Wood samples were collected from four forest districts located in southern Poland (Józefów, Krzeszowice, Siewierz, and Węgierska Górka) between 2015–2019. In each district, 10 stands dominated by Fagus sylvatica L. (Krzeszowice, Małopolskie Province), Picea abies (L.) H. Karst. (Węgierska Górka, Śląskie Province), P. sylvestris (Józefów, Lubelskie Province), and Quercus robur L. (Siewierz, Śląskie Province) were selected, making a total of 40 stands (10 stands for each tree species). The stands were managed and between 35 to 135 years of age.

All sampled stands have temperate climates. Węgierska Górka is located in the lower montane belt of the Western Carpathians (607–896 m a.s.l.) with an average annual temperature and precipitation of 6.5 °C and approximately 950 mm, respectively. The other forest stands are in the Highlands of Poland (217–347 m a.s.l.) with average annual temperature and precipitation of 7–8 °C and approximately 600–800 mm, respectively. Tree-stratum vegetation in Józefów is dominated by P. sylvestris, but also consists of Abies alba Mill., Alnus glutinosa (L.) Gaertn., Betula pendula Roth, P. abies, and Q. robur. In Krzeszowice, F. sylvatica is the dominant tree species, but other species are also present, such as Carpinus betulus L., P. sylvestris, and Q. robur. Siewierz stands are dominated by Q. robur, but also include Acer pseudoplatanus L., A. glutinosa, B. pendula, C. betulus, Larix decidua Mill., P. abies, and P. sylvestris. Finally, vegetation in Węgierska Górka is dominated by P. abies, but also contains A. alba and F. sylvatica.

Soil samples for laboratory analyses were collected from each stand (10 samples per stand, for a total of 400 samples). The samples were collected from the humus A mineral horizon (10 cm deep) after the upper organic O horizon was removed. Freshly collected soil samples were dried and then sieved through a 2 mm mesh sieve. The particle size distribution was analyzed using a laser diffraction method (Analysette 22, Fritsch, Idar-Oberstein, Germany). The pH of soil samples in H₂O and KCl was determined by a potentiometric method (Ostrowska et al. 1991). All stands were characterized by high soil acidity, with the pH ranging from 3.81 to 6.10 (in H₂O) and 2.91 to 5.67 (in KCl). After air-drying, soil samples were sifted through a sieve with a mesh diameter of 2 mm. The particle size distribution was determined using laser diffraction (Analysette 22, Fritsch, Idar-Oberstein, Germany). Soil textures were sandy in 27 stands and silty in the remaining 13 stands.

Fungi were isolated using branches (25 cm × 5 cm × 5 cm) of F. sylvatica, P. abies, P. sylvestris, and Q. robur that were cut along the axes. Healthy branches were taken from trees that represented the dominant species in each stand; for example, in stands dominated by F. sylvatica, only its branches were used. Each branch was autoclaved in a sterile plastic bag and was stored for 1–2 days at a temperature of 5 °C. They were then removed from the bags and immediately placed in the soil. Ten sterilized branches were placed in each stand. Branches were buried in the humus mineral A horizon after the organic O horizon was removed, at random locations in the stands (Suppl. material 1: fig. S1). There is no information about the occurrence of Ophiostomatales species in specific soil levels. We have used the humus mineral horizon (A) because this level is characterized by high thermal and humidity stability (Ekici et al. 2014; Neto et al. 2017). Due to potential fungal infection from roots, the branches were placed 2 m away from tree roots. The branches were buried after the main flight period of the root-feeding bark beetles to avoid colonization by insects carrying other Ophiostomatales species (Jankowiak and Bilański 2013a) and were retrieved two months after they were initially buried. After removal, the branches were placed in separate sterile bags and moved to the laboratory of Robert Jankowiak at the University of Agriculture in Krakow, Poland (Suppl. material 1: fig. S2). A total of 400 samples were collected during the study from every stand type (100 from beech stands, 100 from oak, 100 from pine, and 100 from spruce). No signs of insect presence (adults, larvae, bites, wood holes, galleries) were visible on any branch.

The branches were washed under tap water and dried on blotting paper and covered with cotton wool saturated with 96% ethanol for 15 seconds to sterilize the wooden surfaces. A sterile wood chisel was then used to remove the surface of the wood up to a depth of 2 mm. From each block, six small fragments of discolored wood (4 × 4 mm) were taken with a sterile chisel and placed in Petri dishes containing 2% malt extract agar (MEA; Biocorp Polska Sp. z o.o., Warszawa, 20 g Biocorp malt extract, 20 g Biocorp agar, and 1000 mL sterile water) amended with cycloheximide (200 mg/L, Aldrich-Sigma, St. Louis, Co. LLC.) and tetracycline (50 mg/L, Aldrich-Sigma, St. Louis, Co. LLC). Based on the preliminary morphological investigation, emerging cultures resembling members of the Ophiostomatales were purified by transferring small pieces of mycelium or spore masses from individual colonies to fresh 2% MEA. Cultures were incubated at room temperature in the dark at 22 °C. After two weeks of incubation, the purified fungal cultures were grouped into morphotypes. Depending on the number of isolates that belonged to the same morphotype, 1–12 isolates per morphotype were chosen for molecular identification (Table 1). In the end, the isolates were categorized into ten morphotypes.

The collection details for the Sporothrix species isolated from fallen shoots of P. sylvestris (Table 1) are described in a study by Jankowiak and Kolařík (2011). The cultures are maintained in the culture collection of the Department of Forest Ecosystems Protection, University of Agriculture in Krakow, Poland. The ex-type isolates and representative isolates of the new species described were deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands and in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, South Africa. Dried cultures were deposited as holotype specimens in the Mycological Herbarium (O) of the Natural History Museum at the University of Oslo, Norway.

Morphological characters were examined for selected isolates including the type specimens. Cultures were grown on 2% Malt Extract Agar (MEA) made up of 20 g Bacto malt extract and 20 g Bacto agar powder (Becton Dickinson and Company, Franklin Lakes, USA) in 1 L of deionized water. In attempts to induce ascomata formation, autoclaved twigs of host trees were placed at the centres of agar plates containing 2% MEA. To promote the production of ascomata, single conidial isolates were crossed following the technique described by Grobbelaar et al. (2009). These cultures were incubated at 25 °C and monitored regularly for the appearance of developing structures.

Samples of fungal tissues were placed in 80% lactic acid on glass slides, and developing structures were observed using a Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) with an Invenio 5S digital camera (DeltaPix, Maalov, Denmark) to capture photographic images. Color designations were based on the color charts of Kornerup and Wanscher (1978). For each taxonomically relevant structure, fifty measurements were made, when possible, using the Coolview 1.6.0 software (Precoptic, Warsaw, Poland). Averages, ranges, and standard deviations were presented in the format ‘(min–)(mean–SD)–(mean+SD)(–max)’.

Growth characteristics of the novel species were determined by analyzing the radial growth for two isolates per species. Agar disks (5 mm in diameter) were cut from the actively growing margins of fungal colonies and these disks were placed at the centres of plates containing 2% MEA. Four replicate plates for each isolate of the three putative new species were incubated in the dark. The isolates were grown at 5, 10, 15, 20, 25, 30 and 35 °C. The radial growth was determined 14 days after inoculation, and growth rates were calculated as mm/day.

DNA was extracted using the Genomic Mini AX Plant Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. For fungi that resided in the genus Leptographium, the nuclear large subunit (LSU) region was amplified using the primers LR0R and LR5 (Vilgalys and Hester 1990), the β-tubulin (TUB2) gene was amplified using the primers Bt2a and Bt2b (Glass and Donaldson 1995), and the elongation factor 1-α (TEF1) gene was amplified using the primers EF2F (Marincowitz et al. 2015) and EF2R (Jacobs et al. 2004). For all other fungi, the internal transcribed spacer regions 1 and 2 (ITS), including the 5.8S region, were amplified using the primers ITS1F and ITS4 (White et al. 1990; Gardes and Bruns 1993), the TUB2 gene was amplified using the primers Bt2a and Bt2b (Glass and Donaldson 1995), and the TEF1 gene was amplified using the primers F-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998). In addition, the calmodulin (CAL) gene was amplified with the primer pairs CL1 and CL2a (O’Donnell et al. 2000) or CL3F and CL3R (de Beer et al. 2016) for fungi that reside in the genus Sporothrix. For new Sporothrix species, LSU region was amplified using the primers LR0R and LR5 (Vilgalys and Hester 1990). PCR and sequencing were conducted following the protocols described by Jankowiak et al. (2019c). All sequences obtained in this study were deposited in GenBank. The obtained ITS/LSU sequences were compared with sequences in NCBI GenBank for preliminary identifications and were used to determine generic placement in the Ophiostomatales. For Leptographium and Ophiostoma spp. the TUB2 and TEF1 datasets were analyzed separately for each species complex. For Sporothrix spp., the CAL, TUB2 and TEF1 datasets were analyzed across the entire genus.

Phylogenetic trees were generated independently for each gene. Resulting trees were visually compared for topological incongruences. Genes showing no topological incongruence for Sporothrix spp. were combined and analyzed as a concatenated dataset. Sequence alignments were performed using the online version of MAFFT v7 (Katoh and Standley 2013). The ITS, LSU, TUB2, CAL, and TEF1 datasets were aligned using the E-INS-i strategy with a 200PAM/k=2 scoring matrix, a gap opening penalty of 1.53 and an offset value of 0.00. The alignments were checked manually with BioEdit v.2.7.5 (Hall 1999). Phylogenetic trees were inferred for each of the datasets using three different methods: Maximum likelihood (ML), Maximum Parsimony (MP), and Bayesian inference (BI). For ML and BI analyses, the best-fit substitution models for each aligned dataset were established using the corrected Akaike Information Criterion (AIC) in jModelTest 2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012). ML analyses were carried out with PhyML 3.0 (Guindon et al. 2010), utilizing the Montpelier online server (http://www.atgc-montpellier.fr/phyml/). Node support values and the overall reliability of the ML tree topology were assessed using 1000 bootstrap pseudoreplicates.

MP analyses were performed using PAUP* 4.0b10 (Swofford 2003). Gaps were treated as a fifth state. Confidence levels for the nodes within the inferred tree topologies were determined using 1000 bootstrap replicates. Tree bisection and reconnection (TBR) were selected as the branch swapping option. The tree length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI), and Rescaled Consistency Index (RC) were recorded for each analyzed dataset after the trees were generated.

BI analyses using Markov Chain Monte Carlo (MCMC) methods were carried out with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003). Four MCMC chains were run for 10 million generations applying the best-fit model for each dataset. Trees were sampled every 100 generations, resulting in 100,000 trees. Tracer v1.4.1 (Rambaut and Drummond 2007) was used to determine the burn-in value for each dataset. The remaining trees were used to generate a 50% majority rule consensus tree, which allowed for calculating posterior probability values for the nodes. All alignments and trees were deposited into TreeBASE (Reviewer access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S29855?x-access-code=62dd9f4ad30f131a52104b44860daf9e&format=html).

In total, 623 Ophiostomatales isolates were obtained from 2400 wooden samples (six pieces from each of 400 branches; Table 2). Five hundred and forty-one isolates were collected from pine, 79 isolates were collected from oak, and three isolates were collected from Norway spruce. No isolates were obtained from the beech wooden fragments (Table 2).

Based on morphological observations, the fungal isolates obtained from this study were arranged into 10 species. Five fungal species were isolated from pine while six species were isolated from oak fragments. Only one species (designated as taxon 10; Table 2) was isolated from pine, oak, and spruce samples. The most frequently isolated fungi were taxon 2 and taxon 10 represented by 263 and 174 isolates, respectively. The third most abundant fungus was named as taxon 9, which was isolated 66 times. In addition, 53 isolates of taxon 6 were gathered from buried oak branches (Table 2).

Based on analysis of ITS and LSU sequence data, of the 623 isolates collected in this study, 305, 298, 17 and 3 isolates resided in Sporothrix (Fig. 1), Leptographium (Suppl. material 2: fig. S3), Ophiostoma and Heinzbutinia (Suppl. material 2: fig. S4), respectively. Most of the isolates belonging to Leptographium grouped in the L. procerum species complex, while most of the isolates belonging to Sporothrix nested in the S. inflata species complex. Phylogenetic analyses of these datasets separated the isolates into 11 distinct taxa, eight of which were previously described species and three represented novel species.

Figure 1. 

Phylogram from Maximum Likelihood (ML) analyses of ITS data for Sporothrix spp. Sequences obtained in this study are in bold. Bootstrap values (if ≥ 75%) for ML and Maximum Parsimony (MP) analyses are presented at the nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values < 75%. The tree is drawn to scale (see bar) with branch lengths measured in the number of substitutions per site. Graphilbum fragrans represents the outgroup.

In the genus Heinzbutinia, analyses of TUB2 sequences data (Suppl. material 2: fig. S5) showed that taxon 1 belonged to Heinzbutinia grandicarpa (Kowalski & Butin) Z.W. de Beer & M. Procter. In Leptographium genus, taxon 2 was represented by nine isolates grouping in the L. procerum species complex (Suppl. material 2: fig. S3) and TUB2 and TEF1 sequence analyses confirmed this taxon was conspecific with L. procerum (W.B. Kendr.) M.J. Wingf. (Suppl. material 2: figs S6, S7). Taxon 3 was represented by four isolates that grouped in the L. galeiforme species complex (Suppl. material 2: fig. S3) and TUB2 and TEF1 sequence analyses confirmed that these isolates represented L. radiaticola (J.J. Kim, Seifert & G.H. Kim) M. Procter & Z.W. de Beer (Suppl. material 2: figs S8, S9).

In the genus Ophiostoma taxon 4 was represented by two isolates that did not group in any species complex (Suppl. material 2: fig. S4). Analyses of TUB2 sequence data (sSuppl. material 2: fig. S5) showed that this taxon belongs to O. piliferum (Fr.) Syd. & P. Syd. Taxon 5 was represented by four isolates in the O. ulmi species complex (Suppl. material 2: fig. S4), while TUB2 sequences grouped this taxon with O. quercus (Georgev.) Nannf. (Suppl. material 2: fig. S5).

In the genus Sporothrix, the four isolates of taxon 7 resided in the S. inflata species complex and grouped with the ex-type isolate of S. dentifunda (Aghayeva & M.J. Wingf.) Z.W. de Beer, T.A. Duong & M.J. Wingf. based on the ITS, TUB2, CAL, and TEF1 phylogenies (Figs 1–4). Taxa 9 and 10 also belonged to the S. inflata species complex (Fig. 1) as defined by de Beer et al. (2022) and were represented by four and seven isolates, respectively. Based on the TUB2 phylogeny, taxon 9 was close to S. dimorphospora and ‘S. inflata 2’ and formed a distinct and well-supported clade, while taxon 10 formed a distinct and well-supported clade which included isolates of ‘S. inflata 2’ (Fig. 2). Based on the CAL phylogeny, taxa 9 and 10 formed two distinct and well-supported clades which were close to, but distinct from S. dimorphospora (Fig. 3). Based on the TEF1 sequence data (Fig. 4), both taxa formed distinct and well-supported clades, and thus represented novel species. This was supported by the combined analyses of the ITS, TUB2 and CAL datasets (Fig. 5). Taxon 8 was represented by one isolate and grouped in the S. gossypina & S. stenoceras species complexes (Fig. 1). TUB2, CAL, and TEF1 phylogenies (Figs 2–4) showed that this taxon is S. eucastaneae (R.W. Davidson) Z.W. de Beer, T.A. Duong & M.J. Wingf. Taxon 11 was represented by two isolates collected from fallen pine shoots and did not group in any species complex (Fig. 1). The combined analyses of the ITS, TUB2, and CAL datasets (Fig. 5) showed that this taxon formed a distinct and well-supported clade which was closest to, but clearly distinct from S. macroconidia H.M. Wang, Q. Lu & Zhen Zhang, and thus represented a novel species.

Figure 2. 

Phylogram from Maximum Likelihood (ML) analyses of TUB2 data for Sporothrix spp. Sequences obtained in this study are in bold. Bootstrap values (if ≥ 75%) for ML and Maximum Parsimony (MP) analyses are presented at the nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values < 75%. The tree is drawn to scale (see bar) with branch lengths measured in the number of substitutions per site. Graphilbum fragrans represents the outgroup.

Figure 3. 

Phylogram from Maximum Likelihood (ML) analyses of CAL data for Sporothrix spp. Sequences obtained in this study are in bold. Bootstrap values (if ≥ 75%) for ML and Maximum Parsimony (MP) analyses are presented at the nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values < 75%. The tree is drawn to scale (see bar) with branch lengths measured in the number of substitutions per site. Graphilbum fragrans represents the outgroup.

Figure 4. 

Phylogram from Maximum Likelihood (ML) analyses of TEF1 data for the Sporothrix spp. Sequences obtained in this study are in bold. Bootstrap values (if ≥ 75%) for ML and Maximum Parsimony (MP) analyses are presented at the nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values < 75%. The tree is drawn to scale (see bar) with branch lengths measured in the number of substitutions per site. Graphilbum fragrans represents the outgroup.

Figure 5. 

Phylogram from Maximum Likelihood (ML) analyses of the combined datasets of ITS+BT+CAL for Sporothrix spp. Sequences obtained in this study are in bold. Bootstrap values (if ≥ 75%) for ML and Maximum Parsimony (MP) analyses are presented at the nodes as follows: ML/MP. Bold branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap values < 75%. The tree is drawn to scale (see bar) with branch lengths measured in the number of substitutions per site. Graphilbum fragrans represents the outgroup.

Taxon 6 was represented by nine isolates grouped separately from Sporothrix and belonged to lineage XIX (Fig. 1) as defined by de Beer et al. (2022). Analyses of TUB2 and CAL sequences data (Figs 2, 3) showed that this taxon is Sporothrix brunneoviolacea.