cucullata CBS218.83 AJ538335 T 100 LSU Gro. olivaceapini MUCL 18368 AJ538336 T Gro. olivaceae CBS138.51 AJ538337 T L. lundbergii CMW17264 DQ062068 T Gro. clavigera ATCC18086 AY544613 T L. shansheni CMW44462 MH144097 T L. pinicola CMW2398 DQ062060 T L. wingfieldii CMW2096 AY553398 T Gro. koreana MCC206 AB222065 T L. albopini CMW26 AF343695 L. procerum CMW13 JF279977 T L. terebrantis CBS337.70 JF798477 L. bhutanense CMW18649 EU650187 T L. gracile CMW12398 HQ406840 T L. Lundbergii and L. truncatum CMW28 DQ062052 T L. clavigera complexes L. conjunctum CMW12449 HQ406832 T L. pyrinum CMW169 DQ062072 T L. longiclavatum SL-Kw1436 AY816686 T Gro. yunnanensis CMW5304 AY553415 T L. manifestum CMW12436 HQ406839 T VPRI22395 Taxon 5 Gro. robusta CMW668 AY544619 T Gro. huntii UAMH4997 AY544617 Gro. aurea ATCC16936 AY544610 T Gro. piceaperda CMW660 DQ294392 81 L. zhangii CXY1552 KM236108 T Gro. laricis CBS633.94 NG_064170 T Leptographium s.l. L. aenigmaticum CMW2199 AY553389 T Gro. alacris CBS591.79 JN135313 T Gro. serpens CBS141.36 JN135314 L. yamaokae CBS129732 JN135315 T L. wageneri var. pseudotsugae CMW154 AF343706 L. wageneri var. ponderosum CMW2812 AF343708 L. douglasii CMW2078 AY553381 T L. neomexicanum CMW2079 AY553382 L. reconditum CMW15 AF343690 L. wageneri var. wageneri CMW402 AF343707 L. alethinum CMW3766 AF343685 T L. abietinum C1883 EU177472 74 Gro. penicillata CMW470 DQ294385 T 98 Gro. abiocarpa MUCL18351 AJ538339 L. pistaciae CMW12499 HQ406846 T L. pineti CMW3831 DQ062076 T L. koraiensis CMW44461 MH144096 T L. owenii CMW34448 KF515912 Gro. galeiformis CMW5290 DQ294383 Gro. galeiformis complex VPRI43523 Taxon 6 90 Gro. radiaticola KUC2036 AY744551 T L. seifertii CMW34620 KF515911 T L. taigense ES15_2 JF279980 T R. montetyi MPFN 308 AB496453 T 90 R. quercus-mongolicae RQM04 KF513155 85 R. quercivora MAFF410918 AB496454 T 95 100 R. sulphurea C593 EU177463 T R. amasae CBS116694 EU984295 E. vermicola CNU 120806 EU627684 99 Raffaelea sp. TR25 EU984281 90 R. brunnea C2229 EU177457 R. lauricola C2339 KF515710 T 88 R. seticolle CBS634.66 AF135578 R. tritirachium C2222 EU177464 T R. sulcati C2234 EU177462 T R. subalba C2401 EU177443 T R. santoroi CBS399.67 EU984302 R. albimanens C2223 EU177452 R. canadensis C2233 EU177458 100 R. gnathotrichi C2219 EU177460 T 93 R. arxii C2218 EU177454 T Raffaelea s.str. 79 R. scolytodis CCF3572 AM267270 R. rapaneae CMW40358 KT182934 C2225 EU177453 T 73 86 R. ambrosiae R. subfusca C2335 EU177450 T R. fusca C2394 EU177449 T 90 VPRI43720 Taxon 11 91 R. deltoideospora WIN(M)71-26 (Hausner 1993) R. vaginata CMW40365 KT182932 T 93 F. purpurea CBS133.34 AF096191 F. reniformis CBS134.34 AB189155 Fragosphaeria 70 H. crousii CMW37531 KX396548 H. hibbettii CMW37663 KX396547 80 H. lignivora CMW18600 EF139119 T Hawksworthiomyces H. taylorii CMW20741 KX396546 79 Cop. brevicomi UM1452 EU913683 T Cop. ranaculosa CMW13940 DQ294357 Cop. collifera CBS126.89 EU913681 Ceratocystiopsis sp. 3 SWT1 EU913676 79 Cop. manitobensis CW13792 DQ294358 Ceratocystiopsis sp. 1 WY13TX1-3 EU913667 VPRI43766 Taxon 1 Cop. pallidobrunnea UM51 EU913682 Cop. cf. pallidobrunnea CXY2015 MN892641 Cop. concentrica WIN(M)71-07 AF135571 Ceratocystiopsis A 90 Ceratocystiopsis sp. 2 YCC329 EU913671 Cop. minuta UM1532 EU913656 T 98 Cop. minuta-bicolor CMW1018 DQ294359 Cop. minuta sp. 3 CBS463.77 EU913645 71 Cop. parva UM59 L05805 94 Cop. minima CMW162 DQ294361 Cop. minuta sp. 2 CBS116963 EU913655 UM110 EU913679 98 Cop. rollhanseniana Cop. neglecta CBS100596 MH874319 T Cop. longispora UM48 EU913684 100 Neurospora crassa AF286411 100 Sordaria fimicola AY545728 Podospora decipiens AY780073

0.04 Fig. 2 ML phylogeny of LSU region for isolates residing in Ceratocystiopsis, Leptographium s. lat. and Rafaelea. Sequences generated in this study are printed in bold. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. T ex-type ≥ ≥ = cultures. A Taxon names as described by de Beer and Wingfeld (2013) Trollip et al. IMA Fungus (2021) 12:24 Page 10 of 27

YCC513 EU913712 JPN UM110 EU913758 NOR ITS Cop. rollhanseniana 98 BT YCC294 EU913691 JPN UM113 EU913757 NOR

94 YCC330 EU913710 JPN Ceratocystiopsis sp. 2A CBS116795 EU913727 POL YCC329 EU913711 JPN CBS116963 EU913735 POL CBS117566 EU913694 GBR UM1534 EU913738 POL UM1534 EU913699 POL YCC513 EU913751 JPN 95 CBS116963 EU913696 POL Cop. minuta sp. 2A YCC330 EU913749 JPN CBS116795 EU913688 POL YCC329 EU913750 JPN UM1501 EU913703 CAN YCC294 EU913730 JPN UM1462 EU913704 USA CBS117566 EU913733 GBR Cop. minima 93 UM235 EU913702 CAN CBS117562 EU913728 AUT UM85 EU913701 CAN UM1535 EU913739 POL

CBS145.59 EU913687 USA Ceratocystiopsis sp. UM1532 EU913736 POL T 87 CBS100596 MH862711 T Cop. neglecta CBS116796 EU913734 POL YCC251 EU913692 JPN UM1533 EU913737 POL

YCC139 EU913693 JPN YCC251 EU913731 JPN 95 CBS116796 EU913695 POL YCC139 EU913732 JPN Cop. minuta UM1533 EU913698 POL UM1501 EU913742 CAN 82 UM1535 EU913700 POL UM1462 EU913743 USA 86 UM1532 EU913697 POL T UM85 EU913740 CAN 99 UM214 EU913715 CAN UM235 EU913741 CAN 97 Cop. manitobensis 100 UM237 EU913714 CAN T CBS145.59 EU913726 USA Cop. sp3i (SWT1) EU913716 CAN VPRI43766 A 100 Ceratocystiopsis sp. 3 Cop. sp3ii (SWT3) EU913717 CAN VPRI43836 97 VPRI43834 VPRI43835

99 VPRI43766 Taxon 1: VPRI43834 70 Ceratocystiopsis sp. Cop. sp. 1ii (WY21TX1-2) EU913747 CAN VPRI43836 100 VPRI43835 Cop. sp. 1iii (WY21TX2-2) EU913748 CAN 100 Cop. sp. 1iii (WY21TX2-2) EU913709 CAN Cop. sp.1i (WY13TX1-3) EU913746 CAN 98 A UM237 EU913753 CAN T 83 Cop. sp. 1ii (WY21TX1-2) EU913708 CAN Ceratocystiopsis sp. 1 100 Cop. sp.1i (WY13TX1-3) EU913707 CAN UM214 EU913754 CAN 83 81 CBS216.88 EU913713 USA T Cop. ranaculosa Cop. sp3ii (SWT3) EU913756 CAN 99 88 CBS126.89 MH862160 MEX Cop. collifera Cop. sp3i (SWT1) EU913755 CAN UM1452 EU913722 USA T Cop. brevicomi UM1452 EU913761 USA T 96 94 UM480 EU913705 CAN CBS216.88 EU913752 USA T Cop. minuta-bicolor 100 UM844 EU913706 USA UM480 EU913744 CAN 100 CBS463.77 EU913686 USA Cop. minuta sp. 3A UM844 EU913745 USA 72 UM48 EU913723 CAN Cop. longispora CBS 463.77 EU913725 USA

100 Fragosphaeria purpurea CBS133.34 AB278192 UM48 EU913762 CAN Fragosphaeria reniformis CBS134.34 AB278193 F. purpurea CBS133.34 G

0.2 0.2 Fig. 3 Phylogenetic analysis of isolates residing in Ceratocystiopsis. Sequences generated in this study are printed in bold. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. T ex-type isolates, G sequence retrieved from ≥ ≥ = = genome. ACurrent taxon name as described by De Beer and Wingfeld (2013) brevicomis (Fig. 2). Analysis of ITS and BT regions isolates collected during this survey forming Taxon 2 (Fig. 3) supports this placement and illustrates that the (Table 1) were confrmed as Graphilbum fragrans (Figs. 1, Australian isolates are most closely related to a previously 4). Taxon 3 and 4 (which comprised of four and two iso- undescribed taxon reported as Ceratocystiopsis species lates, respectively; Table 1) were both preliminarily iden- 1 (Cop. minuta-like) from Canada (Plattner et al. 2009). tifed as G. cf. rectangulosporium isolates, with BLASTn Multi-locus analysis suggests the isolates of Ceratocysti- searches suggesting an afliation to previously reported opsis sp. (Taxon 1) represent a novel lineage. isolates from the USA, China, and Europe. Further analy- Tree taxa (Taxa 2, 3 and 4) residing within Graphil- sis of the BT, TEF and CAL regions (Figs. 1, 4, Additional bum were collected during this study (Fig. 1). Reference fle 4: Fig. S1) revealed that Taxon 3 represented a phy- collection isolate DAR84707 and two representative logenetically distinct lineage, forming part of a species Trollip et al. IMA Fungus (2021) 12:24 Page 11 of 27

CBS145814 MN548915 NOR T G. sexdentatum 100 CBS130864 MN548978 ESP T CBS145815 MN548913 NOR 100 CBS130866 MN548979 ESP ITS 74 TEF CBS130864 MN548925 ESP T 90 CBS145822 MN548984 NOR G. crescericum 99 CBS130866 MN548926 ESP 100 CBS145820 MN548980 NOR 99 G. crescericum CBS145814 MN548968 NOR T 99 CBS145822 MN548931 NOR 100 CBS145815 MN548967 NOR G. sexdentatum CBS145820 MN548927 NOR 82 CBS145813 MN548961 NOR 99 CBS145836 MN548960 NOR T G. furuicola CBS145836 MN548906 NOR T 100 G. furuicola CBS145816 MN548963 RUS T CBS145813 MN548907 NOR 98 CBS145818 MN548965 RUS G. interstitiale C2316 GU129997 USA Graphilbum sp. VPRI43761 Taxon 3: VPRI43759 VPRI43762 T Graphilbum ipis- 100 100 VPRI43761 Taxon 3: VPRI43762 T grandicollis, sp. nov. Graphilbum ipis- VPRI43760 VPRI43760 grandicollis, sp. nov. 100 100 VPRI43843 Taxon 4: VPRI43759 VPRI43763 Graphilbum sp. 100 CBS145828 MN548952 POL T 84 CMW41657 MG205668 CHN G. kesiyae CBS145809 MN548947 NOR G. acuminatum CMW41729 MG205669 CHN T CBS145833 MN548953 POL 90 G. carpaticum CBS145816 MN548909 RUS T 100 CBS145835 MN548956 POL T G. interstitiale CBS145818 MN548911 RUS 76 99 KFL49aKFJD KY568501 POL CBS145832 KY568502 POL T G. curvidentis VPRI43758 100 CBS146201 MN548970 POL Taxon 2: CMW12376 FJ434976 CHN CBS146203 MN548972 POL T G. gorcense G. fragrans CBS279.54 AF198248 SWE T 100 CBS405.77 MN548977 USA T CMW54772 MN548976 NOR G. sparsum YCC612 GU134170 JPN G. microcarpum DAR84707 94aLMD KY568108 POL 95 CMW44159 KY568497 POL CMW11778 MG205667 CHN CMW43200 KY568494 POL Taxon 2: G. fragrans 99 CMW43200 KY568103 POL 100 VPRI43758 VPRI43756 DAR84707 G. fragrans 94aLMD KY568499 POL G. microcarpum 89 99 C990 DQ062976 NZL H. lignivorus CMW18600 G T CMW44159 KY568106 POL 0.4 100 VPRI43756 YCC459 AB506676 JPN G. microcarpum VPRI43759 BT Taxon 3: C1496 DQ062977 AUS G. aff. fragrans 97 VPRI43761 Graphilbum ipis- VPRI43760 grandicollis, sp. nov. 100 CBS405.77 MN548924 USA T 88 G. sparsum VPRI43762 T CMW54772 MN548923 NOR 97 CMW41729 MG205713 CHN T 100 G. kesiyae CBS146203 MN548919 POL T CMW41657 MG205714 CHN G. gorcense 100 CBS146201 MN548943 POL CBS146201 MN548917 POL G. gorcense CBS146203 MN548945 POL T TFM:FPH7756 AB242825 JPN T G. rectangulosporium CBS145833 KY568224 POL G. carpaticum 90 CBS163.61 MH858010 USA G. nigrum 100 CBS145835 KY568226 POL T CBS145835 KY568116 POL T 100 VPRI43843 Taxon 4: G. carpaticum Graphilbum sp. CBS145831 MN548905 POL VPRI43763 87 CFCC52632 MH683596 CHN 70 UAMH11701 KJ661745 CAN T G. tsugae CFCC52631 MH683595 CHN T G. anningense 98 99 KFL49aKFJD KY568110 POL CBS145809 MN548933 NOR G. curvidentis CBS145828 MN548938 POL T G. acuminatum CBS145832 KY568111 POL T 100 CMW41942 MG205719 CHN T G. puerense 91 VPRI43763 CMW41667 MG205716 CHN 99 CBS405.77 MN548946 USA T G. sparsum 92 VPRI43843 Taxon 4: C2300 GU393357 USA Graphilbum cf. rectangulosporium CMW11778 MG205710 CHN 99 C2477 GU129987 USA VPRI43758 85 VPRI43756 94 CBS145828 MN548902 POL T DAR84707 Taxon 2: G. acuminatum G. fragrans CBS145809 MN548897 NOR 85 CMW44159 KY568220 POL CMW41942 MG205671 CHN T CMW43200 KY568217 POL 73 G. puerense 76 CMW12376 FJ455600 CHN CMW41667 MG205670 CHN 94aLMD KY568222 POL G. microcarpum CFCC52632 MH555901 CHN 100 H. lignivorus CMW18600 EF139104 T G. anningense 99 CFCC52631 MH555903 CHN T H. lignivorus CMW18598 EF139102 CMW18598 EF127888 100 Hawksworthiomyces lignivorus 0.2 Hawksworthiomyces lignivorus CMW18600 EF127890 T x2

0.08 Fig. 4 Phylogenetic analysis of isolates residing in Graphilbum. Sequences generated in this study are printed in bold with reference collection isolates coloured purple. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. ≥ ≥ T ex-type cultures, G sequence retrieved from genome = = Trollip et al. IMA Fungus (2021) 12:24 Page 12 of 27

complex including G. crescericum, G. furuicola, G. inter- Rafaelea s. str. and the delimitation of this species as R. stitiale, G. kesiyae, and G. sexdentatum. Tis new spe- deltoideospora (Additional fle 5: Fig. S2). cies is described below. Taxon 4 formed a well-supported Tree Sporothrix taxa were obtained during this study clade with two previously undescribed Graphilbum iso- (Taxa 12, 13, and 14; Table 1). Taxon 12 and Taxon 14 lates reported from the USA (Fig. 4). both grouped within the S. gossypina complex (Fig. 1). Within Leptographium s.l., two taxa (Taxa 5 and 6) While analysis of the ITS region gave limited resolu- were collected (Table 1; Fig. 2). Taxon 5 comprised of tion within the S. gossypina complex (Fig. 1), analysis of two reference collection isolates, as well as a single isolate BT and CAL was able to distinguish between the closely collected during this survey. Phylogenetic analysis con- related species (Fig. 7a). Te single isolate of Taxon 12 frmed Taxon 5 as Grosmannia huntii (Figs. 2, 5a). Taxon was identifed as S. euskadiensis (Romón et al. 2014). 6 comprised of three representative isolates which fell Taxon 14 included all reference collection Sporothrix into a clade within the Gro. galeiformis species complex isolates as well as 5 additional representative isolates col- (Fig. 2). Analysis of BT and TEF confrmed the delinea- lected during this study (Table 1). ML analysis placed tion of Taxon 6 as the species Gro. radiaticola (Fig. 5b). isolates of Taxon 14 in a well-supported clade alongside Taxa 7 to 10 resided within Ophiostoma s. lat. with the type strain of S. pseudoabietina (Fig. 7a). Taxon 13 Taxon 9 the only one belonging to a well-recognised comprised of a single isolate collected in this study, with species complex (Fig. 1). Taxon 9 included the reference the ITS region placing the taxon among species belong- collection isolates DAR84692, DAR84817, VPRI42284, ing to a group within Sporothrix recently referred to as VPRI42255 and VPRI43316, along with fve additional “Group G” (De Beer et al. 2016) (Fig. 1). Taxon 13 shared isolates collected during this study (Table 1). Analysis an almost identical ITS sequence with the type sequences of the ITS region identifed Taxon 9 isolates as Ophios- for S. nigrograna and S. zhejiangensis (Fig. 7b). While toma ips (Fig. 1). Despite incongruence with regards to analysis of the BT region was unable to clearly distin- the delimitation of O. ips using ITS alone, BT analysis guish between S. nebularis and S. zhejiangensis (Fig. 7b), confrmed little variation between the Australian isolates, analysis of CAL did show good support for the distinc- and established a clear grouping with several isolates tion of Taxon 13 from S. nebularis (Additional fle 6: Fig. recently confrmed as O. ips (Fig. 6a). Taxa 7, 8 and 10 all S3). A lack of available molecular data for these species grouped peripherally to Ophiostoma s. str. and are regu- limited further phylogenetic comparisons and thus, the larly referred to as Group A/Lineage G (Chang et al. 2017; placement of the Australian taxon. Wang et al. 2020). Taxon 7 included two reference col- lection isolates forming a lineage along with two species, Microascales namely O. angusticollis and O. denticulatum (Fig. 6b). Te single isolate (Taxon 15) residing in Microascales Te currently available molecular data for reference was identifed as a Graphium species (Fig. 8). Analysis specimens within this lineage is lacking for appropri- of ITS and TEF regions revealed that this isolate resides ate taxonomic comparison, and thus clear diferentia- closely to the species of Gra. basitruncatum and Gra. tion between these species is limited. For now, Taxon 7 carbonarium (Fig. 8). While this taxon may represent a is referred to as O. angusticollis. Taxon 8 included a sin- novel lineage, we have chosen not to formally describe it gle isolate collected during this study (Table 1), with ITS until additional specimens and/or reference material can and BT analyses identifying this taxon as O. fasciatum be examined. Taxon 15 is thus referred to as a Graphium (Figs. 1, 6b). Taxon 10 included a single strain preliminar- species. ily identifed as O. pallidulum (Fig. 1). BT analysis further confrmed this identifcation (Fig. 6b). Species of Ophiostomatales and Microascales associated Te single isolate of Taxon 11 grouped within Rafae- with Australian Pinus , verifed by DNA sequence data lea s. str. (Fig. 2). Analysis of the LSU sequence showed Revision of the literature, as well as the database search, that the Australian isolate forms a well-supported line- allowed for the comparison of our results to the historical age with Rafaelea deltoideospora (Fig. 2). Analysis of the records of ophiostomatoid fungi associated with Pinus in ITS region further validated Taxon 11’s placement within Australia (Table 2). While several taxa identifed in the

(See fgure on next page.) Fig. 5 Phylogenetic analysis of BT and TEF for Leptographium s.lat. a. Isolates residing in the L. lundbergii and Gro. huntii species complexes. b. Isolates residing in the Gro. galeiformis species complex. Sequences generated in this study are printed in bold, with reference collection isolates coloured blue. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. T ex-type ≥ ≥ = cultures Trollip et al. IMA Fungus (2021) 12:24 Page 13 of 27

a BT CMW2399 DQ061995 CMW2399 DQ062028 CAN 96 TEF L. pinicola CMW2398 DQ061994 CAN T CMW2398 DQ062027 CAN T 91 MUCL46362 EU502811 CHN MUCL46362 EU502826 CHN 83 Gro. koreana 90 KUC2102 AY707184 KOR T KUC2102 EU502827 KOR T 100 CMW2850 DQ061991 ZAF T CMW1873 DQ062030 JPN L. truncatum 98 CMW30 DQ061988 NZL CMW30 DQ062021 NZL 95 99 99 CMW12473 HQ406879 CHN T CMW2850 DQ062024 ZAF T 71 L. conjunctum CMW12452 HQ406881 CHN CMW12473 HQ406855 CHN T 100

CMW5304 AY707192 CHN T CMW12449 HQ406856 CHN 100 73 Gro. yunnanense CMW5152 DQ062007 CHN CMW5304 AY536209 CHN T CMW39396 KF515863 USA CMW5152 DQ062040 CHN 100 CMW455 KF779134 USA UAMH4997 AY544640 CAN UAMH 4997 AY349023 CAN CMW39396 KF515890 USA 100 82 CMW30682 KF515864 USA CMW30682 KF515889 USA CMW2868 DQ354933 Taxon 5: VPRI43530 99 77 Gro. huntii 100 CMW2824 DQ354932 VPRI43837 VPRI43530 CMW2868 DQ354938 100 100 97 VPRI43837 CMW2824 DQ354937 VPRI22395 VPRI22395 UAMH4825 AY544625 CAN UAMH4825 AY544641 CAN

100 L. lundbergii CMW17264 DQ062002 NT L. lundbergii CMW2190 DQ062033 100 100 L. lundbergii CMW2190 DQ062000 L. lundbergii CMW17264 DQ062035 T 100 L. shansheni CMW44462 MH124293 T L. shansheni CMW44462 MH124373 T

0.02 0.02

b BT 98 CMW9490 JF280015 MEX Grosmannia sp. CMW9490 JF280053 MEX TEF CMW34619 MT637203 USA T L. gordonii CMW34619 MT637207 USA T 70 CMW34479 MT637202 USA T 87 99 CMW34393 KF515881 USA 81 CMW34581 KF515856 USA L. doddsii CMW34479 MT637205 USA T 79 CMW34393 KF515855 USA CMW34581 KF515882 USA CMW9494 JF280011 ZAF CMW34448 KF515884 USA T CMW12323 MG205723 CHN 96 CMW9988 JF280012 SWE CMW34620 KF515885 USA T KUC2036 AY744562 KOR T CMW39393 KF515886 USA VPRI43523 CMW578 JF280054 ZAF VPRI43838 Taxon 6: Gro. radiaticola VPRI43523 MUCL46364 EU502815 CHN CMW9494 JF280055 ZAF 99 MUCL46457 EU502817 CHN CMW12323 MG205764 CHN 94 CMW9478 JF280013 CHL 80 87 CMW9482 JF280014 CHL VPRI43839 72 VPRI43838 VPRI43839 86 CMW578 JF280010 ZAF CMW9482 JF280058 CHL 78 93 100 CMW5290 JF280009 UK T CMW9478 JF280057 CHL Gro. galeiformis CMW23282 JF280007 FIN CMW9988 JF280056 SWE CMW44461 MH124292 CHN T L. koraiensis CMW12686 JF280052 AUT 100 CMW12686 JF280006 AUT Grosmannia sp. CMW5290 JF280060 UK T CMW34448 KF515860 USA T L. owenii CMW23282 JF280059 FIN 78 CMW39393 KF515859 USA L. seifertii CMW44461 MH124372 CHN T CMW34620 KF515858 USA T L. olivaceapini CMW116 JF280038 T L. raffai CMW34451 MT637204 T L. olivaceapini CMW116 JF279995 T L. olivaceum CBS138.51 JF280046 T 100 L. cucullatum CMW1141 JF280000 T L. cucullatum CMW1141 JF280039 T 100 L. olivaceum CBS138.51 JF279997 T L. raffai CMW34451 MT637206 T

0.05 0.09 Fig. 5 (See legend on previous page.) Trollip et al. IMA Fungus (2021) 12:24 Page 14 of 27

89 CMW30680 KF515894 USA CMW30680 KF515870 USA 100 a ITS O. gilletteae 83 BT 84 CMW30681 MT637227 USA T CMW30681 MT637200 USA T 100 CMW135 AY546696 USA T O. adjuncti CBS137.36 AY194956 USA CBS137.36 AY194933 USA O. ips CMW7075 DQ296101 MEX T CMW7075 AY546704 MEX T VPRI42255 VPRI43316 VPRI43851 DAR84817 CMW19371 G USA NT DAR84817 CMW19371 G USA NT VPRI42255 CXY1631 MH324811 CHN VPRI43738 VPRI43743 Taxon 9: ATCC24285 AY194949 USA VPRI43731 O. ips VPRI43731 CMW22835 DQ539541 ESP VPRI42284 VPRI42284 VPRI43734 DAR84692 DAR84692 80 VPRI43734 VPRI43743 UAMH9962 AY194938 USA 79 VPRI43316 VPRI43851 UAMH9962 AY194951 USA 92 VPRI43738 CXY1631 MH324805 CHN CMW9022 AY546714 MEX T 86 CMW30687 KF515868 USA CMW9020 AY546713 MEX O. pulvinisporum ATCC24285 AY194937 USA CMW9020 EU977487 MEX 85 O. ips CMW9022 DQ296100 MEX T 71 CMW30687 KF515896 USA 100 CMW23179 HM031505 RUS CMW23196 HM031563 FIN T 100 CBS492.7 DQ268604 CAN T O. bicolor CMW28019 HM031564 RUS 79 CFCC52683 MK748188 CHN T CMW23179 HM031560 RUS 82 O. pseudobicolor 99 CFCC52684 MK748190 CHN CBS492.7 DQ268635 CAN T 99 99 CMW28019 HM031503 RUS CFCC52684 MN896040 CHN O. fuscum CMW23196 HM031504 FIN T CFCC52683 MN896038 CHN T 98 SS519 HQ413639 CAN CMW13221 DQ296099 USA 100 CMW13221 AY546711 USA O. montium SS519 HQ413487 CAN CMW44468 MH144083 CHN 100 100 CMW44592 MH124282 CHN CMW44592 MH144086 CHN O. japonicum 80 CMW44468 MH124279 CHN YCC099 GU134169 JPN 100 98 CMW41954 MH121662 CHN T CMW41872 MH124464 CHN CMW41872 MH121661 CHN O. manchongi CMW41954 MH124465 CHN T 100 100 O. piceae CMW13239 KU184438 O. piceae CMW25034 KU184312 T O. piceae CMW25034 KU184441 T O. piceae CMW8093 KU184313

0.04 0.2

CMW23278 HM031510 FIN T CMW23278 HM031566 FIN T 92 b ITS 94 Taxon 10: BT CMW23279 HM031509 FIN CMW34941 HM031567 FIN 83 O. pallidulum 74 VPRI43846 81 100 CMW28135 HM031508 FIN VPRI43846 95 CMW34945 HM031507 FIN T O. saponiodorum CMW30883 HM031569 FIN 98 100 CFCC52691 MK748185 CHN T CMW34945 HM031571 FIN T O. lotiforme 70 CFCC52692 MK748201 CHN CFCC52691 MN896036 CHN T 100 CFCC51648 KY094067 CHN T CFCC52692 MN896037 CHN CFCC51649 KY094068 CHN O. massoniana CFCC51648 MH370810 CHN T CMW41812 MG205656 CHN 99 100 CMW41850 MG205657 CHN T O. acarorum CFCC51649 MH370811 CHN 99 CMW40491 MH144094 CHN T CMW41850 MG205686 CHN T 80 O. jilinense CMW40492 MH144095 CHN CMW40491 MH124290 CHN T 100 100 CBS124562 FN546957 ESP S. brunneoviolacea CMW40492 MH124291 CHN CMW37443 FN546959 ESP T 100 CMW26818 HM041877 ZMB 99 CMW26813 HM051412 ZAF T CMW26818 HM051415 ZMB S. fumea CMW26813 HM041878 ZAF T 100 UM56 EU913720 CAN Taxon 8: CBS454.83 KX590773 CHL T VPRI43845 O. fasciatum CBS455.83 KX590767 CHL T CBS454.83 KX590830 CHI T O. valdivianum CMW37443 FN547385 ESP T 98 CBS455.83 KX590826 CHL T S. dombeyi CBS124562 FN547384 ESP 100 Ophi1B AY934520 ESP Ophi1A AY934519 ESP T O. sejunctum VPRI43845 100 ATCC38087 KX590816 USA T UM56 EU913759 CAN 99 O. denticulatum VPRI43764 CBS497.77 KX590758 CAN 77 94 CBS186.86 AY924383 ESP CBS189.86 KX590772 USA 72 Zoq16 EU109671 MEX Taxon 7: 100 CMW650 AY280479 USA T Zoq9 EU109672 MEX O. angusticollis 100 VPRI43765 CMW651 AY280480 USA C1647 DQ062969 CAN 72 O. coronatum VPRI43764 88 CBS497.77 AY924385 CAN ATCC38087 KX590759 USA T 100 97 CBS189.86 AY934523 USA O. tenellum CBS186.86 KX590757 ESP 100 CMW650 AY280489 USA T CMW651 AY280490 USA O. nigricarpum VPRI43765 100 Hawksworthiomyceslignivorus CMW18600 EF127890 T Hawksworthiomyces lignivorus CMW18600 EF139104 T 100 Hawksworthiomyceslignivorus CMW18598 EF127888 Hawksworthiomyces lignivorus CMW18598 EF139102

0.2 0.3 Fig. 6 Phylogenetic analysis of ITS and BT for isolates residing in Ophiostoma. a. Analysis of isolates belonging in the O. ips complex. b. Analysis of isolates belonging in ‘Group A’. Sequences generated in this study are printed in bold, with reference collection isolates coloured orange. Bold branches indicate posterior probability values 0.9, while ML bootstrap values of 70% are recorded at nodes. T ex-type cultures ≥ ≥ = Trollip et al. IMA Fungus (2021) 12:24 Page 15 of 27

VPRI43749 VPRI43751 a BT DAR84899 DAR84897 CAL CMW110 AY280470 USA DAR84899 CFCC52627_MH683599 CHN DAR84900 CMW109 AY280469 USA VPRI43752 DAR84900 Taxon 14: DAR84898 CFCC52626 MH592601 CHN T VPRI43751 S. pseudoabietina DAR84706 VPRI43749 DAR84897 CFCC52627 MH592602 CHN 100 VPRI43752 DAR84706 DAR84898 VPRI43721 89 VPRI43721 CMW26269 JQ511963 CHN CFCC52626 MH683598 CHN T CMW26262 JQ511964 CHN 94 100 CMW27318 EF396344 ESP T CMW22310 JQ511966 MEX T CMW27898 GU566608 ESP Taxon 12: CMW9489 JQ511960 MEX 99 S. euskadiensis S. cf. abietina CMW9487 JQ511961 MEX VPRI43754 92 CMW22310 KX590755 MEX T S. abietina CMW9491 JQ511958 MEX 96 CMW26262 EU785434 CHN S. cf. abietina CMW9488 JQ511959 MEX 97 S. cf. abietina CMW26269 EU785433 CHN CMW27898 JQ438832 ESP 85 CMW1116 KX590761 USA T S. gossypina VPRI43754 CMW1468 AY280468 CAN S. cf. abietina CMW27318 JQ438830 ESP T CBS251.88 KX590770 POL T S. prolifera CBS541.84 JQ511968 CHL CMW1118 KX590754 USA T CMW1116 KX590789 USA T 76 S. rossi CMW9968 AY280461 AZE T CMW1118 JQ511972 USA T CMW7131 AY280464 AUT S. fusiformis CMW39767 KF951541 ESP 100 CBS541.84 KX590777 CHL ‘S. curviconia 2’ CMW39766 KF951540 ESP T 91 92 CMW28030 HM031568 RUS CMW28030 JQ511969 RUS 94 CMW39766 KF951544 ESP T S. cantabriensis CBS251.88 KX590797 POL T CMW39767 KF951545 ESP CMW10563 JQ511970 AUT T 98 CMW10564 AY280467 AUT CMW9968 JQ511967 AZE T 92 CMW10563 AY280466 AUT T S. lunata CMW7131 JQ511971 AUT CMW40316 KU639616 ZAF P S. uta CMW19362 KX590783 ZAF T 74 82 CMW19362 DQ396800 ZAF T S. aurorae CBS424.77 KX590781 USA T CBS424.77 KX590753 USA T S. eucastanea CMW23051 KX590813 ZAF T CMW23051 DQ821539 ZAF T S. variecibatus CMW40316 KU639605 ZAF P 100 S. stenoceras CMW3202 DQ296074 T S. stenoceras CBS798.73 KX590782 100 S. stenoceras CBS798.73 KX590756 S. stenoceras CMW3202_JQ511956 T

0.05 0.05

CMW14487 KX590825 JPN T VPRI43755 b ITS Taxon 13: BT VPRI43755 S. nigrograna CFCC52166 MH397731 CHN CFCC52165 MH397730 CHN T CFCC52166 KY094072 CHN S. zhejiangensis CMW41816 MG205685 CHN CMW11791 MG205646 CHN S. nebularis 100 CMW11791 MG205682 CHN CFCC52165 KY094071 CHN T S. zhejiangensis CMW41776 MG205684 CHN 87 CMW27319 KX590824 ESP T 99 S. nebularis CMW41762 MG205683 CHN 78 CMW41762 MG205647 CHN CMW27319 KX590766 ESP T CBS959.73 KX590835 CIV T S. curviconia CBS959.73 KX590776 CIV T CMW38930 KR051115 ZAF T 100 S. thermara CMW26814 HM041875 ZAF 100 85 CMW38929 KR051114 ZAF 75 CMW26813 HM041878 ZAF T CBS573.63 KX590817 ARG T S. epigloea CMW38929 KR051102 ZAF 99 CBS474.91 FN546965 BRA T 84 CMW38930 KR051103 ZAF T S. bragantina CBS430.92 FN546964 BRA 100 CBS139899 KX273395 AUS T 100 CBS139899 KR476721 AUS T CMW44295 KX273390 ZAF S. eucalyptigena CMW44295 KU865588 ZAF CBS474.91 FN547387 BRA T CMW26813 HM051412 ZAF T 100 CBS430.92 FN547386 BRA S. fumea CMW26814 HM051413 ZAF CBS573.63 KX590760 ARG T

CMW3202 AF484462 T CBS798.73 KX590756 100 S. stenoceras S. stenoceras 100 S. stenoceras CBS798.73 AF484475 S. stenoceras CMW3202 DQ296074 T

0.04 0.3 Fig. 7 Phylogenetic analysis of isolates residing in Sporothrix. a. Analysis of the BT and CAL regions for the S. gossypina complex. b. Analysis of the ITS and BT regions for Sporothrix ‘Group G’. Sequences generated in this study are printed in bold, with reference collection isolates coloured blue. Bold branches indicate posterior probability values 0.9. ML bootstrap values of 70% are recorded at nodes. T ex-type cultures ≥ ≥ = Trollip et al. IMA Fungus (2021) 12:24 Page 16 of 27

99 CMW503 AY148186 ZAF T ITS Gr. pseudormiticum TEF 85 CMW12285 HM630608 CHN CMW503 HM630586 ZAF T 99 CMW5601 AY148183 AUT T 96 Gr. laricis CMW12285 HM630587 CHN CMW5603 AY148182 AUT 98 100 841EW2-1 GQ266158 KOR CMW5601 HM630588 AUT T Graphium sp. 99 841EW1-1 GQ266157 KOR CMW5603 HM630589 AUT UAMH10638 DQ268588 CAN Graphium sp. 100 CMW5605 HM630590 FRA T CMW5605 AY148177 FRA T 98 Gr. fimbriisporum CMW5606 AY148180 AUT CMW5606 HM630591 AUT 100 UAMH10636 DQ268586 CAN Graphium sp. CMW30626 HM630592 MDG T UAMH10637 DQ268587 CAN 100 100 CMW30626 GQ200616 MDG T CMW30627 HM630593 MDG Gr. fabiforme CMW30627 GQ200617 MDG CMW12420 HM630603 CHN T 100 JCM7440 AB038424 UK Graphium sp. CMW12418 HM630602 CHN 100 CMW12420 FJ434979 CHN T Gr. carbonarium CMW12418 FJ434980 CHN CBS320.72 KJ131248 SLB 98 76 JCM8083 AB038425 JPN 79 Gr. basitruncatum VPRI43844 CBS320.72 AB038427 SLB CMW30620 HM630597 ZAF VPRI43844 Taxon 15: Graphium sp. 100 100 CCF3566 AM267264 CRI T CMW30618 HM630598 ZAF T 100 Gr. scolytodis CCF3570 AM267265 CRI CMW30628 HM630595 MDG T CMW5295 HQ335311 CZE 100 Gr. penicillioides 100 CMW5292 HQ335310 CZE CMW30629 HM630594 MDG 100 CMW30620 GQ200613 ZAF CMW5295 HM630601 CZE Gr. adansoniae 100 CMW30618 GQ200611 ZAF T CMW5292 HM630600 CZE CMW30628 GQ200619 MDG T 100 Gr. madagascariense CMW30625 GQ200618 MDG Petriellasordida CBS145121 MK442701

99 Petriellasordida CBS145121 MK442609 0.3 83 Petriellasordida CBS124169 GQ426957 100 Petriellopsisafricana CBS311.72 AJ888425 Parascedosporium putredinis CMW352 HQ335312

0.2 Fig. 8 ML phylogenies of Graphium isolates generated from ITS and TEF sequence data. Sequences generated in this study are printed in bold type. Bold branches indicate posterior probability values 0.9. ML bootstrap values of 70% are recorded at nodes. T ex-type cultures ≥ ≥ = current study represent frst records for Australia, molec- the S. pseudoabietina strain, VPRI34531. Te GC con- ular sequence data has verifed the previous morphologi- tent had a mean of 57%, with a standard deviation of 3% cal records of a Ceratocystiopsis sp. (Stone and Simpson from this mean. Gene predictions resulted in an average 1987) and a Graphium sp. (Vaartaja 1967). Notably how- estimate of 7800 Open Reading Frames (ORFs), with a ever, fve previous records still require molecular confr- gene density ranging from 240 to 341 ORFs/Mb. All draft mation and their current status should be treated with genomes had a high BUSCO completeness assessment care due to the numerous taxonomic re-evaluations that score ranging between 93.48 and 98.24%. All representa- have taken place since their initial identifcation (Table 2; tive draft genomes were made publicly available on Gen- footnotes). Bank with Accession details summarised in Table 3.

Draft genomes of representative isolates Taxonomy of ophiostomatoid fungi from Australian pine plantations Graphilbum ipis-grandicollis C. Trollip, Q. Dinh, & Jac- Genome summary statistics of the representative draft queline Edwards, sp. nov. genomes produced in this study are summarised in MycoBank: MB840696. Table 3 (see Additional fle 3: Table S3 for extended com- (Fig. 9) parison). Genomes were assembled to an average size of Etymology: ipis-grandicollis (Latin), referring to Ips 28 Mb and were represented by a mean scafold number grandicollis, the bark beetle vector of this species. of 148. Te N50 ranged from 208,570 to 1,285,428 bp, Diagnosis: Graphilbum ipis-grandicollis is phylogeneti- with the longest contig of 3,412,636 bp generated for cally distinct from all morphologically similar species, Trollip et al. IMA Fungus (2021) 12:24 Page 17 of 27

Table 2 Current status list of Australian ophiostomatoid fungi associated with Pinus Genus Species/taxon recorded State/s Verifed GenBank accession References

Ophiostomatales Ceratocystiopsis Ceratocystiopsis sp.a NSW Table 1 Stone and Simpson (1987, 1990), Cur- + rent study Ceratocystiopsis minuta NSW NA Stone and Simpson (1990) − Graphilbumb Pesotum af. fragrans SA DQ062977 Harrington et al. (2001); Thwaites et al. + (2005) Graphilbum fragrans NSW Table 1 Carnegie et al. (2019), Current study + Graphilbum ipis-grandicollis, sp. NSW Table 1 Current study nov. + Graphilbum cf. rectangulosporium NSW Table 1 Current study + Ophiostoma Ophiostoma pilifera VIC Eckersley (1934), Rawlings (1960) − Ophiostoma ips NSW, QLD, SA, VIC Table 1 Vaartaja 1967, Zhou et al. 2007, Carn- + egie et al. 2019, Current study Ophiostoma foccosum SA NA Harrington et al. (2001) − Ophiostoma quercus SA NA Harrington et al. (2001) − Ophiostoma angusticollis NSW Table 1 Carnegie et al. (2019), Current study + Ophiostoma pallidulum NSW, TAS Table 1 Carnegie et al. (2019) Current study + Ophiostoma fasciatum NSW Table 1 Current study + Leptographium s.l Grosmannia huntii NSW, VIC, TAS Table 1 Jacobs et al. (1998), Carnegie et al. + (2019), Current study Leptographium sp.c TAS NA Griggs, J.A. thesis (1998) − Grosmannia radiaticola SA, TAS Table 1 Current study + Rafaelea Rafaelea deltoideospora NSW Table 1 Current study + Sporothrix Sporothrix pseudoabietina NSW, VIC Table 1 Carnegie et al. (2019), Current study + Sporothrix euskadiensis NSW Table 1 Current study + Sporothrix cf. nigrograna NSW Table 1 Current study + Microascales Graphiumd Graphium sp. NSW Table 1 Vaartaja (1967) + Species identifed in the current study are presented in bold, and their accession details can be found in Table 1 a We speculate that the taxon reported as Ceratocystiopsis sp. by Stone and Simpson (1987, 1990) is likely the same taxon recorded in the current study. Refer to discussion for more information b Stone and Simpson (1990) reported a Graphilbum sp. associated with Ips grandicollis in NSW. This taxon could refer to any of the four taxa currently confrmed using molecular data c Griggs, J.A. recorded Leptographium lundbergii in association with Hylastes ater infesting P. radiata in TAS. This ID has not been verifed molecularly and should be treated with caution considering the taxonomic re-evaluation of L. lundbergii by Jacobs et al. (1998) d Vaartaja (1967) identifed several species of Graphium. This descriptor is somewhat ambiguous and could refer to species in both the Ophiostomatales and Microascales

from which it can be readily distinguished using molec- sometimes in groups, (102–)128–213(–263) µm long ular sequence data for the ITS, beta-tubulin, elongation including conidiogenous apparatus, (14–)20–38(–45) µm factor 1-alpha, and calmodulin regions (Fig. 4, Additional wide at the base; conidiogenous cells (17–)19–26(–31) fle 4: Fig. S1). long; conidia hyaline, single-celled, smooth, cylindrical to Type: Australia: New South Wales: Moss Vale, Belan- oblong, (3–)4–6(–8) × (2–)2–3(–3) µm. Mononematous glo State Forest (Compartment 119), from Ips grandicol- morphs: hyalorhinocladiella-like, arising directly from lis gallery on Pinus radiata, 21 Aug. 2019, A. J. Carnegie mycelium; conidiophores, simple to strongly branched, (Holotype VPRI43762, stored in a metabolically inactive hyaline, (27–)45–136(–170) µm long; conidiogenous state; ex-holotype VPRI43762). cells (5–)13–27(–36) long; conidia hyaline, single-celled, Description: Sexual morph not observed. Asexual smooth, oblong, often tapering at truncated base, (4–)4– morphs observed both synnematous and mononematous 8(–13) × (2–)2–3(–4) µm. morphs. Synnematous morph: pesotum-like, macronema- Culture characteristics: Colonies hyaline, circular with tous, hyaline or pale yellow, erect, clavate, often singular, smooth growing edge on MEA. Mycelia submerged or Trollip et al. IMA Fungus (2021) 12:24 Page 18 of 27

Table 3 Genome summary statistics of representative ophiostomatoid isolates sequenced in this study Species Ceratocystiopsis Graphilbum Leptographium s. l.. Ophiostoma s. l. Ceratocystiopsis G. fragrans G. ipis- G. cf. Gro. huntii Gro. radiaticola O. angusticollis sp. grandicollis sp. rectangulosporium nov

Taxon Taxon 1 Taxon 2 Taxon 3 Taxon 4 Taxon 5 Taxon 6 Taxon 7 Sequenced strain VPRI43766 VPRI43528 VPRI43762 VPRI43763 VPRI43530 VPRI43523 VPRI43764 GenBank Acces- JADHKF010000000JAD- JAD- JADHKI010000000 JADHKJ010000000JAD- JAD- sion HKG010000000 HKH010000000 HKK010000000 HKL010000000 Total reads after 27,063,242 50,135,289 49,429,518 46,778,484 94,294,776 114,599,394 10 454 088 QC Number of scaf- 79 237 178 117 254 85 317 folds Longest contig 1,540,000 999,956 1,555,217 1,343,814 1,099,032 2,583,828 651 020 (bp) Est. genome size 20.45 34.04 24.02 23.61 28.05 27.56 23.80 (Mb) N50 (bp) 471,680 323,198 601,306 298,270 343,842 883,760 236 362 L50 12 32 13 22 25 11 34 # N’s per 100 kbp 10 7 14 6 8 5 14 GC (%) 61.48 55.75 55.53 60.91 54.63 57.09 57.89 Avg. coverage 197 218 305 296 493 619 61 depth No. of predicted 6967 9034 7221 7270 7836 7867 8022 genes Est. gene density 341 265 301 308 279 286 337 Complete BUSCO 95.20 97.10 95.60 95.70 97.40 96.40 96.60 (%) Complete BUSCO 3636 3706 3647 3651 3716 3680 3686 (n) Complete—single 3631 3699 3644 3648 3710 3673 3680 Complete—dupli- 5 7 3 3 6 7 6 cated Fragmented 17 23 29 18 17 21 18 Missing 164 88 141 148 84 116 113 Species Ophiostoma s. lat. Rafaelea Sporothrix Graphium O. fasciatum O. ips O. pallidulum R. S. euskadiensis S. S. Graphium sp. deltoideospora cf. nigrograna pseudoabietina

Taxon Taxon 8 Taxon 9 Taxon 10 Taxon 11 Taxon 12 Taxon 13 Taxon 14 Taxon 15 Sequenced VPRI43845 VPRI43529 VPRI43846 VPRI43720 VPRI43754 VPRI43755 VPRI43531 VPRI43844 strain GenBank Acces- JAD- JAD- JAD- JAD- JAD- JAD- JAD- JAD- sion HKM010000000 HKN010000000 HKO010000000 HKP010000000 HKQ010000000 HKR010000000 HKS010000000 HKT010000000 Total reads after 51,287,508 37,261,588 36,666,452 35,927,882 29,627,312 37,781,794 36,719,768 28,160,580 QC Number of scaf- 36 208 473 120 92 125 51 490 folds Longest contig 2,360,849 1,117,630 543,815 1,965,135 1,881,930 1,307,617 3,412,636 1,008,604 (Mb) Est. genome size 22.54 26.01 32.66 30.95 36.13 27.34 35.20 31.65 (Mb) N50 (bp) 966,108 283,044 126,120 416,364 790,753 398,989 1,285,428 190,968 L50 9 29 84 25 16 23 9 49 # N’s per 100 4 8 12 18 11 12 8 12 kbp Trollip et al. IMA Fungus (2021) 12:24 Page 19 of 27

Table 3 (continued) Species Ophiostoma s. lat. Rafaelea Sporothrix Graphium O. fasciatum O. ips O. pallidulum R. S. euskadiensis S. S. Graphium sp. deltoideospora cf. nigrograna pseudoabietina

GC (%) 65.26 56.88 57.93 54.55 52.70 59.54 53.27 50.12 Avg. coverage 341 209 167 173 122 205 154 132 depth No. of predicted 7498 7470 8757 7428 9348 7908 9190 9482 genes Est. gene density333 287 268 240 259 289 261 300 Complete 96.60 96.90 97.20 94.90 98.10 97.20 97.80 96.60 BUSCO (%) Complete 3688 3696 3710 3621 3743 3710 3734 3686 BUSCO (n) Complete— 3685 3690 3705 3613 3739 3703 3729 3679 single Complete— 3 6 5 8 4 7 5 7 duplicated Fragmented 16 16 28 30 10 23 12 40 Missing 113 105 79 166 64 84 71 91

fat on MEA and WA, with colonies reaching approx. Inverell, Copeton Dam, from Ips grandicollis gallery on 90 mm diam after 14, and 21 d, respectively. Sporula- P. radiata, 25 Jul. 2019, A. J. Carnegie (VPRI43759 – tion evident after one week of growth on pine needle culture); Tumut, Buccleuch State Forest (Compartment amended WA. Initial formation of hyalorhinocladiella- 1129), from I. grandicollis gallery on P. radiata, 2 Jun. like morphs submerged in agar, with sparse formation 2019, D. Sargeant (VPRI43760 – culture). on the agar surface. Tis is followed by formation of the pesotum-like morphs, frst forming on the pine needle Discussion between 7 and 14 d, and eventually observed sparsely Tis study was undertaken to review and update the sta- on the agar surface after 4–6 wk. Aerial hyphae bear- tus of ophiostomatoid fungi associated with pine and ing conidiophores, mycelial balls, and white to yellow pine bark beetles in plantations in south-eastern Aus- synnemata-like clusters were also randomly observed tralia. Tis was achieved by reviewing reference isolates on the two media. available from historic collections lodged in Australian Ecology: Isolated from beetles and beetle galleries collections, as well as including a total of 120 new isolates found on various Pinus hosts. Host trees: Pinus radiata, collected through routine forest health surveillance dur- P. elliottii and P. caribaea x elliottii hybrid (Additional ing the 2019–20 period. Multi-locus phylogenetic analy- fle 2: Table S2). Insect vector: Ips grandicollis. sis using whole genome sequencing of 46 representative Distribution: Currently known only from New South isolates revealed a greater than expected diversity of Wales, Australia. ophiostomatoid fungi, including 14 species from six gen- Notes: Graphilbum ipis-grandicollis forms part of era in Ophiostomatales and a single species residing in . an expanding species complex in Graphilbum, which While most species reported in this study were already includes G. crescericum, G. furuicola, G. interstitiale, G. known, our study includes seven frst reports and three kesiyae, and G. sexdentatum. Using morphology alone verifcations for Australia, including the identifcation of makes distinction between these closely related spe- three previously undescribed lineages, viz. Graphilbum cies difcult, as they share considerable similarities in ipis-grandicollis sp. nov. (Taxon 3), Ceratocystiopsis sp. the size and shapes of conidia, conidiogenous appara- (Taxon 1) and a Graphium sp. (Taxon 15). Draft genomes tus, and the asexual morphs recorded (Jankowiak et al. of representative isolates for each taxon are also provided 2020). here to contribute to a curated reference database of Additional specimens examined: Australia: New ophiostomatoid fungi for Australian biosecurity. South Wales: Moss Vale, Belanglo State Forest (Com- Of the fve ophiostomatoid genera previously recorded partment 123), from I. grandicollis gallery on P. radiata, from pine in Australia, isolates were available for Ophios- 21 Aug. 2019, A. J. Carnegie (VPRI43761 – culture); toma, Graphilbum, Leptographium s. lat., and Sporothrix. Trollip et al. IMA Fungus (2021) 12:24 Page 20 of 27

Fig. 9 Graphilbum ipis-grandicollis (VPRI43762). a Fourteen-d culture on MEA. b–d Pesotum-like macronematal asexual morph formed on pine needle mounted in WA. e–f Conidiogenous cells (e) and conidia (f) of Pesotum-like macronematal asexual morph. g. Hyalorhinocladiella-like asexual morph. h, i Conidiogenous cells (h) and conidia (i) of Hyalorhinocladiella-like asexual morph. Bars: b 500 µm; c, d 50 µm; e–i 10 µm = = =

Results of the collection database searches allowed for morphological identifcation of taxa belonging to Cerato- the inclusion of reference isolates of G. fragrans (Taxon cystiopsis (Stone and Simpson 1987, 1990), no reference 2), Gro. huntii (Taxon 5), O. angusticollis (Taxon 7), O. material was available of this genus. Similarly, with the ips (Taxon 9), and several initially identifed as Sporothrix recent detection of O. pallidulum (Carnegie and Nah- species (Carnegie and Nahrung 2019; Carnegie et al. rung 2019; Carnegie et al. 2019), no isolates were readily 2019). Sporothrix isolates obtained from the Austral- available for inclusion in this study. ian reference collections were putatively identifed as S. Detections made during the current survey included cf. abietina or O. nigrocarpum based on BLAST results seven taxa not previously recorded in Australia. Four of the ITS during routine diagnostics (Carnegie et al. were identifed as known species: specifcally, Gro. 2019). However, our results revealed that these isolates radiaticola (Taxon 6), O. fasciatum (Taxon 8), R. del- were all a single species, identifed here as S. pseudo- toideospora (Taxon 11), and S. euskadiensis (Taxon 12). abietina. Although historical records also included the Taxon 4 and 13 are tentatively identifed here as G. cf. Trollip et al. IMA Fungus (2021) 12:24 Page 21 of 27

rectangulosporium and S. cf. nigrograna, respectively. Graphilbum isolates collected from New Zealand and Both these taxa require further taxonomic revision due Australia, which show diferences in morphological com- to a lack of available reference data in each case. Te parisons and sequence analysis of the ITS (Harrington remaining frst records included the detection of the et al. 2001; Twaites et al. 2005). Te G. fragrans iso- novel species Graphilbum ipis-grandicollis sp. nov., as lates collected in the present study shared high sequence well as an undescribed lineage in Ceratocystiopsis and similarity with the type strain CBS279.54 from Sweden Graphium, respectively. For both, our record here serves and are clearly distinguishable from the single sequence as a frst verifcation of presence made using molecular available for the putative taxon reported as G. af. fra- data. Tese new detections were considered in a biosecu- grans from Australasia in 2005 (Harrington et al. 2001; rity context, following guidelines in the Emergency Plant Twaites et al. 2005; De Beer and Wingfeld 2013). Pest Response Deed (Plant Health Australia EPPRD 2020; Graphilbum ipis-grandicollis sp. nov. (Taxon 3) https://​www.​plant​healt​haust​ralia.​com.​au/​wp-​conte​nt/​ grouped with several species residing in an evidently uploa​ds/​2020/​09/​EPPRD-2-​Septe​mber-​2020.​pdf), and expanding complex of bark beetle associates isolated determined not to be signifcant pathogens nor feasible from Europe and China (Chang et al. 2017; Jankowiak to eradicate. et al. 2020). Multi-locus phylogenetic analysis suggests Isolates of Ceratocystiopsis (Taxon 1) from this study Taxon 3 is most closely related to a clade comprised of grouped closely to the Cop. ranaculosa-brevicomis com- G. crescericum, G. furuicola, G. interstitiale, G. kesiyae, plex, with morphological and molecular sequence data and G. sexdentatum. While ITS sequence data suggests suggesting that it is most closely related to a previously a close relationship to a previously undescribed Graphil- undescribed North American taxon, Ceratocystiopsis bum isolate from North America (GU129997; Fig. 4.) sp. 1 (Kim et al. 2005a, b; Lee et al. 2006; Plattner et al. further investigations are required to postulate as to the 2009). As mentioned, previous reports of Ceratocys- true origin of this novel taxon. Te species within this tiopsis in Australia were based on morphology alone and complex are mainly distinguishable using molecular included the putative identifcation of two taxa—one of sequence data, with only minor morphological difer- which was recorded as Cop. minuta (Stone and Simpson ences observed in characteristic features such as conidia 1987). A preceding study by the same authors, however, or the production of mononematous conidiophores only referred to the Australian isolates in this taxon as observed for only a couple of species (Jankowiak et al. Ceratocystiopsis sp. (Stone and Simpson 1990). Inter- 2020). Isolates of the Graphilbum sp. (Taxon 4) shared estingly, the morphological descriptions made by those an identical ITS sequence with Graphilbum isolates pre- authors correlate with the morphological description of a viously reported as G. cf. rectangulosporium in the USA Ceratocystiopsis sp. 1 identifed in North America, which (Kim et al. 2011). Te US isolates were described as ster- were described as Cop. minuta-like (Plattner et al. 2009). ile and shown to share high levels of sequence similarity It is possible therefore to speculate that the isolates col- with the type strain of G. rectangulosporium from Japan lected in this study represent this same taxon reported by (AB242825; Ohtaka et al. 2006; Kim et al. 2011). Cultures Stone and Simpson. Most of the Ceratocystiopsis isolates of the isolates in the current study did not produce either collected in the current survey were isolated from I. gran- sexual or asexual characters, an observation that further dicollis beetles collected from P. ponderosa, P. caribaea x validates the association with the US isolates. Te lack of elliottii and P. taeda in northern NSW, the same region morphologically distinguishing characteristics in culture, that Stone and Simpson collected from. as well as limited availability of alternative barcoding loci Te genus Graphilbum has recently been expanded to currently restricts further taxonomic placement, and so include 20 formally described species, which are char- we refer to this taxon as G. cf. rectangulosporium. acterised by synnematous pesotum-like and/or mon- In Leptographium s.lat., isolates of Grosmannia huntii onematous hyalorhinocladiella-like asexual morphs (Taxon 5) and Gro. radiaticola (Taxon 6) were collected (Seifert et al. 2013; Jankowiak et al. 2020). Of the three in this study. Gro. huntii was frst reported in Australia Graphilbum taxa from Australian pine plantations, one in 1998, when it was believed to have been introduced was identifed as G. fragrans (Taxon 2). G. fragrans can along with its insect vector, H. ater (Jacobs et al. 1998). be considered the most common species of the genus Until now, the known distribution within Australia and is known to have a global distribution, including included Victoria and NSW in association with H. ater reports from Europe, Asia, North and South America, and Hy. ligniperda. Te isolations made in the current as well as Australasia (Harrington et al. 2001; Twaites study expand the known distribution to include Tasma- et al. 2005; Seifert et al. 2013; Chang et al. 2017; Jankow- nia, where it was isolated from stumps in recently har- iak et al. 2020). Previous studies have suggested that G. vested pine plantations infested by the root-feeding bark fragrans comprises potentially cryptic species, based on beetle, Hy. ligniperda. Taxon 6 included three isolates of Trollip et al. IMA Fungus (2021) 12:24 Page 22 of 27

Gro. radiaticola which were collected from P. radiata undescribed lineages previously referred to as either S. cf. samples infested with H. ater in South Australia, and abietina or Sporothrix sp., which included isolates from Hy. ligniperda in Tasmania. Tese are the frst records of the USA, Mexico, South Africa, Poland and China (Zhou Gro. radiaticola for Australia. Gro. radiaticola forms part et al. 2004a; Min et al. 2009; Romón et al. 2014; Jankow- of the Gro. galeiformis species complex, and has been iak et al. 2018). In 2019, this taxon was formally described previously reported across Eurasia (Kim et al. 2005a, b; as S. pseudoabietina, with the type specimen originating Linnakoski et al. 2012; Jankowiak and Bilański 2013a, b; in China (Wang et al. 2019). Our results confrm that S. Chang et al. 2017) and throughout the Southern Hemi- pseudoabietina is a commonly isolated fungus from Aus- sphere, including South America, South Africa, and New tralian-grown pine which was frst detected in 2019 (Car- Zealand (Zhou et al. 2001, 2006; Twaites et al. 2013; de negie et al. 2019). Te second species belonging to this Errasti et al. 2018). complex was identifed as S. euskadiensis, associated with Of the four species in our study residing within Ophi- I. grandicollis. S. euskadiensis was frst described from ostoma s. lat., only O. ips (Taxon 9) grouped in a well- P. radiata in Spain, where isolates were associated with recognised species complex. Te remaining three species, Hylurgops palliatus and Hylastes attenuatus (Romón O. angusticollis (Taxon 7), O. fasciatum (Taxon 8) and O. et al. 2014). pallidulum (Taxon 10), currently group within smaller Te third species of Sporothrix identifed during this lineages that sit peripherally to Ophiostoma s. str. and are study, Taxon 13, sits within species complex ‘G’ (De Beer commonly referred to as ‘Group A’ (Chang et al. 2017; et al. 2016). Molecular analysis and taxonomic placement Wang et al. 2020). Species residing in Group A are con- for this isolate exemplifes some of the major challenges sistently recorded in low numbers and known to be highly for diagnostics of ophiostomatoid fungi. Our single strain phoretic (non-permanent interaction for the purpose of shared an identical ITS sequence with the type specimens travel) in their association with insects (Chang et al. 2017). of both S. nigrograna and S. zhejiangensis (De Beer et al. Ophiostoma fasciatum was frst described in Canada in 2016; Wang et al. 2018). LSU sequences for our strain 1972 from Pseudotsuga menziesii (as Ceratocystis fasciata; also shared high sequence similarity to those available for Olchowecki and Reid 1974) and P. banksiana (as Cerato- both S. nebularis and S. nigrograna (De Beer et al. 2016). cystis spinifera; Olchowecki and Reid 1974). Te single Te lack of available sequence data for other molecu- isolate of O. fasciatum (Taxon 8) collected in this survey lar regions of S. nigrograna limits further comparison to came from I. grandicollis collected from a P. caribaea x this species, and therefore analysis of the BT region was elliottii hybrid in northern NSW and has not been previ- only possible for sequences from S. nebularis and S. zhe- ously reported in Australia. O. pallidulum and O. angusti- jiangensis. Using BT alone would delimit our strain as S. collis species were recently detected in NSW in 2016 and zhejiangensis. Morphologically these species can only be 2017, respectively (Carnegie and Nahrung 2019). Te sin- distinguished by the presence or absence of a sheath on gle isolate of O. pallidulum from our current survey was ascospores (Masuya et al. 2003; Wang et al. 2019). Until collected from a Hy. ligniperda beetle sampled in Tasma- appropriate taxonomic comparison is possible, we refer to nia and serves as a frst report outside of NSW. this isolate as S. cf. nigrograna due to its initial placement Te single isolate of Rafaelea deltoideospora (Taxon with S. nigrograna and its distinction from S. nebularis. 11) was isolated directly from galleries of I. grandicollis A single isolate (Taxon 15) residing within Graphium found on a P. caribaea x elliottii hybrid from northern (Microascales) was collected from an I. grandicollis gal- NSW. R. deltoideospora was originally described from lery originating from P. elliottii in NSW. ML analysis of our isolates collected from the wood of several pine species strain revealed a potentially distinct lineage that is closely in Canada (Olchowecki and Reid 1974). Later records related to Gra. basitruncatum and Gra. carbonarium. Gra. have found this species associated with cerambycid pupal basitruncatum was frst described from soil in the Solo- chambers in the USA and China (Wingfeld 1987; Wang mon Islands (Okada et al. 2000), while Gra. carbonarium et al. 2018). R. deltoideospora has also been reported was isolated from Pissodes beetles on Salix babylonica in from P. pinaster in the Iberian Peninsula (Villarreal et al. Yunnan, China (Paciura et al. 2010). With only this single 2005). Tis is a frst report for this fungus in Australia. isolate obtained we refer to this taxon as Graphium sp. Results of our study revealed three species of Sporo- until more isolates can be collected and studied. thrix present in Australian pine plantations. Two species Although the relationship of ophiostomatoid fungi belonged to the S. gossypina species complex, namely, S. and arthropod vectors has been extensively studied, the pseudoabietina (Taxon 14) and S. euskadiensis (Taxon precise role of each taxonomic group within these sys- 12). Species within this complex are commonly isolated tems and the specifcity of these associations, are yet from bark beetle and mite associates (De Beer et al. to be clearly defned (Chang et al. 2017; Wingfeld et al. 2016). Taxon 14 showed close association to several 2017b). All taxa isolated in the current study form part Trollip et al. IMA Fungus (2021) 12:24 Page 23 of 27

of species complexes and/or groupings that are consist- Ceratocystiopsis sp. VPRI43766 (Taxon 1; 6967 ORFs) ently associated with bark beetles or other insect vec- when compared to that of Cop. minuta (7786 ORFs). tors (De Beer et al. 2016; Wingfeld et al. 2017b). While In the modern era, fungal taxonomy relies more heav- our goal was to assess the diversity of ophiostomatoid ily on an integrative approach where genealogical con- fungi associated with pine bark beetles and beetle galler- cordance is combined with morphological examination ies collected during routine forest health surveillance, I. to recognise and delimit species (Lücking et al. 2020). For grandicollis was the more commonly encountered beetle taxonomists and diagnosticians looking to delineate taxa species during the current surveillance period. Tis was a of ophiostomatoid fungi, this could include analysing any- somewhat expected observation as, historically, I. gran- thing from two to ten diferent gene regions (De Beer and dicollis is more commonly caught in NSW than either Wingfeld 2013; De Beer et al. 2014, 2016) all while com- H. ater or Hy. ligniperda (Stone et al. 2010). While this paring morphological characters that can prove extremely could explain the dominance observed for some of the difcult to distinguish (Jankowiak et al. 2020). Another ophiostomatoid species isolated, such as Ophiostoma ips major challenge that was exemplifed several times in and Sporothrix pseudoabietina, our results highlight the this study is the inconsistency of recovering sequence potential phoresy of these associations with I. grandicol- data for specifc taxa. As the cost of sequencing contin- lis being linked to seven of the taxa recovered during this ues to decrease, the feasibility for future taxonomic sur- study (Additional fle 2: Table S2). While we were able to veys to include whole genome sequences should become make a few general observations regarding the patterns more readily attainable. As shown in this study, future of isolations, a more in-depth systematic review would taxonomic surveys could strive to include whole genome be required for an improved understanding and descrip- sequence data published alongside their identifcations tion of these fungus-vector associations across Australia. and/or descriptions of novel taxa. Currently, in the Ophios- More targeted surveys, particularly studies focused on tomatales the number of available genomes encompasses the insect vectors present, are likely to reveal an even 38 species across 11 genera. Expanding these genomic greater diversity, for example, the isolates of Gro. radia- resources provides a fundamental platform on which diag- ticola and Gro. huntii were only recovered from samples nostic and biosecurity capacity can be developed. that came from H. ater and Hy. ligniperda infestations. Genome assemblies for the isolates chosen as repre- Conclusions sentatives for each taxon collected in this study resulted in Te results of this study have uncovered a higher than the addition of 12 draft genomes to the Ophiostomatales, expected diversity for ophiostomatoid fungi associated and the release of the frst draft genome publicly available with pine and pine bark beetles in south eastern Aus- for an isolate residing in the Graphiaceae (Microascales). tralia. Te current status of ophiostomatoid fungi in Aus- Te genome assembly statistics of the Ophiostomatales tralian pine plantations confrmed using molecular data isolates collected during this study mirror those available has been expanded from 7 previously confrmed taxa to for species residing in Ophiostoma, Sporothrix, Graphil- now include 15 verifed species across six genera in the bum, Leptographium s.l., Ceratocystiopsis and Rafaelea Ophiostomatales, as well as a single taxon identifed in (DiGuistini et al. 2011; Forgetta et al. 2013; Haridas et al. the Graphiaceae (Microascales). As demonstrated several 2013; Teixeira et al. 2014; van der Nest et al. 2014; Wing- times in this study, a major challenge for accurate fungal feld et al. 2015a, b, 2016, 2017a, 2018; D’Alessandro et al. diagnostics and species delimitation is the availability of 2016; Huang et al. 2016; Shang et al. 2016; Jeon et al. 2017; multi-locus sequence data for reference specimens. With Vanderpool et al. 2018; Liu et al. 2019). Comparisons of the ever-decreasing costs of sequencing, as well as the genome statistics, specifcally estimated size, GC content need for multi-locus sequence data, our study provides and number of predicted genes, generally correlate with an early example of WGS replacing standard PCR-based the taxonomic placement of each species (Additional approaches. Future taxonomic studies could begin to fle 3: Table S3). Tis is evident, for example, when com- look in earnest at the opportunities of providing the full paring the genomes of Gro. galeiformis and Gro. radia- complement of DNA sequence data along with the results ticola, or S. euskadiensis and S. pseudoabietina. In both of a given taxonomic survey. Tis would ensure that taxo- cases the size, GC content and number of predicted open nomic studies continue to improve upon the availability reading frames (ORFs) vary marginally. Tere are how- of molecular data while rapidly expanding on the num- ever slight deviations evident within some genera. For bers of sampled taxa. Results of the current survey, cou- example, in Ceratocystiopsis the genome sizes range from pled with other recent detections in Australia, illustrates 20.45 to 21.30 Mb, and the number of predicted ORFs the need for continued surveillance of ophiostomatoid are somewhat lower for Cop. brevicomis (6884 ORFs) and fungi. Tis not only provides an important platform for recognising the underlying diversity of these fungi but Trollip et al. IMA Fungus (2021) 12:24 Page 24 of 27

allows for the establishment of an improved ophiostoma- through funding from the Australian Government Department of Agricul- ture as part of its Rural R&D for Proft Program, with funding from 16 partner toid-specifc database which will continue to develop the organisations, including Grains Research & Development Corporation, Sugar diagnostic capabilities for Australian biosecurity. Research Australia, Cotton Research & Development Corporation, Wine Australia, AgriFutures Australia, Forest and Wood Products Australia, and Agri- culture Victoria. CT is supported through a La Trobe University Postgraduate Abbreviations Research Scholarship and a La Trobe University Full Fee Research Scholarship BI: Bayesian inference; BT: β-Tubulin; CAL: Calmodulin; DAR: The NSW Plant at La Trobe University, Victoria, Australia. Funding organisations had no role in Pathology and Mycology Herbarium; ITS: The internal transcribed spacer; LSU: the study design, data collection, analysis, and interpretation, or the writing of The large ribosomal subunit (28S); MEA: Malt extract agar; ML: Maximum likeli- the manuscript. hood; NCBI: National Center for Biotechnology Information; NSW: New South Wales; s.lat.: Sensu lato; s.str.: Sensu stricto; TEF: Translation elongation factor Availability of data and materials 1-α; VPRI: The Victorian Plant Pathology Herbarium; WA: Water agar; WGS: All data generated or analysed during this study is included in this published Whole genome sequencing. article [and its supplementary information fles] and/or is available from the corresponding author upon reasonable request. Supplementary Information Declarations The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s43008-​021-​00076-w. Ethics approval and consent to participate Not applicable. Additional fle 1. Table S1. Sampling information. Adherence to national and international regulations Additional fle 2. Table S2. Host association and isolation frequencies of Not applicable. ophiostomatoid fungi obtained during this study. Additional fle 3. Table S3. Extended genome summaries of ophios- Consent for publication tomatoid fungi which correspond with all genera obtained during current Not applicable. study. Competing interests Additional fle 4. Figure S1. ML phylogeny of the CAL region for isolates The authors declare that they have no competing interests. residing in Graphilbum. Sequences generated in this study are printed in bold type with reference collection isolates coloured purple. Bold Author details branches indicate posterior probability values 0.9. ML bootstrap values 1 ≥ School of Applied Systems Biology, La Trobe University, Bundoora, VIC 3083, of 70% are recorded at nodes. T ex-type isolates. 2 ≥ = Australia. Department of Jobs, Precincts and Regions, Agriculture Victoria Additional fle 5. Figure S2. ML phylogeny of ITS region for repre- Research, AgriBio Centre, Bundoora, VIC 3083, Australia. 3 Forest Science, NSW sentative species of Leptographium, Rafaelea and Hawksworthiomyces. Department of Primary Industries – Forestry, Parramatta, NSW 2150, Australia. Sequences generated in this study are printed in bold type. Bold branches 4 Department of Jobs, Precincts and Regions, Biosecurity and Agricultural indicate posterior probability values 0.9. ML bootstrap values of 70% Services, Agriculture Victoria, Cranbourne, VIC 3977, Australia. are recorded at nodes. T ex-type isolates.≥ ≥ = Received: 25 October 2020 Accepted: 31 July 2021 Additional fle 6. Figure S3. ML phylogeny of CAL for isolates residing in Sporothrix. Sequences generated in this study are printed in bold type. Bold branches indicate posterior probability values 0.9. ML bootstrap values of 70% are recorded at nodes. T ex-type isolates.≥ ≥ = References Acknowledgements Alamouti SM, Kim J, Breuil C (2006) A new Leptographium species associated We would like to thank and acknowledge the following for their assistance with the northern spruce engraver, Ips perturbatus, in western Canada. across various aspects of this study. David Sargeant, Matthew Nagel, Rudi Hof- Mycologia 98(1):149–160. https://​doi.​org/​10.​1080/​15572​536.​2006.​ man, Karl Wotherspoon and Nita Ramsden for their eforts in feld collections 11832​722 during the forest health surveillance period. The Crop Health Services diag- Brasier CM, Webber JF (2019) Is there evidence for post-epidemic attenua- nosticians at Agriculture Victoria, specifcally Isabel Valenzuela-Gonzalez for tion in the Dutch elm disease pathogen Ophiostoma novo-ulmi? Plant her assistance in beetle identifcations and Ramez Aldaoud and Soheir Salib Pathol 68(5):921–929. https://​doi.​org/​10.​1111/​ppa.​13022 for the technical assistance provided during sample reception and processing. Bushnell B (2014) BBMap: a fast, accurate, splice-aware aligner. Lawrence Robyn Brett, Karren Cowan, Jordan Bailey and Roger Shivas for the review, pro- Berkeley National Lab.(LBNL), Berkeley, CA (United States), vision and maintenance of isolates in the VPRI, DAR and BRIP. Tom May for his Carnegie AJ, Nahrung HF (2019) Post-border forest biosecurity in Australia: advice and guidance in determining the appropriate Latin nomenclature for response to recent exotic detections, current surveillance and ongoing the species described herein. The authors would also like to acknowledge the needs. Forests 10(4):336 growers whose plantations samples were collected from: Forestry Corporation Carnegie AJ, Cant RG, Eldridge RH (2008) Forest health surveillance in New of NSW, HVP Plantations, OneFortyOne, and Sustainable Timber Tasmania. South Wales, Australia. Aust for 71(3):164–176. https://​doi.​org/​10.​1080/​ 00049​158.​2008.​10675​031 Authors’ contributions Carnegie AJ, Lawson S, Wardlaw T, Cameron N, Venn T (2018) Benchmarking CT, JE, BR and AJC conceived the idea of the study, and all authors contributed forest health surveillance and biosecurity activities for managing Aus- to the study design. CT performed isolations, sequencing and data analysis tralia’s exotic forest pest and pathogen risks. Aust for 81(1):14–23 with assistance and advice from JK, QD, RM and JE. QD performed microscopy Carnegie AJ, Daniel R, Lidbetter F, Daley A, Laurence M, Nagel M, Sargeant D, for the species description and edited the descriptions text. CT wrote the Duong TA, Wingfeld MJ (2019) Pathogenicity and new records of exotic manuscript, with editing and revision by JE, BR and AJC to produce the fnal blue stain fungi on Pinus in Australia. In: Abstracts of Australasian Plant version. All authors read and approved the fnal manuscript. Pathology Society conference 2019, Melbourne, 25–28 November 2019. Chang R, Duong TA, Taerum SJ, Wingfeld MJ, Zhou XD, De Beer ZW (2017) Funding Ophiostomatoid fungi associated with conifer-infesting beetles and This project has been funded through iMapPESTS. iMapPESTS is a research, their phoretic mites in Yunnan, China. Mycokeys 28:19–64. https://​doi.​ development and extension (RD&E) project supported by Hort Innovation, org/​10.​3897/​mycok​eys.​28.​21758 Trollip et al. IMA Fungus (2021) 12:24 Page 25 of 27

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